UNIVERSIDADE DE LISBOA
FACULDADE DE CIÊNCIAS
DEPARTAMENTO DE BIOLOGIA ANIMAL
The Intermediate Mesoderm,
a new player in the establishment of organ laterality?
Dalila Maria Neves Silva
Mestrado em Biologia Evolutiva e do Desenvolvimento
Dissertação orientada por:
Leonor Saúde
(Instituto de Medicina Molecular)
Sólveig Thorsteinsdóttir
(Faculdade de Ciências da Universidade de Lisboa)
2017
II
Acknowledgements
Parece que cheguei ao fim! Uns neurónios a menos, umas dores nas costas a mais, mas já está!!
E Até parece que foi difícil… por favor… de onde é que tiraram essa ideia?! Mas sabem que mais?
Porque por muito que os dias possam ter sido longos ou que as noites mal dormidas, que quase nada
tenha funcionado à primeira (but hey, that’s science baby ;D), ou que tenha “panicado” algumas vezes
(vá… quase sempre xD), estive sempre acompanhada pelas melhores pessoas que podia ter! E agora
chegou a altura de lhes agradecer toda a paciência e disponibilidade que tiveram comigo.
Quero começar por agradecer à chefe mais FANTÁSTICA! que poderia existir. Muito obrigada
Leonor por me ter deixado fazer parte deste grupo cheio de pessoas incríveis e que me ensinaram tanto
não só durante a minha primeira aventura pelo mundo da Ciência como durante a minha primeira estadia
cá à 3 anos. Obrigada por toda a paciência que teve, por toda a disponibilidade e sabedoria que partilhou
comigo.
À Professora Solveig não posso deixar de agradecer o facto de ter sido minha orientadora interna
durante este ano, como também por me ter ajudado na escolha deste mestrado como durante o meu
primeiro ano. Tenho ainda que lhe agradecer, juntamente com a Professora Gabriela, todo o entusiasmo
que transmitem durante as vossas aulas. Foram sem dúvida peças chave na minha escolha.
Um ENORME (porque ela é GIGANTE!!) e especial obrigada à minha querida MINI BOSS!!!
:D Sem ti, I don’t know… Obrigada por toda a paciência para as perguntas parvas, por me deixares
“roubar” as tuas coisas (incluindo a tua cadeira confortável *.*), por me ensinares todas as tuas manias,
por me ajudares com as contas e com o inglês (See?? I’m so much better now :3). Obrigada por te
armares em psicóloga e me tentares motivar, muitas vezes sem sucesso, quando estava mais em baixo,
aaaa….. o que é que eu posso dizer…. obrigada … I don’t know, for EVERYTHING!!!!! Ah! E desculpa
lá qualquer coisinha  (just in case I will always have puppy photos on my phone eheh!)
Guida e Isaura durante este ano a vossa boa disposição foi contagiante, e refrescante. Obrigada
pelos almoços e cafés, por me terem feito companhia durante 1h na sala da matança do Serial Killer
eheh :P Vocês tornaram este ano muito mais animado . Obrigada Lara por me teres acolhido tão bem
na fish da primeira vez que estive aí, pelas conversas quando os peixes não colaboravam comigo
(continuo a achar que alguns deles não gostavam muito de mim), por me ajudares a cortar caudas e por
me ensinares como funciona uma facillity . Minha querida Aida! Obrigada pela tua simpatia e pelos
boost de confiança às quartas. Sem ti não tinham embarcado nesta aventura, obrigada por seres a relações
publicas e por me “meteres a cassete” que era ai que devia fazer o mestrado  Ah e é claro, um obrigada
à Mariana . Caríssimo Diogo, obrigada não só pelas cantorias no lab e pelas belas tostas no bar como
também por levar o meu cartão a passear enquanto eu ficava a trabalhar :P. Zé, obrigada por me teres
deixado ensinar-te o pouco que sei . É claro que também tenho que agradecer ao restante grupo, Sofia,
Rita, Ana e Tiago por terem estado lá e por me terem ajudado em diversos momento ao longo deste
ano. E como a ciência não se faz sozinha, quero agradecer aos Grupos de Bioimaging e Histologia,
por toda a ajuda e brainstorming, à Fish Facillity, por terem me ajudado com os peixes mais difíceis e
cuidado tão bem deles, ao Grupo do Domingos Henrique, por toda a partilha e disponibilidade, ao
Grupo da Susana Lopes, e em especial à Raquel Jacinto por me ter acolhido durante 1 semana no
vosso laboratório, ao Grupo do António Jacinto, por me ter incluído nos seus programas não sei muito
bem porquê ;), e por último aos meus amigos!
Não posso deixar de agradecer a um dos grandes pilares da minha vida, à minha família! Muito
obrigada por todos os sacrifícios que fazem e por apoiarem e ajudarem sempre. Eu sei que nem não sou
fácil de aturar muitas vezes e que nem sempre demonstro o quanto gosto de vocês, mas quero que saibam
que sem vocês a minha vida não fazia sentido! Por último queria agradecer ao meu namorado. Jorge,
obrigada por me apoiares, ajudares e motivares “even when the skies get rough”, não só nisto mas em
tudo.
…À Pua e ao Pantufa!
I
II
Resumo
A simetria bilateral é uma característica comum nos vertebrados. No entanto, os nossos órgãos
internos, tais como o coração, intestino, pâncreas e fígado, estão assimetricamente posicionados no que
diz respeito ao eixo Esquerda-Direita (ED) de uma forma conservada chamada “situs solitus”.
O estabelecimento da assimetria ED é um passo crucial na organogénese e envolve uma série
de eventos moleculares e morfogenéticos. Caso ocorram anomalias durante o estabelecimento da
assimetria, estas podem levar a situações incompatíveis com a vida. No peixe-zebra, este processo
começa com a quebra da simetria, na qual, os transportadores de iões, que estão assimetricamente
distribuídos durante a clivagem no embrião, criam diferenças no potencial de voltagem da membrana
assim como no pH entre o lado esquerdo e direito. Depois disto, o organizador ED, que se chama
vesicula de Kupffer (KV) no peixe-zebra, é formado, num processo que se inicia entre as 4 e as 6 horaspós-fertilização (hpf), a partir de um grupo de células localizadas numa zona adjacente ao escudo
embrionário, as dorsal forerunner cells (DFC). Quando a KV está formada, uma libertação assimétrica
de cálcio ativa a cascata Nodal (sinalização TGF-β). A primeira proteína a ser expressa assimetricamente
é a proteína secretada Dand5, que pertence à família Cerberus/Dan, que é uma conhecida inibidora de
TGF-β. Esta proteína está expressa do lado direito da KV e antagoniza Spaw (Nodal-related) do lado
direito que vai viajar desde a KV em direção à Mesoderme Lateral (LPM) esquerda através de
mecanismos ainda não conhecidos entre os estádios de 10 e 12 somitos. Na LPM esquerda, Spaw vai
induzir a sua própria expressão, por feedback positivo, bem como a dos seus reguladores negativos, os
genes lefty, e o factor de transcrição homeobox pitx2. Os mecanismos morfogenéticos que acontecem
downsteam de Pitx2 vão promover o estabelecimento da assimetria dos órgãos.
Normalmente consideramos que os eventos que acontecem na LPM controlam a assimetria do
coração, intestino, fígado e pâncreas. No entanto apenas o coração deriva exclusivamente da LPM,
enquanto os restantes órgãos previamente enumerados provêm maioritariamente de uma associação
entre a LPM e a endoderme. No peixe-zebra, o coração é o primeiro órgão a tornar-se assimétrico de
forma a adquirir a sua conformação e função. Para que isso aconteça, dois eventos específicos de
assimetria ocorrem, um entre as 24 e as 36 hpf (o jog do coração), e o segundo imediatamente depois
até as 48 hpf, denominado de loop do coração. Estes processos estão, respetivamente, direta e
indiretamente sob a influência da sinalização Nodal. Neste organismo, a torção do intestino dá-se entre
as 26 e as 30 hpf na zona que corresponde aos somitos 1 a 3. Para que este processo ocorra a LPM tem
que migrar assimetricamente levando à torção do intestino.
No entanto ainda não é conhecida como é que os sinais ED são transferidos desde a KV até à
LPM esquerda. Neste projeto, nós propomos que a Mesoderme Intermédia (IM), um tecido embrionário,
e/ou os Pronéferos (PN), um dos derivados da IM, possam ter um papel durante o estabelecimento da
assimetria ED, no peixe-zebra. Propomos que este papel se dê através da ação da proteína de adesão
Caderina-11 (Cdh11), um tipo de molécula já implicada no estabelecimento da assimetria ED em galinha
e na Drosophila. A nossa hipótese assume a IM como um possível mediador da transferência de Spaw
da KV para a LPM entre os estádios de 10 a 12 somitos. Relativamente aos PN, nós propomos que
possam atuar como uma estrutura de suporte para a migração assimétrica da LPM que tem como
finalidade a torção do intestino para a esquerda.
Quando se elimina com o auxilio de um morfolino (MO) a Cdh11, podemos ver que, na maioria
dos embriões afetados, parece não ter havido quebra de simetria no que diz respeito ao coração, e que a
assimetria do intestino foi revertida. Este resultado sugere que o estabelecimento da assimetria ED ao
longo do eixo Anterior-Posterior pode ser promovida em diferentes módulos.
Quando analisamos a expressão de spaw e pitx2 em embriões injetados com o MO da Cdh11
verificamos que estes estão maioritariamente expressos na LPM direita (expressão revertida). Sabe-se
que a migração assimétrica da LPM que leva à torção do intestino está dependente de sinais utilizados
durante o estabelecimento da assimetria ED. Esta expressão de spaw na LPM direita consegue explicar
a reversão do intestino observada nesta condição, mas não consegue explicar o fenótipo observado no
coração. No entanto, propomos que a Cdh11 possa estar a influenciar a expansão anterior de spaw e de
pitx2. A falta da expressão destes genes nos territórios da LPM anterior, que vão dar origem ao coração,
não irão permitir a quebra da simetria deste órgão. Durante este projeto nós não fomos capazes de
identificar esta molécula nos tecidos associados à assimetria ED, no entanto, esta hipótese levanta a
III
questão da presença da Cdh11 na KV. Mais ensaios têm que ser feitos, de forma a responder de forma
definitiva a esta questão.
Uma vez que os resultados obtidos com recurso a MOs tem sido questionada, resolvemos
analisar um mutante para a Cdh11 como técnica complementar. Para isso encomendamos do European
Zebrafish Resource Center um mutante no qual uma mutação pontual no 454º aminoácido que leva à
formação de um codão STOP prematuro e por consequência, caso a proteína seja formada, esta será
truncada. Ao analisar este mutante não fomos capazes de reproduzir nenhum dos fenótipos obtidos
previamente publicados, nem como os obtidos no decorrer deste trabalho usando o MO para Cdh11.
Esta análise levantou algumas questões não só em relação à especificidade do MO, como se o mutante
é nulo ou não. De forma a perceber em que cenário nos encontramos, iremos realizar alguns testes, tais
como “rescues”, Microarray ou RNA sequencing, e também gerar um novo mutante para a Cdh11.
Apesar de não termos ainda provas do papel da IM no estabelecimento da assimetria ED através
da Cdh11, não queremos deixar esta ideia de lado. Utilizando outros genes e técnicas, tais como a análise
de mutantes simples para pax2a, pax8 e ors e mutantes duplos para pax2 e pax8, e ainda gerar e analisar
uma linha de ablação para Caderina 17, pretendemos avaliar a necessidade deste tecido no
estabelecimento da assimetria ED.
Palavra-chave: Mesoderme Intermédia; Assimetria Esquerda-Direita; Caderina-11; Peixezebra; Morfolino; Mutante.
IV
Abstract
External bilateral symmetry in vertebrates hides internal organ asymmetries. The establishment
of these asymmetries is a crucial initial step in organogenesis and results from a series of molecular and
morphogenetic events. In zebrafish, the establishment of the Left-Right (LR) asymmetry occurs
essentially in four main steps: the breaking of bilateral symmetry during the cleavage stages, the
formation of the Kupffer's vesicle (KV) at early somitogenesis, the activation of the Nodal Cascade in
the lateral plate mesoderm (LPM), and the left- or right-specific morphogenesis of the organs. However,
the processes through which communication is established between the KV and the LPM and the
morphogenetic dynamics that potentiate, liver, pancreas and gut asymmetries are still poorly understood.
In this work, we propose that the Intermediate Mesoderm (IM), an embryonic tissue that lays
between the LPM and the KV, or its derivative, the Pronephros (PN), might play a role in the
establishment of LR asymmetry in zebrafish. We propose that this role could be done through the action
of Cadherin-11 (Cdh11) protein. This type of adhesion molecules has already been implicated in the LR
asymmetry in other organisms. Our hypothesis assumes that the IM could work as a mediator of the
transfer of Spaw from the KV to the LPM at 10 to 12-somite stages. Regarding the PN, we envision that
they could work as stable structure to sustain the LPM asymmetric migration that ultimately leads to the
initial shift of the gut tube to the left side of the body.
A loss-of-function approach to knockdown Cdh11 revealed that the majority of the affected
embryos exhibit laterality defects in the gut (reversed phenotype) and in the heart (No Loop). The lack
of concordance between the laterality of the gut and heart suggest that the establishment of LR
asymmetry along the AP axis reinforces the idea that laterality is promoted by different modules. It is
known that asymmetric LPM migration is dependent on the normal LR pathway cues and is essential to
impose the laterality of the gut. The analysis of the expression of the conserved LR genes (spaw and
pitx2), that are expressed, in the majority of the morphant embryos, on the right LPM instead of the left
LPM, can explain the reversion observed in the gut in this condition but cannot explain the one detected
in the heart. We propose that Cdh11 could influence the anterior expression of spaw and consequentially
pitx2 in the LPM, and that the lack of these genes in the anterior heart prospective territory would not
allow the break of symmetry. During this project we were not able to detect the Cdh11 in any of the
known LR patterning tissues, however this result raises the question that Cdh11 could work at the KV
level. More assays need to be done in order to address this question.
We also ordered and analyzed a cdh11 mutant as a complementary approach. We were not able
to reproduce any of the phenotypes observed with the cdh11MO. This assay raises questions about the
cdh11 MO specificity and the quality of the null cdh11 mutant. To understand which scenario is true, we
will perform a series of tests such as “rescues”, microarray or RNA sequencing and even generate a new
cdh11 mutant.
Even though we were not able to produce any convincing evidence that the IM plays a role in
the establishment of LR asymmetry, through the expression of Cdh11, we want to further investigate
the necessity of this tissue in LR asymmetry using other approaches. Therefore, we will analyze IM
mutants as well as generate a targeted cell ablation line under the control of the Cdh17 promoter.
Keywords: Intermediate Mesoderm; Left-Right Asymmetry; Cadherin-11; Zebrafish;
Morpholino; Mutant.
V
Abbreviations
AP – Anterior-Posterior;
DV – Dorsal-Ventral;
LR – Left-Right;
LRO – Left-Right Organizer;
KV – Kupffer's vesicle;
DFC – Dorsal Forerunner Cells;
hpf - hours post-fertilization;
TGF- β - transforming growth factor-β;
Spaw – Southpaw;
LPM - lateral plate mesoderm;
aPKC - atypical protein kinase C;
ZO-1 - zonula occludens 1;
IM - Intermediate Mesoderm;
PN – Pronephros;
i.e – That is;
Cdh – Cadherin;
EC – Extracellular Domain;
TM - Transmembrane Domain;
IC – Intracellular Domain;
Cdh2 – N-cadherin;
HH - Hamburger and Hamilton;
DM – Dorsal Mesentery;
Cdh11 – Cadherin-11;
WT – Wild Type;
EZRC - European Zebrafish Resource Center;
gDNA – genomic Deoxyribonucleic acid;
PK – Proteinase K;
RT – room temperature;
PCR – Polymerase Chain Reaction;
Fw – Forward;
Rv – Reverse;
MO - Morpholino oligonucleotides;
cdh11MO - cdh11 splice blocking MO;
CtrMO - Standard Control MO;
DIG – Dioxigenin;
WISH - Whole mount in situ hybridization;
s – seconds;
h – hours;
Pre-Hybmix - pre-Hybridization Mix;
PBS - Phosphate-buffered saline;
PFA – Paraformaldehyde;
MetOH – Methanol;
Abs/Ab – Antiboies/Antibody;
BSA - Bovine Serum Albumin;
qPCR – quantitative PCR;
CRISPR - Clustered Regularly Interspaced Short Palindromic Repeat;
gRNA - single-guide RNA;
Cas9 - CRISPR associated protein 9;
NTR – Nitroreductase;
Mtz – Metronidazole.
VI
Index
Acknowledgements
I
Resumo
III
Abstract
V
Abbreviations
VI
Chapter 1 – Introduction
1.1 - Symmetry and Asymmetry
1.2 - Left-Right Asymmetry
1.3 – Left-Right Asymmetry in Zebrafish
1.3.1 - Step 1- The breaking of symmetry
1.3.2 - Step 2 - The Left-Right Organizer formation
1.3.3 - Step 3- The Nodal Cascade
1.3.4 - Step 4 - Establishment of organ asymmetry
1.4 - The Intermediate Mesoderm, a new player in the Left-Right asymmetry?
1.5 – Cadherins
1.5.1 – Cadherins in Left-Right asymmetry
1.5.2 – Cadherin-11 in zebrafish
1.6 – Objectives
1
1
1
2
2
3
3
4
6
6
7
9
10
Chapter 2 – Experimental Procedures
2.1 - Zebrafish maintenance and care
2.2 - Zebrafish genotyping
2.2.1 - Caudal fin amputation and Phenol-chloroform genomic DNA (gDNA) extraction
2.2.2 - Polymerase Chain Reaction (PCR)
2.3 - cdh11 Knockdown
2.3.1 - Morpholino oligonucleotides (MO)
2.3.2 - MO preparation
2.3.3 - Embryo microinjections
2.4 - Anti-sense mRNA probe synthesis for Whole mount in situ hybridization (WISH)
2.4.1 - Transformation of competent Escherichia coli bacteria
2.4.2 - DNA Plasmid linearization
2.4.3 - in vitro transcription
2.4.4 - Probe preparation
2.5 - Whole mount in situ hybridization
2.5.1 - in situ hybridization imaging
2.6 - Embryo embedding, preparation of cryostat sections and immunostaining - Protocol
optimization
2.6.1 - Embryo embedding
2.6.2 – Cryosectioning
2.6.3 - Immunostaining protocol
2.6.4 - Immunostaining imaging
2.6.5 – Notes
2.7 - Cdh11 protein pattern analysis
2.7.1 - Cdh11 specific antibodies (Abs)
2.7.2 - Immunostaining protocol
2.7.3 - Western Blot
2.8 - Agarose gel electrophoresis
12
12
12
12
13
13
13
13
13
14
14
14
14
14
14
15
Chapter 3 – Results
3.1 - Analysis of the cdh11 morphant phenotype
19
19
15
15
16
16
16
16
16
16
17
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18
VII
3.1.1 - cdh11 knockdown affects Left-Right positioning of the organs.
3.1.2 - cdh11 does not seem to be expressed in any of the known Left-Right patterning tissues
3.1.3 – Intermediate Mesoderm formation does not seem to be affected in cdh11 morphants
3.1.4 - Do the pronephric ducts provide a stable structure for the asymmetric migration of the
underlying LPM?
3.1.5 - Conserved Nodal cascade components, spaw and pitx2, became expressed on the right
LPM in cdh11 morphants
3.2 - Analysis of the cdh11 mutant phenotype, a complementary approach.
3.2.1 – cdh11 mutants are homozygous viable
3.2.2 - cdh11 mutants do not exhibit the previously phenotypes observed in cdh11 morphants
3.2.3 - The Intermediate Mesoderm is formed properly in cdh11 mutants
3.3 – Does the lack of Cdh11 activates a genetic compensation program in cdh11 mutants?
The possible roles of Cdh6 and Cdh17 in Left-Right asymmetry
19
21
23
Chapter 4 – Discussion and Future Directions
4.1 - cdh11 morphants exhibit laterality problems
4.1.1 - A new example of uncoupling laterality of the gut and heart
4.1.2 – The affected conserved LR genes can explain the organ laterality defects observed
4.1.3 – Does Cdh11 influence the KV fluid flow and dand5 expression?
4.1.3.1 – Is Cdh11 expressed in the KV?
4.2 – Mutant vs Morpholino
4.2.1 - Which one is the best approach?
4.2.2 – A new cdh11 mutant
4.3 – Alternative ways to understand the role of the intermediate mesoderm in Left-Right
asymmetry
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34
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36
Chapter 5 - Bibliography
38
Attachments
A1 – Recipes
A2 – Supplementary Tables
A3 – Supplementary Figures
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49
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25
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27
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VIII
Chapter 1 – Introduction
1.1 - Symmetry and Asymmetry
During development of a new organism, a number of molecular cues are used by the embryo to
specify the Anterior-Posterior (AP) and Dorsal-Ventral (DV) axes [1,2]. During this body plan
specification, symmetry is used as a guide line and, it has also been suggested that this feature is used
as a measurement of genetic fitness of a potential mate during sexual selection [3]. Vertebrates belong
to the phyla bilateria, since they only have one plane of symmetry, however this only occurs on the
outside of the body. In these animals, internal organs such as the heart, gut, liver, pancreas and spleen,
the nervous system (particularly among higher mammals) and the asymmetric development of paired
organs (such as the lungs) use the preexisting positional cues, to organize themselves asymmetrically
with respect to the Left-Right (LR) axis in a conserved way called situs solitus (Figure 1) [1-7].
Evolutionarily, several reasons for the appearance of asymmetry in organisms have been
proposed, and one of these indicates that asymmetry came from the necessity that individuals had to
prevent malformations and to maximize the surface area of their tubes in order to maximize the
absorption of ingested nutrients [3]. This hypothesis describes that, during evolution, due to the lack of
space to accommodate the entire gut’s length, this organ had to be packaged asymmetrically. Thus, the
looping and consequentially, the function of the remaining organs had to be rearranged to occupy the
space left in order to perform their functions in a normal way [3].
Situs solitus
Right isomerism
Situs inversus
Dextrocardia
Left isomerism
Heterotaxy
R
L
Figure 1 – Human laterality disorders adapted. Schematic representation of normal Left Right body
asymmetry (situs solitus) and five laterality defects: situs inversus, Left isomerism (polysplenia), Right
isomerism (asplania), Dextrocardia and Heterotaxy. R – Right; L- Left. Adapted from [8].
1.2 - Left-Right Asymmetry
Understanding how LR asymmetry is established is not only an important subject in the field
of developmental biology, but also, further investigation of this event can assist in clinical therapies.
1
The formation of the LR axis is a crucial initial step during organogenesis that normally does not
undergo alterations, however, 1 in 8.000 live births show one of the several basic types of abnormalities
in the establishment of LR asymmetry [1,2]. Situs inversus is the only condition that is not lifethreatening, and it is characterized by a complete inversion of the internal organs disposition along the
LR axis and occur in 1 in 20.000 human births (Figure 1) [1-5]. The other cases are: Isomerism, in
which there is a loss of asymmetry, and it can be divided into left isomerism (polysplenia), where the
organism has a duplication of some of the single organs, or right isomerism (asplenia), that shows a
complete loss of single organs such as the spleen; Dextrocardia, that consists on reversed heart
positioning and morphology; and Heterotaxy, characterized by the independent decision of each organ
regarding their position relatively to the LR axis, being also the most life threatening condition (Figure
1) [1,2,4,6].
1.3 – Left-Right Asymmetry in Zebrafish
The mechanisms that act during the establishment of LR asymmetry are highly conserved across
the different vertebrate model organisms [6,9,10]. In zebrafish, the establishment of the LR asymmetry
pattern occurs essentially in four main steps: the breaking of bilateral symmetry during the cleavage
stages, the formation of the Left-Right Organizer (LRO) at early somitogenesis stages, the activation of
the Nodal Cascade in the LPM and, the left- or right-specific morphogenesis in the organs occurring
downstream of the Nodal cascade (Figure 2) [9].
1.3.1 - Step 1- The breaking of symmetry
During cleavage stages, an asymmetric membrane polarization promotes the accumulation of
LR determinants, such as serotonin, through directional transport involving gap-junction channels. This
is caused by ion transporters, such as H+/K+ ATPases, that are asymmetrically distributed in the embryo
and create differences in the pH and in the membrane voltage potential between the left and right side
(Figure 2A) [6,11].
C
B
Figure 2 – The establishment of Left-Right asymmetry in zebrafish. A - Ion transporters are asymmetrically distributed
in the embryo and create differences in the pH and in the membrane voltage potential between the left and right side; B After KV formation, the directional flow will impact on LR according to “Two cilia model” or the “Morphogen Gradient
Model”; C- Nodal Cascade genes are activated on the left side of the embryo. Spaw (green) reaches the left LPM without
the antagonism of Dand5 (red). Green arrow- Leftward flow; Red arrow – Ca2+ release; Stars – Determinant molecule.
Adapted from [6,7,9].
2
1.3.2 - Step 2 - The Left-Right Organizer formation
The formation of the LRO, called Kupffer's vesicle (KV) in zebrafish, begins with the
specification of a group of 20 to 30 dorsal surface epithelial cells that will originate the dorsal forerunner
cells (DFC), in the region adjacent to the embryonic shield, between 4 to 6 hours-post-fertilization (hpf),
in response to Nodal signaling. By late gastrulation, DFC form a cluster that migrates towards the vegetal
pole, in a process controlled by different cell adhesion processes (collective migration). The DFC cluster
undergoes compaction and becomes a bottle-shaped polarized structure, later giving rise to a multiple
3D rosette structures. At early somitogenesis (4 to 6-somite stages), the formation of the lumen begins.
At this point, the 3D structures rearranges into a single rosette structure (Figure 3) [9].
The KV organogenesis ends with lumen formation followed by ciliogenesis [6,9]. On the
anterior-left side of the KV, the flow created by
the motile cilia, present in KV epithelia, is
stronger [12]. This flow is necessary and
sufficient to activate the Nodal Cascade on the left
side of the embryo (Step 3 of the LR patterning)
(Figures 2B,C) [6,9,13]. How this flow impacts
on LR asymmetry is still controversial. According
to the “Two cilia model” the mechanosensory
cilia present in KV epithelia sense the directional
fluid flow created by motile cilia and may trigger
the asymmetric Ca2+ release on the left side of the
KV. On the other hand, the “Morphogen Gradient
Model” describes that the flow transports a
determinant molecule towards the left side where
its accumulates and, once more may trigger the
asymmetric Ca2+ release (Figures 2B) [7]. Recent
findings suggest that intraciliary Ca2+ signaling is
necessary for the LR asymmetry and that it
depend on ciliary motility and polycystin-2, a
cation channel required for sensing ciliary
motility [14].
Figure 3 – From DFC to KV. This process is divided into
three steps: DFC specification, Collective migration and
KV organogenesis. Adapted from [9].
1.3.3 - Step 3- The Nodal Cascade
The Nodal cascade is a well conserved pathway known to be present in both protostomes,
deuterostomes and possibly in their last common ancestor, the urbilateria (Supplementary Figure 1). So
far, this cascade has been shown to be required in the establishment of LR asymmetry in all characterized
organisms, however the specific mechanisms that activate this pathway differ among them [3,6,9]. In
the zebrafish, the transforming growth factor-β (TGF-β) effector involved in this cascade is southpaw
(spaw) which is expressed bilaterally, between 4 to 6-somite stages, in the cells that surround the KV
(Figure 2C) [9,15-17]. Other important protein required in the establishment of LR asymmetry, and the
first to become asymmetric, is the secreted protein Dand5, member of the Cerberus/Dan family and a
known TGF-β inhibitor [15,17]. At 5 to 6-somite stages, dand5 is expressed bilaterally in the KV cells
with a horseshoe-shaped pattern. Later, at 7-somite stage, dand5 expression begins to be restricted to
the right side of the KV. It is unknown if this switch is due to an upregulation of the dand5 levels on the
right side or if it corresponds to a downregulation on the left side of the KV [9,15]. After this period
(from 8-somite stages) the embryos show a right asymmetric expression of this gene [9,15]. Between
10 to 12-somite stages, Spaw will diffuse from the KV towards the LPM, through an unknown
3
mechanism. However, because Spaw is inhibited by Dand5, Spaw can only reach the left lateral plate
mesoderm (LPM) [9,18]. There, Spaw will induce a positive feedback on itself and amplify its own
expression from posterior to anterior until the 22-somite stage [6,9,18]. Simultaneously, Spaw will also
activate its own negative regulators, the lefty genes. Lefty1 is a diffusible inhibitor that will act on the
midline as a barrier while Lefty2 will confine the domain of spaw expression to the left LPM [6,9].
Afterwards, Spaw will also induce the homeobox transcription factor Pitx2, which becomes detectable
at 13-somite stage and its expression propagates anteriorly similar to spaw with a 2-somite stage delay
[18]. The morphogenetic mechanisms downstream of Pitx2 will promote the establishment of organ
asymmetry (Figure 2C) [6,9]. Disruptions in this cascade, such as Spaw knockdown using a morpholino
oligonucleotide technology, results in loss of asymmetric genes expression and organ laterality defects
[16,19,20].
1.3.4 - Step 4 - Establishment of organ asymmetry
It is assumed that the events that take place in the LPM controls the heart, gut, liver and pancreas
laterality. However, the heart is only one that derives exclusively from the LPM, while the gut, liver and
pancreas are mainly originated from the LPM in association with the endoderm.
During vertebrate development, the heart is the first organ to undergo asymmetric migrations to
achieve, in zebrafish, its S-shaped conformation, and ultimately its function. In this organism two
specific asymmetric events have to occur, the heart jogging and looping, that are known to be,
respectively, directly and indirectly influenced by Nodal signaling [20,22,23]. First, at 16 hpf, the
cardiac precursors are specified and differentiated (Figure 4A). At 22 hpf, a disk-shaped structure called
the cardiac cone is converted into a linear primitive heart tube (Figure 4B). Later, between 24 to 36 hpf,
the linear heart tube is positioned under the left eye (jogging) (Figures 4C,D). The final heart
conformation is achieved through the displacement of the ventricle towards the mid-line in a process
called cardiac looping, which is a conserved process in all vertebrates, and that starts at 36 hpf and
finishes at 48 hpf in zebrafish (Figure 4E) [20,22-24].
Figure 4 - Zebrafish heart development. A- Specification and differentiation of the cardiac precursors; B- Cardiac cone
structure; C and D – jogging, the linear heart tube is localized under the left eye; E – Cardiac looping. Normal D-shaped
heart conformation. Adapted from [21].
To acquire the correct asymmetric position of the pancreas and liver, the primitive intestine has
to loop. This event occurs between 26 to 30 hpf (Figure 5). In zebrafish, the digestive organs are
originated from a solid rod of endodermal cells localized in the midline. Surrounding this structure, and
at the same DV level, both LPMs are symmetrical U-shaped structures with strong apical localization
of atypical protein kinase C (aPKCs) in the middle of this U structure. aPKCs protein is usually colocalized with the tight junction protein zonula occludens (ZO-1), which indicates that this complex is
associated with a mature and polarized epithelium (Figures 5E,E’) [19,25,26]. At 26 hpf, both LPMs
start to migrate medially, with the left LPM moving dorsally and the right LPM ventrolateral to the
endoderm specifically at the gut looping region (between the first and the third somites) (Figures 5F,F’)
4
[19,27]. At this point, both LPMs are asymmetrically positioned. As a consequence of these movements,
the endodermal rod is constrained and still localized in the midline. Four hours later (30 hpf), the LPM
migration is complete. By this time, the gut has shifted to the left and shows up as a compact mass of
cells with little to no polarization and weak expression of aPKCs. Regarding the LPMs, the left one is
dorsal to the endoderm and exhibits columnar cells on the ventral arm of the U, while the right one is
placed in a ventrolateral position in relation to the endoderm and displays its columnar cells on the dorsal
side of the U arm (Figures 5G-I, 5G’-I’) [19].
Figure 5 – LPM asymmetric migrations promote gut looping in zebrafish between 26 and 30 hpf. Whole mount in situ
hybridization using foxA3 probe (endodermal marker) Before (A) and after (B) gut looping; C - Schematic representation
of the looped gut at 30 hpf, the blue lines indicate the transversal sections in G to I; D – Key for the schemes E’ to I’; E to
I – transversal sections through the endoderm and LPM of a sox17:EGFP transgenic embryo (red – aPKC, green – phalloidin
488, dorsal to the top); E’ to I’ – Schemes of the relative positions of the LPM and endoderm in E to I. Adapted from [19].
It was demonstrated, through the analysis of mutant lines like bonnie and clyde (a line with a
reduced number of endodermal cells – Supplementary Figures 2A,B), heart and soul (a line with defects
in asymmetric organ morphogenesis – Supplementary Figure 2D) and nagie oko (a line that fails to
establish the epithelial polarity - Supplementary Figure 2E) that the asymmetric migration of the LPM
is necessary for correct gut looping and is independent of the endoderm. It was also shown, using
morpholino antisense oligonucleotide technology that both LPM migration and gut looping depend on
normal LR positional cues. This suggest that the dynamic asymmetric migration of the LPM over the
endoderm and downstream of the Nodal cascade leads to the gut displacement. However, the
mechanisms that drive LPM polarization and migration are not fully understood [19].
5
1.4 - The Intermediate Mesoderm, a new player in the Left-Right
asymmetry?
To establish organ asymmetry during development, different morphogenetic mechanisms have
to take place downstream of Pitx2 (Nodal cascade). However, how the LR signals are transferred from
the LRO to the left LPM remains a mystery. The Intermediate Mesoderm (IM) is an embryonic tissue
originated from a group of ventral mesodermal cells that lays between the KV and the LPM
(Supplementary Figure 3) [28], and thus possibly could play a role during this signal relay.
During zebrafish development, the IM gives rise to both blood cells and kidney [28]. The
pronephros (PN) is the first kidney to be formed during development and in teleost fish, such as the
zebrafish, the PN is the functional kidney of early larval life [28]. This structure is composed by three
segments: the glomerulus, the tubule and the duct (Figure 6) [28]. Each segment is formed through the
action of a combination of genes in a pre-patterned IM that will determine the fate of each cell during
nephrogenesis. Between 8 to 10-somite stages, the IM adjacent to the somite 1 to 4 expresses wt1 (wilms
tumor 1) and the IM near somite 4 onwards expresses sim1 (single-minded family bHLH transcription
factor 1). Overlapping with the expression domains of these two genes is pax2a (paired box 2a), which
is expressed from somite 2 onwards. During nephrogenesis, the IM region near the somites 1 and 2 (wt1
only) will give rise to the glomerulus, the one adjacent to somites 3 and 4 (wt1 and pax2a) will originate
the tubule and the remaining IM (pax2a and sim1) will develop into the duct (Figure 6) [28,29]. Beside
the IM, the PN could also have a role in the establishment of LR asymmetry. At later stages in
development, the PN could be used as a stable structure for the asymmetric migration of the LPM during
gut looping. In fact, at the time this migration occurs (i.e. 26 to 30 hpf), the PN, the migrating LPM and
the gut are localized in the same region (i.e. first to third somite level) [19,27,30]. Despite their
localization, neither the IM nor the PN have been implicated in organ laterality.
Figure 6 – Pre-patterned Intermediate Mesoderm will determine the cell fate during nephogenesis. A – Expression
pattern of wt1 orange), pax2.1 (blue) and sim1 (brown) in the IM at 8 to 10 somite stages; B – Schematic representation of
a mature PN indicating the final position of the segment – Glomerulus (wt1) in yellow, tubule (wt1 and pax2.1) in green
and duct (pax2.1 and sim1) in horizontal lines. Adapted from [29].
1.5 - Cadherins
The proposed involvement of the IM, and its derivative, the PN, could derive from local action
of cell-adhesion molecules like cadherins, which would determine the stiffness of these tissues.
6
Cadherins (Cdh) are calcium-dependent cell-cell adhesion molecules, known to be key players
in distinct events during development, such as the separation or fusion of different tissue layers,
formation of boundaries, tissue architecture (through cell rearrangements), conversion between
histological cell states, cell migration and neuronal processes [31]. These molecules can be subdivided
into classical cadherins (most common) type I or II, desmosomal cadherins, protocadherins and
cadherin-related molecule [32,33]. The classic
cadherins are composed by five extracellular
domains (EC) highly homologous (from EC1 (Nterminus) to EC5), a transmembrane domain (TM)
and an intracellular domain (IC) that are associated
with cytoplasmic proteins, such as catenins, that
mediate the interaction between the cadherins and the
actin cytoskeleton (Figure 7). These cell-cell
interactions can lead to an intercellular activation of
cellular pathways [32,33]. Cadherins normally form
adherens junctions as a result of interactions between
extracellular domains of identical cadherins present
on the membranes of the neighboring cells
(homophilic interactions). Nonetheless, there are
some cadherins, such as N- or E-Cadherin, that can
Figure 7 - Structure of classical cadherin and their
also bind to different cadherins, mediating
interaction with cytoplasmic proteins. Adapted from
heterophilic interactions [33].
[33].
1.5.1 – Cadherins in Left-Right asymmetry
Cadherins, especially N-Cadherin (Cdh2), are molecules that have been described to be
implicated in the establishment of LR asymmetry both early and later in development [5, 34-38].
In the chicken embryo, N-Cadherin is symmetrically expressed in the node at stage HH4, and
then shifts its expression to the right side at stage HH5 (Supplementary Figure 4). On the right side of
the node, N-Cadherin stops the leftward movement of cells observed at stage HH4. Using a
photoconvertion technique, that allows the labeling of a specific group of cells, it was demonstrated that
when N-Cadherin activity is blocked, using an anti-N-Cadherin antibody at stage HH3+, the transient
leftward movement of cells observed between stages HH4 and HH5 does not stop and continues past
the midline during the stages HH5 and HH6 (Figure 8) [37]. Stopping this movement is important to
stabilize the molecular asymmetries created in the node allowing the correct asymmetric information to
be transferred to the appropriate side of the LPM and to correctly position the heart (Supplementary
Figure 5 ) [35,37]. Both studies also suggest that adhesion or migratory functions controlled by Ncadherin are either in parallel or following Nodal signaling, because when this molecule is inhibited,
both pitx2 and snail expression patterns are altered but lefty and nodal do not undergo any change in
expression [35,37].
Later in development, both in chicken and mouse, N-Cadherin acts downstream of Pitx2 on the
left side of the dorsal mesentery (DM) in order to create asymmetries in the cellular architecture of this
structure that will determine the chirality of the midgut (Figure 9). Here, together with Shroom3, NCadherin promotes cell elongation and apical constriction. Besides that, the epithelium assumes a
columnar shape and the accumulation of this adhesion molecule on the left side will change the
production and deposition of the extracellular matrix promoting the condensation of DM cells. This will
cause a trapezoidal shape on DM, that will provide a tilt in the developing midgut and consequentially
a Left-Right biased gut rotation (Figure 9) [34,36,38].
7
Control
F
Anti-N-cadherin
B
C
D
E
G
H
I
J
Figure 8 - N-cadherin plays a role in LR patterning in the chicken embryo. A – Diagram of a stage HH4/HH4+ embryo
showing the photoconverted cells (red dots) on the right side of the node; F – Diagram on a stage HH5 embryo where
photoconverted cells at stage HH4/HH4+ (light red dots) moved to the left, and new cells were photoconverted (dark red
dots) on the right side of the node; B, C, G and H - The position of the photoconverted cells in a control at stage HH4 (B
and C) and stage HH5 (G and H); D, E, I and J - The position of the photoconverted cells in anti-N-cadherin-treated embryo
at stage HH4 (D and E) and stage HH5 (I and J). Adapted from [37].
B
A
Mouse
L
R
Chicken
Figure 9 – N-cadherin acts on the dorsal mesentery creating asymmetries in the cellular architecture determine the
chirality of the midgut. Model for the directional looping of the gut tube in mouse (A) and in Chicken (B) embryos.
L – Left, R- Right. Adapted from [34,36,38].
8
1.5.2 – Cadherin-11 in zebrafish
Cadherin-11 (Cdh11) is a classic type II cadherin expressed in a set of tissues during zebrafish
development including the Intermediate Mesoderm (List of tissues in Supplementary Table 1) (Figure
10) [39]. This molecule is described as a player in different processes
during zebrafish development [39-43].
It has been shown that otolith assembly in zebrafish is
promoted by cadherin-mediated vesicle adhesion mechanisms. The
authors demonstrated that the Cdh11 is synthesized in the otic
epithelial cells and afterwards packed into vesicles. These packages
are exported and will promote otolith assembly and growth. This
process is possible due to the adhesive properties of Cdh11 that will
guarantee that the minerals and proteins necessary for otolith’s growth
are delivered more efficiently (Figure 11) [40].
Another study describes Cdh11 as an element in retinal
Figure 10 – Cdh11 expression at 5
somite stage embryo. Dorsal view
differentiation. When this protein is reduced due to cdh11 morpholino
IM - intermediate mesoderm, NK injection, morphant embryos exhibit less retinal ganglion cells at 72
Neural keel. Adapted from [35].
hpf as showed in Figure 12 using immunostaining for neuronal RNAbinding proteins (HU). Analyzing both lenses and retinal tissues size and organization it was visible that
these structures were reduced in cdh11 knockdown embryos (Figure 13). Lastly, this study demonstrates
that the retinotectal axon projection is also affected when cdh11 function is blocked. In this case the
axon is extended from the eye until it reaches the brain to form the optic chiasm, however the optic
nerve is thinner (Figure 14) [41].
Finally, unpublished data from our lab also shows a role for Cdh11 protein, this time in LR
establishment in zebrafish. Using a morpholino to knockdown we demonstrated that the lack of Cdh11
leads to organ laterality defects in zebrafish embryos at 48 hpf (See Result 3.1.1 – Figures 18-20).
Figure 11 – Cdh11 promotes efficient otolith formation. Left panel – Control situation – otic vesicles containing Cdh11
are exported. Efficient otolith assembly is mediated by Cdh11 in these extracellular vesicles; Right panel –The lack of
Cdh11 does not interfere with vesicle exportation but with the adhesion process. Blue arrows - Vesicles adhere to otolith;
Blue arrows with red X - Vesicles do not adhere to otolith). Adapted from [40].
9
Figure 12 – cdh11 loss of function affects retinal differentiation at 72 hpf. Immunostaining for HuC/D (detects neuronal
RNA-binding proteins - HU) show less retinal ganglion cells in morphant embryos when compered in Control embryos.
Adapted from [41].
Figure 13 – Cdh11 is involved in lenses development at 48 hpf. Live images of using differential interference contrast
microscope show a disorganized and smaller lenses and retinal tissues in embryos injected with cdh11 morphants. Ventral
view. onl – outer nuclear layer; inl – inner nuclear layer; rgl – retinal ganglion cell layer; ipl – inner plexiform layer.
Adapted from [41].
Figure 14 – Retinotectal axon projections are reduced in cdh11 morphants. 48 hpf cdh11 morphant embryos (B)
labeled with acetylated tubulin exhibit axons extended from the eye towards the brain where it forms the chiasm but a
reduced optic nerve when compared with control embryos (A). Arrow – optic chiasm. Adapted from [41].
1.6 - Objectives
In this project, we propose that the IM, and/or its derivative the PN, might play a role in the
establishment of LR asymmetry in zebrafish through the action of Cdh11. Our hypothesis is that the IM
could be used as mediator of the transfer of Spaw from the KV towards the LPM at the 10 to 12-somite
stages. Regarding the PNs, we hypothesize that this IM derivative can provide a stable structure for the
LPM asymmetric migration between 26-30 hpf, that ultimately leads to the shift of the gut to the left.
This can be possible due to the fact that these structures (PN, gut and LPM) are all spatially and
temporally co-localized (Figure 15) .
10
In this work we aim to:
1. Confirm the absence of Cdh11 (transcript and protein) from the known LR patterning
tissues during zebrafish development;
2. Study the heart and gut positioning and the formation of the IM both in cdh11 morphant
and in cdh11 mutants as a complementary approach;
3. Analyze the expression of the conserved Nodal genes (spaw and pitx2) in cdh11
morphants;
4. Evaluate the role of the PN during the LPM asymmetric migration;
5. Analyze, by candidate approach, if the lack of Cdh11 activates a genetic compensation
program in cdh11 mutants.
Figure 15 – Schematic representation of the localization of the gut looping region, future heart territory and LPM
regarding the IM position. LPM – lateral plate mesoderm; IM intermediate mesoderm; S – somites. Anterior to the top.
Adapted from [29,30,44,45].
11
Chapter 2 – Experimental Procedures
2.1 - Zebrafish maintenance and care
Adult zebrafish (Danio rerio) and embryos used in this project were maintained and bred under
standard laboratory conditions [46]. During this project, the embryonic stages were confirmed according
to Kimmel et al. (1995) [47].
The cadherin-11 (cdh11) knockdown characterization (see below cdh11 Knockdown) was
performed using embryos from wild-type (WT) AB strains and transgenic Tg(sox17:EGFP) line,
established at Instituto de Medicina Molecular (iMM) [48].
As a complementary approach, we used the mutant line cdh11sa14413 [49] obtained from the
European Zebrafish Resource Center (EZRC) that was generated within the TILLING project context
[50]. This line produces a truncated protein in the Cdh-Cdh interaction domain (fourth EC) due to a
single nucleotide nonsense mutation (T to A) in the 454th amino acid (aa). This change leads to the
formation of a premature STOP codon (TTA) instead of a Leucine (Leu) aa (TTT) (Figure 16). Because
this mutant line has no associated phenotype, it was imported to sequence the fish in order to detect
which ones carried the mutation (see Zebrafish genotyping protocol below). After this, the fish were
divided into three categories: the ones that have the WT nucleotide (T/T) were named WT control
(cdh11+/+); the fish that carried both WT and mutated alleles (T/A) were classified as heterozygous
(cdh11+/sa14413); and the fish that carried the mutated allele (A/A) were called homozygous (cdh11sa14413).
Figure 16 - cdh11 mutant line. A - WT Cdh11 protein structure, the 455th aa is a Leucine (Leu); B - Mutant Cdh11
truncated protein structure, with premature STOP codon. SP - signal peptide; EC1-5 - Extracellular Domain 1-5; TM Transmembrane Domain; IC- Intracellular domain.
2.2 - Zebrafish genotyping
2.2.1 - Caudal fin amputation and Phenol-chloroform genomic DNA (gDNA)
extraction
Adult zebrafish were anesthetized using 1x Tricaine solution to proceed with the caudal fin
amputation. To extract the gDNA, the caudal fin was incubated in 199μL of digestion buffer and 1μL
of Proteinase K (PK) (20mg/mL) overnight at 55°C with 400rpm. Each zebrafish was separated and
kept in a labeled box during this process until the results from the DNA sequencing were obtained. On
the following day each tube content was homogenized by inversion. 200μL of
Phenol:Chloroform:Isoamyl alcohol (25:24:1) (Sigma) was added and each tube content homogenized
again by inversion and centrifuged at 650g for 10 minutes at room temperature (RT). The upper phase
12
was transferred to a new tube, and mixed with half of the volume transferred of ammonium acetate
4,5M, and 2,5x the volume transferred of absolute ethanol. The samples were homogenized and
incubated for 30 minutes at -80°C, followed by 15 minutes centrifuge at 13000g, at 4°C. The supernatant
was discarded and washed with previously cooled 70% ethanol. The tubes were centrifuged for 15
minutes at 13000g, at 4°C. The supernatant was discarded and the pellet was air dried. It was then
ressuspended in 20μL of UltraPure™ DNase/RNase-Free Distilled Water (Gibco). The samples were
quantified using the NanoDrop 2000 spectrophotometer and stored at -20°C.
2.2.2 - Polymerase Chain Reaction (PCR)
To genotype the cdh11 mutants, we used the Ensembl database to obtain the mRNA sequences
[51]. Forward (Fw) and reverse (Rv) primers were designed using NCBI primer blast [52] and
synthesized by STABVida. The annealing temperature was calculated using Tm Calculator by
ThermoFisher Scientific™ (Supplementary Table 2) [53].
All the reaction specifications were performed according with Thermo Scientific Phusion HighFidelity DNA Polymerase product information. For a final volume of 50μL it was used 150ng/50μL of
gDNA template, 10μL of 5x Phusion HF Buffer (ThermoFisher Scientific™), 1μL of 10mM dNTP mix
(ThermoFisher Scientific™), 2,5μL of 10μM primer mix, 0,5μL Phusion DNA polymerase
(ThermoFisher Scientific™) and water. Cycle sequencing was performed with conditions described in
Supplementary Table 3. The DNA was purified using the manufacturer's instructions of the DNA clean
& concentrator™- 5 kit (Zymo Research) and sent to STABVida for sequencing.
2.3 - cdh11 Knockdown
2.3.1 Morpholino oligonucleotides (MO)
To Knockdown the cdh11 gene, we used a cdh11 specific splice blocking MO, the MO3-cdh11
(5' - TGTCACGCACCTCTGTTGTCCTTGA - 3') (cdh11MO) [40], and a Standard Control MO (CtrMO)
as a negative control (5' - CCTCTTACCTCAgTTACAATTTATA - 3') (GeneTools).
2.3.2 - MO preparation
100 nM of CtrMO and 300 nM of cdh11MO sterile salt-free lyophilized powder were mixed,
separately, with RNase-Free Water to produce a stock solution of 3mM and 2,5mM respectively, and
stored at -20°C until use. Injection mixture was prepared fresh by diluting the MOs in RNase-Free water
to reach a 0,2mM injection concentration.
2.3.3 - Embryo microinjections
Adult zebrafish of interest (lines WT AB) were kept overnight in a breeding cage. In the
morning, using a micrometer and mineral oil, a loaded injection needle was clipped using forceps and
calibrated each time to produce a consistent injection volume. The embryos were then collected, aligned
to a microscope slide in a Petri dish with a pipette and injected at one-cell stage into the cell cytoplasm.
1,4nL of 0,2mM MO solution (0,23ng) was injected per embryo.
For each experiment, both MOs were injected into sibling embryos from two to three
independent batches, and incubated in 1x Embryo Medium at 28°C until the desired developmental stage
was reached.
13
2.4 - Anti-sense mRNA probe synthesis for Whole mount in situ hybridization
(WISH)
The mRNA probes used during the course of this project were synthesized from plasmid
templates pre-existing in our laboratory or sent to us by other groups (see Supplementary Table 4 for
more information about each probe used).
2.4.1 - Transformation of competent Escherichia coli bacteria
Frozen aliquots of competent Escherichia coli bacteria previously prepared in our lab (DH5α
strain, kept at -80°C), were thawed on ice. 100μL of cells were incubated on ice for 30 minutes with 1
to 3μL of plasmid DNA (followed by 40 seconds (s) heat shock at 42°C and a 2 minutes cooldown on
ice. 900μL of SOB solution was added to the mixture and left for incubation, for 45 to 90 minutes, at
37°C with agitation. After this, 50 to 100μL of this mixture was plated on LB agar medium (containing
ampicillin 100μg/mL of LB agar medium) and left at 37°C overnight. On the next day, an isolated colony
was inoculated on a 15mL falcon with LB media and left overnight at 37°C with agitation. DNA was
purified according to the manufacturer's instructions of the GeneJET Plasmid Miniprep Kit
(ThermoFisher Scientific™). DNA concentration was determined by spectrophotometry using
NanoDrop 2000 Spectrophotometer (ThermoFisher Scientific™).
2.4.2 - DNA Plasmid linearization
For a final volume of 50μL, 3μg of DNA plasmid, 1μL of the appropriated restriction enzyme,
5μL of the respective buffer (10x) and water were mixed together to linearize the plasmid DNA (see
Table 3). The reaction mixture was incubated 1 hour (h) at 37°C. The efficiency of digestion was
visualized on an agarose gel (see Experimental Procedures 2.8), and the DNA was purified according to
the manufacturer's instructions of the DNA clean & concentrator™- 5 kit (Zymo Research) and
quantified (NanoDrop 2000).
2.4.3 - in vitro transcription
The anti-sense transcripts were produced using 1μg of the purified linearized plasmid DNA, 1
μL of the appropriated RNA polymerase (1U), 2,5μL of transcription buffer (10x) (Roche Life Science),
1μL of dithiothreitol (DTT) (100mM Promega), 2,5μL of Digoxigenin (DIG) labeling Mix (10x) (Roche
Life Science), 1μL of RNasin® Ribonuclease Inhibitors (Promega) and water in a final volume of 25μL.
The mixture was incubated at 37°C for 3 hours. The probe size was monitored on a 1% agarose gel (see
Experimental Procedures 2.8) and the sample was purified according to the manufacturer's instructions
of the illustra™ Probe Quant™G-50 Micro Columns (GE Healthcare Life Sciences) and stored at -20°C.
2.4.4 - Probe preparation
The probes were diluted (specific concentration for each probe) in 1mL of pre-Hybridization
Mix (pre-Hybmix) with 50μg/mL heparin and 500μg/mL tRNA mixture and stored at -20°C.
2.5 - Whole mount in situ hybridization
Zebrafish embryos were collected at specific developmental stage and fixed in a 4%
paraformaldehyde solution prepared in 1x Phosphate-buffered saline (1x PBS) (4% PFA) during 4 to 5
hours at RT or overnight at 4°C. The embryos were then stored at -20°C after dehydrate washes
performed with increasing concentrations of methanol (MetOH) diluted in 0,1% Tween 20 in 1x PBS
(0,1% PBT) (two washes with 0,1% PBT, 50% MetOH and 100% MetOH).
14
Day 1: The stored embryos were rehydrated by successive washes with 75%, 50%, 25% MetOH
in 0,1% PBT, and four times in 0,1% PBT, for 5 minutes each. Chorions were removed using forceps.
Each set of embryos was incubated with PK (Roche) (10μg/mL) in 0,1% PBT (incubation period
according to embryo stage - see Table 1) and immediately re-fixed in 4% PFA for 20 minutes at RT and
washed five times with 0,1% PBT (5 minutes each). For 2 to 5 hours, the samples were incubated at
70°C in 500μL of pre-Hybmix, followed by an overnight incubation with 200μL of probe at 70°C,
having this one been previously heated for 10 minutes at 70°C.
Day 2: The probe solution was recovered in the next day and the embryos washed at 70°C with
pre-heated solutions of 100% pre-Hybmix for 10 minutes, 25%, 50% and 75% 2x SSC in pre-Hybmix
and 100% 2x SSC for 15 minutes. After that the embryos were washed at RT twice in 0,2x SSC for 15
minutes each, once in 50% 0,2x SSC in 0,1% PBT and two times in 0,1% PBT, each one lasting 10
minutes. Embryos were incubated in 500μL of blocking solution for in situ at least for 1 hour at RT and
after that incubated with Anti-DIG-AP (Roche Life Science) in blocking solution (1:5000) overnight at
4°C.
Day 3: On the last day, the embryos were washed six times with 0,1% PBT for 15 minutes each,
and three times in Staining Buffer for 5 minutes each. To reveal the probe, the embryos were incubated
with 500μL of purple AP substrate (Roche Life Science) in the dark at RT. The revelation process was
monitored with a dissecting microscope, and stopped by changing the substrate for 0,1% PBT followed
by a fixation in 4% PFA for 20 minutes at RT and a wash in 0,1% PBT. To store the embryos at 4°C in
100% glycerol, a series of washes in glycerol and 0,1% PBT (20%, 50% glycerol in 0,1% PBT) were
performed.
Table 1 - Appropriate PK incubation period according to embryo developmental stage.
Stage
Early somitogenesis
(until 8 somites)
18 – 20 hpf
(19-23 somites)
24 hpf
36-48 hpf
Incubation time
1 minutes
5 minutes
15 minutes
30 minutes
2.5.1 - in situ hybridization imaging
The embryos were photographed with a LEICA 26APO stereoscope coupled to a LEICA
DFC490 camera. The images were processed using the FIJI® software.
2.6 - Embryo embedding, preparation of cryostat sections and
immunostaining - Protocol optimization
2.6.1 - Embryo embedding
The embryos were collected and fixed overnight in 4% PFA at 4°C. On the next day, they were
washed in 1x PBS and incubated, on ice with agitation for approximately 15 to 30 minutes in 15%
sucrose in 1x PBS solution. Later, the samples were incubated for 1h at 42°C in a previously heated
7.5% gelatin and 15% sucrose in 1x PBS solution. The bottom of a plastic mold was filled with the same
15
solution and allowed to harden at RT. Finally, the embryos were disposed with the correct orientation
for transversal sectioning and embedded. The gelatin cubes were fast freeze using 2-Methylbutane
(isopentane) (Sigma) solution, previously cooled at -40°C, using dry ice, and stored at -80°C.
2.6.2 - Cryosectioning
20μm thick transversal sections cut with a Cryostat LEICA CM 3050S were mounted into
microscope slides by the Histology and Comparative Pathology Laboratory at iMM and stored at -20°C.
2.6.3 - Immunostaining protocol
The frozen microscope slides were thawed for 30 minutes at RT. and circled with an immunopen. The samples were washed 4 times with a pre-heated 1x PBS in a water bath at 42°C for 5 minutes
and 1 time with 0,1% Triton-X in 1x PBS (PBS-T) at RT. 400μL of Blocking solution was added and
incubated for 1 hour. Later, the slides were left o.n at 4°C in a humidified chamber with the antibody
solution (1:1000 ZO-1 Ms MAb Alexa Fluor® 594 phalloidin (Life Technologies); 1:200 Alexa Fluor®
488 phalloidin (Invitrogen) in Blocking solution). On the following day, a series of washes (0,1% PBST for 5 times and with 1x PBS for 2 times) were performed for 5 minutes each at RT. The excess liquid
was removed and 80μL of mowiol was added. The slides was covered with a cover slip #1,5 and left to
dry overnight at RT and afterwards stored at 4°C.
2.6.4 - Immunostaining imaging
The images were obtained using the confocal point-scanning microscope Zeiss LSM 710. All
images were acquired using a 20x/0.8 numerical aperture dry objective, and processed and analyzed
using the FIJI® software.
2.6.5 - Notes
During this optimization, sections with different thickness were also tested (10, 50, 100, 150,
300μm, the last two obtained with the Leica VT1200S vibrating blade microtome). The transgenic line
Tg(hand2:EGFP) [27] alone or crossed with sox17:EGFP, Alexa Fluor® 594 phalloidin (Life
Technologies) and different ZO-1 concentrations were also tested (see Supplementary Table 5 for more
details).
2.7 - Cdh11 protein pattern analysis
2.7.1 - Cdh11 specific antibodies (Abs)
To determine the expression of Cdh11 protein, two antibodies were specifically designed and
ordered from Abcam (created in Goat), to recognized two different regions on the Cdh11 protein. The
Ab1 recognizes the 19 aa sequence (VSATDKDEMAHRQHFHFSL) present on the fifth extracellular
domain. The
Ab2 identifies
the
last 50 aa
of
the
intracellular
domain
(GRGSIAGSLSSLESVTTDSDLDYDYLQSWGPRFKKLADLYGTKDSVDDNS) (Figure 17).
Figure 17 – Sites in the Cdh11 protein to which the antibodies were raised. SP - Signal peptide; EC1-5 - Extracellular
Domain 1-5; TM - Transmembrane Domain; IC- Intracellular domain
16
2.7.2 - Immunostaining protocol
WT embryos (8-somite stage) were collected and fixed with 2% PFA for 4 hours at RT. The
samples were washed overnight in 0,5% Tween 20 diluted in 1x PBS (0,5% PBT) at 4°C with agitation.
On the following day, the embryos were dechorionated and washed in a permeabilizing solution for 1
hour at RT 10 minutes 0,5% PBT wash was performed succeeded by blocking solution incubation at RT
for as long as possible. The embryos were incubated overnight at 4°C with the Cdh11 primary Ab (Ab1
or Ab2) in blocking solution (1:100, or 1:500, or 1:1000) with agitation. On the next day, 4 washes, at
least for 30 minutes each at RT, were performed with blocking solution before an overnight incubation
with agitation at 4°C with a secondary Donkey α-Goat Ab (1:200, or 1:400). The washing step with
blocking solution after an Ab incubation was repeated. The embryos were re-fixed in PFA 4% for 30
minutes at RT and stored at 4°C in 0,5% PBT.
Due to the fact that this protocol had not worked, some alterations were performed according to
the whole mount immunostaining troubleshooting tips [54]:

To make sure that the egg membrane is permeabilized, the embryos were dechorionated
before fixing;

Sometimes the fixative that is used is not the best one for the Ab, therefore the fixation
with 2% PFA was substituted for one with MetOH (Dent's fixative) followed by a MetOH rehydration
(decreasing concentrations of MetOH diluted in 0,5% Triton-X in 1x PBS 75% MetOH, 50% MetOH
and 25% MetOH);

The antigen retrieval protocol could destroy the tissues, however in some samples this
protocol was performed (after the first overnight wash with 0,5% PBT, the embryos were washed two
times (10 minutes each) at 37°C with 1x PBS, followed by a 10 minutes incubation with the antigen
retrieval buffer at 95°C. The samples were cooled down for 5 minutes and washed with 1x PBS for 5
minutes Standard Immunostaining protocol was continued at the permeabilization solution step);

To allow full permeabilization of the reagents and Ab through the whole sample, 0,5%
Tween 20 was substituted by 0,5% Triton-X in all the steps (including 0,5% PBT);

To ensure that the incubation time for the primary Ab was sufficient, 3 day incubation
at RT was also tested.
Other troubleshooting guides [55,56] also indicated that some primary Ab could not be suitable
for IHC and that they must be tested a Western Blot assay to check if the Ab is not damaged.
2.7.3 - Western Blot
WT embryos were collected at the 8-somite stage and dechorionated, rinsed 3 times in Calcium
free Cold Ringer’s solution, and immediately washed with 1mL of Cold Ringer’s solution with EDTA
and PMSF. The embryos were dissociated and the mixture was homogenized at 1100rpm for 5 minutes
at 10°C, flowed by a centrifuge at 300g for 30 seconds, and the supernatant discarded. This process was
repeated twice using only Cold Ringer’s solution. After the final wash, the liquid was fully removed.
Afterwards, the embryos were defrosted, centrifuged for 2 minutes at 300g and excess liquid was
removed. It was added 1μL of 2x SDS sample Buffer per embryo, and the mixture was homogenized
and incubated at 95ºC for 10 minutes. After that the tubes were placed on ice for 2 minutes and
centrifuged at 1300rpm for 1 minute. The supernatant was transferred to a new tube. Both Revolving
and Stacking (12% or 8%) gels were prepared and polymerized. The setup was assembled, and the
samples were loaded (final volume corresponding to 10 embryos per well) into the stacking gel through
which they ran at 70V before being separated in the running gel for about 80 minutes at 120V in 1X
running buffer. Proteins were then transferred into 0,2µm Immun-Blot® PVDF Membrane (BIO-RAD)
previously treated briefly with MetOH, for 5 minutes with milliQ water, and 1x transfer buffer. The
transference from the gel to the membrane was performed in 1X transfer buffer for 70 minutes at 75V.
17
The membrane was then washed for 1 minute with MetOH, milliQ water and incubated with Ponceau S
for a few seconds, and washed once more time with milliQ water, MEtOH, milliQ water and finally
TBS-T. Finally, the membrane was blocked with 5% Bovine Serum Albumin (BSA) in TBS-T at 4°C
during overnight with agitation. On the following day, the membrane was washed for 10 minutes in
TBS-T with agitation at RT and incubated overnight with the primary Abs (Ab1 and Ab2) diluted in 1%
BSA in TBS-T at 4°C with agitation (1:50, or 1:100, or 1:500, or 1:1000). On the next day, it was washed
3 times, 5 minutes each, with TBS-T at RT with agitation. The AP-conjugated secondary Ab solution
(Donkey anti-goat HRP (Jackson ImmunoResearch) in 1% dried milk in TBS-T) (diluted 1:5000, or
1:10000) was incubated for 1 hour at RT with agitation. Before developing in the Chemidoc XRS+ with
Clarity™ Western ECL Blotting Substrates (BIO-RAD), the membrane was washed twice (5 minutes
each) in TBS-T, and once in TBS.
2.8 - Agarose gel electrophoresis
To access the linearization of the plasmid DNA, PCR product size and probe synthesis, gels
were prepared by heating agarose dissolved in 1x TAE buffer mixed with RedSafe™ Nucleic Acid
Staining Solution (Intron) (5μL per 100mL TAE 1x). The samples mixed with Orange G (Loading
Buffer) at a minimum of 1μL per 5μL of DNA sample and applied to the gel. A 1Kb Plus DNA ladder
(Invitrogene) was used to evaluate samples size. The electrophoresis was performed in TAE 1x buffer
for 15 minutes at 100V.
18
Chapter 3 – Results
3.1 - Analysis of the cdh11 morphant phenotype
3.1.1 - cdh11 knockdown affects Left-Right positioning of the organs.
To understand if Cdh11 has a role in the establishment of Left-Right asymmetry in zebrafish,
we used a splice blocking MO (cdh11MO) [40] to knockdown the expression of cdh11 during the early
stages of development. Given the fact that it was already published that cdh11 knockdown reduces the
number of otoliths in zebrafish embryos, our group started this project by trying to reproduce this
phenotype in order to access the MO concentration to be used (data not shown) [40]. We choose to use
the lowest concentration (2,3ng) that gave us a reproducible phenotype.
The position of the gut and direction of the cardiac looping in morphant embryos injected with
2,3ng of cdh11MO at 1-cell stage was accessed at 50 hpf (Figures 18,19). In WT embryos the liver is
present on the left side whereas the pancreas takes its position on the right side. To address whether the
cdh11 loss-of-function leads to alterations in the position of the gut, the cdh11MO was injected in the
transgenic line Tg(sox17:EGFP), which labels the endoderm, and analyzed live at 50 hpf (Figure 18)
[48].
Figure 18 – Cdh11 knockdown causes gut laterality defects at 50 hpf. A- Schematic representation of normal gut loop
(Normal) and five gut laterality defects (Reversed, Bilateral 1, 2, 3 and 4); B- Percentages of normal (blue), reversed (red),
MO
MO
bilateral 1 (green), bilateral 2 (purple), bilateral 3 (yellow) and bilateral 4 (Orange) in Ctr (n=109) and cdh11 (n=122)
injected embryos analyzed at 50 hpf. Lv-liver, Pc-Pancreas, L-Left, R-Right, A-Anterior, P-Posterior. Unpublished data from
Sara Fernandes
When compared with the embryos injected with the Standard Control MO (CtrMO) (n=109), the
morphant embryos (n=122), showed approximately 25% of defects regarding the normal gut looping
and the position of the liver and the pancreas (Figure 18B). Most of the affected embryos (17,22%)
exhibit the liver on the right and the pancreas on the left, a conformation we called Reversed. A small
percentage of these (6,6%) had a more complex conformation regarding the pancreas and liver
positioning (Figure 18): there were embryos with two livers and pancreas on the left (Bilateral 1);
19
embryos with these organs centered (Bilateral 2); others with no liver and the pancreas in the middle
(Bilateral 3); and embryos that had the liver on the right side with a centered pancreas (Bilateral 4)
(Figure 18A). To assess if the lack of Cdh11 caused any heart laterality defects, we performed a WISH
using a myosin light chain 7 regulatory (myl7) probe in 50 hpf morphant embryos. The myl7 probe was
used because it labels the heart myosin light chain and allows us to see the morphology acquired by this
organ (Figure 19). The heart of the control embryos (CtrMO, n=66) exhibited a normal WT D-loop shape
(Figure 19A). However, we observed in the morphant embryos (n=81), not only the normal D-loop
shape (in 51,85% of the embryos) but also other shapes such as L-loop (11,11%, Figure 19C) or Noloop (37,04%, Figure 19B), that were indicative of a problem in the heart laterality (Figure 19D). This
unpublished data from our lab, was indicative that Cdh11 seemed to have a role in the establishment of
LR asymmetry.
D
Figure 19 – Morphants embryos exhibit heart laterality defects
at 50 hpf. A to C - Ventral view of the direction of the cardiac
MO
MO
looping in Ctr
or cdh11
injected embryos at 50 hpf after in
situ hybridization for myl7 probe (which lables the heart) (Aembryo with a WT conformation (D-loop); B- Embryo with no
looped heart; C- embryo with inverted heart (L-loop)); B –
Percentages of D loop (blue), No loop (red), L loop (green) and
MO
MO
deformed hearts (purple) in Ctr
(n=66) and cdh11
(n=81)
injected embryos. R-Right, L-Left. Unpublished data from Sara
Fernandes.
When I join this project, a new batch of the cdh11MO had to be purchased and it was important
to reproduce the phenotypes observed before in our laboratory. Two concentrations were tested, namely
0,2mM (2,3ng) and 0,3mM (3,5ng). The morphants injected with the higher concentration exhibited
malformations in contrast to the ones injected with 2,3ng of cdh11MO (data not shown). Therefore, we
choose to use the same concentration as used before in our laboratory (0,2mM), Figure 20. With this
new cdh11MO we only characterize the LR phenotype of the heart because it was easier to quantify the
LR asymmetries. When injecting the CtrMO (n=133), we obtain in 94,7% of the embryos a normal Dloop conformation (Figure 20A), in contrast to the morphants (n=189) that displayed this conformation
in only 58,2%. The L-loop phenotype was only visible in 2,3% of the control embryos whereas this
conformation was showed in 4,2% of the embryos injected with cdh11MO (Figure 20C). The hearts that
did not loop during development (No Loop) were seen in 11,6% of the morphants and in approximately
0,8% of the Control embryos (Figure 20B). A difference that came up when compared to the results
obtained before in our laboratory and the ones obtained now is the category Mild (2,3% in Control and
25,9% in the morphants, Figure 20D) that did not appear in our previous classification. We concluded
that on the last analysis we were more conservative with the classification and that the majority of the
embryos that we classified as Mild were classified as No Loop on our first analysis. Given the fact that,
the cdh11MO was tested two times by two different people with two different batches of MO gave us a
20
very similar result, it is possible to conclude that the cdh11MO gives a consistent LR phenotype in
zebrafish.
Figure 20– New operator and new cdh11
MO
MO
vial produces the same heart laterality defects previously describe at 50
MO
hpf. A-D - Ventral view of a Ctr or cdh11 injected embryos at 50 hpf after myl7 hybridization (A- embryo with a WT
conformation (D-loop); B- Embryo with a centered heart (No-Loop); C- embryo with inverted heart (L-loop); D– Example
of three embryos with Mild heart loops ); E – Percentages of D loop (blue), No loop (red), L loop (green) and Mild loop
hearts (purple) in Ctr
MO
(n=133) and cdh11
MO
(n=189) injected embryos. R-Right, L-Left.
3.1.2 - cdh11 does not seem to be expressed in any of the known Left-Right
patterning tissues
As we showed before, Cdh11 has a role in the establishment of the LR asymmetry. It became
therefore important to characterize the Cdh11 expression pattern, especially during the stages when LR
asymmetry is being promoted. Through a bibliographic search we found that the presence of Cdh11
(transcript and protein) has been reported in numerous tissues during zebrafish development
(summarized in Supplementary Table 1) but no record of this protein in the known LR patterning tissues.
Therefore, it was important to verify whether, in fact, this protein is not present in these tissues or if its
expression has not been reported.
We began by looking for the expression of cdh11 in the first tissue involved in LR asymmetry,
the KV in WT embryos at the 8-somite stage (Figure 21A). This transcript was not detected in the
zebrafish LRO but rather in the neural tube and the IM as already published (Figure 21A’) [35]. To
analyze the expression of cdh11 in the LPM (another important tissue in the LR asymmetry), we
hybridized WT embryos at 20-somite stage with cdh11 probe (Figure 21B). At this stage, the transcript
was observed in the otic vesicle, as previously described (Figure 21B’), and, regarding the LPM, we did
not find significant levels of the cdh11 expression (Figure 21B).
21
Figure 21 – cdh11 expression pattern in WT embryos. WISH for cdh11 in 8-somite stage (A- whole-mount embryo
(dorsal view), A’- transversal section through KV), 20- somite stage (B- whole-mount embryo (dorsal view), transversal
section through otic vesicle region (B’) and 24 hpf (C – upper view of a whole-mount embryo, D - whole-mount embryo
(dorsal view), D’- transversal section through inner ear level and) WT embryos. IM- Intermediate mesoderm, NT- Neural
Tube, KV- Kupffer’s vesicle, Ov- Otic vesicle, LPM- Lateral Plate Mesoderm, IE- Inner Ear, HB- Hind brain, MB- Mid
brain, dorsal view, anterior to the top. WISH done by Sara Fernandes, Photos taken by me and Sara.
Because we could not find cdh11 transcript in any of the known LR tissues, at least at a
detectable level, we addressed the question whether or not cdh11 mRNA was indeed not expressed in
those tissues or if this transcript is unstable. To answer this question, we proposed to determine the
expression pattern of the Cdh11 protein (molecular weight is approximately 89 KDa – Figure 22 Dashed
line) using two custom made antibodies that recognize the extracellular or the intracellular domain of
this protein (see Experimental Procedures 2.7.1). To test the Cdh11 antibodies we tested different
immunochemistry protocols using WT embryos at 8-somite stage (see Experimental Procedures 2.7.2).
Nonetheless, none of the performed protocols showed a specific expression (data not shown). To
overcome this issue, the Cdh11 antibodies were tested in a Western Blot assay using pools of 10 WT
embryos at 8-somite stage (Figure 22). Lanes A and C display the results obtained with Cdh11 Ab1
diluted 1:50 and 1:100, respectively (Figure 22). Lane B shows the results obtained with the secondary
antibody control, in which the membrane was just incubated with the secondary Antibody to guarantee
that the results obtained were specific for the Cdh11 antigen. The results obtained in this Lane indicate
that the secondary antibody is working properly (Figure 22 Lane B). In Figure 22, it is visible that, in
Lanes A and C, we have a strong band at the expected Cdh11 size. However this band might not
correspond to Cdh11 protein but to yolk proteins that are known to show a similar molecular weight
[57]. It is important to mention that the results presented in Figure 22 refer to Ab1, and that they were
representative of the results obtained using the Cdh11 Ab2.
We thus concluded that the cdh11 transcript does not seem to be found in any of the LR
patterning tissues. Regarding the protein expression pattern further optimizations need to done and
perhaps different protocols need to be tested in order to completely demonstrate that in fact Cdh11 is
not present in any of the LR patterning tissues.
22
Figure 22 – Optimization of Cdh11 Antibodies by
Western Blot. Cdh11 Ab tested in a 8% SDS-Page gel
using different Ab concentrations in a protein extract
from pools of ten WT embryos at 8-somite stage. Lane
A – Cdh11 Ab1 diluted 1:50 in blocking solution;
Lane B- Secondary Ab control (No primary Ab, the
membrane was incubated only in blocking solution);
Lane C - Cdh11 Ab1 diluted 1:100 in blocking
solution. Protein marker (PM): Precision Plus
Protein™ All Blue Prestained Protein Standards (BIORAD) (The results presented here were obtained using
Cdh11 Ab1 and using secondary Ab at a concentration
of 1:5000 and are representative of the results obtained
using the Cdh11 Ab2 and the remaining Ab
concentrations and gels percentage).
3.1.3 – Intermediate Mesoderm formation does not seem to be affected in cdh11
morphants
According to our results, despite the LR phenotypes, the adhesion molecule Cdh11 is apparently
not expressed in the LR tissues, but instead is expressed in the IM and in the PN during the symmetrybreaking period. This suggests that, surprisingly, the IM could have a role in the LR asymmetry
establishment. Considering this hypothesis, we wanted to know if the laterality defects that we observed
in Cdh11 knockdown, described above, results from the absence of the IM or a subtler regulation
performed in or through this tissue. To evaluate if the IM is formed and to evaluate its morphology
when Cdh11 is knockdown, we performed a WISH using a pax2a probe, an IM marker [28,29], in WT
(n=49), Ctr MO (n=67) and cdh11 MO (n=71)
injected embryos at 8-somite stage (Figures
23A-C). Analyzing all three conditions we
were not able to find any differences between
them, which means that all embryos had a
normal shaped IM (Figure 23). These results
indicate that the LR phenotype observed upon
Cdh11 knockdown does not result from
absence of the IM.
Figure 23 – The IM formation and morphology is not
changed in absence of Cdh11. A-C – Posterior part of
8- somite stage embryos after pax2a hybridization in WT
MO
MO
(A, n=49), Ctr
(B, n=67) and cdh11
(C, n=71)
injected embryos. Anterior; P- Posterior; ss- somite
stage.
23
3.1.4 - Do the pronephric ducts provide a stable structure for the asymmetric
migration of the underlying LPM?
We showed that the Cdh11 morphants form a normal IM, as far as we can tell. According, the
LR phenotype observed can be a consequence of the absence of Cdh11 in the PN and not in the IM
itself. A study done in mouse shows that this adhesion molecule can regulate the tissue mechanics and
the synthesis of extracellular matrix, such as collagen and elastin [58].
Thus, we also propose a role for the PN during the LPM asymmetric migration, that has been
shown to be necessary to bend the gut to the left from 24 to 30 hpf in zebrafish [19]. In our hypothesis
the lack of Cdh11 could change the PN itself or its extracellular matrix components, which could prevent
or alter this asymmetric migration. To study this process, we optimized an immunostaining protocol that
allowed us to reproduce the images describing the LPM migration obtained previously (see
Experimental Procedures 2.6) [19].
During the optimization process, we had to overcome three major difficulties:
sample orientation after sectioning (to distinguish the left and right sides), best thickness for sectioning,
and best combination of antibodies and transgenic lines (see Supplementary Table 5 and Supplementary
Figure 6 for more details). In the end, the best approach to observe LPM migration, and in the future
characterize in detail this process in absence of Cdh11, is to use 20μm thickness sections of sox17:EGFP
transgenics, to label the endoderm, and to immunostaining these embryos using a mix of ZO-1 (in red),
to label the tight junctions in epithelial cells and reveal polarity, and phalloidin 488 (in green) to stain
the F-actin and reveal the shape of the cells (Figure 24). Due to time constrains, we were not able to
perform a detailed characterization in morphant embryos.
Figure 24 – Does the PN provide a stable structure for the asymmetric migration of the underlying LPM? - Protocol
optimization. Confocal Laser point-scanning image from Tg(sox17:EGFP) 20μm sections immunostained for ZO-1 (in
red) and phalloidin 488 (in green) using 20x magnification. (scale – 30μm). LPM – lateral plate mesoderm, NT – neural
tube, E – endoderm, L- left, R – Right.
24
3.1.5 - Conserved Nodal cascade components, spaw and pitx2, became expressed on
the right LPM in cdh11 morphants
One of our hypotheses proposes that the IM might serve as a mediator in the molecular
communication between the LRO (KV) and the LPM (12 to 14 hpf). After this initial period, the
establishment of organ asymmetry continues through the Nodal cascade on the left side of the LPM.
Taking into account that our previous analyses with the cdh11 morphant embryos exhibited a heart and
gut phenotypes, we next determined if this conserved cascade was also perturbed in the LPM (Figure
25).
Figure 25 – Left-Right markers spaw and pitx2 are affected in morphants at late somite stages. A to H - WholeMO
MO
mount in situ hybridization in Ctr or cdh11 injected embryo using spaw probe at 20-somite stage (A to D) and pitx2
probe at 24-somite stage (E to H) (spaw and pitx2 expression on the left side (WT) (A, E), right (B, F), bilateral (C, G)
or absent (D, H); I-J - Percentages of Left (blue), Right (red), Bilateral (purple) and Absent (green) expression of spaw
MO
MO
(I) and pitx2 (J) in WT (spaw n=24; pitx2 n=63), Ctr (spaw n=70; pitx2 n=88) and cdh11 (spaw n=62; pitx2 n=145)
injected embryos analyzed at 20- and 24-somite stage respectively. L-Left, R-Right, A-Anterior, P-Posterior, ss- somite
stage.
25
For that we injected the cdh11 MO and Ctr MO in WT embryos at 1-cell stage and analyzed spaw
expression at 20-somite stage (Figures 25A-D,I) and pitx2 at 24-somite stage (Figures 25E-H,J) by
WISH assay. In normal conditions, we observed that, both WT (n=24) and Ctr MO (n=70) injected
embryos showed spaw expressed on the left LPM (Figures 25A,I), while the expression of spaw in
morphant embryos (n=62) seemed to be randomized (Figure 25I). In this condition, 39,7% of the
embryos showed a normal left side expression (Figures 25A,I) although in another 39,7% this expression
is reverted to the right side (Figures 25B,I). The remaining 20% was distributed between absence of
spaw in the LPM (14,3% - Figures 25D,I) and expression on both sides (Bilateral, 6,5% - Figures 25C,I).
Regarding pitx2, we also observed a restricted expression of this LR marker on the left LPM
under normal conditions (98,4% in WT (n=63) and 95,5% in Ctr MO (n=88)) (Figures 25E,J). However,
in these two conditions, we could also observe 1,6% of the WT embryos and 2,3% of the Ctr MO injected
embryos with pitx2 expressed on the right side. In 2,3% of the control embryos we were able to see pitx2
expressed on both sides. It was visible, once more a pitx2 randomization in morphant embryos (n=145).
In this condition, pitx2 is expressed on the left side in 43,4% of the time (Figures 25E,J), on the right
side 33,1% of the time (Figures 25F,J), in 5,5% of the embryos pitx2 absent (Figures 25H,J), and in
17,9% it is expressed on both sides (Figures 25G,J). Given the fact that we observed alterations in the
expression pattern of spaw and pitx2 in morphants embryos, these results indicate that Cdh11 is acting
upstream of the Nodal Cascade.
3.2 - Analysis of the cdh11 mutant phenotype, a complementary approach.
3.2.1 – cdh11 mutants are homozygous viable
To validate the results obtained so far with the cdh11 MO, we ordered a Cdh11 mutant line
(cdh11sa14413, EZRC) that incorporates a premature codon STOP in the fourth Cdh-Cdh interaction
domain originating a truncated Cdh11 protein with no transmembrane or intracellular domain (see more
on Experimental Procedures 2.1). So far no phenotype has been associated to this line and therefore to
determine which fish carried the mutation, they were genotyped (sequenced). From the approximately
120 embryos received, 60 reached adulthood (3 months old - Figure 26A). Using the genotyping
protocol described in Experimental
Procedures 2.2, we were able to find
WT control fish (cdh11+/+) and
Heterozygous
(cdh11+/sa14413)
(Figure 26B). To generate a line
which carries the mutation on the
cdh11 gene in homozygosity, we
did an incross of heterozygous fish
and genotyped the adult progeny
(Figure 26C). This time we found
not only WT control and
Heterozygous fish but also
Homozygous (cdh11sa14413 - Figure
26D), meaning that fish with no
Cdh11 are viable.
Figure 26 – Details on the Cdh11 mutant line. A- Embryos received from EZRC to adult (blue arrow), ready to genotype.
B- Genotype results (orange arrow), WT control fishes and Heterozygous (one female and seven males). C- Heterozygous
fishes were incrossed (blue arrow), after 3 months the progeny obtained were ready to genotype (orange arrow). D- Results
obtained after sequencing: WT control, Heterozygous and Homozygous fishes.
26
3.2.2 - cdh11 mutants do not exhibit the previously phenotypes observed in cdh11
morphants
To characterize the cdh11 mutant line we began by analyzing some of the previously published
phenotypes observed in morphant embryos (Figures 27,28) [40,41]. As mention before, one of the
phenotypes described in morphant embryos was a reduced number or absence of otoliths [40]. We
analyzed this feature at 50 hpf using live WT control (n=85) and cdh11 mutant embryos (n=200). In
both conditions, the majority of the embryos (96,47% on the left side and 97,65% on the right side of
WT control embryos, and 97,5% on the left side and 100% on the right side of mutant embryos) showed
a normal otolith number (Figures 27A,B). In some cases (3,53% on the left side and 2,35% on the right
side of the WT control embryos, and 2% on the left side of the mutant embryos), we could also observe
embryos with three otoliths (Figures 27A,B). On the left side, we observed 0,5% of the cdh11 mutant
embryos with one otolith (Figure 27A). Although the size of the otoliths was not quantified, in some
cases the normal conformation, in which the posterior is larger than the anterior one, was not observed
(Figures 27D,E).
Figure 27 – Otoliths number in Cdh11 mutants seem to be normal at 50 hpf. A – Quantification of otoliths number
+/+
on the left and right side in cdh11
+/+
and cdh11
sa14413
(0 (green), 1 (yellow), 2 (purple) and 3 (red)); B to E - Bright field
sa14413
visualization of cdh11
or cdh11
otoliths (B– Normal otolith shape, size and number; C – three otoliths; D and
E - Normal otolith number but problems on shape/size. P- Posterior; A- Anterior.
In the absence of Cdh11 function we also detected problems in the morphology and size of the
eyes [41]. To see possible problems on the eyes we analyze optic fissure closure in Cdh11 mutants
(Figure 28). We observed that in WT control embryos only 2,35% showed problems on the optic fissure
closure on the left eye and 1,17% on the right eye (Figures 28A,C,C’). In cdh11 mutant embryos, the
defects in the optic fissure closure increased to 14,29% on the left eye and 11,11% on the right eye
(Figures 28A,C,C’).
27
Figure 28– Optic fissure closure is slight affected in Cdh11 mutants at 50 hpf. A – Optic fissure closure quantification
+/+
sa14413
cdh11 and cdh11
on the left and right eye (opened (red) and closed (blue)); B and C- Bright field view of an eye
with closed (B) and opened (C) optic fissure (red dashed lines); B’ and C’- Correspond to a zoom of the black square (B
and C). P- Posterior; A- Anterior.
We continued to characterize the cdh11 mutant line regarding the LR phenotypes observed in
our laboratory (Figures 29,30). The heart phenotype was analyzed, after myl7 hybridization, at two time
points, 30 hpf (to determine the jog) and 50 hpf (to determine the loop) (Figure 29). During the first
asymmetric event to form the heart (jogging), in the majority of the WT control (88,4%) and cdh11
mutant embryos (95,4%) we could observe a normal heart conformation (Left Jog – Figures 29A,D). In
both conditions, WT control (n=199) and cdh11 mutant embryos (n=216), we could also observe
alternative conformations. In cdh11+/+ embryos, 4% of the hearts did not jog (No jog -Figures 29B,D)
and 7,5% displayed a reversed jog (Right Jog -Figures 29C,D). The same alterations were observed in
cdh11 mutants, 2,8% of the embryos showed No Jog (Figures 29B,D) and 1,9% exhibit a Right Jog
(Figures 29C,D).
The final S-shaped conformation (D Loop) is complete at 50 hpf. While analyzing this process
we observed a similar distribution to the one we saw during the heart jogging (Figures 29E-H). In WT
control embryos (n=229) we have a distribution in which 90,4% of the embryos exhibit a D Loop
(Figures 29E,H), 4,8% a L Loop (Figures 29G,H), 3,1% do not have a looped heart (No Loop - Figures
29F,H) and 1,8% have a mild phenotype (Figure 29H). Regarding the cdh11 mutants (n=199) 97% of
the embryos have a normal heart (Figures 29D,H), 1% did not loop (Figures 29F,H) and 2% showed a
reversed heart loop (L Loop - Figures 29G,H).
28
Figure 29 – cdh11
+/+
sa14413
embryos do not exhibit heart jog or loop phenotype observed in morphants. Ventral view of
sa14413
a cdh11 or cdh11
embryos at 30 hpf (A to C) and at 50 hpf (E to G) after WISH for myl7 (A- embryo with a WT
conformation (Left Jog); B- Embryo with a centered heart (No Jog); C- embryo with inverted heart (Right Jog); E- embryo
with a WT conformation (D Loop); F- Embryo with no heart Loop; G- embryo with L Loop; D- Percentages of Left Jog
(blue), Right Jog (red) and No Jog (green) and in cdh11
+/+
(n=199) and cdh11
sa14413
(n=216) embryos. H – Quantifications
of D Loop (blue), L Loop (red), No Loop (green) and Mild Loop (purple) in cdh11
embryos. R-Right, L-Left.
+/+
(n=229) and cdh11
sa14413
(n=199)
To analyze the asymmetric morphology of the gut we decided to generate a line in which the
sox17:EGFP transgene was present in a cdh11 mutant background. For that we crossed cdh11
heterozygous fish with sox17:EGFP transgenics. We then sequenced the progeny and created two lines,
cdh11+/+ and cdh11+/sa14413, both positive for GFP (sox17:EGFP+/-) (Figure 30A). To assess the position
of the liver and pancreas, cdh11+/sa14413 GFP positive (GFP+/-) male fish were crossed with cdh11 mutant
females (cdh11sa14413) to guarantee no possible maternal contribution. The posture was selected for GPF
positive embryos, which according to the Mendelian ratio will correspond to 50% of the population Experimental embryos (Figure 30C purple box). Once more, according to the Mendelian ratio, in these
50% GFP positive embryos, 50% will be cdh11 mutants (cdh11sa14413) and the other 50% will be
heterozygous (cdh11+/sa14413) (Figure 30C purple box). As a control we used a similar approach. This
time we crossed cdh11+/sa14413 GFP positive (GFP+/-) male fish with cdh11+/+ (WT control) fish, and once
more select the GFP positive progeny embryos (50% of the population). The control embryos include
50% of WT embryos (cdh11+/+) and 50% that had just one of the cdh11 alleles (cdh11+/ sa14413) (Figure
30B yellow box). Both experimental and control embryos were analyzed live at 50 hpf. In the
experimental condition, we expected to see gut laterality defects in 50% of the population that would
correspond to the cdh11 mutants (cdh11sa14413). Instead we found that only 13,6% of the guts were
perturbed (3,4% showed a reversed gut, 2,3% exhibited liver on the right and pancreas in the middle
29
(Bilateral 5) and the rest of the embryos were distributed evenly (1,1% each) through the remaining
conformations (Bilateral 3, 4, 6, 7, 8, 9, 10)) (Figures 30D,E). Comparing these results to the Control
condition we saw a higher percentage of defects regarding the gut loop (16,7%) (Figures 30D,E). In this
case 13,9% of the embryos show a reversed gut conformation (with the liver on the right and the pancreas
on the left), 2,7% of the embryos display a bilateral conformation (Bilateral 3, 4 and 6 – 0,9% each)
(Figures 30D,E). Together, this data shows that this particular cdh11 mutant does not present any of the
otoliths, eye or LR phenotypes observed in the morphants.
Figure 30 – Liver and pancreas placed in the normal configuration in Cdh11 mutants at 50 hpf. A- Creating a line
in which Cdh11 mutation was on a sox17:EGFP background; B and C- Generating the control (B) and the experimental
(C) embryos; D- Schematic representation of normal gut loop (Normal) and nine gut laterality defects observed (Reversed,
Bilateral 3, 4, 5, 7, 8, 9 and 10); E- Percentages of control (n=108) and experimental (n=80) embryos analyzed live at 50
hpf with normal (blue), reversed (red), bilateral 3 (green), bilateral 4 (purple), bilateral 5 (yellow), bilateral 6 (orange),
bilateral 7 (black), bilateral 8 (Brown), bilateral 9 (pink) and bilateral 10 (gray). Blue arrow – progeny; Orange arrowSequencing results, green arrow- GFP Selecting GFP, yellow box- control embryos, purple box- experimental embryos,
A– Anterior, P- Posterior, L- Left, R- Right, Lv- Liver, Pc- Pancreas, N.I.- Non identified.
30
3.2.3 - The intermediate mesoderm is formed properly in cdh11 mutants
As previously analyzed in the morphants, we wanted to check the formation of the IM in the
cdh11 mutants. We used 8-somite stage WT control and cdh11 mutant embryos for WISH using pax2a
probe (Figure 31). As before, the IM does not seem to be affected by the lack of Cdh11 function (Figure
31B) when compared to WT control embryos (Figure 31A).
Figure 31 – In Cdh11 mutants the formation
and morphology of IM does not seem to be
affected. A-B – Posterior end of 8- somite stage
embryos accessed with an in situ hybridization for
pax2a in cdh11
+/+
(WT, n=49) and cdh11
(Cdh11 mutant line, n=67) (A- cdh11
+/+
sa14413
(WT)
sa14413
embryo; B- cdh11
(Cdh11 mutant line)
embryo. A- Anterior; P- Posterior, ss - somite
stage.
3.3 – Does the lack of Cdh11 activates a genetic compensation program in
cdh11 mutants? The possible roles of Cdh6 and Cdh17 in Left-Right
asymmetry
So far we have conflicting results regarding the data acquired with the cdh11MO and with the
cdh11 mutant line (Results 3.1 and 3.2). In 2015, Rossi and colleagues [59] described the possibility
that morphants and mutants might have different phenotypes due to the activation of a genetic
compensation program in mutant lines. With this in mind and by candidate approach, we have identified
two other cadherins, Cdh6 [60] and Cdh17 [61], that are also expressed in the IM and the PN during
zebrafish development. To investigate the possible role of these two cadherins, we performed a WISH
for cdh6 (Figures 32A,B) and cdh17 (Figures 32C-G) at 8-somite stage in mutant embryos. We could
not detect any significant differences in expression between mutants and WT control embryos. Taking
the fact that in situ hybridization is not the best method for quantitative comparisons, we cannot draw
any conclusions from this experiment and more studies have to be done.
31
Figure 32 – Does the lack of Cdh11 activate some genetic compensation program in Cdh11 mutants? Posterior end
+/+
of 8-somite stage embryos hybridized with cdh6 (A- cdh11
and cdh17 probes (C and D cdh11
+/+
(WT) embryo n=94 and B- Cdh11 mutant embryo n=98)
embryos n=102, E to G- cdh11
sa14413
embryos n=107). A- Anterior; P- Posterior.
32
Chapter 4 – Discussion and Future Directions
Unraveling the mysteries that occur during development is a fundamental question in
developmental biology. In this project we aimed to study a possible role of the IM and its derivative the
PNs, in the establishment of LR asymmetries using Cdh11 as our protein of interest.
4.1 - cdh11 morphants exhibit laterality problems
4.1.1 - A new example of uncoupling laterality of the gut and heart
In this study we showed that when we knockdown Cdh11, using a previously published splice
blocking MO (cdh11MO) [40], both gut and heart position were affected. Regarding gut laterality, the
majority of the affected embryos displayed a reversed gut conformation (Figure 18), which indicates
that symmetry within this prospective territory was broken but in a reversed manner. Regarding defects
observed in the heart, most morphant embryos acquired a No Loop phenotype (Figures 19,20),
suggesting that the symmetry in this prospective territory was not broken. The no concordance between
the laterality of the gut and the heart suggests that the establishment of LR asymmetry along the AP axis
might be promoted by different modules. Ours results constitute an interesting new example of the
uncoupling of laterality of the thoracic organs (such as the heart), the abdominal organs (such as the gut)
and, possibly, the brain [15,62] that has already been reported.
4.1.2 – The affected conserved LR genes can explain the organ laterality defects
observed
Since organ laterality is established early in development, we decided to look for the expression
of the conserved LR pathway genes, spaw and pitx2. In embryos injected with the cdh11MO we observed
a concordant reverse expression of both spaw and pitx2 in the right LPM (Figures 25I,J). However, it
was also possible to see that the percentage of bilateral expression of spaw and pitx2 shows a lack of
concordance. This has already been described in different experimental conditions and animal models,
suggesting that an additional activator of pitx2 in the LPM might exist [15,63,64].
As already described, the asymmetric LPM migration that ultimately leads to the gut looping is
dependent on the normal LR pathway cues [19]. Therefore, we propose that the abnormal expression of
the conserved LR genes in the right LPM might underlie the reversed gut loop phenotype that we
observed in our morphant embryos (Figure 18). Our prediction is that the asymmetric LPM migration
is affected in the absence of Cdh11, because the right LPM acquires the identity of the left LPM, as also
seen in other studies [19]. To confirm this hypothesis, we aim to use our optimized immunostaining
protocol (see Experimental Procedures 2.6 and Figure 24) in morphant embryos and analyze the
asymmetric LPM migration in this condition.
Although the expression of spaw and pitx2 in the right LPM can explain the laterality defects in
the gut, it cannot explain the no breaking of symmetry in the heart (Figures 19,20). We hypothesize that
Cdh11 could influence the anterior expansion of spaw expression and therefore its downstream target
pitx2 in the right LPM. The lack of spaw and pitx2 expression in this anterior heart prospective territory
would not allow the break of symmetry. In order to verify our hypothesis, and to test if spaw expression
does not reach the prospective heart territory, we propose to look at the combined expression of spaw
and a marker for heart progenitors, such as nkx2.5, myl7 or lefty2, by WISH at 24-somite stage morphant
embryos.
33
4.1.3 – Does Cdh11 influence the KV fluid flow and dand5 expression?
According to the current models, the expression of spaw in the right LPM, would be a
consequence of an abnormal expression of its antagonist Dand5 in the left side of the KV instead of the
right side, as in normal conditions. To investigate our prediction we propose to characterize the
expression of dand5 by performing a WISH in 8-somite stage morphant embryos.
If dand5 expression in knockdown embryos is indeed on the left side of the KV, then we would
expect that Cdh11 would act within the KV in order to change the fluid flow that ultimately will lead to
alterations in dand5 expression. The KV fluid flow can be influenced by a change in the structure or
size of the KV, which we intend to analyze by performing an immunostaining for phalloidin 488 and
ZO-1 (in red) in morphant embryos at 8-somite stage. It is also possible to influence KV fluid flow by
modifying cilia length and/or number that we will analyze by immunostaining for acetylated α-tubulin,
once more, in 8-somite stage cdh11MO injected embryos.
4.1.3.1 – Is Cdh11 expressed in the KV?
Nonetheless, if we find a phenotype in terms of dand5 expression and in KV morphology in
cdh11 morphants, this will still be puzzling because, cdh11 expression seems to be only expressed in
the IM during these early stages (Figure 21). Because of that it will be important to clearly demonstrate
where Cdh11 mRNA and protein are expressed. For that we propose to retest our antibodies (see
Experimental Procedures 2.7.1) using other immunostaining protocols as well as an improved Western
Blot assay. For this last assay we will use the de-yolking protocol that has been demonstrated to benefit
the resolution of the Western Blot since it avoids the overloading effects of the yolk proteins [57].
As a complementary approach we will perform a quantitative PCR (qPCR) using sorted KV
enriched cell extracts, a technique that will be used to quantify cdh11 mRNA levels. In this experiment
we will resort to the Tg(sox17:EGFP) and Tg(foxj1a:EGFP) [65] because they are expressed in KV
cells. Both transgenic lines are not ideal for our assay because these genes are also expressed in other
tissues. For example, besides the KV, foxj1a:EGFP is expressed during development in the DFC, floor
plate, IM, the pronephric duct and spinal cord [65-67]. Regarding sox17:EGFP, it is also expressed in
the DFC as well as endodermal cells, pancreas and liver [12,48,68,69]. If we find significant levels
of cdh11 mRNA in the cells sorted from both transgenic lines, it will mean that cdh11 is most probably
expressed in the KV because this is, to my knowledge, the only structure that is common between them.
Other approach that we can use to verify if Cdh11 is acting within the KV is by injecting cdh11
MO
at 3 hpf embryos in order to specifically target the KV progenitors, the DFC. If cdh11 is not expressed
in the KV then we expect that the injected embryos would not have laterality problems.
4.2 – Mutant vs Morpholino
4.2.1 - Which one is the best approach?
In the last few years there has been some controversy between knockdown and knockout
phenotypes in zebrafish [70,71]. Because of that we needed to verify if the phenotypes that we observe
using cdh11MO would stand in a cdh11 mutant line. As we mentioned before in Experimental Procedures
2.1, we ordered for EZRC the mutant line cdh11sa14413 [49], in which the formation of a premature STOP
codon due to a single nucleotide nonsense mutation, leads to a Cdh11 truncated protein in the Cdh-Cdh
interaction domain (Figure 16). However, using this line, we were not able to reproduce any of the
phenotypes associated to the morphants (Figures 29,30).
The analysis of these results can lead us to three possible scenarios:
A - cdh11 MO is not specific and the phenotypes observed are due to nonspecific effects [70-72];
34
B- cdh11sa14413 is not a null mutant and the Cdh11 truncated protein still maintains some of its
function;
C- cdh11MO is specific and the cdh11 mutant is a null mutant and the results observed are due
to the activation of a compensatory mechanism in the cdh11sa14413 embryos [59].
To evaluate the cdh11MO specificity (scenario A), we will use a classic mRNA “rescue” approach
to verify the ability to reverse the effects caused by this MO in WT embryos. For that we will inject
synthetic cdh11 capped mRNA together with cdh11MO into 1 cell-stage embryos. In this experiment we
will perform controls for both delivery and concentration of the synthetic cdh11 mRNA and cdh11MO
[72]. We will also address the specificity of this MO by injecting it into the cdh11 mutant. This
experiment can be performed because our mutant does not produce any phenotype and our MO
recognizes a sequence that is localized posterior to the mutated nucleotide (Figure 33 and Supplementary
Figure 7). If the embryo produces a protein, it will be truncated and we expect it to be non-functional
(Figure 16). So if cdh11MO is specific we are not expecting to see any phenotype when this MO is
injected into cdh11sa14413 embryos, because the premature STOP codon, that will produce the affected
protein, appears before the sequence recognized by the MO (Supplementary Figure 7).
Figure 33 – Schematic representation of the Cdh11 protein structure. A- Local in which premature STOP codon is
localized; B- Region recognized by the cdh11MO. SP - signal peptide; EC1-5 - Extracellular Domain 1-5; TM Transmembrane Domain; IC- Intracellular domain.
In scenario B, we hypothesize that we are not able to reproduce any of the phenotypes observed
in the morphant embryos because the Cdh11 mutants produce a truncated protein that could still be
functional. In cdh11sa14413 line, the premature STOP codon is localized at EC4, which means that this
truncated protein still has several of the Cdh11 interactions motifs including the EC1 which are known
to be responsible for homophilic interactions (Figure 16B) [33]. This scenario raises the question of
whether or not this truncated protein is still functional. To test this, we intend to inject a cloned version
of the truncated protein into morphant embryos and analyze the heart phenotype by WISH at 50 hpf
using a myl7 probe. If we are able to “rescue” the phenotype in these embryos, this experiment will
produce a convincing evidence that this truncated protein is functional and is still capable of producing
interactions.
Cdh11 is a cell adhesion molecule also known to engage in heterophilic interactions namely
with Cdh2 [73]. In this context this could suggest that the Cdh11 protein, even truncated, uses the Cdh2
IC to signal in the IM, where they are both expressed (Figure 34). To verify if Cdh11 is acting upstream
of Cdh2, we will use a Cdh2 MO in cdh11 mutant embryos and see if these embryos reproduce the
phenotype observed in cdh11 morphant embryos. As a control, these results will be compared to
both cdh11 and cdh2 morphant embryos to see if additive or different phenotype.
35
Figure 34 – Is Cdh11 signaling through Cdh2? - Left side - In WT embryos the Cdh11 protein (orange) interacts with
other Cdh11 protein (homophilic interactions) or with Cdh2 (blue) in heterophilic interaction. These cell-cell interactions
both cells (black squares) can lead to an intercellular activation of cellular pathways. Both cells are also attached to each
other through the adhesion function of these proteins. Right side - If the mutant embryos produce a truncated Cdh11
protein, the homophilic interactions between this protein would not lead to the activation of any cellular pathways or
contribute to the adhesion between cells because this protein would not have the TM or the IC. Regarding Heterophilic
interactions with Cdh2, the adhesion function would be compromised, once more because the truncated protein does not
have TM or the IC. However, the interaction between the truncated Cdh11 protein and the WT Cdh2 protein can activate
the normal cellular pathways in the cells that express Cdh2. TM – Transmembrane Domain; IC- Intracellular Domain.
For the last possible scenario (C), we consider the possibility that our mutant does not reproduce
the phenotypes observed in morphant embryos because it activates a genetic compensation program as
seen in a previous study [59]. We analyzed this hypothesis by using a candidate approach. We examined
the expression of cdh6 and cdh17 (cadherins also expressed in the some of the domains that Cdh11 is
expressed such as the IM) at 8-somite stage by WISH in mutant embryos. From this experiment we
concluded that, we could not detect any differences in expression and that this method was not the best
one for quantitative comparisons (Figure 32). Therefore, we propose to reanalyze this case by
performing a Microarray or a RNA sequencing in mutant embryos to verify if there is a gene that is
considerably upregulated in this context.
4.2.2 – A new cdh11 mutant
If cdh11 mutant proves not be a null mutant, we will generate a new cdh11 mutant line using
Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) genome editing system [74-76].
This technology is an efficient gene-targeting system, used in a variety of in vivo organisms, that
facilitates multiplex gene-targeting because the
binding of this protein is guided by single basepair complementarities between the engineered
single-guide RNA (gRNA) and the target gDNA
sequence [74-76]. Our idea is to target three
relevant functional domains of the Cdh11 protein:
the signal peptide, the EC3 (a Cdh-Cdh interaction
domain) and Cdh11s’ transmembrane domain
(Figure 35). For that we will produce three
customized small RNA molecules, gRNA. Each
one will be injected into 1-cell stage embryos,
together with the CRISPR associated protein 9
(Cas9)
endonuclease. With this system, the
Figure 35 – Three relevant functional domains that
we will target in cdh11 gene. Adapted from [33].
36
gRNAs direct the Cas9 protein to the target region where it will produce site-specific cleavages. This
will be done in WT and sox17:EGFP backgrounds. Once more, these lines will be used to confirm the
organ laterality phenotype and to evaluate the involvement of Cdh11 in asymmetric tissue
morphogenesis.
4.3 – Alternative ways to understand the role of the Intermediate Mesoderm
in Left-Right asymmetry
So far, we were not able to produce any convincing evidence that the IM plays a role in the
establishment of LR asymmetry, through the expression of Cdh11. Therefore, it is important to prove
the necessity of this tissue in LR asymmetry using other genes and approaches. We will start by
searching for genes that are asymmetrically expressed in the IM. To identify such genes, we will use a
study from 2014 that allow us to analyze a high-resolution genome-wide 3D atlas of gene expression in
the zebrafish embryo at three developmental stages [77]. If any gene fits into this category, we will
search, for mutants or morpholino, that have already been used, to analyze the mutant or morphant
regarding possible LR phenotypes.
To address the necessity of the IM in the LR asymmetry, we propose to use mutant lines to
remove the IM and its derivative, the PN. In these mutants, we will analyze the heart phenotype at 50
hpf by WISH for myl7 and the LPM asymmetric migration between 26 and 30 hpf by immunostaining.
In mouse and Xenopus, it was demonstrated, using mutant lines and morphant embryos that, pax2 and
pax8 have a redundant function and that they are necessary for the specification of the nephric lineage.
It was also shown that, when using double mutants (pax2 and pax8), the embryos fail to form the nephric
duct, which means that none of the embryonic kidneys are formed [78]. In zebrafish, the analysis of the
no isthmus mutant (noi - pax2a mutant) suggests a role for this gene in the definition of the glomerular
and tubule progenitor boundary [78,79]. Therefore, we propose to use a pax8 and a pax2 mutant, such
as the noi, as well as a double mutant for these genes to analyze whether the IM is needed to establish
LR asymmetry. Another study shows that, the odd gene is not required for the IM specification in mouse
embryos, but it controls IM differentiation [80]. Thus, we also propose to analyze odd homologue mutant
in zebrafish, like the osr mutant.
Another way to evaluate the contribution of these tissues during the establishment of LR
asymmetry in zebrafish is to generate a targeted cell ablation line using the
Nitroreductase (NTR)/Metronidazole (Mtz) system [81]. In this technique, the cells that express the
NTR enzyme are capable of converting the non-toxic prodrug Mtz, into a cytotoxic compound that
causes the death of these cells without affecting the surrounding cells. This system is both spatially and
temporally specific since NTR is under the control of a tissue-specific promoter and the cytotoxic
compound is only formed when Mtz is added into the water. Usually, a fluorescent protein is fused into
this construct to confirm the success of the ablation [81]. In our case, we could take advantage of the
cdh17 promoter, which is only activated in the IM and PN territories, and generate a construct in which
both the NTR and the fluorescent protein sequences would be under the control of this promoter [61].
37
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41
Attachments
A1 – Recipes
25x Tricane (VF=1L)
 2g Tricaine powder;
 500mL milliQ Water;
 10mL Tris 1M (pH 9);
 Adjust to pH 7.
Embryo medium 50x (VF=1L)
 14,69g NaCl;
 0,63g KCl;
 2,43g CaCl2.2H2O;
 4,07g MgSO4.7H2O;
 Up to 1L with osmosis reverse water.
Embryo medium 1x (VF=10L)
 200mL 50X Embryo Medium;
 1mL Methylene Blue Solution;
 Up to 10L with osmosis reverse water.
1x Tricane (VF=25mL)
 24mL Embryo medium 1x;
 1mL Tricane 25x.
PK (VF=1mL)
 20mg PK;
 1mL water.
Digestion buffer (VF=5mL)
 125μL NaCl;
 50μL EDTA;
 50μL Tris;
 0,25mL SDS;
 Up to 5mL with DNase-Free water.
20x SSC (VF=1L)
 175.3g of NaCl;
 88.23g of Tri-sodium citrate –dehydrate;
 In 800mL of distilled water;
 Adjust the pH to 7.0 with a few drops of 1M HCl;
 Adjust the volume to 1L with additional distilled water.
42
Pre-Hybridization Mix (VF=500mL)
 250mL Formamide 100%;
 125mL 20x SSC;
 500μL Tween 20;
 ~4,6 mL Citric Acid 1M (ajust to final pH 6.0);
 up to 500mL with milliQ water.
10x PBS (VF=2L)
 160g NaCl;
 4g KCl;
 53,6g Na2HPO4-7H2O ;
 4,8g KH2PO4;
 Adjust pH 7,4 with HCl;
 Up to 2L with milliQ water.
1x PBS (VF=1L)
 100mL of PBS 10x;
 Adjust the volume to 900ml milliQ water.
4% PFA (VF=500mL)
 50ml of PBS 1X;
 20g of paraformaldehyde powder;
 Up to 500mL of PBS 1x.
2x SSC (VF=1L)
 100mL 20x SSC;
 900mL distilled water.
0,2x SSC (VF=1L)
 10mL 20x SSC;
 990mL distilled water.
Blocking solution for in situ (VF=10mL)
 200μL sheep serum;
 0.02g Bovine Serum Albumin (BSA);
 up to 10 mL with 0,1% PBT.
Staining Buffer (VF=50mL)
 2,5mL Tris 2M pH 9,5;
 1,25mL MgCl2 2M;
 1mL NaCl 5M;
 50μL Tween 20;
 up to 50mL with milliQ water.
43
Blocking solution (VF=150mL)
 1,5g BSA;
 0,75mL Tween 20;
 0,75mL Triton-X;
 1,5mL Dimethyl sulfoxide (DMSO);
 Up to 150mL with 1x PBS.
Permeabilizant solution (VF=15mL)
 0,075mL Tween 20;
 0,075mL Triton-X;
 0,11g Glycine;
 Up to 15mL with 1x PBS.
2% PFA (VF=20mL)
 2,5mL 16% PFA
 17,5mL 1x PBS
Dent’s Fixative (VF=10mL)
 8mL MetOH;
 2mL DMSO.
Antigene retrievel buffer (EDTA buffer) (VF=100mL)
 200μL EDTA;
 50 μL Tween 20;
 Up to 100mL with Distilled water.
Calcium free Cold Ringer’s solution (VF=50mL)
 5,8mL 1M NaCl;
 145µL 1M KCl;
 500µL 0.5M HEPES, pH 7.0-7.2;
 Up to 50mL sterile water.
Cold Ringer’s solution with EDTA and PMSF (VF=10mL)
 30µL PMSF 100mM;
 1mL of EDTA 10mM;
 10mL of Calcium free Cold Ringer’s solution.
20% SDS (VF=500mL)
 100g SDS poder;
 Up to 500mL with milliQ water.
10% SDS (VF=200mL)
 100mL 20% SDS;
 100mL milliQ water.
44
2x SDS sample Buffer (VF=10mL)
 1mL 1M Tris-HCl pH 6.8;
 4mL 10% SDS;
 2mL Glycerol;
 2,5mL β-mercaptoethanol;
 500µL 1% Bromophonol blue;
 Up to 10mL with Distilled water.
Stacking Gel
Water
30% Acrylamide
Tris 1,5M (pH=8,8)
10% SDS
Ammonium Persulfate (PSA) 10%
TEMED
12%
(20mL)
6,6
8,0
5,0
0,2
0,2
0,008
8%
(20mL)
9,3
5,3
5,0
0,2
0,2
0,012
Resolving Gel
Water
30% Acrylamide
TRIS 1,0M (PH=6,8)
10% SDS
PSA 10%
TEMED
4mL
2.7
0.67
0.5
0.04
0.04
0.004
5x Running buffer (VF=500mL)
 15,1g Tris-Base;
 94g Glycine;
 900mL milliQ water;
 50mL 10% SDS.
1x Running buffer (VF=1L)
 200mL Running buffer 5x;
 800mL milliQ water.
10x Tranfer Buffer (VF=1L)
 30,3g Tris-Base;
 144g Glycina;
 800mL distilled water;
 Adjust the pH 8,1-8,4;
 Up to 1L distilled water.
45
1x Tranfer Buffer (VF=1L)
 100mL 10x Transfer Buffer;
 200mL MetOH;
 Up to 1L distilled water.
TBS-T (VF=1L)
 20mL TrisHCl 1M (pH 7.6);
 27.5mL NaCl 5M;
 1mL Tween 20;
 up to 1L milliQ water.
TBS (VF=1L)
 20mL TrisHCl 1M (pH 7.6);
 27.5mL NaCl 5M;
 up to 1L milliQ water.
50x TAE (VF=500mL)
 121g TrisBase;
 28,55mL Glacial acetic acid;
 100mL EDTA 0,5M pH8;
 Up to 500mL with MilliQ water.
1x TAE (VF=2L)
 40mL 50x TAE
 Up to 2L with milliQ water
46
A2 – Supplementary Tables
Supplementary Table 1 – List of tissues in which Cadherin-11 (transcript and protein) is known to be expressed during
development in zebrafish. ss – somite stage, hpf – hours post fertilization, Prim-5 – 24 hpf, Prim-15 – 30 hpf, Protrudingmouth – 72 hpf, Long pec- 48 hpf.
Developmental
Stages
5-ss
Organ/Tissue
Neural Keel, IM
Mid brain, Diencephalon, otic vesicle,
ventral neural tube, hindbrain, eye, tail bud
Cleithrum, pectoral fin bud proximal region
Inner ear
Otolith
Brain, inner ear, whole organism
Cranial nerve II, lens, optic tectum, optic
vesicle, retinal, retinal ganglion cell
PN
Reference
[39]
20-ss
Prim-5 to Day 5
Prim-15
Protruding-mouth
Prim-5 to Adult
[43]
14-19-ss to Long pec
[41]
Long pec
[82]
[40]
Supplementary Table 2 – Primers used for cdh11 mutants genotyping.
Primer
Name
sa14413_Fw1
sa14413_Rv1
Sequence
# Bases
CCTTTATGGCTCCCAGCTAC
AGGTTTACCGAGTGCCTTGAT
20
21
Annealing
Temperature
Product
length
62.9°C
1151 bp
Supplementary Table 3 – Primers used for cdh11 mutants genotyping.
Cycle step
Initial denaturation
Denaturation
Annealing
Extension
Final extension
Temperature
98°C
98 °C
62.9°C
72°C
72°C
4°C
Time
30s
10s
30s
30s
10 min
Hold
Cycles
1
30
1
47
Supplementary Table 4 - Appropriate restriction enzyme and RNA polymerase for each probe.
Probe
Restriction Enzyme
myl7
NotI
(New England Biolabs,
10U/μL)
pax2a
cdh11
BamHI
(New England Biolabs,
20U/μL)
HindIII
(New England Biolabs,
20U/μL)
Buffer
RNA polymerase
References
NEBuffer3.1
T3
(Roche Life
Science)
L. Saúde's
Laboratory
NEBuffer3.1
T7
(Roche Life
Science)
L. Saúde's
Laboratory
NEBuffer 2.1
T7
L. Saúde's
Laboratory
cdh6
XhoI
(Promega, 10U/μL)
BufferD
Sp6
(Roche Life
Science)
S. Hans et al
2013
cdh17
NotI
NEBuffer3.1
Sp6
E. Butko et al
2015
CutSmart®Buffer
T7
L. Saúde's
Laboratory
CutSmart®Buffer
T7
L. Saúde's
Laboratory
spaw
pitx2
SacI
(New England Biolabs,
20U/μL)
SpeI
(New England Biolabs,
10U/μL)
Supplementary Table 5 – Combinations tested during the immunostaining protocol optimization for the LPM
migration assay.
Lines
Antibody combination
Tg(hand2:EGFP)
Tg(hand2:EGFP)
Tg(hand2:EGFP)
WT AB
Tg(sox17:EGFP)
WT AB
Tg(hand2:EGFP)
Tg(sox17:EGFP)
Tg(hand2:EGFP)
Tg(hand2:EGFP)
Tg(sox17:EGFP)
Tg(sox17:EGFP)
Phalloidin 488
(1:200)
Phalloidin 488
(1:200)
Phalloidin 488
(1:200)
Phalloidin 488
(1:200)
Phalloidin 594
(1:200)
Phalloidin 488
(1:200)
Thickness (μm)
10, 50, 100, 200,
300
Anti-GFP
(1:2000)
ZO-1 594
(1:1000)
20
48
A3 – Supplementary Figures
Supplementary Figure 1 - Phylogenetic tree of the major animal clades. Outline in red are the phyla in which Nodal
cascade genes were identified. Adapted from [3].
A
Supplementary Figure 2 – LPM asymmetric
migration is independent on the endoderm existence
and is necessary for the correct gut loop. A and B –
Asymmetric LPM migration occur in the absence of
endoderm, transverse sections through the gut-looping
region of bon mutants. C to E – Transversal sections on
has (D) and nok (E) mutants embryos with gut looping
defects. Adapted from [19].
B
C
D
E
49
A
B
C
LPM
IM
KV
Supplementary Figure 3 - Origins of the Intermediate Mesoderm. A and B – The IM origin in zebrafish; C- IM during
development. IM – intermediate mesoderm; KV – Kupffer's vesicle; LPM – Lateral plate mesoderm; Adapted from [28].
Supplementary Figure 4 - cdh2 is asymmetric
express in Hensen’s Node at HH5. Whole mount in
situ hybridization using cdh2 probe in A- HH4 and
B- HH5 embryos. Adapted from [37].
Supplementary Figure 5 – N-cadherin affects the direction of
the heart loop in chicken embryos. A- Normal heart loop in
Control embryos B- Inverted heart loop in Anti-N-cadherintreated embryos. L – left, R- right. Adapted from [35].
50
A
Somite
Right LPM
Gut
R
L
NT
B
Somite
Left LPM
Gut
Right LPM
Supplementary Figure 6 – Does the PN provide a stable structure for the asymmetric migration of the underlying
LPM? Confocal Laser point-scanning image sections from A- Tg(hand2:EGFP) outcross at 30hpf immunostained for and
phalloidin 488 (in green) (10μm thickness) and B- Tg(hand2:EGFP) immunostained for phalloidin 594 (in red) and GFP
(in green) (sections with 20μm thickness). (scale – 30μm). LPM – lateral plate mesoderm, NT – neural tube, L- left, R –
Right.
51
Supplementary Figure 7 –WT and mutant when injected with cdh11MO. WT - The pre-mRNA, transcribed from DNA
sequence, and processed (splicing) in order to become a mature mRNA. Afterwards this mRNA will be translated and
produce a functional protein. WT with cdh11MO - When a splicing blocking MO, such as the cdh11MO, is injected into a
WT embryos, the pre-mRNA processing is inhibit, and this will allow the incorporation of an intron, in this case the 10 th
intron. This will not allow the production of a functional protein. Mutant - In cdh11 mutant line, we have a premature
STOP codon due to a single nucleotide nonsense mutation that produces a truncated and we expect it not to be functional.
Mutant with cdh11MO – If cdh11MO is specific , Cdh11 would not be functional because the premature codon STOP,
that will produce the affected protein, appears before the sequence recognized by the MO. T/U – WT nucleotide; A –
Mutated nucleotide; purple rectangle – Schematic representation of the cdh11MO; Arrows – mRNA Splicing.
52