3754 RESEARCH ARTICLE
STEM CELLS AND REGENERATION
Development 140, 3754-3764 (2013) doi:10.1242/dev.098798
© 2013. Published by The Company of Biologists Ltd
Transcriptional components of anteroposterior positional
information during zebrafish fin regeneration
Gregory Nachtrab1, Kazu Kikuchi1,*, Valerie A. Tornini1 and Kenneth D. Poss1,2,‡
SUMMARY
Many fish and salamander species regenerate amputated fins or limbs, restoring the size and shape of the original appendage.
Regeneration requires that spared cells retain or recall information encoding pattern, a phenomenon termed positional memory. Few
factors have been implicated in positional memory during vertebrate appendage regeneration. Here, we investigated potential
regulators of anteroposterior (AP) pattern during fin regeneration in adult zebrafish. Sequence-based profiling from tissues along
the AP axis of uninjured pectoral fins identified many genes with region-specific expression, several of which encoded transcription
factors with known AP-specific expression or function in developing embryonic pectoral appendages. Transgenic reporter strains
revealed that regulatory sequences of the transcription factor gene alx4a activated expression in fibroblasts and osteoblasts within
anterior fin rays, whereas hand2 regulatory sequences activated expression in these same cell types within posterior rays. Transgenic
overexpression of hand2 in all pectoral fin rays did not affect formation of the proliferative regeneration blastema, yet modified the
lengths and widths of regenerating bones. Hand2 influenced the character of regenerated rays in part by elevation of the vitamin
D-inactivating enzyme encoded by cyp24a1, contributing to region-specific regulation of bone metabolism. Systemic administration
of vitamin D during regeneration partially rescued bone defects resulting from hand2 overexpression. Thus, bone-forming cells in a
regenerating appendage maintain expression throughout life of transcription factor genes that can influence AP pattern, and differ
across the AP axis in their expression signatures of these and other genes. These findings have implications for mechanisms of
positional memory in vertebrate tissues.
INTRODUCTION
Many recent studies have employed cell transplantation or genetic
fate-mapping approaches to identify the cellular sources of tissues
that arise during injury-induced regeneration (Buckingham and
Meilhac, 2011; Tanaka and Reddien, 2011). Upon defining sources
of regeneration through these experiments, a key priority is then to
understand how these stem/progenitor cells and differentiated cell
types successfully restore complex tissues of the correct size, pattern
and function after organ damage.
Limbs and fins are complex three-dimensional structures
composed of numerous tissue types. Remarkably, many fish and
salamanders retain the ability to regenerate amputated appendages
throughout their adult lives. To do this, cells within the appendage
stump must retain and recall detailed patterning information, which
is commonly referred to as positional memory. A regulator of
regenerative positional memory is expected to possess two main
characters: (1) presence in a gradient or restricted pattern within the
intact and regenerating adult appendage; and (2) its overexpression
or blockade impacts the regenerative pattern. In planarians, which
are invertebrates that undergo vigorous cellular turnover and
regenerate through a stem cell population known as neoblasts,
recent evidence indicates that the adult pattern is actively
1
Department of Cell Biology and Howard Hughes Medical Institute, Duke University
Medical Center, Durham, NC 27710, USA. 2Davis Center for Regenerative Biology
and Medicine, Mount Desert Island Biological Laboratory, Salisbury Cove, ME 04672,
USA.
*Present address: Developmental and Stem Cell Biology Division, Victor Chang
Cardiac Research Institute, Darlinghurst, NSW 2010, Australia
‡
Author for correspondence ([email protected])
Accepted 26 June 2013
maintained by the regionalized expression of developmental
pathway regulators. Upon injury, these same factors help restore
tissue and reinstate pattern (Reddien et al., 2007; Gurley et al., 2008;
Petersen and Reddien, 2011; Roberts-Galbraith and Newmark,
2013).
During amphibian limb regeneration, retinoic acid (RA) has been
implicated in positional memory, as RA treatment causes wrist-level
amputations to sprout shoulder-level regenerates (Maden, 1982).
Although this finding is remarkable, the role of endogenous RA in
positional memory is unclear as there is no discernible
proximodistal (PD) gradient of RA in the intact limb. A second
candidate factor is Prod1, a proposed receptor for the newt blastemal
mitogen Anterior gradient (da Silva et al., 2002; Kumar et al.,
2007b). Prod1 is induced by exogenous RA and expressed at
slightly higher levels in proximal intact limb regions as compared
with distal regions (Kumar et al., 2007a). Although its in vivo
function is unknown, inhibition of Prod1 in cultured blastemas
blocks the characteristic in vitro behavior of proximal blastemas,
and blastemal cells electroporated with excess Prod1 distribute
proximally compared with control electroporations (da Silva et al.,
2002; Echeverri and Tanaka, 2005). Potential links between RA and
Prod1 could be provided by Meis proteins, which are important for
the proximalizing effects of RA on regeneration and may regulate
Prod1 (Mercader et al., 2005; Shaikh et al., 2011). In summary,
although positional memory is a crucial aspect of regeneration, there
remains much to learn about how it is encoded and enacted during
vertebrate appendage regeneration.
How pattern is initially established in the developing appendages
of vertebrate embryos has been intensely studied (Duboc and Logan,
2009; Towers and Tickle, 2009; Zeller et al., 2009). In particular,
several regulators of embryonic limb anteroposterior (AP) patterning
have been identified based on their restricted expression patterns and
Development
KEY WORDS: Regeneration, Anteroposterior patterning, Zebrafish, Fin, Blastema, Hand2, Vitamin D, Positional memory
RESEARCH ARTICLE 3755
Patterning fin regeneration
MATERIALS AND METHODS
Zebrafish
Males of several zebrafish strains show defects in pectoral fin regeneration
(Nachtrab et al., 2011), necessitating the use of females for these
experiments. All animals were between 4 and 12 months of age and in an
outbred Ekkwill (EK) strain background. Pectoral fins were amputated
proximal to the first bifurcation point at approximately one-third of their
original length using iridectomy scissors. Fin lengths and widths were
measured from images using Leica Application Suite V3.6 software. Widths
of the segment proximal to the first bifurcation of the ray were measured in
uninjured fins, and the second ray segment distal to the amputation plane
was measured in fin regenerates. Heat-shock experiments were performed
by giving transgenic and clutchmate controls a daily 38°C heat shock as
described (Wills et al., 2008). A Vivo-Morpholino (Gene Tools) was directly
microinjected into the posterior region of 3- and 4-dpa pectoral fin
regenerates. The translation-blocking hand2 morpholino was described
previously (Maves et al., 2009). Fins were collected and dissected 12 hours
after the second morpholino injection, at ~4.5 dpa. 1α,25-dihydroxyvitamin
D3 (Sigma, D1530) was dissolved in ethanol to make a 10 µM stock solution
and stored at −20°C. For intraperitoneal injections, this stock solution was
diluted 1:10 with water and 10 µl was injected per fish. For quantitative
PCR assays after heat shocks in transgenic animals during regeneration,
fins were collected for RNA isolation 6 hours after the heat shock at 4 dpa
unless otherwise indicated. hand2 mutant embryos were collected along
with clutchmates from hand2s6 heterozygous crosses and identified visually
at 4 dpf for RNA isolation (Yelon et al., 2000).
Construction of transgenic animals
osx:EGFP-CAAX was generated by subcloning an EGFP-CAAX cassette that
had been amplified from Tol2kit plasmid #384 (Kwan et al., 2007)
downstream of published promoter sequences of medaka osterix (Renn and
Winkler, 2009). The full name of this transgenic line is Tg(osterix:EGFPCAAX)pd51. For alx4a:DsRed2, the first exon of alx4a in the BAC clone
CH211-107P11 was replaced with a DsRed2 cassette at the translational
initiation site by Red/ET recombineering (GeneBridges). The full name of
this transgenic line is Tg(alx4a:DsRed2)pd52. For tbx18:EGFP, the first exon
of tbx18 in the BAC clone CH211-197L9 was replaced with an EGFP cassette
at the translational initiation site by Red/ET recombineering (GeneBridges).
The full name of this transgenic line is Tg(tbx18:EGFP)pd21. For hsp70l:alx4a,
hsp70l:id4, hsp70l:lhx9 and hsp70l:hand2, full-length cDNAs were cloned
from adult pectoral fins and then subcloned downstream of the inducible
hsp70l promoter (Halloran et al., 2000). The full names of these transgenic
lines are Tg(hsp70l:alx4a)pd53, Tg(hsp70l:id4)pd54, Tg(hsp70l:lhx9)pd55 and
Tg(hsp70l:hand2)pd56. An α-crystallin:EGFP cassette was inserted in reverse
orientation to make lens fluorescence an identifier of transgenic animals
(Waxman et al., 2008). Purified plasmid or BAC DNA was co-injected with
I-SceI into single-cell embryos.
RNA isolation and quantitative PCR (qPCR)
For gene expression analysis, fin regions from three fish were dissected and
pooled for each sample. RNA was isolated using Tri Reagent (Sigma).
cDNA was synthesized from 1 μg total RNA using the Roche First-Strand
Synthesis Kit. qPCR was performed using the Roche LightCycler 480 and
SYBR Green I Master Mix. All samples were analyzed in biological
triplicate and technical duplicate, and all reactions were performed with an
annealing temperature of 60°C. The analysis was performed using the ΔΔCT
method as previously described (Yin et al., 2008). Primers are listed in
supplementary material Table S1.
RNA sequencing (RNA-Seq)
The two most anterior and posterior rays (AP1 and AP5) were collected
and pooled from pectoral fins of 20 6- to 8-month-old zebrafish in duplicate.
RNA was isolated using Tri Reagent. Samples were then submitted to the
Duke Genome Sequencing and Analysis Core for library preparation and
run on an Illumina HiSeq2000. The data were analyzed using TopHat,
Bowtie and Cufflinks according to described protocols (Trapnell et al.,
2012). Ensembl Zv9.70 was used for genome annotation. Gene ontology
analysis was performed using the Princeton University Lewis-Sigler
Institute for Integrative Genomics website (http://go.princeton.edu/).
Immunofluorescence and BrdU incorporation
Fins were removed and fixed in 4% paraformaldehyde at room temperature
for 1 hour. Staining of fin cryosections was performed as described (Johnson
and Weston, 1995; Wills et al., 2008) using a p63 (Tp63) antibody (mouse
4A4, Santa Cruz Biotechnology) at 1:200 or Zns-5 (ZIRC) at 1:200.
Imaging and colocalization analysis were performed using a Zeiss LSM
700 confocal microscope. Quantification of pectoral fin BrdU incorporation
was as described (Nachtrab et al., 2011).
RESULTS
Regionalized gene expression in adult zebrafish
pectoral fins
The skeletal components of fins, a set of cylindrical segmented rays,
are composed of dermal bone that is formed by the deposition of
collagen and other proteins as well as mineral components. Fin rays
are connected by intraray mesenchyme and covered by epidermis,
and encase fibroblasts, nerves, blood vessels and pigment cells.
There are clear differences in fin ray lengths and widths along the
AP axes of pectoral fins. For instance, the third (anterior) ray is on
average 47% longer and 65% wider than the eighth (posterior) ray
at the base where segmentation begins (Fig. 1A; supplementary
material Fig. S1). Fin regeneration in zebrafish is initiated by the
formation of a blastema at the healed distal tip of each ray by 2-4
days post-amputation (dpa). Fin ray regeneration proceeds by
maintenance of proliferative blastemal tissue just distal to a
patterning zone, in which osteoblasts align and mineralize bone
(Akimenko et al., 2003; Poss et al., 2003). We analyzed ray
morphology and osteoblast differentiation events during
regeneration, aided by a transgenic reporter strain visualizing
expression of the osteoblast transcription factor osterix (osx or sp7).
Differences in the patterns of aligned osteoblasts among rays on the
AP axis began to manifest during a period from 5-7 dpa, and the
AP ray pattern was restored by 10 dpa (Fig. 1A,B).
To identify genes that might be responsible for AP differences in
ray pattern, we sequenced RNAs collected from the most anterior
(AP1) and most posterior (AP5) regions of pectoral fins (Fig. 1C;
Development
the robust effects of their gain- or loss-of-function on limb patterning
(Riddle et al., 1993; Qu et al., 1997; Charité et al., 2000; Zákány et al.,
2004). For instance, the transcription factor Hand2 is localized in the
posterior region of developing pectoral appendages, and its
overexpression causes developmental transformations along the AP
axis. Despite abundant experimental data on AP patterning in
embryonic limbs, positional memory of regenerating appendages has
focused instead on PD regulation.
Adult zebrafish regenerate amputated fins with speed and
precision. Recent work has produced cellular and molecular models
for blastema formation and regenerative outgrowth during fin
regeneration, but positional memory remains largely unexplained
(Knopf et al., 2011; Blum and Begemann, 2012). Here, we used
RNA sequencing to identify factors with potential roles in positional
memory along the AP axis in zebrafish pectoral fins. We found
many genes with AP region-specific expression in uninjured fins,
several of which are transcription factor genes with known roles in
AP patterning of embryonic pectoral appendages. Transgenic
overexpression of one of these genes, hand2, modified bone
patterning during regeneration, in part through regulation of vitamin
D metabolism and signaling. These experiments identify a factor
with the characteristics of a positional memory component, and
provide evidence that adult zebrafish fins maintain positional
information via the sustained regional restriction of key embryonic
patterning genes.
3756 RESEARCH ARTICLE
Development 140 (18)
sequencing data are deposited in the NCBI SRA database, accession
SRP027598). We found 235 predicted genes with significantly
different expression in these two regions, representing ~0.8% of the
transcriptome (supplementary material Fig. S2A). These 235
predicted genes comprised 195 annotated genes and 40 putative
genes. Of the 195 AP genes, 105 were elevated in anterior rays and
90 in posterior rays. To assess whether certain categories or classes
of genes were enriched in our AP dataset, we performed a gene
ontology (GO) search. Using the GO category ‘biological process’,
we found our AP genes to be enriched for general terms such as
‘developmental process’ and ‘biological regulation’. However, we
also saw enrichment for the more specific terms ‘fin development’,
‘nervous system development’ and ‘regulation of transcription,
DNA-dependent’ (supplementary material Fig. S2B).
For several reasons, we focused our initial analysis on
differentially expressed transcription factor genes. First,
transcription factors are central to many developmental programs;
thus, their regionalized expression has the potential to impact
multiple downstream genes. Second, as mentioned above, a rich
field of embryonic limb development has detailed the relationship
between transcription factors and AP skeletal patterning (Qu et al.,
1997; Charité et al., 2000; Fernandez-Teran et al., 2000; Zákány et
al., 2004; Tzchori et al., 2009; Galli et al., 2010; Sheth et al., 2012).
Third, our dataset revealed many examples of developmental
transcription factors with expression differences along the AP axis.
In particular, the transcription factor hand2, which is crucial for
pectoral fin development and expressed in the posterior region of
developing pectoral fin and forelimb buds (Charité et al., 2000;
Fernandez-Teran et al., 2000; Yelon et al., 2000), showed the most
polarized expression of all genes enriched in posterior fin rays
(Table 1). Additionally, the embryonic anterior patterning factors
alx4a and lhx9 were fifth and eighth on the list of genes enriched in
anterior rays (Table 1).
To define transcription factor expression signatures in pectoral
fins, we performed quantitative RT-PCR (qPCR) using tissues
across five different regions of the AP axis. This approach
Development
Fig. 1. Regeneration of pattern and underlying differential gene expression in pectoral fins. (A) Zebrafish pectoral fins possess and regenerate an
anteroposterior (AP) skeletal pattern. Early osteoblasts marked by osx:EGFP-CAAX (green) are present in the regenerate prior to the onset of AP
differences, which arise between 5 and 7 days post-amputation (dpa). Arrowheads indicate amputation plane. BF, bright field. (B) Zebrafish robustly
regenerate the AP bone pattern. Quantification of the relative ratios between the width of the uninjured portion of anterior ray 3 (blue) and the widths
of regenerating rays across the AP axis. n=12; bar indicates mean. (C) Diagram of a pectoral fin defining the two regions used for RNA-Seq (circled AP1
and AP5) and the five regions (AP1-AP5) used for subsequent qPCR validations. (D) Each AP region of uninjured zebrafish pectoral fins expresses a
unique AP code of patterning transcription factor genes. Results shown reflect qPCR confirmation of RNA-Seq data, normalized to β-actin 1 (actb1)
levels. n=3. (E) The AP regionalized expression of transcription factor genes is maintained during regeneration. qPCR expression profiles at 4 dpa,
normalized to actb1 levels. n=3; mean ± s.e.m.; *P<0.05, **P<0.005, Student’s t-test.
RESEARCH ARTICLE 3757
Table 1. The top 20 genes enriched in the fin anterior or fin
posterior
Gene
Anterior-enriched genes
rspo4
zgc:165423
mpz
rspo1
alx4a
CR925813.1
BX927244.2
lhx9
chodl
fcgbp
si:ch211-36i19.1
tyrp1b
slc7a8 (2 of 2)
plcxd3
scinla
Posterior-enriched genes
hand2
scpp7
ucp1
si:dkey-22i16.3
and2
tbx4
asip
and3
si:dkey-189n19.4
hoxd12a
and1
hoxd11a
frem2a
hoxd13a
cyp24a1
Anterior
(FPKM)
Posterior
log2
(FPKM) (fold-change)
P-value
1.478
1.353
20.003
2.937
5.257
3.400
40.493
8.475
0.940
1.193
1.297
23.081
2.980
1.010
55.695
0.000 –1.79769e+308 1.2E–04
0.018
–6.259
1.9E–05
0.757
–4.724
3.8E–08
0.113
–4.705
1.0E–13
0.221
–4.575
0.0E+00
0.163
–4.380
1.5E–07
1.993
–4.345
3.9E–13
0.424
–4.320
3.2E–09
0.059
–3.985
1.2E–04
0.077
–3.947
2.8E–08
0.087
–3.900
1.1E–04
1.598
–3.852
0.0E+00
0.222
–3.749
9.3E–08
0.085
–3.572
4.1E–06
4.687
–3.571
0.0E+00
0.097
0.287
0.105
0.520
0.410
0.026
2.140
0.572
5.103
0.983
4.981
1.125
0.143
0.674
6.780
18.979
50.526
16.550
35.554
12.387
0.780
36.870
8.420
66.136
12.602
60.058
12.779
1.575
5.491
52.182
7.614
7.462
7.294
6.095
4.918
4.901
4.107
3.880
3.696
3.680
3.592
3.506
3.459
3.026
2.944
0.0E+00
0.0E+00
2.3E–04
0.0E+00
0.0E+00
6.9E–06
8.0E–11
1.8E–12
4.8E–05
8.7E–12
0.0E+00
0.0E+00
0.0E+00
1.0E–12
0.0E+00
Among the posterior-enriched genes, hand2 displays the greatest differential
expression. P-values by Student’s t-test.
FPKM, fragments per kb of transcript per million map reads.
confirmed restriction of hand2 to posterior fin rays, and also verified
region-specific expression for eight other transcription factor genes:
alx4a, lhx9, id4, pax9, tbx2a, hoxc8a, hoxd11a and hoxd13a. Six of
these eight transcription factors have roles in AP patterning during
limb or fin development (Qu et al., 1997; Harrelson et al., 2004;
Zákány et al., 2004; McGlinn et al., 2005; Tzchori et al., 2009),
whereas id4 has been implicated in bone homeostasis (Tokuzawa
et al., 2010). The anterior expression of the Hoxc genes has been
described during pectoral fin development (Molven et al., 1990),
and HOXC8 has specifically been shown to be expressed in the
anterior region of developing chick wings (Nelson et al., 1996).
Gauged by the expression of these nine factors, each set of two rays
across the AP axis displayed a unique gene signature (Fig. 1D).
Notably, hand2, hoxd13a, alx4a and lhx9 displayed regional
expression in adult fins similar to their reported expression in
embryonic forelimb or pectoral fin buds (Qu et al., 1997; Charité et
al., 2000; Ahn and Ho, 2008; Wang et al., 2011). If important for
patterning regenerating structures, these transcription factors would
be expected to retain their regionalized character during
regeneration. Indeed, at 4 dpa, all nine factors had a profile similar
to that in uninjured fins (Fig. 1E).
In summary, adult zebrafish pectoral fins retain region-specific
signatures of transcription factor genes important for AP patterning
of embryonic pectoral appendages, consistent with potential roles in
patterning and positional memory.
Fin fibroblasts and osteoblasts differ across the
AP axis in the expression of patterning
transcription factor genes
To visualize the differential expression of anterior and posterior
factors, we generated a BAC transgenic reporter line
Tg(alx4a:DsRed2)pd52 and also assessed reporter expression in the
Tg(hand2:EGFP)pd24 BAC transgenic line (Kikuchi et al., 2011).
Using these lines, we found that the alx4a region extended from the
most anterior to the third rays of the pectoral fin. Conversely, the
hand2 domain extended from the sixth ray to the posterior edge of
the fin (Fig. 2A). In uninjured fins, both regulatory sequences
activated expression along the entire PD axis of the fin, and
similarly during regeneration to the near distal tips (Fig. 2A-C).
These adult expression domains were reminiscent of their profiles
in developing embryonic fin buds (Fig. 2D). We visualized in the
same way the expression of a third transcription factor, tbx18, which
is known to be expressed along the entire AP axis of developing
zebrafish pectoral fins (Liu and Stainier, 2010). The BAC transgenic
reporter line Tg(tbx18:EGFP)pd21 revealed fin-wide expression of
tbx18 in adult zebrafish pectoral fins, a result verified by qPCR
(supplementary material Fig. S3A,B).
To assess expression differences among fins, we also examined
alx4a-, hand2- and tbx18-driven transgenic reporter expression in
other adult zebrafish fins. This revealed a similar pattern of alx4a,
hand2 and tbx18 expression in pelvic fins as in pectoral fins. In the
anal and dorsal fins, alx4a was expressed in the most anterior
marginal ray, whereas hand2 was expressed weakly in the most
caudal ray. In the caudal fin, alx4a was expressed only in the most
ventral ray, and hand2 expression was not detectable. tbx18 was
expressed weakly in these unpaired fin types (supplementary
material Fig. S3C,D). These observations indicated that all adult
zebrafish fins maintain the expression of transcription factors
associated with their initial development, with different expression
domains in differentially patterned appendages.
To determine which cells contained AP regionalized expression
of hand2 and alx4a, we histologically assessed pectoral fins from
the reporter strains. Longitudinal and transverse sections of
regenerates revealed that the expression of hand2 and alx4a was
limited to the mesenchymal compartment of fins (Fig. 2C,E).
Although the majority of cells in the mesenchyme are fibroblasts,
we also found that many osteoblasts lining the bone rays were
distinctly positive for either of these transcription factors (Fig. 2F).
Thus, AP patterning transcription factors are maintained in unique
expression domains in the bone-forming cells of uninjured and
regenerating fins. Although differentiated osteoblasts from different
fin ray regions appear identical and have been considered as such in
models of regeneration, it is now clear that they have different gene
expression signatures.
hand2 overexpression alters bone lengths and
widths in regenerating fins
Next, we examined whether the regional restriction of transcription
factors is required for normal patterning during fin regeneration.
We generated transgenic zebrafish permitting heat-inducible
expression of the anterior genes alx4a, lhx9 or id4 or of the posterior
gene hand2. This approach enables inducible expression of a
candidate factor across the entire AP axis of the adult pectoral fin.
We reasoned that the overexpression of potential positional memory
components would alter regenerative patterning and be manifested
in changes in the lengths and widths of fin rays. This approach is
analogous to the overexpression techniques that defined key regions
and factors for embryonic limb patterning in mouse and chick, prior
Development
Patterning fin regeneration
3758 RESEARCH ARTICLE
Development 140 (18)
to the development of many conditional knockout models (Maccabe
et al., 1973; Riddle et al., 1993; Charité et al., 2000).
We identified inducible transgenic lines for the anterior genes
alx4a, id4 and lhx9 [Tg(hsp70l:alx4a)pd53, Tg(hsp70l:id4)pd54 and
Tg(hsp70l:lhx9)pd55] that enabled ~13-, 10- and 13-fold increases,
respectively, in the expression of these genes in posterior rays after
a single heat shock at 4 dpa (supplementary material Fig. S4A). We
then examined whether this overexpression could alter regenerative
pattern by amputating pectoral fins in these lines and administering
a daily heat shock. At 7 dpa, there was no gross change in the
appearance of the fins (supplementary material Fig. S4B). We
quantified the lengths and widths of regenerated rays to ascertain
whether there were any subtle alterations. Heightened id4 levels
during regeneration produced an 11% increase in posterior fin ray
lengths (supplementary material Fig. S4C). Elevated alx4a
increased relative medial and posterior ray widths by 4% and 6%,
respectively, and there was a 6% increase in the anterior and medial
regions caused by id4 overexpression (supplementary material Fig.
S4D). These modest changes in patterning suggested that the
anterior factors we examined are not individually sufficient to
influence patterning in regenerating pectoral fin rays.
We next assessed the effects of hand2 overexpression
[Tg(hsp70l:hand2)pd56], which was increased 58-fold in anterior fin
regions after a single heat shock at 4 dpa (Fig. 3A), a change that
reflects more the negligible endogenous levels of hand2 mRNA in
anterior rays, rather than the sheer magnitude of hand2 induction
(which is similar to that of alx4a, lhx9 or id4 after heat-shock
induction of the respective transgenes) (Fig. 1D,E; supplementary
material Fig. S5A). Seven days of hand2 induction initiated after
amputation was sufficient to produce moderate to severe defects in
the patterns of regenerating fin rays (Fig. 3B). Overall, there was a
42% decrease in the lengths of regenerated anterior rays at 7 dpa
and a 56% decrease in lengths of posterior rays (Fig. 3C). We also
found reductions in width ratios across pectoral fins ranging from 14
to 22% (Fig. 3D). To assess whether the hand2 overexpression
phenotype was the result of a transient delay in regeneration, we
extended the experiment to 30 dpa, approximately twice the time
within which regeneration is normally completed. We observed
Development
Fig. 2. Visualization of AP region-specific transcription factor expression in fin fibroblasts and osteoblasts. (A) Expression of fluorescent
transgenic reporters in adult pectoral fins. The alx4a:DsRed2 domain ranges from the most anterior to the third ray. The hand2:EGFP domain extends
from the posterior edge to the sixth ray of the fin. (B) The AP expression characteristics of alx4a:DsRed2 and hand2:EGFP are maintained throughout
regeneration. Arrowheads indicate amputation plane. (C) Longitudinal sections at 4 dpa confirm reporter expression to nearly the distal tip of the fin.
Arrowheads indicate amputation plane. (D) Expression of fluorescent reporters in embryonic pectoral fins. At 4 days post-fertilization (dpf ), both
reporters display region-specific expression. The expression is similar to that of the adult, but the double-negative medial region is not yet defined.
(E) Transverse sections indicate that the expression of each reporter is restricted to the fin mesenchyme. The antibody against p63 marks fin epidermis
adjacent to the mesenchymal compartment. Arrowheads indicate amputation plane. (F) Transverse sections indicating that both alx4a:DsRed2 and
hand2:EGFP are expressed in a population of fin osteoblasts identifiable by Zns-5 immunoreactivity. Arrowheads indicate amputation plane.
Patterning fin regeneration
RESEARCH ARTICLE 3759
shortened regenerates with smaller rays and fewer segments in the
hsp70l:hand2 transgenic fish at 30 dpa, similar to phenotypes at 7
dpa (Fig. 3E-G), indicating that the patterning phenotype was not
the result of a transient regenerative delay.
Overexpression of hand2 in developing mammalian limbs results
in striking phenotypes, including polydactyly and anterior-toposterior digit conversions (Charité et al., 2000). However,
overexpression of hand2 during zebrafish pectoral fin regeneration
caused a less striking phenotype – an increase in the posterior
character of all fin rays. There are several reasons that might explain
this result. First, unlike an embryonic fin bud, mammalian limb bud
or newt limb blastema, in which a single primordium is patterned by
signaling molecules, an adult fin regenerate is a composite of
individual ray primordia. The overall fin shape is a cumulative
readout of the patterning of the individual ray blastemas to give each
ray a length and width, and it is unlikely that the overexpression of
a single factor such as hand2 would produce a phenotype similar to
that seen in other contexts. Second, hand2 is only one of several
transcription factors with differential AP expression in adult pectoral
fins, and overexpression of hand2 did not cause similar significant
alternations in the expression of other AP transcription factors
(supplementary material Fig. S5B). Third, heat shock raises
expression without altering the underlying endogenous asymmetry,
leading to a shallower but still significant AP gradient of hand2
across the AP axis (supplementary material Fig. S5C). Finally, as
many adult fin rays have no detectable hand2 expression, there is
unlikely to be a striking change in length and width caused by
changing only Hand2 levels.
To identify a critical window for the effects of hand2
overexpression, we controlled the timing and number of heat shocks
with respect to amputation. When hand2 was induced only during
the first 3 days after amputation and prior to significant bone
Development
Fig. 3. Overexpression of hand2 during fin
regeneration alters ray patterning. (A) hand2
expression is induced 58-fold in anterior regions of
hsp70l:hand2 pectoral fins 4 hours after a single heat shock
at 4 dpa. Values are normalized to actb1 levels and relative
to wild-type controls. n=3; mean ± s.e.m. (B) Appearance
of hsp70l:hand2 and wild-type clutchmate fins at 7 dpa
after a series of daily heat shocks. hand2 overexpression
generates shorter rays with a reduced number of bone
segments. Phenotypes range from moderate (upper right)
to severe (lower right). Representative regenerative
growths of wild-type rays 3 and 8 are denoted by dotted
lines. (C) Overexpression of hand2 reduces the lengths of
regenerating rays across the AP axis of pectoral fins. n=16
(wild type) and n=15 (hsp70l:hand2). (D) Overexpression of
hand2 during regeneration reduces the widths of
regenerating fin rays across the AP axis. Transgenic fish
that displayed a moderate phenotype were quantified.
n=13 (wild type) and n=6 (hsp70l:hand2). (E) Appearance
of hsp70l:hand2 and wild-type clutchmate fins at 30 dpa
after daily heat shocks. hsp70l:hand2 regenerates remain
stunted with shorter and thinner fin rays. Representative
regenerative growths of wild-type rays 3 and 8 are
denoted by dotted lines. (F) Quantification of
hsp70l:hand2 ray lengths at 30 dpa. n=12.
(G) Quantification of hsp70l:hand2 segment numbers at 30
dpa. n=12. (H) Quantification of hsp70l:hand2 ray widths at
30 dpa. n=12. *P<0.05, **P<0.005, Student’s t-test; bar
indicates mean. Arrowheads (B,E) indicate amputation
plane. Scale bars: 0.5 mm.
3760 RESEARCH ARTICLE
Development 140 (18)
deposition, there was no significant change in the lengths of
regenerating fins (Fig. 4A,B). In agreement with this, daily hand2
induction had no significant effect on indices of blastemal cell
proliferation at 3 dpa, as compared with heat-shocked wild-type
zebrafish (Fig. 4C,D). However, when daily hand2 induction was
initiated at 3 dpa, there was a 20% decrease in the lengths of
regenerated anterior rays and an 8% decrease in ray widths by 7 dpa
(Fig. 4E-G). These results indicated that elevating the levels of the
normally posterior-restricted hand2 throughout the fin does not
affect early regenerative growth, but alters ray patterning by
reducing ray lengths and widths.
Together, our data indicate that the maintenance of hand2 in the
mesenchyme of posterior rays contributes to the restoration of
posterior ray characteristics during regeneration.
Hand2 effects on vitamin D metabolism in
zebrafish fins
hand2 regulates the localization of sonic hedgehog (shh) in the
posterior region of developing embryonic forelimbs, wings or
pectoral fins (Charité et al., 2000; Fernandez-Teran et al., 2000;
Yelon et al., 2000; Galli et al., 2010). However, shha is expressed
in epidermal cells at the distal tip of each uninjured and regenerating
adult fin ray, regardless of whether that ray expresses hand2 (Quint
et al., 2002; Lee et al., 2009). We examined our RNA-Seq dataset
for possible alternative targets of hand2 during regeneration, with a
focus on genes known to be involved in bone formation or
homeostasis. A significant AP asymmetry was observed for
cyp24a1, which encodes a vitamin D-inactivating enzyme (Knutson
and DeLuca, 1974), which was expressed at higher levels in
posterior rays, like hand2 (Table 1, Fig. 5A). Vitamin D is a known
regulator of calcium and bone homeostasis in mammals and has
also been shown to influence bone formation in zebrafish larvae
(Gardiner et al., 2000; Fleming et al., 2005; Baldock et al., 2006).
Although levels of cyp24a1 expression were reduced during
regeneration, the AP distribution of expression was maintained
(Fig. 5B). To test whether hand2 regulates the posterior expression
domain of cyp24a1 during fin regeneration, we measured the effects
of transgenic hand2 elevation at 4 dpa. Increased hand2 expression
raised anterior cyp24a1 expression 7.5-fold, while also elevating
posterior cyp24a1 levels and maintaining an AP gradient (Fig. 5C;
supplementary material Fig. S6A). Experimental induction of alx4a,
lhx9 or id4 during regeneration had no detectable effect on posterior
cyp24a1 expression (supplementary material Fig. S6B).
To further test this relationship, we injected a translation-blocking
morpholino known to phenocopy hand2 loss-of-function mutations
into 3- and 4-dpa regenerates (Maves et al., 2009). These injections
caused a 28% decrease in cyp24a1 levels, consistent with Hand2
regulation of cyp24a1 during fin regeneration (Fig. 5D). We also
observed a 32% decrease in cyp24a1 levels in 4-dpf hand2 mutant
embryos compared with their clutchmates (supplementary material
Fig. S6C).
We next examined whether posterior cyp24a1 expression levels
correlated with differential vitamin D signaling across the AP axis
of pectoral fins. calb2a, bglap and sparc, which are known targets
of vitamin D signaling in mammals (McDonnell et al., 1989;
Wasserman and Fullmer, 1989; zur Nieden et al., 2003), were
induced in uninjured pectoral fins after intraperitoneal injection of
vitamin D, indicating that they are also vitamin D-responsive in
zebrafish (supplementary material Fig. S6D). The expression of
each of these genes was significantly higher in the cyp24a1-low
anterior regions of uninjured pectoral fins (supplementary material
Fig. S6E), suggesting differential vitamin D signaling across the fin
AP axis. Additionally, hand2 overexpression in regenerating
pectoral fins reduced calb2a, bglap and sparc levels by 64%, 57%
Development
Fig. 4. hand2 overexpression exerts its
effects during later stages of regeneration.
(A,B) No difference in the appearance of
hsp70l:hand2 and wild-type clutchmate fins at 3
dpa after a series of daily heat shocks. n=16.
(C,D) Blastemal proliferation as measured by
BrdU incorporation is also unaffected by hand2
overexpression. n=13. (E) Stunted regeneration
in an hsp70l:hand2 pectoral fin compared with
a wild-type clutchmate fin at 7 dpa after a series
of daily heat shocks beginning at 3 dpa.
Representative regenerative growth of wildtype ray 3 is denoted by a dotted line.
(F,G) Significant reductions in anterior ray
lengths and widths manifest when hand2 is
overexpressed during later stages of
regeneration. n=8. *P<0.05, Student’s t-test; bar
indicates mean. Arrowheads (A,C,E) indicate
amputation plane.
Patterning fin regeneration
RESEARCH ARTICLE 3761
and 22%, respectively, consistent with a model in which Hand2
regulates vitamin D signaling in pectoral fin rays (supplementary
material Fig. S6F).
We also examined whether Hand2 influences a second class of
posterior genes involved in bone formation and fin ray pattern. The
actinodin genes are structural components of actinotrichia, which
are unmineralized fibrils found in developing pectoral fins (Zhang
et al., 2010). Unexpectedly, the entire gene family, and1-4, was
elevated in the posterior regions of uninjured adult pectoral fins, as
detected by RNA-Seq and qPCR (Table 1; supplementary material
Fig. S7A). In contrast to cyp24a1, daily induction of hand2 during
regeneration did not significantly increase the levels of actinodin
family genes in the anterior regions of pectoral fins at 4 dpa
(supplementary material Fig. S7B). This result indicates that Hand2
controls a subset of genes that show AP regionalized expression and
potentially contribute to posterior ray characteristics.
To define the significance of vitamin D signaling as a target of
Hand2, we supplemented zebrafish with vitamin D and assessed
pectoral fin regeneration. Daily vitamin D injection had no effects on
pectoral fin regeneration in wild-type fish. By contrast, this systemic
regimen led to 24% and 20% increases in the lengths of regenerating
anterior and posterior rays, respectively, during hand2 overexpression
(Fig. 5E,F). Accompanying the length increase was an ~11% increase
in the relative widths of the rays across the AP axis (Fig. 5G).
These gene expression and pharmacological rescue experiments
together support a model in which Hand2 controls patterning in
regenerating pectoral fins by modulating vitamin D metabolism and
signaling (Fig. 6).
DISCUSSION
A defining feature of appendage regeneration is the maintenance
and recall of positional information, evident in the restoration of
Development
Fig. 5. AP regionalization of vitamin D signaling controlled by Hand2. (A) cyp24a1, which encodes a vitamin D-inactivating enzyme, is more highly
expressed in posterior regions of uninjured pectoral fins. (B) Posterior enrichment of cyp24a1 is maintained during regeneration at 4 dpa. (C) hand2
induction daily during regeneration elevates anterior cyp24a1 expression ~7.5-fold at 4 dpa relative to heat-shocked wild-type clutchmate controls.
(D) hand2 morpholino injection into pectoral fin regenerates reduces posterior cyp24a1 expression by ~28% relative to vehicle-injected clutchmate
controls. (A-D) Expression is normalized to actb1. *P<0.05, **P<0.005, Student’s t-test. n=3; mean ± s.e.m. (E) Appearance of 7-dpa fins from hsp70l:hand2
and wild-type clutchmates given a daily heat shock and either daily vehicle or vitamin D injections. Vitamin D injection had little effect on wild-type
regeneration, but partially suppressed the effects on bone patterning caused by hand2 overexpression. Representative regenerative growth of
hsp70l:hand2 vehicle-injected rays 3 and 8 is denoted by dotted lines. Arrowheads indicate amputation plane. (F) Quantification of the effects of daily
vitamin D injection on ray lengths, indicating improvement in hsp70l:hand2 animals. (G) Daily vitamin D injection increases ray widths in regenerating
hsp70l:hand2 fins. (F,G) n=20-28; bar indicates mean. **P<0.05, Student’s t-test. Scale bars: 0.6 mm.
Fig. 6. Model for Hand2 influences on positional memory. Adult
zebrafish pectoral fins maintain region-specific expression of transcription
factor genes. Levels of the posterior transcription factor gene hand2 can
help control bone patterning during pectoral fin regeneration. Hand2
levels regulate bone formation during regeneration by direct or indirect
regulation of the vitamin D-inactivating enzyme encoded by cyp24a1,
restricting bone formation in the posterior rays of pectoral fins.
correctly patterned structures. Here we discovered a transcription
factor gene expression signature and a downstream signaling
pathway that might underlie AP positional memory in zebrafish
pectoral fins. Many genes displayed region-specific expression
across the AP axis of uninjured adult pectoral fins, and we examined
the impact of four of them on patterning during regeneration. One
of these transcription factor genes, hand2, displayed the expected
aspects of a regulator of positional memory. First, hand2 expression
is maintained in an AP region-specific manner in bone-forming cells
of uninjured and regenerating fins. That is, anterior ray osteoblasts
have a different transcription factor signature to posterior ray
osteoblasts, as defined minimally for hand2 and alx4 expression.
Second, hand2 overexpression changes the AP patterning of bone
during fin regeneration. Furthermore, our data indicate that hand2
regulates posterior bone formation in part through local control of
the activity of a systemic signal – vitamin D.
Our findings reveal that zebrafish pectoral fins maintain a basal
level of transcription factors in preferential domains throughout all
life stages, as opposed to turning off expression after patterning and
differentiation. We suggest that this concept is likely to be central to
positional memory in zebrafish fins, and potentially other
regenerative vertebrate tissues. The expression of embryonic
signaling factors in uninjured adult structures has been indicated in
other examples of appendage regeneration (Nicolas et al., 2003;
Schnapp et al., 2005; Wills et al., 2008; Poss, 2010), and
mammalian fibroblasts are known to express region-specific Hox
gene codes (Chang et al., 2002; Rinn et al., 2006).
Although positional memory factors are likely to recapitulate
aspects of the embryonic developmental program, they are also
likely to regulate pattern by mechanisms that are distinct from
embryogenesis. Relevant to this second idea, our data do not
indicate that Hand2 influences pattern via Shh signaling in adult
pectoral fins as it does in embryonic pectoral appendages. Rather,
they implicate the control of vitamin D signaling as an important
regulatory mechanism, an interaction that to our knowledge has not
previously been reported. This mechanism might be relevant to
other tissues in which Hand2 is of influence, such as the developing
craniofacial structures. Previous work has demonstrated the
Development 140 (18)
complex role of vitamin D in mammalian bone homeostasis (Lieben
and Carmeliet, 2013). Although implanting vitamin D-soaked beads
was shown to have somewhat minor effects on axolotl limb
regeneration (Washabaugh and Tsonis, 1995), to our knowledge
there have been no reports that vitamin D signaling helps define AP
patterning in regenerating appendages. This lack of evidence can
be explained in part by the fact that only when signaling was
compromised by hand2 overexpression was a vitamin D-related
phenotype observed (Fig. 5E-G). Aside from appendage
regeneration, vitamin D has been shown to influence liver, axon and
skeletal muscle regeneration (Ethier et al., 1990; Chabas et al., 2008;
Stratos et al., 2013). It will be interesting to examine whether the
regulation of vitamin D signaling is important in additional
regenerative contexts.
By what mechanism(s) do adult cells maintain the regionalized
expression of patterning transcription factors? There are at least two
important gene regulatory components of regeneration that might on
the surface appear contradictory. First, cells must be capable of rapid
gene expression flux to enact major changes such as dedifferentiation in response to injury. Second, as shown here, cells
also lock in the expression of key developmental regulators in a
region-specific manner throughout life. Novel epigenetic regulation,
possibly at the chromatin level, is likely to underlie this versatility.
Although we found differential AP expression of numerous
transcription factors, functional manipulation of just one of these
produced clear patterning effects during regeneration. This might
reflect the limitations of the overexpression technologies currently
available for use in adult zebrafish fins. Alternatively, it is possible
that multiple transcription factors act in concert as a code to
specify positional information (Gebelein et al., 2004; Dasen et al.,
2005; Tümpel et al., 2009). Consistent with this notion, we also
found posterior-enriched genes that were not influenced by the
overexpression of hand2 in anterior rays. Moreover, complete
reprogramming of positional memory is likely to require
simultaneous increases and decreases in gene expression affecting
multiple genes. Emerging genetic toolsets for zebrafish and other
highly regenerative vertebrates should enable the discovery of
core modules of positional memory (Meng et al., 2008; Bedell et
al., 2012; Dahlem et al., 2012), bringing us closer to an
understanding of how regeneration recalls and replicates complex
tissues.
Acknowledgements
We thank J. Burris, A. Eastes, P. Williams and N. Blake for zebrafish care; the
Duke Genome Sequencing and Analysis Core for RNA sequencing; and K.D.P.
laboratory members for comments on the manuscript.
Funding
V.A.T. was supported by a Graduate Research Fellowship [1106401] from the
National Science Foundation. This work was supported by a grant from the
National Institutes of Health [GM074057] to K.D.P. K.D.P. is a Howard Hughes
Medical Institute Early Career Scientist. Deposited in PMC for release after 6
months.
Competing interests statement
The authors declare no competing financial interests.
Author contributions
G.N. conducted the experiments and analyzed the data, G.N. and K.D.P.
designed the experiments and wrote the paper, K.K. generated the
hand2:EGFP and tbx18:EGFP reporter lines, and V.A.T. performed RT-PCR
experiments.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.098798/-/DC1
Development
3762 RESEARCH ARTICLE
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