RESEARCH ARTICLE 1205
Development 137, 1205-1213 (2010) doi:10.1242/dev.041251
© 2010. Published by The Company of Biologists Ltd
PHOX2A regulation of oculomotor complex nucleogenesis
Khaleda B. Hasan1, Seema Agarwala2 and Clifton W. Ragsdale1,*
SUMMARY
Brain nuclei are spatially organized collections of neurons that share functional properties. Despite being central to vertebrate
brain circuitry, little is known about how nuclei are generated during development. We have chosen the chick midbrain oculomotor
complex (OMC) as a model with which to study the developmental mechanisms of nucleogenesis. The chick OMC comprises two
distinct cell groups: a dorsal Edinger-Westphal nucleus of visceral oculomotor neurons and a ventral nucleus of somatic oculomotor
neurons. Genetic studies in mice and humans have established that the homeobox transcription factor gene PHOX2A is required for
midbrain motoneuron development. We probed, in forced expression experiments, the capacity of PHOX2A to generate a spatially
organized midbrain OMC. We found that exogenous Phox2a delivery to embryonic chick midbrain can drive a complete OMC
molecular program, including the production of visceral and somatic motoneurons. Phox2a overexpression was also able to
generate ectopic motor nerves. The exit points of such auxiliary nerves were invested with ectopic boundary cap cells and, in four
examples, the ectopic nerves were seen to innervate extraocular muscle directly. Finally, Phox2a delivery was able to direct ectopic
visceral and somatic motoneurons to their correct native spatial positions, with visceral motoneurons settling close to the
ventricular surface and somatic motoneurons migrating deeper into the midbrain. These findings establish that in midbrain, a
single transcription factor can both specify motoneuron cell fates and orchestrate the construction of a spatially organized
motoneuron nuclear complex.
INTRODUCTION
A characteristic feature of the vertebrate brain is that its neuronal cell
bodies are organized into clusters. These clusters, known as nuclei,
serve to organize much of the circuitry of the brain (Nieuwenhuys
et al., 1998). Key attributes of nuclei include a spatial arrangement
of neurons, often with multiple cell types, which have distinct inputs,
axonal projections and neurotransmitter phenotypes. Only limited
progress has been made in understanding how neuronal progenitor
cells are allocated to different nuclear fates, and the molecular
mechanisms that coordinate cell type composition, spatial
arrangement and nucleus size remain largely unknown (Agarwala
and Ragsdale, 2002; Diaz et al., 1998; Kawauchi et al., 2006).
The chick midbrain oculomotor complex (OMC) is an attractive
model system for studying brain nucleogenesis. In vertebrates, the
OMC supplies somatic motoneuron input to extraocular muscles and
preganglionic autonomic input to the ciliary ganglia. The somatic
and visceral midbrain motoneurons form separate pools, although
the pool of visceral motoneurons in rodents is not sharply
distinguished in Nissl-stained preparations or by gene expression.
By contrast, the chicken shares with all birds a cleanly segregated
OMC with discrete subnuclei that innervate specific autonomic and
ocular muscle targets (Evinger, 1988; Gamlin et al., 1984; Heaton
and Wayne, 1983). Indeed, in birds the preganglionic motoneurons
arise exclusively from the Edinger-Westphal (EW) nucleus, which
is the dorsal component of the OMC (Gamlin and Reiner, 1991;
Reiner et al., 1991). The segregation of neurons in the developing
chick OMC into discrete subnuclei occurs in a series of migrations,
as documented in the classic histological studies of Levi-Montalcini
1
Department of Neurobiology, The University of Chicago, Chicago, IL 60637, USA.
Section of Neurobiology, University of Texas at Austin, Austin TX 78712, USA.
2
*Author for correspondence ([email protected])
Accepted 26 January 2010
(Levi-Montalcini, 1963) and Puelles (Puelles, 1978). The cellular
and molecular factors that mediate these migrations are unknown
(Guthrie, 2007).
Genetic studies over the past ten years have identified the
PHOX2A homeobox gene as an important regulator of midbrain
oculomotor development. Zebrafish embryos with a single amino
acid mutation in the Phox2a homeodomain exhibit a reduction in
oculomotor nucleus precursor cells (Guo et al., 1999). In Phox2a–/–
mice, midbrain oculomotor neurons are absent (Pattyn et al., 1997).
In humans, PHOX2A mutations are linked to severe defects in
extraocular muscle movement and in pupillary light reflexes (Bosley
et al., 2006; Nakano et al., 2001), and magnetic resonance imaging
has established that the oculomotor, or third, cranial nerve is missing
in these patients (Bosley et al., 2006). The closely related homeobox
gene Phox2b, which is required for the development of hindbrain
branchiomotor and visceromotor neurons, is also expressed in
midbrain oculomotor neurons but is dispensable for their production
(Pattyn et al., 2000; Pattyn et al., 1997). Recent findings, however,
have shown some degree of shared competency between the
Phox2a/b genes in motoneuron development. Knock-in of Phox2b
coding sequence into the Phox2a locus allows for the development
of some midbrain oculomotor neurons (Coppola et al., 2005). Gainof-function studies in chick spinal cord have demonstrated that
exogenous Phox2a/b are able to upregulate the expression of
motoneuron markers and induce ectopic axons that can contribute
to existing nerve roots (Hirsch et al., 2007; Pattyn et al., 1997).
Interestingly, these axons exit the cord through dorsal exit points, a
feature of hindbrain branchiomotor and visceromotor neurons that
distinguishes them from somatic motoneurons. This evidence
establishes that Phox2a/b are required for brainstem motoneuron
development and can, when misexpressed, induce motoneurons that
issue axons that exit the nerve cord.
In this study, we extended the analysis of PHOX2A motoneuron
induction to midbrain in order to address a very different kind of
question: to what extent can a single transcription factor drive the
DEVELOPMENT
KEY WORDS: Brain nuclei, Midbrain, Motoneurons, Extraocular muscles, Chick
1206 RESEARCH ARTICLE
production of a spatially organized nuclear complex that contains
different cell types? First, we report on normal OMC development,
showing that the visceral and somatic motoneurons of the chick
OMC are generated in an ‘inside-out’ fashion, with the final position
of the OMC neurons dependent on their time of origin. Second, we
demonstrate that exogenous Phox2a can induce a complete OMC
molecular program, including the production of both visceral and
somatic motoneurons. Crucially, we document that the ectopic
visceral and somatic oculomotor neurons segregate along the
ventricular-pial axis, mimicking the architecture of the native OMC.
Third, we demonstrate that Phox2a-induced motoneurons make
ectopic cranial nerves that recruit boundary cap cells and, in some
instances, target extraocular muscles directly. These results,
combined with previous findings in mouse and human, establish that
a single transcription factor can act as a primary developmental
determinant for a brain nucleus and its constituent cell types.
MATERIALS AND METHODS
Chicken embryos
Fertilized Babcock B-300 White Leghorn chicken eggs (Gallus gallus
domesticus) were supplied by Phil’s Fresh Eggs (Forreston, IL, USA). The
first day of incubation was designated embryonic day 0 (E0). Staging of
embryos followed Hamburger and Hamilton (Hamburger and Hamilton,
1951). Our chick embryo protocols were approved by the University of
Chicago IACUC.
Expression plasmids
The human EF1a (EEF1A1) promoter expression construct pEFX was
generated from pEF1/Myc-His C (Invitrogen) by excising a 2.2 kb PvuII
fragment containing an f1 origin, neomycin resistance gene and SV40
elements. The rat Phox2a (rArix) expression construct pEFX-rArix was
engineered in pEFX by insertion of a 1.6 kb cDNA containing the complete
rat Phox2a coding sequence (Zellmer et al., 1995). pEFX-mPhox2b contains
a 1.3 kb full-length mouse Phox2b cDNA isolated from E12.5 brain RNA
by RT-PCR using the forward primer 5⬘-GCCATCCAGAACCTTTTCAATG-3⬘ and the reverse primer 5⬘-CCGTCTCTCCCTATCACCTACTTG3⬘.
Development 137 (7)
situ hybridization was carried out with digoxigenin-, DNP- and fluoresceinlabeled riboprobes synthesized from chick cDNA plasmids for CDH7,
CHAT, CX36, EGR2 (KROX20), EVX1, ISL1, ISL2, NKX6.1, NRG1, PAX6,
PHOX2A, PHOX2B, ROBO2, SLIT3, TBX20 and VT and from plasmids for
rat Phox2a and mouse Phox2b. For light microscopy, labeled RNA duplexes
were detected with antibody-alkaline phosphatase conjugates (Roche
Diagnostics) and using the distinguishable formazans of NBT and TNBT
(Grove et al., 1998). For fluorescence microscopy, antibody-horseradish
peroxidase conjugates were substituted and peroxidase activity was
demonstrated by fluorescent tyramide signal amplification using FITCtyramide for fluorescein-labeled probes, cyanine 3-tyramide for
digoxigenin-labeled probes and cyanine 5-tyramide for DNP-labeled probes
(TSA Plus Fluorescence Systems Kit, PerkinElmer).
BrdU experiments
5-bromo-2-deoxyuridine (BrdU) can be very toxic to chicken embryos,
particularly before E2.5 (Gould et al., 1999). Extensive scout experiments
were carried out to identify a BrdU dose that elicits clear nuclear labeling
while allowing embryos to survive without obvious morphological defects
to E12. We found success with BrdU (Sigma) prepared as a 75 mg/ml
solution in PBS containing 0.01% Fast Green and delivered as a 50 ml
aliquot through a small hole made in the vitelline membrane near the heart.
Twenty embryos with BrdU injection at st 8, 9 and 11-23 were harvested at
E12 with PFA-PBS fixation. Serial sections (40 mm) of midbrain were first
processed for TNBT gene expression histochemistry with digoxigeninlabeled ISL1 riboprobes to identify OMC structure. Sections were then
treated with 2M HCl for 30 minutes to denature nuclear DNA and, after
washing, incubated in a 1:50 dilution of monoclonal FITC-conjugated antiBrdU antibody (Becton Dickinson) in 2% lamb serum. The anti-BrdU
antibody was detected with the anti-fluorescein phosphatase conjugate and
NBT histochemistry employed in our gene expression experiments.
Images
Tissue was photographed with a Zeiss AxioCam digital camera attached to
a Zeiss Axioskop 50 upright microscope or a Leica MZ FLIIII
stereomicroscope. Images were captured through the AxioVision 4.5
software system and processed with Adobe Photoshop. Images requiring
increased depth of focus were assembled with Helicon Focus 4.6 Pro
software.
For the forced expression of Phox2a/b, we employed expression plasmid
delivery by in ovo microelectroporation (Agarwala et al., 2001; Momose et
al., 1999), targeting, in most experiments, the rostrolateral right ventral
midbrain. Negative electrodes were fabricated from 0.025 mm tungsten wire
and positive electrodes from 0.063 mm platinum wire (Goodfellow).
Plasmid DNA solution (50-250 nl at 1-3 mg/ml in endotoxin-free water
containing 0.02% Fast Green) was pressure-injected from a glass capillary
into the midbrain vesicle of Hamburger and Hamilton stage (st) 9-24
embryos. The negative electrode was placed in the midbrain lumen and the
positive electrode just outside the midbrain. Electric fields were generated
with a Model 2100 Isolated Pulse Stimulator (A-M Systems) delivering
three 25-millisecond pulses of 9 V, with an inter-pulse period of 1 second.
Electroporated embryos were incubated for a further 1-4 days. Our analyses
are based on gene expression in 704 embryos successfully electroporated
with the pEFX-rArix (n=433) and pEFX-mPhox2b (n=271) constructs. Of
these embryos, 316 were co-electroporated with pEFX-EGFP to assess the
zone of misexpression before processing. In 23 embryos, we coelectroporated the alkaline phosphatase reporter plasmid pEFX-AP to label
midbrain efferent axons. For the remaining experiments, the reproducible
accuracy of plasmid delivery was confirmed through parallel
electroporations of pEFX-AP, which was demonstrated by histochemistry,
or of pEFX-rArix or pEFX-mPhox2b, which were detected by Phox2a/b
transgene in situ hybridization.
Nucleic acid histology
Harvested embryos were fixed in 4% paraformaldehyde (PFA) in phosphatebuffered saline (PBS). Dissected embryonic brains were processed as wholemounts or cryoprotected and sectioned at 40 mm on a sledge microtome. In
RESULTS
Molecular and cellular development of the avian
OMC
The avian OMC is a spatially organized nuclear complex comprising
a dorsal EW nucleus and a ventral somatic motor nucleus (Evinger,
1988; Heaton and Wayne, 1983). The EW nucleus contains the
midbrain visceral motoneurons, which innervate the ciliary ganglion
and control pupillary constriction, accommodation and choroidal
blood flow (Gamlin et al., 1982). The somatic oculomotor nucleus
harbors the midbrain somatic motoneurons, which in birds are
discretely organized into subnuclear pools, each innervating distinct
extraocular muscle targets (Fig. 1A). In mammals, the somatic
oculomotor neurons aggregate to form a single nucleus, and in many
species, including mouse, the EW nucleus is challenging to identify
(Weitemier et al., 2005). The anatomical clarity of the avian OMC
coupled with the ease of experimental manipulation makes the
chicken embryo attractive for investigations of midbrain
motoneuron development.
To identify genes that reflect the connectional heterogeneity of the
OMC subnuclei, we screened chick midbrain sections at E12, an age
at which the OMC subnuclei are readily distinguished. Genes
encoding known motoneuron markers, such as the LIMhomeodomain protein ISL1, were expressed by all oculomotor
subnuclei (Fig. 1A; see Fig. S1 in the supplementary material) (Jessell,
2000). Three markers, however, identified visceral and somatic
DEVELOPMENT
In ovo electroporation
Fig. 1. Gene expression markers distinguish the somatic and
visceral motoneuron subnuclei of the chick oculomotor complex
(OMC). Coronal sections of E12 chick midbrain processed for in situ
hybridization to identify the somatic motoneurons of the oculomotor
nucleus (yellow arrowhead) and the visceral motoneurons of the EdingerWestphal nucleus (EW, green arrowhead). (A) The motoneuron marker
ISL1 identifies all OMC motoneurons. (B) Gene expression for the
neuropeptide VT selectively labels the visceral motoneurons.
(C,D) The gap junction subunit gene CX36 (C) and the T-box transcription
factor TBX20 (D) are expressed by the motoneurons of all somatic
subnuclei. Within the DM subnucleus, TBX20 is strongly expressed in its
lateral division, but is absent from its medial division (red arrow). DL,
dorsolateral; DM, dorsomedial; VM, ventromedial. Scale bar: 0.2 mm.
subnuclei specifically. The EW nucleus was labeled by expression of
the neuropeptide gene for vasotocin (VT) (Muhlbauer et al., 1993).
Somatic OMC subnuclei expressed the gap junction subunit CX36 and
the T-box transcription factor TBX20 genes (Fig. 1C,D).
We constructed a timeline of OMC molecular and cellular
development to relate the expression of the motoneuron subtype
markers to other aspects of OMC maturation (Fig. 2). To study
oculomotor nerve development, we electroporated an alkaline
phosphatase-encoding expression vector into E2 midbrain and
processed the embryos for phosphatase histochemistry 1-4 days
later. We found that the axons are first issued from ventral midbrain
at st 16 and reach the ipsilateral orbit over the next 2 days, contacting
the inferior oblique (IO) muscle by st 25 (data not shown). This
schedule matches that described by Chilton and Guthrie, who
employed anti-neurofilament antibody staining to mark axons
(Chilton and Guthrie, 2004).
We found that OMC marker onset readily fell into two groups:
early onset genes that were expressed before oculomotor nerve exit
and which principally encode transcription factors, and late onset
genes that were expressed after nerve exit but before target
innervation (Fig. 2).
The homeodomain transcription factor gene NKX6.1 was the first
OMC marker that we detected in medial ventral midbrain, but it is
not selective for midbrain motoneurons as it is also expressed in red
nucleus progenitor domains and more lateral midbrain territories (P.
M. Garfin, PhD thesis, University of Chicago, 2004).
PHOX2A, which is restricted to OMC cells, is detected at st 12+.
PHOX2B is expressed in prospective OMC cells immediately
thereafter, at st 13–, and the LIM homeobox genes ISL1 and ISL2 are
detected by st 14+ (Fig. 2) (Varela-Echavarria et al., 1996).
The late onset genes included the motoneuron neurotransmitter
enzyme choline acetyltransferase (CHAT; st 24), the axon guidance
regulator ROBO2 (st 25) and the specific markers that distinguish
visceral and somatic motoneuron identity. The visceral oculomotor
RESEARCH ARTICLE 1207
Fig. 2. Schedule of OMC development. Gene expression onset and
timing of key events in chick oculomotor nerve (OMN) development,
illustrated with reference to Hamburger-Hamilton (HH) stages (see text
for details). Expression of the ligand-encoding SLIT3 and neuregulin-1
(NRG1) genes and of the SLIT receptor ROBO2 was detected by st 13+,
st 17 and st 25, respectively. Timing of functional innervation is from
Martinov et al. (Martinov et al., 2004). IO, inferior oblique.
neuron marker VT was first detected at st 20 (E3.5). The onset of the
somatic oculomotor neuron markers CX36 and TBX20 occurred
later, at st 25, which is when the oculomotor nerve makes initial
contact with extraocular muscle (Fig. 2, black arrow). Although
there might be other subtype-restricted molecules that are expressed
earlier in OMC development, the differential time of onset of the
visceral and somatic motoneuron markers raised the possibility that
OMC visceral motoneurons are born before the OMC somatic
motoneurons.
Inside-out pattern of OMC development
The embryonic ventral midbrain is organized into a series of arcs, with
the PHOX2A-rich OMC arising in the most medial arc, arc 1
(Agarwala and Ragsdale, 2002; Sanders et al., 2002). Thus, unlike the
somatic and visceral motoneurons of hindbrain (Guthrie, 2007), the
midbrain somatic and visceral motoneurons appear, by gene
expression, to be generated from a common progenitor pool, that is,
from the ventricular zone overlying arc 1. To test for a timing
difference in the neurogenesis of visceral and somatic oculomotor
neurons, we delivered BrdU to embryos between E2 and E4.5 and
collected them at E12, when the OMC subnuclei are easily
recognized. BrdU birthdating in chick is problematic because
delivered BrdU is not a true pulse as it is in mammals, but is
continuously available in the egg up to 22 hours after delivery (Gould
et al., 1999). Consequently, we were not able to determine precisely
when neurogenesis begins, only when it ends. Heavy labeling,
however, is predicted to mark cells undergoing their final divisions.
We found clear evidence for a temporal difference in the birth order
of the oculomotor subtypes. Neurogenesis begins first in visceral
oculomotor neurons (Fig. 3A). The neurons of the EW nucleus were
most heavily labeled when BrdU was applied at st 11 and 12 (Fig. 3A;
data not shown), close to the time of PHOX2A expression onset (st
12+). Most visceral oculomotor neuron progenitors have undergone
their final cell division by st 18, when only a few medial EW cells still
incorporated BrdU (Fig. 3C), and visceral motor neurogenesis was
largely complete by st 19 (data not shown). By contrast, somatic
oculomotor neuron progenitors continued to divide in large numbers
through st 18 (Fig. 3B,C). BrdU labeling in the somatic subnuclei was
heaviest when BrdU was delivered at st 17 (data not shown),
DEVELOPMENT
Midbrain oculomotor nucleogenesis
Development 137 (7)
Fig. 3. Inside-out gradient of OMC neurogenesis. Chick midbrains
were processed at E12 for BrdU immunohistochemistry (blue) and ISL1
gene expression (brown/pink) after BrdU delivery to st 8-23 embryos.
Coronal sections (top) and corresponding BrdU chartings (bottom)
illustrate the dorsal-to-ventral (D}V) progression of OMC neurogenesis.
Panels illustrate the left OMC, with the midline to the right. OMC
subnuclei are identified in D. (A) Heavy BrdU labeling in the EW nucleus
indicates that neurogenesis of visceral oculomotor neurons precedes
that of somatic oculomotor neurons. (B) Extensive BrdU labeling is seen
throughout the OMC. (C,D) Neurogenesis is nearly complete for the EW
nucleus by st 18 (C) and for the somatic motoneurons by st 23 (D).
Scale bar: 0.1 mm.
Fig. 4. OMC motoneuron subtype segregation at the end of OMC
neurogenesis. (A-D) Coronal sections of chick midbrains of the indicated
stages processed for two-color fluorescent in situ hybridization. The
ventricle is to the top and the pial surface is to the bottom. For st 21 (A)
and st 24 (B), before TBX20 gene expression onset, VT (green) was
employed to label visceral motoneurons and ISL1 (red) served to identify
all OMC neurons. Because ISL1 co-labels VT-expressing cells, many
visceral motoneurons appear yellow. St 25 (C) and st 26 (D) midbrains
probed for VT (green) to mark visceral motoneurons and TBX20 (red) to
label somatic motoneurons. At st 21, somatic motor production and
migration is still underway and OMC subtypes appear intermixed (A).
Once somatic motoneuron production has ended (B,C), few visceral
motoneurons are found in the prospective somatic motoneuron territory
(arrows). By st 26, the motoneuron classes are fully segregated, with a
gap between the populations (D). Scale bar: 0.1 mm.
suggesting that somatic motoneuron neurogenesis has begun by this
stage and somewhat overlaps with the end of visceral motoneuron
generation. By st 21, somatic oculomotor neurogenesis was largely
confined to medial cells in the ventromedial subnucleus (data not
shown) and was nearly complete by st 23 (Fig. 3D). Thus, there is a
clear temporal difference in the birth of the oculomotor neuron
subtypes in the chick embryo and this difference is part of a general
gradient of OMC neurogenesis that extends from dorsolateral EW
neurons to the ventromedial somatic subnuclei (Fig. 3A-D).
Interestingly, the four-stage separation in neurogenesis completion for
the visceral (st 19) versus somatic (st 23) motoneurons proved to be
close to the five-stage separation in the onset of our visceral (VT) and
somatic (CX36, TBX20) differentiation markers.
In the mature OMC, the visceral and somatic motor nuclei are
segregated in the dorsal-ventral axis, which corresponds to the
ventricular-pial axis of the embryonic ventral midbrain. We were
interested in whether visceral and somatic motoneurons in their
initial allocation to the prospective OMC prepatterned their eventual
ventricular-pial segregation, or were at first fully intermixed. Our
specific somatic motoneuron markers are not expressed until st 25.
At st 25, TBX20-expressing somatic cells were already largely
segregated from the VT-expressing EW cells, but there was some
mixing of motoneuron subtypes (Fig. 4C; see Movie 1 in the
supplementary material). Because of the inside-out generation of the
OMC neurons, some TBX20-expressing cells in the prospective EW
nucleus could be in transit, but there were also a few VT-expressing
cells in the TBX20-positive territory. One stage later (st 26), nearly
all VT-expressing neurons occupied a territory that was entirely
separate from that of the TBX20-expressing somatic neurons, with
a clear gap between the two populations (Fig. 4D).
To study the spatial allocations of OMC neurons before st 25, we
employed VT (visceral) and ISL1 (visceral and somatic) gene
expression. Between st 21 and 23, when motoneuron neurogenesis
is still underway and somatic motoneurons must be transiting the VT
territory, the prospective somatic (expressing ISL1 but not VT) and
visceral (expressing both VT and ISL1) motoneurons were more
intermixed along the ventricular-pial axis (Fig. 4A). By st 24,
however, when motoneuron production is complete, there was a
clear ISL1-rich territory that contained only a few cells expressing
both VT and ISL1 (Fig. 4B). We conclude from these results that
although the motoneuron subtypes are not fully segregated along the
ventricular-pial axis during OMC neurogenesis and migration, by
the end of motoneuron production the numbers of mislocated cells
are small and these errors are rapidly corrected by remedial
migration or cell death between st 24 and 26.
Phox2a drives a molecular OMC program
Previous work has shown that the signaling molecule sonic
hedgehog can produce motoneurons in chick midbrain (Agarwala et
al., 2001; Bayly et al., 2007; Wang et al., 1995). We were interested
in the extent to which OMC transcription factors could, singly or in
combination, induce OMC cell fates. PHOX2A was the clear choice
for initial study: it is required for midbrain oculomotor neuron
development in mouse and human (Nakano et al., 2001; Pattyn et
al., 1997); in the chick embryo it is the first transcription factor we
can identify that is selectively restricted to prospective OMC cells;
and, most importantly, Dubreuil et al. (Dubreuil et al., 2000;
Dubreuil et al., 2002) and Hirsch et al. (Hirsch et al., 2007) have
shown that Phox2a/b can induce motoneuron markers, including
ISL1 and CHAT, in chick spinal cord. We found that in chick
DEVELOPMENT
1208 RESEARCH ARTICLE
Midbrain oculomotor nucleogenesis
RESEARCH ARTICLE 1209
developmental regulatory gene expression, the arcuate organization
of the ventral midbrain extends into the adjoining subthalamic
region of forebrain (Agarwala and Ragsdale, 2009; Sanders et al.,
2002). Our Phox2a misexpression data indicate that the subthalamus
also shares inductive properties with the ventral midbrain.
The neuropeptide hormone gene VT selectively labels visceral
oculomotor neurons in the developing OMC. We found that Phox2a
is able to induce this subset of oculomotor neurons (Fig. 5F). We
were unable to detect convincing ectopic induction of the somatic
oculomotor neuron marker CX36 owing to the extensive expression
of endogenous CX36 laterally in the ventral midbrain. However, we
were able to demonstrate that Phox2a can induce TBX20, our second
marker for somatic motoneurons (Fig. 5G; see Table S1 in the
supplementary material).
midbrain, electroporation of full-length rat Phox2a (Arix) induced
the early and late genes identified in Fig. 2, including motoneuron
transcription factor, signaling and axon guidance genes (Fig. 5C-E;
see Fig. S2 and Table S1 in the supplementary material). Induction
of OMC markers was efficient, with 269/365 electroporated
embryos demonstrating ectopic OMC gene expression. Similar
efficiencies were seen in the induction of these markers by mouse
Phox2b gene delivery (see Fig. S3 in the supplementary material).
To test for spatial restrictions to midbrain motoneuron induction,
we performed two-color in situ hybridization on Phox2aelectroporated brains harvested at E6 and probed for ISL1 and the
lateral midbrain markers PAX6 and EVX1 (Fig. 5E; see Fig. S4 in the
supplementary material). These data establish that Phox2a induction
of motoneuron markers extends across the rostral-caudal extent of
the ventral midbrain, from the ventral midline at least as far lateral
as the EVX1 stripe. We also found that Phox2a could induce ISL1 in
the adjoining subthalamic region of forebrain (Fig. 5E; see Fig. S4
in the supplementary material). By contrast, we were unable to elicit
motoneuron markers in dorsal midbrain following either Phox2a or
Phox2b gene delivery (not shown). By morphology and
PHOX2A regulation of nerve biogenesis
The central feature of motoneurons is that their axons leave the
nervous system and innervate peripheral targets. To study whether
Phox2a misexpression can drive ectopic nerve production, we coelectroporated the rat Phox2a expression plasmid with an alkaline
phosphatase expression vector to label cranial nerves. Phox2a
overexpression generated ectopic nerve rootlets exiting from the
midbrain, often several in each electroporated embryo. Table S2 in
DEVELOPMENT
Fig. 5. Phox2a induction of OMC gene expression markers.
(A) Open-book preparation of E6 chick brainstem whole-mount
demonstrates native PHOX2A gene expression in the midbrain OMC
and the rostral hindbrain trochlear nucleus (Tr). Rostral is to the top,
and the ventricular surface faces the viewer. (B-G) Expression plasmids
for rat Phox2a (Arix) were electroporated into E2 chick ventral midbrain
and embryos harvested for whole-mount in situ hybridization at E5 (D)
and E6 (B,C,E-G). (B) A typical pattern of plasmid delivery by
electroporation illustrated in the right midbrain by transgene expression
(arrow). (C) Phox2a misexpression elicits induction of endogenous chick
PHOX2A (arrow). (D) Phox2a forced expression induces other OMC
markers, including the axon guidance molecule SLIT3 (arrow). (E) Lateral
arcuate markers PAX6 (P6) and EVX1 (E1) (brown) illustrate the capacity
of Phox2a to induce ISL1 (blue) throughout the ventral midbrain (red
arrows) and rostrally in caudoventral diencephalon (green arrow).
(F,G) Phox2a misexpression induces molecular markers (arrows) of
visceral (VT, F) and somatic oculomotor neurons (TBX20, G). Asterisks
(A,C,G) mark the contralateral migration of the prospective superior
rectus somatic motoneurons (see Chilton and Guthrie, 2004). IS,
isthmus. Scale bar: 0.2 mm.
PHOX2A autonomously regulates the migration of
visceral and somatic oculomotor neurons
The mature OMC is organized with visceral motoneurons in the
dorsally located EW nucleus and somatic motoneurons occupying
distinct subnuclei ventrally (Fig. 1A). By E6, the visceral and
somatic motoneuron markers in the native developing OMC identify
two distinct populations of cells that are segregated along the
ventricular-pial axis, with the VT-expressing visceral oculomotor
neurons lying closer to the ventricle (Fig. 4D, Fig. 6B). We
employed two-color detection to distinguish the VT-expressing and
TBX20-expressing population subtypes in Phox2a-electroporated
chick embryos. We identified, in the whole-mounts of four brains,
36 ectopic clusters of cells with both VT and TBX20 expression (Fig.
6A). The spatial organization of these clusters was studied in serial
cross-section. Two of these clusters showed some intermixing of the
VT-expressing and TBX20-expressing cells. The other 34
overlapping clusters presented full segregation of the VT-expressing
and TBX20-expressing cells, with the VT-positive cell cluster
invariably closer to the ventricular surface. In 26/34 clusters, the
segregation featured a gap between the two populations (Fig. 6C,D).
In addition to this induction of ‘mini’-OMC clusters, we saw
many isolated examples of ectopic cells (Fig. 6E,F, green arrows).
In these examples, even when ectopic expression of only one of the
two motoneuron class-specific markers was detected, the VTexpressing ectopic neurons appeared to lie closer to the ventricle
than the TBX20-expressing ectopic neurons (Fig. 6F). To test this
directly, we analyzed the arrangement of the isolated ectopic VTexpressing (n=111) and TBX20-expressing (n=43) cells along the
ventricular-pial axis by measuring the distances from the base of the
ventricular zone to each isolated cell or cell cluster. Because these
distances were non-normally distributed (Fig. 7), we compared them
with the two-sample Wilcoxon rank sum test. This analysis
established that the isolated TBX20-expressing cells migrated to
significantly greater tissue depths than the VT-expressing cells
(mean, 87.2 versus 39.4 mm; median, 75.9 versus 32.9 mm;
P<0.0001). We conclude that the spatial arrangement of the classspecific ectopic oculomotor neurons precisely follows that of the
native motoneuron classes in the OMC.
Fig. 6. Phox2a induction of mini-OMCs. Chick ventral midbrains
electroporated with rat Phox2a plasmids at E2 and collected at E6.
Tissue was processed to demonstrate visceral (VT, brown) and somatic
(TBX20, blue) motoneurons. (A) Two-color in situ hybridization wholemount, oriented and labeled as in Fig. 5A, demonstrates the ectopic
induction of low-density fields of VT-expressing cells (green arrow) and
clusters containing both VT-expressing and TBX20-expressing cells (red
arrow). In this sample, the overlap of the VT-expressing and TBX20expressing cells in the native OMC is not seen because of the density of
TBX20 labeling, and the extent of the VT-positive territory is
exaggerated by whole-mount flattening. (B) Entopic arrangement of
the E6 OMC demonstrated in coronal cross-section, with the ventricle
to the top (asterisk). VT-expressing visceral motoneurons sit closer to
the ventricle, whereas TBX20-expressing somatic motoneurons lie
deeper, closer to the pial surface, with a gap between the subtypes
(arrowhead). (C-F) Coronal cross-sections through the ventral midbrain
show the segregation of ectopic motoneuron classes along the
ventricular-pial axis (ventricle marked by asterisk). A clear gap is often
(C,D, arrowheads), but not always (E, red arrow), seen between the
two classes of ectopic motoneurons. Forced Phox2a expression also
produced isolated VT-expressing (E,F) and TBX20-expressing (F) cells in
low-density fields (green arrows). Scale bars: 0.1 mm.
the supplementary material details the statistics on the behavior of
Phox2a-induced rootlets for a sample of 19 electroporated embryos.
These ectopic axons (Fig. 8) projected directly from ectopic spots
within the ventral midbrain without aligning with the oculomotor
nerve. Some projections stopped abruptly in the subjacent
mesenchyme (n=16/49) (Fig. 8A), but many axons found their way
to nearby cranial nerves III and IV, which carry oculomotor axons
(III, n=14/49; IV, n=15/49) (Fig. 8B). The most striking observation
was that four ectopic nerves were found to travel directly to the
extraocular muscles of the orbit, bypassing the cranial nerves
altogether (Fig. 8C). Finally, the timing of exit of these ectopic axons
adhered closely to that of the native oculomotor nerve (st 16, n=3/7
tested). Thus, misexpressed Phox2a can produce axonal projections
that exit the neuroepithelium on schedule and innervate natural
targets.
Boundary cap cells (BCCs) are neural crest derivatives that
accumulate at prospective nerve exit points (Vermeren et al., 2003;
Yaneza et al., 2002). In the chick embryo, BCCs have been shown
Development 137 (7)
Fig. 7. Ectopic visceral and somatic motoneurons have intrinsic
migration programs. (A) Statistics on ectopic VT and TBX20 gene
expression in four chick midbrains studied in serial section. In this
sample, ectopic VT-expressing cells appeared more frequently (58%)
without adjacent TBX20-expressing cells than in mini-OMCs (19%).
Isolated Phox2a-induced TBX20-expressing cells accounted for 23% of
the sample. (B) For each occurrence of cells expressing only VT or
TBX20, the distance from the ventricular zone along the ventricular-pial
axis was measured. These distances were compared using the twosample Wilcoxon rank sum test. The horizontal line in the box
represents the median value, and the edges of the box are the first and
third quartiles. The lines extending from the edges of the box end at
the last value within 1.5 times the interquartile range (third minus first
quartile) from the edge of the box. Values beyond this distance are
considered outliers and are plotted individually. Distances from the
ventricular zone were statistically significantly greater for TBX20expressing than for VT-expressing cells (see text).
to express high levels of EGR2 (KROX20) and cadherin-7 (CDH7),
and CDH7-positive BCCs in particular have been demonstrated at
oculomotor axon exit points (Nakagawa and Takeichi, 1998;
Niederlander and Lumsden, 1996). We asked whether these neural
crest derivatives are also found at Phox2a-induced ectopic axon exit
points. In control brains, we found EGR2 and CDH7 expression just
outside the neuroepithelium, where native oculomotor axons exit
(data not shown). EGR2 expression in the mesenchyme subjacent to
the ventral midbrain neuroepithelium was most readily detected
between E3 and E4, whereas CDH7 expression was present at high
levels between E3 and E5 (Fig. 9). In Phox2a-electroporated heads,
EGR2 and CDH7 expression was seen at ectopic locations, as well
as at native oculomotor axon exit points (Fig. 9A,D). Cross-sections
of experimental midbrains demonstrated that the CDH7-expressing
cells populated the ectopic nerve exits (Fig. 9B,C). Our results
establish that Phox2a induction of motor nerves constitutes a
sufficient trigger for BCCs to populate axon exit points. Since
ectopic exit points are unconstrained in their ventral midbrain
distributions (not shown), the ectopic axons must release a BCC
attractant (Bron et al., 2007; Mauti et al., 2007) or induce a BCC
identity in local head crest cells.
DISCUSSION
PHOX2 transcription factors appear to be exceptionally effective in
driving neuronal cell type identities. Dubreuil et al. (Dubreuil et al.,
2000) have shown that exogenous Phox2b misexpression in chick
spinal cord, a tissue in which PHOX2A/B are not normally expressed
in motoneurons, can trigger motoneuron-specific gene expression
and act as a neurogenic factor driving progenitor cells out of the cell
cycle (Dubreuil et al., 2002; Pattyn et al., 2000). To explore the
motoneuron inductive potential of PHOX2A/B in more detail, we
studied the consequences of forced Phox2a/b expression in chick
DEVELOPMENT
1210 RESEARCH ARTICLE
Fig. 8. Phox2a misexpression in ventral midbrain drives the
production of ectopic cranial nervelets. Co-electroporation of
alkaline phosphatase-expressing vector with rat Phox2a plasmid into
ventral chick midbrain at E2 demonstrates ectopic axon outgrowth 4
days later, at harvesting. Mesenchyme was partially dissected away to
show alkaline phosphatase-labeled nerves. Arrows point to ectopic
axons. (A) Lateral view of an E6 embryo illustrating ectopic rootlets
projecting out of ventral midbrain and ending in the subjacent
mesenchyme. (B) Some ectopic axons join the oculomotor (III) and
trochlear (IV) cranial nerves. (C) An ectopic nerve is seen to travel
directly to the orbit, reaching the superior rectus muscle and bypassing
nearby cranial nerves altogether. hb, hindbrain; OMN, oculomotor
nerve; TrN, trochlear nerve; v.mb, ventral midbrain. Scale bars: 0.2 mm.
midbrain, where PHOX2A/B are natively expressed and restricted
to motoneuron lineages. We found that Phox2a delivery can drive a
full program of oculomotor neuron cellular and molecular fates,
including the generation of a spatially organized nuclear complex.
Nuclei are a central feature of vertebrate brain architecture, but little
is known about the mechanisms of their production. These
mechanisms will undoubtedly be multiple and vary across brain
regions and cell types, but our findings with PHOX2A provide
evidence for one type of mechanism that is arguably the simplest.
Upstream signals drive the expression of PHOX2A in the progenitor
tissue of arc 1 in ventral midbrain (Agarwala and Ragsdale, 2002;
Agarwala et al., 2001). PHOX2A drives the production of both
visceral and somatic motoneurons. The visceral and somatic
motoneurons autonomously migrate to appropriate positions along
the ventricular-pial axis, producing a spatially organized nuclear
complex.
Spatial allocation of OMC cell types
In BrdU birthdating studies, we found an inside-out pattern of
neurogenesis in the chick OMC. Inside-out patterns of brain
development have been most extensively studied in the formation
of the layers of the mammalian cerebral cortex (Rakic, 1974). In an
influential series of transplantation studies, McConnell and
colleagues have demonstrated that the neocortical inside-out
patterning is not a passive process, in which cells simply migrate
past earlier-born cells before settling (Frantz and McConnell, 1996;
McConnell, 1991). Rather, cortical progenitor cells acquire specific
layer fate identity before they leave the ventricular zone of neuronal
progenitors. These transplantation experiments were conducted with
cortex donor tissue transplanted to cortex host tissue. Consequently,
the execution of the laminar fate program was tested within native
cerebral cortex, where the cues used by transplanted cells could be
layer-intrinsic signals or molecules (McConnell, 1991). In our
Phox2a-misexpression experiments, we have been able to drive
ectopic production of isolated early-born (visceral motor) and lateborn (somatic motor) neurons in lateral midbrain, that is, in
neuroepithelium without a native OMC template. Thus, the ectopic
singletons must employ intrinsic mechanisms, such as counting cells
or measuring distances, as they migrate to their appropriate depths,
or must draw on general cues that are deployed throughout ventral
midbrain. A particularly attractive cueing mechanism would be a
RESEARCH ARTICLE 1211
Fig. 9. Phox2a-induced motoneurons organize boundary cap cells
(BCCs) at ectopic motor nerve exits. (A-D) Rat Phox2a was delivered
to the right side of E2 chick ventral midbrain. Heads were harvested 1-3
days later and processed for whole-mount in situ hybridization for the
BCC markers CDH7 (A-C) and EGR2 (D). (A,D) Lateral views of right side
of E3 (A) and E4 (D) heads. CDH7 (A) and EGR2 (D) labeling identifies
BCCs at the native oculomotor nerve exit points (green arrows) and
ectopic motor exit points (red arrows). (B,C) E5 Phox2a-electroporated
heads probed for CDH7 expression and studied in cross-section.
Sections cut transverse (B) and parallel (C) to the third cranial nerve
demonstrate CDH7 expression at native (green arrows) and ectopic (red
arrows) motor exit points. Ventricle in C is indicated by an asterisk.
dien, diencephalon; fb, forebrain; hb, hindbrain; v.mb, ventral midbrain.
Scale bars: 0.1 mm.
positional signaling system that supplies newborn neurons with
positional information along the ventricular-pial axis. Such a system
would be generally useful for arranging brain nuclei in three
dimensions and, unlike a cell-counting mechanism, could
accommodate brain growth. Our findings with native OMC
development, particularly the (limited) intermixing of cell types seen
at st 25, indicate that secondary correction mechanisms, such as cell
sorting or deletion, contribute to OMC morphogenesis as well.
Oculomotor neuron diversification mechanisms
A striking finding in the current work is that Phox2a forced
expression is able to drive the production of both visceral and
somatic motoneurons broadly in ventral midbrain. The molecular
mechanisms underlying the diversification of these motoneuron
subtypes are unknown (Song and Pfaff, 2009). One possibility is that
the ectopic cells take advantage of endogenous signaling systems,
such as the NOTCH pathway, that mediate binary cell fate decisions
(Mizuguchi et al., 2006; Peng et al., 2007). A second possible
mechanism for somatic and visceral motoneuron diversification is
the action of cell-autonomous temporal specification factors
(Maurange et al., 2008; Zhu et al., 2006). A requirement for both
these mechanisms is that they be resident throughout the territory
competent for OMC induction or that they be triggered by Phox2a
misexpression. A third possibility is that PHOX2A levels themselves
directly specify visceral and somatic motoneuron fates. In vivo
plasmid electroporation does not tightly control the gene dose
delivered, and it is likely that different transgenic cells will express
different levels of exogenous proteins. In studies of hematopoietic
lineages, varying transcription factor levels have been found to
regulate cell fate specification differentially (DeKoter and Singh,
DEVELOPMENT
Midbrain oculomotor nucleogenesis
2000). A similar scenario for PHOX2A in OMC cell fate
determination would be that low (or high) levels of PHOX2A in the
earliest progenitors drive visceral motoneuron production, and that
progressive accumulation (or depletion) of PHOX2A protein leads
to somatic motoneuron production. Testing this PHOX2A dose
hypothesis is likely to entail the development of stem cell culture
methods that permit tight regulation of PHOX2A protein delivery.
PHOX2A is a primary regulator of OMC
nucleogenesis
The extensive work on the specification of spinal motoneurons and
hindbrain serotonin cells has emphasized the combinatorial nature
of transcription factor regulation of neuronal identity (Cheng et al.,
2003; Jessell, 2000; Novitch et al., 2001; Pattyn et al., 2004). The
properties of PHOX2A in midbrain development, however, fit better
with non-combinatorial models of cell type specification, such as
those emerging from hematopoietic system studies, in which the
networks of cell type control can be analyzed, at least in some cases,
as hierarchies of primary and secondary regulators (Orkin, 2000;
Singh, 2007). In this analysis, a regulator of a cell fate can be called
primary if it (1) is required for that cell fate, (2) induces secondary
regulators, such as other transcription factors, that are also required
for that cell fate, (3) is activated at the time and site of the cell fate
decision, and (4) is by itself sufficient to produce the cell fate. Our
data, together with the extensive published work of others, establish
PHOX2A is a primary regulator of oculomotor cell fates. First,
genetic studies have demonstrated that PHOX2A is required for the
production of midbrain motoneurons (Guo et al., 1999; Nakano et
al., 2001; Pattyn et al., 1997). Second, the present study and those of
others have shown that Phox2a/b induce the expression of secondary
regulators of OMC cell fate, such as the transcription factor ISL1,
which is required for all motoneuron development (Dubreuil et al.,
2000; Pfaff et al., 1996). Third, BrdU birthdating and gene
expression schedules in the chick (this study) and mouse (Pattyn et
al., 1997) have established that PHOX2A is expressed in prospective
oculomotor cells in the ventral midbrain just at the time when this
cell fate decision is likely to be made. Fourth, this report
demonstrates that forced expression of Phox2a is sufficient to
produce ectopic midbrain motoneurons with the cellular and
molecular features of OMC cell fates. It is possible that primary
regulators of nuclear cell fate decisions will prove rare for vertebrate
brain development. These results, though, establish that in at least
one important example, a single transcription factor can act not only
as a primary determinant of neuron identity, but also as a primary
regulator of brain nucleus formation.
Acknowledgements
We thank May Tran and Melinda Drum for their help and Harinder Singh for
discussions. Nucleic acid reagents were provided by V. Berthoud, M. Dixon, C.
Goridis, M. Goulding, R. Ivell, T. Jessell, E. Laufer, E. Lewis, G. Martin, J.
Rubenstein and M. Takeichi. This work was supported by a NEI/NIH grant to
C.W.R. Deposited in PMC for release after 12 months.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.041251/-/DC1
References
Agarwala, S. and Ragsdale, C. W. (2002). A role for midbrain arcs in
nucleogenesis. Development 129, 5779-5788.
Agarwala, S. and Ragsdale, C. W. (2009). Midbrain patterning. In Encyclopedia
of Neuroscience, Vol. 5 (ed. L. R. Squire), pp. 879-888. Amsterdam: Elsevier.
Development 137 (7)
Agarwala, S., Sanders, T. A. and Ragsdale, C. W. (2001). Sonic Hedgehog
control of size and shape in midbrain pattern formation. Science 291, 21472150.
Bayly, R. D., Ngo, M., Aglyamova, G. V. and Agarwala, S. (2007). Regulation
of ventral midbrain patterning by Hedgehog signaling. Development 134, 21152124.
Bosley, T. M., Oystreck, D. T., Robertson, R. L., al Awad, A., Abu-Amero, K.
and Engle, E. C. (2006). Neurological features of congenital fibrosis of the
extraocular muscles type 2 with mutations in PHOX2A. Brain 129, 2363-2374.
Bron, R., Vermeren, M., Kokot, N., Andrews, W., Little, G. E., Mitchell, K. J.
and Cohen, J. (2007). Boundary cap cells constrain spinal motor neuron somal
migration at motor exit points by a semaphorin-plexin mechanism. Neural Dev.
2, 21.
Cheng, L., Chen, C. L., Luo, P., Tan, M., Qiu, M., Johnson, R. and Ma, Q.
(2003). Lmx1b, Pet-1, and Nkx2.2 coordinately specify serotonergic
neurotransmitter phenotype. J. Neurosci. 23, 9961-9967.
Chilton, J. K. and Guthrie, S. (2004). Development of oculomotor axon
projections in the chick embryo. J. Comp. Neurol. 472, 308-317.
Coppola, E., Pattyn, A., Guthrie, S. C., Goridis, C. and Studer, M. (2005).
Reciprocal gene replacements reveal unique functions for Phox2 genes during
neural differentiation. EMBO J. 24, 4392-4403.
DeKoter, R. P. and Singh, H. (2000). Regulation of B lymphocyte and
macrophage development by graded expression of PU.1. Science 288, 14391441.
Diaz, C., Puelles, L., Marin, F. and Glover, J. C. (1998). The relationship between
rhombomeres and vestibular neuron populations as assessed in quail-chicken
chimeras. Dev. Biol. 202, 14-28.
Dubreuil, V., Hirsch, M., Pattyn, A., Brunet, J. and Goridis, C. (2000). The
Phox2b transcription factor coordinately regulates neuronal cell cycle exit and
identity. Development 127, 5191-5201.
Dubreuil, V., Hirsch, M. R., Jouve, C., Brunet, J. F. and Goridis, C. (2002). The
role of Phox2b in synchronizing pan-neuronal and type-specific aspects of
neurogenesis. Development 129, 5241-5253.
Evinger, C. (1988). Extraocular motor nuclei: location, morphology and afferents.
In Neuroanatomy of the Oculomotor System (ed. J. A. Buttner-Ennever), pp. 81117. Amsterdam: Elsevier.
Frantz, G. D. and McConnell, S. K. (1996). Restriction of late cerebral cortical
progenitors to an upper-layer fate. Neuron 17, 55-61.
Gamlin, P. D. and Reiner, A. (1991). The Edinger-Westphal nucleus: sources of
input influencing accommodation, pupilloconstriction, and choroidal blood flow.
J. Comp. Neurol. 306, 425-438.
Gamlin, P. D., Reiner, A. and Karten, H. J. (1982). Substance P-containing
neurons of the avian suprachiasmatic nucleus project directly to the nucleus of
Edinger-Westphal. Proc. Natl. Acad. Sci. USA 79, 3891-3895.
Gamlin, P. D., Reiner, A., Erichsen, J. T., Karten, H. J. and Cohen, D. H. (1984).
The neural substrate for the pupillary light reflex in the pigeon (Columba livia). J.
Comp. Neurol. 226, 523-543.
Gould, T. W., Burek, M. J., Sosnowski, J. M., Prevette, D. and Oppenheim, R.
W. (1999). The spatial-temporal gradient of naturally occurring motoneuron
death reflects the time of prior exit from the cell cycle and position within the
lateral motor column. Dev. Biol. 216, 611-621.
Grove, E. A., Tole, S., Limon, J., Yip, L. and Ragsdale, C. W. (1998). The hem of
the embryonic cerebral cortex is defined by the expression of multiple Wnt
genes and is compromised in Gli3-deficient mice. Development 125, 23152325.
Guo, S., Brush, J., Teraoka, H., Goddard, A., Wilson, S. W., Mullins, M. C. and
Rosenthal, A. (1999). Development of noradrenergic neurons in the zebrafish
hindbrain requires BMP, FGF8, and the homeodomain protein soulless/Phox2a.
Neuron 24, 555-566.
Guthrie, S. (2007). Patterning and axon guidance of cranial motor neurons. Nat.
Rev. Neurosci. 8, 859-871.
Hamburger, V. and Hamilton, H. L. (1951). A series of normal stages in the
development of the chick embryo. J. Morphol. 88, 49-92.
Heaton, M. B. and Wayne, D. B. (1983). Patterns of extraocular innervation by
the oculomotor complex in the chick. J. Comp. Neurol. 216, 245-252.
Hirsch, M.-R., Glover, J. C., Dufour, H. D., Brunet, J.-F. and Goridis, C. (2007).
Forced expression of Phox2 homeodomain transcription factors induces a
branchio-visceromotor axonal phenotype. Dev. Biol. 303, 687-702.
Jessell, T. M. (2000). Neuronal specification in the spinal cord: inductive signals
and transcriptional codes. Nat. Rev. Genet. 1, 20-29.
Kawauchi, D., Taniguchi, H., Watanabe, H., Saito, T. and Murakami, F.
(2006). Direct visualization of nucleogenesis by precerebellar neurons:
involvement of ventricle-directed, radial fibre-associated migration. Development
133, 1113-1123.
Levi-Montalcini, R. (1963). Growth and differentiation in the nervous system. In
The Nature of Biological Diversity (ed. J. M. Allen), pp. 261-295. New York:
McGraw-Hill.
Martinov, V. N., Mochida, H., Hansen, K., Rinde, A. and Glover, J. C. (2004).
Development of functional synaptic connections between oculomotor
motoneurons and extraocular muscles in the chicken embryo. Neuroscience
DEVELOPMENT
1212 RESEARCH ARTICLE
2004 Abstracts. San Diego, CA: Society for Neuroscience (http://sfn.org/
index.cfm?pagename=abstracts_archive&task=view&controlID=15573&year=20
04).
Maurange, C., Cheng, L. and Gould, A. P. (2008). Temporal transcription factors
and their targets schedule the end of neural proliferation in Drosophila. Cell 133,
891-902.
Mauti, O., Domanitskaya, E., Andermatt, I., Sadhu, R. and Stoeckli, E. T.
(2007). Semaphorin6A acts as a gate keeper between the central and the
peripheral nervous system. Neural. Dev. 2, 28.
McConnell, S. K. (1991). The generation of neuronal diversity in the central
nervous system. Annu. Rev. Neurosci. 14, 269-300.
Mizuguchi, R., Kriks, S., Cordes, R., Gossler, A., Ma, Q. and Goulding, M.
(2006). Ascl1 and Gsh1/2 control inhibitory and excitatory cell fate in spinal
sensory interneurons. Nat. Neurosci. 9, 770-778.
Momose, T., Tonegawa, A., Takeuchi, J., Ogawa, H., Umesono, K. and
Yasuda, K. (1999). Efficient targeting of gene expression in chick embryos by
microelectroporation. Dev. Growth Differ. 41, 335-344.
Muhlbauer, E., Hamann, D., Xu, B., Ivell, R., Udovic, B., Ellendorff, F. and
Grossmann, R. (1993). Arginine vasotocin gene expression and hormone
synthesis during ontogeny of the chicken embryo and the newborn chick. J.
Neuroendocrinol. 5, 281-288.
Nakagawa, S. and Takeichi, M. (1998). Neural crest emigration from the neural
tube depends on regulated cadherin expression. Development 125, 2963-2971.
Nakano, M., Yamada, K., Fain, J., Sener, E. C., Selleck, C. J., Awad, A. H.,
Zwaan, J., Mullaney, P. B., Bosley, T. M. and Engle, E. C. (2001).
Homozygous mutations in ARIX(PHOX2A) result in congenital fibrosis of the
extraocular muscles type 2. Nat. Genet. 29, 315-320.
Niederlander, C. and Lumsden, A. (1996). Late emigrating neural crest cells
migrate specifically to the exit points of cranial branchiomotor nerves.
Development 122, 2367-2374.
Nieuwenhuys, R., ten Donkelaar, H. J. and Nicholson, C. (1998). The Central
Nervous System of Vertebrates. Berlin: Springer-Verlag.
Novitch, B. G., Chen, A. I. and Jessell, T. M. (2001). Coordinate regulation of
motor neuron subtype identity and pan-neuronal properties by the bHLH
repressor Olig2. Neuron 31, 773-789.
Orkin, S. H. (2000). Diversification of haematopoietic stem cells to specific
lineages. Nat. Rev. Genet. 1, 57-64.
Pattyn, A., Morin, X., Cremer, H., Goridis, C. and Brunet, J. F. (1997).
Expression and interactions of the two closely related homeobox genes Phox2a
and Phox2b during neurogenesis. Development 124, 4065-4075.
Pattyn, A., Hirsch, M., Goridis, C. and Brunet, J. F. (2000). Control of hindbrain
motor neuron differentiation by the homeobox gene Phox2b. Development 127,
1349-1358.
RESEARCH ARTICLE 1213
Pattyn, A., Simplicio, N., van Doorninck, J. H., Goridis, C., Guillemot, F. and
Brunet, J. F. (2004). Ascl1/Mash1 is required for the development of central
serotonergic neurons. Nat. Neurosci. 7, 589-595.
Peng, C. Y., Yajima, H., Burns, C. E., Zon, L. I., Sisodia, S. S., Pfaff, S. L. and
Sharma, K. (2007). Notch and MAML signaling drives Scl-dependent
interneuron diversity in the spinal cord. Neuron 53, 813-827.
Pfaff, S. L., Mendelsohn, M., Stewart, C. L., Edlund, T. and Jessell, T. M.
(1996). Requirement for LIM homeobox gene Isl1 in motor neuron generation
reveals a motor neuron-dependent step in interneuron differentiation. Cell 84,
309-320.
Puelles, L. (1978). A Golgi-study of oculomotor neuroblasts migrating across the
midline in chick embryos. Anat. Embryol. 152, 205-215.
Rakic, P. (1974). Neurons in rhesus monkey visual cortex: systematic relation
between time of origin and eventual disposition. Science 183, 425-427.
Reiner, A., Erichsen, J. T., Cabot, J. B., Evinger, C., Fitzgerald, M. E. and
Karten, H. J. (1991). Neurotransmitter organization of the nucleus of EdingerWestphal and its projection to the avian ciliary ganglion. Visual Neurosci. 6, 451472.
Sanders, T. A., Lumsden, A. and Ragsdale, C. W. (2002). Arcuate plan of chick
midbrain development. J. Neurosci. 22, 10742-10750.
Singh, H. (2007). Shaping a helper T cell identity. Nat. Immunol. 8, 119-120.
Song, M.-R. and Pfaff, S. L. (2009). Motor neuron specification in vertebrates. In
Encyclopedia of Neuroscience, Vol. 5 (ed. L. R. Squire), pp. 1015-1022.
Amsterdam: Elsevier.
Varela-Echavarria, A., Pfaff, S. L. and Guthrie, S. (1996). Differential expression
of LIM homeobox genes among motor neuron subpopulations in the developing
chick brain stem. Mol. Cell. Neurosci. 8, 242-257.
Vermeren, M., Maro, G. S., Bron, R., McGonnell, I. M., Charnay, P., Topilko, P.
and Cohen, J. (2003). Integrity of developing spinal motor columns is regulated
by neural crest derivatives at motor exit points. Neuron 37, 403-415.
Wang, M. Z., Jin, P., Bumcrot, D. A., Marigo, V., McMahon, A. P., Wang, E. A.,
Woolf, T. and Pang, K. (1995). Induction of dopaminergic neuron phenotype
in the midbrain by Sonic hedgehog protein. Nat. Med. 1, 1184-1188.
Weitemier, A. Z., Tsivkovskaia, N. O. and Ryabinin, A. E. (2005). Urocortin 1
distribution in mouse brain is strain-dependent. Neuroscience 132, 729-740.
Yaneza, M., Gilthorpe, J. D., Lumsden, A. and Tucker, A. S. (2002). No
evidence for ventrally migrating neural tube cells from the mid- and hindbrain.
Dev. Dyn. 223, 163-167.
Zellmer, E., Zhang, Z., Greco, D., Rhodes, J., Cassel, S. and Lewis, E. J. (1995).
A homeodomain protein selectively expressed in noradrenergic tissue regulates
transcription of neurotransmitter biosynthetic genes. J. Neurosci. 15, 81098120.
Zhu, S., Lin, S., Kao, C. F., Awasaki, T., Chiang, A. S. and Lee, T. (2006).
Gradients of the Drosophila Chinmo BTB-zinc finger protein govern neuronal
temporal identity. Cell 127, 409-422.
DEVELOPMENT
Midbrain oculomotor nucleogenesis