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 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.

KEY WORDS: Brain nuclei, Midbrain, Motoneurons, Extraocular muscles, Chick

INTRODUCTION (Levi-Montalcini, 1963) and Puelles (Puelles, 1978). The cellular A characteristic feature of the vertebrate brain is that its neuronal cell and molecular factors that mediate these migrations are unknown bodies are organized into clusters. These clusters, known as nuclei, (Guthrie, 2007). serve to organize much of the circuitry of the brain (Nieuwenhuys Genetic studies over the past ten years have identified the et al., 1998). Key attributes of nuclei include a spatial arrangement PHOX2A homeobox gene as an important regulator of midbrain of neurons, often with multiple cell types, which have distinct inputs, oculomotor development. Zebrafish embryos with a single amino axonal projections and neurotransmitter phenotypes. Only limited acid mutation in the Phox2a homeodomain exhibit a reduction in progress has been made in understanding how neuronal progenitor oculomotor nucleus precursor cells (Guo et al., 1999). In Phox2a–/– cells are allocated to different nuclear fates, and the molecular mice, midbrain oculomotor neurons are absent (Pattyn et al., 1997). mechanisms that coordinate cell type composition, spatial In humans, PHOX2A mutations are linked to severe defects in arrangement and nucleus size remain largely unknown (Agarwala extraocular muscle movement and in pupillary light reflexes (Bosley and Ragsdale, 2002; Diaz et al., 1998; Kawauchi et al., 2006). et al., 2006; Nakano et al., 2001), and magnetic resonance imaging The chick midbrain oculomotor complex (OMC) is an attractive has established that the oculomotor, or third, cranial nerve is missing model system for studying brain nucleogenesis. In vertebrates, the in these patients (Bosley et al., 2006). The closely related homeobox OMC supplies somatic motoneuron input to extraocular muscles and gene Phox2b, which is required for the development of hindbrain preganglionic autonomic input to the ciliary ganglia. The somatic branchiomotor and visceromotor neurons, is also expressed in and visceral midbrain motoneurons form separate pools, although midbrain oculomotor neurons but is dispensable for their production the pool of visceral motoneurons in rodents is not sharply (Pattyn et al., 2000; Pattyn et al., 1997). Recent findings, however, distinguished in Nissl-stained preparations or by . have shown some degree of shared competency between the By contrast, the chicken shares with all birds a cleanly segregated Phox2a/b in motoneuron development. Knock-in of Phox2b OMC with discrete subnuclei that innervate specific autonomic and coding sequence into the Phox2a locus allows for the development ocular muscle targets (Evinger, 1988; Gamlin et al., 1984; Heaton of some midbrain oculomotor neurons (Coppola et al., 2005). Gain- and Wayne, 1983). Indeed, in birds the preganglionic motoneurons of-function studies in chick spinal cord have demonstrated that arise exclusively from the Edinger-Westphal (EW) nucleus, which exogenous Phox2a/b are able to upregulate the expression of is the dorsal component of the OMC (Gamlin and Reiner, 1991; motoneuron markers and induce ectopic axons that can contribute Reiner et al., 1991). The segregation of neurons in the developing to existing nerve roots (Hirsch et al., 2007; Pattyn et al., 1997). chick OMC into discrete subnuclei occurs in a series of migrations, Interestingly, these axons exit the cord through dorsal exit points, a as documented in the classic histological studies of Levi-Montalcini feature of hindbrain branchiomotor and visceromotor neurons that distinguishes them from somatic motoneurons. This evidence establishes that Phox2a/b are required for brainstem motoneuron 1Department of Neurobiology, The University of Chicago, Chicago, IL 60637, USA. development and can, when misexpressed, induce motoneurons that 2Section of Neurobiology, University of Texas at Austin, Austin TX 78712, USA. issue axons that exit the nerve cord. In this study, we extended the analysis of PHOX2A motoneuron *Author for correspondence ([email protected]) induction to midbrain in order to address a very different kind of

Accepted 26 January 2010 question: to what extent can a single transcription factor drive the DEVELOPMENT 1206 RESEARCH ARTICLE Development 137 (7) production of a spatially organized nuclear complex that contains situ hybridization was carried out with digoxigenin-, DNP- and fluorescein- different cell types? First, we report on normal OMC development, labeled riboprobes synthesized from chick cDNA plasmids for CDH7, showing that the visceral and somatic motoneurons of the chick CHAT, CX36, EGR2 (KROX20), , ISL1, ISL2, NKX6.1, NRG1, PAX6, OMC are generated in an ‘inside-out’ fashion, with the final position PHOX2A, PHOX2B, ROBO2, SLIT3, TBX20 and VT and from plasmids for of the OMC neurons dependent on their time of origin. Second, we rat Phox2a and mouse Phox2b. For light microscopy, labeled RNA duplexes were detected with antibody-alkaline phosphatase conjugates (Roche demonstrate that exogenous Phox2a can induce a complete OMC Diagnostics) and using the distinguishable formazans of NBT and TNBT molecular program, including the production of both visceral and (Grove et al., 1998). For fluorescence microscopy, antibody-horseradish somatic motoneurons. Crucially, we document that the ectopic peroxidase conjugates were substituted and peroxidase activity was visceral and somatic oculomotor neurons segregate along the demonstrated by fluorescent tyramide signal amplification using FITC- ventricular-pial axis, mimicking the architecture of the native OMC. tyramide for fluorescein-labeled probes, cyanine 3-tyramide for Third, we demonstrate that Phox2a-induced motoneurons make digoxigenin-labeled probes and cyanine 5-tyramide for DNP-labeled probes ectopic cranial nerves that recruit boundary cap cells and, in some (TSA Plus Fluorescence Systems Kit, PerkinElmer). instances, target extraocular muscles directly. These results, BrdU experiments combined with previous findings in mouse and human, establish that 5-bromo-2-deoxyuridine (BrdU) can be very toxic to chicken embryos, a single transcription factor can act as a primary developmental particularly before E2.5 (Gould et al., 1999). Extensive scout experiments determinant for a brain nucleus and its constituent cell types. were carried out to identify a BrdU dose that elicits clear nuclear labeling while allowing embryos to survive without obvious morphological defects MATERIALS AND METHODS to E12. We found success with BrdU (Sigma) prepared as a 75 mg/ml Chicken embryos solution in PBS containing 0.01% Fast Green and delivered as a 50 ml Fertilized Babcock B-300 White Leghorn chicken eggs (Gallus gallus aliquot through a small hole made in the vitelline membrane near the heart. domesticus) were supplied by Phil’s Fresh Eggs (Forreston, IL, USA). The Twenty embryos with BrdU injection at st 8, 9 and 11-23 were harvested at first day of incubation was designated embryonic day 0 (E0). Staging of E12 with PFA-PBS fixation. Serial sections (40 mm) of midbrain were first embryos followed Hamburger and Hamilton (Hamburger and Hamilton, processed for TNBT gene expression histochemistry with digoxigenin- 1951). Our chick embryo protocols were approved by the University of labeled ISL1 riboprobes to identify OMC structure. Sections were then Chicago IACUC. treated with 2M HCl for 30 minutes to denature nuclear DNA and, after washing, incubated in a 1:50 dilution of monoclonal FITC-conjugated anti- Expression plasmids BrdU antibody (Becton Dickinson) in 2% lamb serum. The anti-BrdU The human EF1a (EEF1A1) promoter expression construct pEFX was antibody was detected with the anti-fluorescein phosphatase conjugate and generated from pEF1/-His C (Invitrogen) by excising a 2.2 kb PvuII NBT histochemistry employed in our gene expression experiments. fragment containing an f1 origin, neomycin resistance gene and SV40 elements. The rat Phox2a (rArix) expression construct pEFX-rArix was Images engineered in pEFX by insertion of a 1.6 kb cDNA containing the complete Tissue was photographed with a Zeiss AxioCam digital camera attached to rat Phox2a coding sequence (Zellmer et al., 1995). pEFX-mPhox2b contains a Zeiss Axioskop 50 upright microscope or a Leica MZ FLIIII a 1.3 kb full-length mouse Phox2b cDNA isolated from E12.5 brain RNA stereomicroscope. Images were captured through the AxioVision 4.5 by RT-PCR using the forward primer 5Ј-GCCATCCAGAACCTTTTCA - software system and processed with Adobe Photoshop. Images requiring ATG-3Ј and the reverse primer 5Ј-CCGTCTCTCCCTATCACCTACTTG- increased depth of focus were assembled with Helicon Focus 4.6 Pro 3Ј. software.

In ovo electroporation For the forced expression of Phox2a/b, we employed expression plasmid RESULTS delivery by in ovo microelectroporation (Agarwala et al., 2001; Momose et Molecular and cellular development of the avian al., 1999), targeting, in most experiments, the rostrolateral right ventral midbrain. Negative electrodes were fabricated from 0.025 mm tungsten wire OMC and positive electrodes from 0.063 mm platinum wire (Goodfellow). The avian OMC is a spatially organized nuclear complex comprising Plasmid DNA solution (50-250 nl at 1-3 mg/ml in endotoxin-free water a dorsal EW nucleus and a ventral somatic motor nucleus (Evinger, containing 0.02% Fast Green) was pressure-injected from a glass capillary 1988; Heaton and Wayne, 1983). The EW nucleus contains the into the midbrain vesicle of Hamburger and Hamilton stage (st) 9-24 midbrain visceral motoneurons, which innervate the ciliary ganglion embryos. The negative electrode was placed in the midbrain lumen and the and control pupillary constriction, accommodation and choroidal positive electrode just outside the midbrain. Electric fields were generated blood flow (Gamlin et al., 1982). The somatic oculomotor nucleus with a Model 2100 Isolated Pulse Stimulator (A-M Systems) delivering harbors the midbrain somatic motoneurons, which in birds are three 25-millisecond pulses of 9 V, with an inter-pulse period of 1 second. discretely organized into subnuclear pools, each innervating distinct Electroporated embryos were incubated for a further 1-4 days. Our analyses extraocular muscle targets (Fig. 1A). In mammals, the somatic are based on gene expression in 704 embryos successfully electroporated oculomotor neurons aggregate to form a single nucleus, and in many 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 species, including mouse, the EW nucleus is challenging to identify zone of misexpression before processing. In 23 embryos, we co- (Weitemier et al., 2005). The anatomical clarity of the avian OMC electroporated the alkaline phosphatase reporter plasmid pEFX-AP to label coupled with the ease of experimental manipulation makes the midbrain efferent axons. For the remaining experiments, the reproducible chicken embryo attractive for investigations of midbrain accuracy of plasmid delivery was confirmed through parallel motoneuron development. electroporations of pEFX-AP, which was demonstrated by histochemistry, To identify genes that reflect the connectional heterogeneity of the or of pEFX-rArix or pEFX-mPhox2b, which were detected by Phox2a/b OMC subnuclei, we screened chick midbrain sections at E12, an age transgene in situ hybridization. at which the OMC subnuclei are readily distinguished. Genes Nucleic acid histology encoding known motoneuron markers, such as the LIM- Harvested embryos were fixed in 4% paraformaldehyde (PFA) in phosphate- homeodomain ISL1, were expressed by all oculomotor buffered saline (PBS). Dissected embryonic brains were processed as whole- subnuclei (Fig. 1A; see Fig. S1 in the supplementary material) (Jessell,

mounts or cryoprotected and sectioned at 40 mm on a sledge microtome. In 2000). Three markers, however, identified visceral and somatic DEVELOPMENT Midbrain oculomotor nucleogenesis RESEARCH ARTICLE 1207

Fig. 1. Gene expression markers distinguish the somatic and Fig. 2. Schedule of OMC development. Gene expression onset and visceral motoneuron subnuclei of the chick oculomotor complex timing of key events in chick oculomotor nerve (OMN) development, (OMC). Coronal sections of E12 chick midbrain processed for in situ illustrated with reference to Hamburger-Hamilton (HH) stages (see text hybridization to identify the somatic motoneurons of the oculomotor for details). Expression of the ligand-encoding SLIT3 and neuregulin-1 nucleus (yellow arrowhead) and the visceral motoneurons of the Edinger- (NRG1) genes and of the SLIT ROBO2 was detected by st 13+, Westphal nucleus (EW, green arrowhead). (A) The motoneuron marker st 17 and st 25, respectively. Timing of functional innervation is from ISL1 identifies all OMC motoneurons. (B) Gene expression for the Martinov et al. (Martinov et al., 2004). IO, inferior oblique. 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 neuron marker VT was first detected at st 20 (E3.5). The onset of the subnuclei. Within the DM subnucleus, TBX20 is strongly expressed in its lateral division, but is absent from its medial division (red arrow). DL, somatic oculomotor neuron markers CX36 and TBX20 occurred dorsolateral; DM, dorsomedial; VM, ventromedial. Scale bar: 0.2 mm. 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 subnuclei specifically. The EW nucleus was labeled by expression of earlier in OMC development, the differential time of onset of the the neuropeptide gene for vasotocin (VT) (Muhlbauer et al., 1993). visceral and somatic motoneuron markers raised the possibility that Somatic OMC subnuclei expressed the gap junction subunit CX36 and OMC visceral motoneurons are born before the OMC somatic the T-box transcription factor TBX20 genes (Fig. 1C,D). motoneurons. We constructed a timeline of OMC molecular and cellular development to relate the expression of the motoneuron subtype Inside-out pattern of OMC development markers to other aspects of OMC maturation (Fig. 2). To study The embryonic ventral midbrain is organized into a series of arcs, with oculomotor nerve development, we electroporated an alkaline the PHOX2A-rich OMC arising in the most medial arc, arc 1 phosphatase-encoding expression vector into E2 midbrain and (Agarwala and Ragsdale, 2002; Sanders et al., 2002). Thus, unlike the processed the embryos for phosphatase histochemistry 1-4 days somatic and visceral motoneurons of hindbrain (Guthrie, 2007), the later. We found that the axons are first issued from ventral midbrain midbrain somatic and visceral motoneurons appear, by gene at st 16 and reach the ipsilateral orbit over the next 2 days, contacting expression, to be generated from a common progenitor pool, that is, the inferior oblique (IO) muscle by st 25 (data not shown). This from the ventricular zone overlying arc 1. To test for a timing schedule matches that described by Chilton and Guthrie, who difference in the neurogenesis of visceral and somatic oculomotor employed anti-neurofilament antibody staining to mark axons neurons, we delivered BrdU to embryos between E2 and E4.5 and (Chilton and Guthrie, 2004). collected them at E12, when the OMC subnuclei are easily We found that OMC marker onset readily fell into two groups: recognized. BrdU birthdating in chick is problematic because early onset genes that were expressed before oculomotor nerve exit delivered BrdU is not a true pulse as it is in mammals, but is and which principally encode transcription factors, and late onset continuously available in the egg up to 22 hours after delivery (Gould genes that were expressed after nerve exit but before target et al., 1999). Consequently, we were not able to determine precisely innervation (Fig. 2). when neurogenesis begins, only when it ends. Heavy labeling, The homeodomain transcription factor gene NKX6.1 was the first however, is predicted to mark cells undergoing their final divisions. OMC marker that we detected in medial ventral midbrain, but it is We found clear evidence for a temporal difference in the birth order not selective for midbrain motoneurons as it is also expressed in red of the oculomotor subtypes. Neurogenesis begins first in visceral nucleus progenitor domains and more lateral midbrain territories (P. oculomotor neurons (Fig. 3A). The neurons of the EW nucleus were M. Garfin, PhD thesis, University of Chicago, 2004). most heavily labeled when BrdU was applied at st 11 and 12 (Fig. 3A; PHOX2A, which is restricted to OMC cells, is detected at st 12+. data not shown), close to the time of PHOX2A expression onset (st PHOX2B is expressed in prospective OMC cells immediately 12+). Most visceral oculomotor neuron progenitors have undergone thereafter, at st 13–, and the LIM homeobox genes ISL1 and ISL2 are their final cell division by st 18, when only a few medial EW cells still detected by st 14+ (Fig. 2) (Varela-Echavarria et al., 1996). incorporated BrdU (Fig. 3C), and visceral motor neurogenesis was The late onset genes included the motoneuron neurotransmitter largely complete by st 19 (data not shown). By contrast, somatic enzyme choline acetyltransferase (CHAT; st 24), the axon guidance oculomotor neuron progenitors continued to divide in large numbers regulator ROBO2 (st 25) and the specific markers that distinguish through st 18 (Fig. 3B,C). BrdU labeling in the somatic subnuclei was

visceral and somatic motoneuron identity. The visceral oculomotor heaviest when BrdU was delivered at st 17 (data not shown), DEVELOPMENT 1208 RESEARCH ARTICLE Development 137 (7)

Fig. 3. Inside-out gradient of OMC neurogenesis. Chick midbrains Fig. 4. OMC motoneuron subtype segregation at the end of OMC were processed at E12 for BrdU immunohistochemistry (blue) and ISL1 neurogenesis. (A-D) Coronal sections of chick midbrains of the indicated gene expression (brown/pink) after BrdU delivery to st 8-23 embryos. stages processed for two-color fluorescent in situ hybridization. The Coronal sections (top) and corresponding BrdU chartings (bottom) ventricle is to the top and the pial surface is to the bottom. For st 21 (A) } illustrate the dorsal-to-ventral (D V) progression of OMC neurogenesis. and st 24 (B), before TBX20 gene expression onset, VT (green) was Panels illustrate the left OMC, with the midline to the right. OMC employed to label visceral motoneurons and ISL1 (red) served to identify subnuclei are identified in D. (A) Heavy BrdU labeling in the EW nucleus all OMC neurons. Because ISL1 co-labels VT-expressing cells, many indicates that neurogenesis of visceral oculomotor neurons precedes visceral motoneurons appear yellow. St 25 (C) and st 26 (D) midbrains that of somatic oculomotor neurons. (B) Extensive BrdU labeling is seen probed for VT (green) to mark visceral motoneurons and TBX20 (red) to throughout the OMC. (C,D) Neurogenesis is nearly complete for the EW label somatic motoneurons. At st 21, somatic motor production and nucleus by st 18 (C) and for the somatic motoneurons by st 23 (D). migration is still underway and OMC subtypes appear intermixed (A). Scale bar: 0.1 mm. 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 To study the spatial allocations of OMC neurons before st 25, we stage and somewhat overlaps with the end of visceral motoneuron employed VT (visceral) and ISL1 (visceral and somatic) gene generation. By st 21, somatic oculomotor neurogenesis was largely expression. Between st 21 and 23, when motoneuron neurogenesis confined to medial cells in the ventromedial subnucleus (data not is still underway and somatic motoneurons must be transiting the VT shown) and was nearly complete by st 23 (Fig. 3D). Thus, there is a territory, the prospective somatic (expressing ISL1 but not VT) and clear temporal difference in the birth of the oculomotor neuron visceral (expressing both VT and ISL1) motoneurons were more subtypes in the chick embryo and this difference is part of a general intermixed along the ventricular-pial axis (Fig. 4A). By st 24, gradient of OMC neurogenesis that extends from dorsolateral EW however, when motoneuron production is complete, there was a neurons to the ventromedial somatic subnuclei (Fig. 3A-D). clear ISL1-rich territory that contained only a few cells expressing Interestingly, the four-stage separation in neurogenesis completion for both VT and ISL1 (Fig. 4B). We conclude from these results that the visceral (st 19) versus somatic (st 23) motoneurons proved to be although the motoneuron subtypes are not fully segregated along the close to the five-stage separation in the onset of our visceral (VT) and ventricular-pial axis during OMC neurogenesis and migration, by somatic (CX36, TBX20) differentiation markers. the end of motoneuron production the numbers of mislocated cells In the mature OMC, the visceral and somatic motor nuclei are are small and these errors are rapidly corrected by remedial segregated in the dorsal-ventral axis, which corresponds to the migration or cell death between st 24 and 26. ventricular-pial axis of the embryonic ventral midbrain. We were interested in whether visceral and somatic motoneurons in their Phox2a drives a molecular OMC program initial allocation to the prospective OMC prepatterned their eventual Previous work has shown that the signaling molecule sonic ventricular-pial segregation, or were at first fully intermixed. Our hedgehog can produce motoneurons in chick midbrain (Agarwala et specific somatic motoneuron markers are not expressed until st 25. al., 2001; Bayly et al., 2007; Wang et al., 1995). We were interested At st 25, TBX20-expressing somatic cells were already largely in the extent to which OMC transcription factors could, singly or in segregated from the VT-expressing EW cells, but there was some combination, induce OMC cell fates. PHOX2A was the clear choice mixing of motoneuron subtypes (Fig. 4C; see Movie 1 in the for initial study: it is required for midbrain oculomotor neuron supplementary material). Because of the inside-out generation of the development in mouse and human (Nakano et al., 2001; Pattyn et OMC neurons, some TBX20-expressing cells in the prospective EW al., 1997); in the chick embryo it is the first transcription factor we nucleus could be in transit, but there were also a few VT-expressing can identify that is selectively restricted to prospective OMC cells; cells in the TBX20-positive territory. One stage later (st 26), nearly and, most importantly, Dubreuil et al. (Dubreuil et al., 2000; all VT-expressing neurons occupied a territory that was entirely Dubreuil et al., 2002) and Hirsch et al. (Hirsch et al., 2007) have separate from that of the TBX20-expressing somatic neurons, with shown that Phox2a/b can induce motoneuron markers, including

a clear gap between the two populations (Fig. 4D). ISL1 and CHAT, in chick spinal cord. We found that in chick DEVELOPMENT 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).

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 Fig. 5. Phox2a induction of OMC gene expression markers. (A) Open-book preparation of E6 chick brainstem whole-mount ventricular-pial axis, with the VT-expressing visceral oculomotor demonstrates native PHOX2A gene expression in the midbrain OMC neurons lying closer to the ventricle (Fig. 4D, Fig. 6B). We and the rostral hindbrain trochlear nucleus (Tr). Rostral is to the top, employed two-color detection to distinguish the VT-expressing and and the ventricular surface faces the viewer. (B-G) Expression plasmids TBX20-expressing population subtypes in Phox2a-electroporated for rat Phox2a (Arix) were electroporated into E2 chick ventral midbrain chick embryos. We identified, in the whole-mounts of four brains, and embryos harvested for whole-mount in situ hybridization at E5 (D) 36 ectopic clusters of cells with both VT and TBX20 expression (Fig. and E6 (B,C,E-G). (B) A typical pattern of plasmid delivery by 6A). The spatial organization of these clusters was studied in serial electroporation illustrated in the right midbrain by transgene expression cross-section. Two of these clusters showed some intermixing of the (arrow). (C) Phox2a misexpression elicits induction of endogenous chick VT-expressing and TBX20-expressing cells. The other 34 PHOX2A (arrow). (D) Phox2a forced expression induces other OMC overlapping clusters presented full segregation of the VT-expressing markers, including the axon guidance molecule SLIT3 (arrow). (E) Lateral arcuate markers PAX6 (P6) and EVX1 (E1) (brown) illustrate the capacity and TBX20-expressing cells, with the VT-positive cell cluster of Phox2a to induce ISL1 (blue) throughout the ventral midbrain (red invariably closer to the ventricular surface. In 26/34 clusters, the arrows) and rostrally in caudoventral diencephalon (green arrow). segregation featured a gap between the two populations (Fig. 6C,D). (F,G) Phox2a misexpression induces molecular markers (arrows) of In addition to this induction of ‘mini’-OMC clusters, we saw visceral (VT, F) and somatic oculomotor neurons (TBX20, G). Asterisks many isolated examples of ectopic cells (Fig. 6E,F, green arrows). (A,C,G) mark the contralateral migration of the prospective superior In these examples, even when ectopic expression of only one of the rectus somatic motoneurons (see Chilton and Guthrie, 2004). IS, two motoneuron class-specific markers was detected, the VT- isthmus. Scale bar: 0.2 mm. expressing 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 VT- midbrain, electroporation of full-length rat Phox2a (Arix) induced expressing (n=111) and TBX20-expressing (n=43) cells along the the early and late genes identified in Fig. 2, including motoneuron ventricular-pial axis by measuring the distances from the base of the transcription factor, signaling and axon guidance genes (Fig. 5C-E; ventricular zone to each isolated cell or cell cluster. Because these see Fig. S2 and Table S1 in the supplementary material). Induction distances were non-normally distributed (Fig. 7), we compared them of OMC markers was efficient, with 269/365 electroporated with the two-sample Wilcoxon rank sum test. This analysis embryos demonstrating ectopic OMC gene expression. Similar established that the isolated TBX20-expressing cells migrated to efficiencies were seen in the induction of these markers by mouse significantly greater tissue depths than the VT-expressing cells Phox2b gene delivery (see Fig. S3 in the supplementary material). (mean, 87.2 versus 39.4 mm; median, 75.9 versus 32.9 mm; To test for spatial restrictions to midbrain motoneuron induction, P<0.0001). We conclude that the spatial arrangement of the class- we performed two-color in situ hybridization on Phox2a- specific ectopic oculomotor neurons precisely follows that of the electroporated brains harvested at E6 and probed for ISL1 and the native motoneuron classes in the OMC. lateral midbrain markers PAX6 and EVX1 (Fig. 5E; see Fig. S4 in the supplementary material). These data establish that Phox2a induction PHOX2A regulation of nerve biogenesis of motoneuron markers extends across the rostral-caudal extent of The central feature of motoneurons is that their axons leave the the ventral midbrain, from the ventral midline at least as far lateral nervous system and innervate peripheral targets. To study whether as the EVX1 stripe. We also found that Phox2a could induce ISL1 in Phox2a misexpression can drive ectopic nerve production, we co- the adjoining subthalamic region of forebrain (Fig. 5E; see Fig. S4 electroporated the rat Phox2a expression plasmid with an alkaline in the supplementary material). By contrast, we were unable to elicit phosphatase expression vector to label cranial nerves. Phox2a motoneuron markers in dorsal midbrain following either Phox2a or overexpression generated ectopic nerve rootlets exiting from the

Phox2b gene delivery (not shown). By morphology and midbrain, often several in each electroporated embryo. Table S2 in DEVELOPMENT 1210 RESEARCH ARTICLE 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 two- sample Wilcoxon rank sum test. The horizontal line in the box represents the median value, and the edges of the box are the first and Fig. 6. Phox2a induction of mini-OMCs. Chick ventral midbrains third quartiles. The lines extending from the edges of the box end at electroporated with rat Phox2a plasmids at E2 and collected at E6. the last value within 1.5 times the interquartile range (third minus first Tissue was processed to demonstrate visceral (VT, brown) and somatic quartile) from the edge of the box. Values beyond this distance are (TBX20, blue) motoneurons. (A) Two-color in situ hybridization whole- considered outliers and are plotted individually. Distances from the mount, oriented and labeled as in Fig. 5A, demonstrates the ectopic ventricular zone were statistically significantly greater for TBX20- induction of low-density fields of VT-expressing cells (green arrow) and expressing than for VT-expressing cells (see text). clusters containing both VT-expressing and TBX20-expressing cells (red arrow). In this sample, the overlap of the VT-expressing and TBX20- expressing cells in the native OMC is not seen because of the density of TBX20 labeling, and the extent of the VT-positive territory is to express high levels of EGR2 (KROX20) and cadherin-7 (CDH7), exaggerated by whole-mount flattening. (B) Entopic arrangement of and CDH7-positive BCCs in particular have been demonstrated at the E6 OMC demonstrated in coronal cross-section, with the ventricle oculomotor axon exit points (Nakagawa and Takeichi, 1998; to the top (asterisk). VT-expressing visceral motoneurons sit closer to Niederlander and Lumsden, 1996). We asked whether these neural the ventricle, whereas -expressing somatic motoneurons lie TBX20 crest derivatives are also found at Phox2a-induced ectopic axon exit deeper, closer to the pial surface, with a gap between the subtypes (arrowhead). (C-F) Coronal cross-sections through the ventral midbrain points. In control brains, we found EGR2 and CDH7 expression just show the segregation of ectopic motoneuron classes along the outside the neuroepithelium, where native oculomotor axons exit ventricular-pial axis (ventricle marked by asterisk). A clear gap is often (data not shown). EGR2 expression in the mesenchyme subjacent to (C,D, arrowheads), but not always (E, red arrow), seen between the the ventral midbrain neuroepithelium was most readily detected two classes of ectopic motoneurons. Forced Phox2a expression also between E3 and E4, whereas CDH7 expression was present at high produced isolated VT-expressing (E,F) and TBX20-expressing (F) cells in levels between E3 and E5 (Fig. 9). In Phox2a-electroporated heads, low-density fields (green arrows). Scale bars: 0.1 mm. 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 the supplementary material details the statistics on the behavior of cells populated the ectopic nerve exits (Fig. 9B,C). Our results Phox2a-induced rootlets for a sample of 19 electroporated embryos. establish that Phox2a induction of motor nerves constitutes a These ectopic axons (Fig. 8) projected directly from ectopic spots sufficient trigger for BCCs to populate axon exit points. Since within the ventral midbrain without aligning with the oculomotor ectopic exit points are unconstrained in their ventral midbrain nerve. Some projections stopped abruptly in the subjacent distributions (not shown), the ectopic axons must release a BCC mesenchyme (n=16/49) (Fig. 8A), but many axons found their way attractant (Bron et al., 2007; Mauti et al., 2007) or induce a BCC to nearby cranial nerves III and IV, which carry oculomotor axons identity in local head crest cells. (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 DISCUSSION extraocular muscles of the orbit, bypassing the cranial nerves PHOX2 transcription factors appear to be exceptionally effective in altogether (Fig. 8C). Finally, the timing of exit of these ectopic axons driving neuronal cell type identities. Dubreuil et al. (Dubreuil et al., adhered closely to that of the native oculomotor nerve (st 16, n=3/7 2000) have shown that exogenous Phox2b misexpression in chick tested). Thus, misexpressed Phox2a can produce axonal projections spinal cord, a tissue in which PHOX2A/B are not normally expressed that exit the neuroepithelium on schedule and innervate natural in motoneurons, can trigger motoneuron-specific gene expression targets. and act as a neurogenic factor driving progenitor cells out of the cell Boundary cap cells (BCCs) are neural crest derivatives that cycle (Dubreuil et al., 2002; Pattyn et al., 2000). To explore the accumulate at prospective nerve exit points (Vermeren et al., 2003; motoneuron inductive potential of PHOX2A/B in more detail, we

Yaneza et al., 2002). In the chick embryo, BCCs have been shown studied the consequences of forced Phox2a/b expression in chick DEVELOPMENT Midbrain oculomotor nucleogenesis RESEARCH ARTICLE 1211

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 Phox2a nerve; TrN, trochlear nerve; v.mb, ventral midbrain. Scale bars: 0.2 mm. Fig. 9. -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 midbrain, where PHOX2A/B are natively expressed and restricted BCC markers CDH7 (A-C) and EGR2 (D). (A,D) Lateral views of right side to motoneuron lineages. We found that Phox2a delivery can drive a of E3 (A) and E4 (D) heads. CDH7 (A) and EGR2 (D) labeling identifies full program of oculomotor neuron cellular and molecular fates, BCCs at the native oculomotor nerve exit points (green arrows) and including the generation of a spatially organized nuclear complex. ectopic motor exit points (red arrows). (B,C) E5 Phox2a-electroporated Nuclei are a central feature of vertebrate brain architecture, but little heads probed for CDH7 expression and studied in cross-section. is known about the mechanisms of their production. These Sections cut transverse (B) and parallel (C) to the third cranial nerve mechanisms will undoubtedly be multiple and vary across brain demonstrate CDH7 expression at native (green arrows) and ectopic (red regions and cell types, but our findings with PHOX2A provide arrows) motor exit points. Ventricle in C is indicated by an asterisk. dien, diencephalon; fb, forebrain; hb, hindbrain; v.mb, ventral midbrain. evidence for one type of mechanism that is arguably the simplest. Scale bars: 0.1 mm. 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 positional signaling system that supplies newborn neurons with visceral and somatic motoneurons. The visceral and somatic positional information along the ventricular-pial axis. Such a system motoneurons autonomously migrate to appropriate positions along would be generally useful for arranging brain nuclei in three the ventricular-pial axis, producing a spatially organized nuclear dimensions and, unlike a cell-counting mechanism, could complex. accommodate brain growth. Our findings with native OMC development, particularly the (limited) intermixing of cell types seen Spatial allocation of OMC cell types at st 25, indicate that secondary correction mechanisms, such as cell In BrdU birthdating studies, we found an inside-out pattern of sorting or deletion, contribute to OMC morphogenesis as well. neurogenesis in the chick OMC. Inside-out patterns of brain development have been most extensively studied in the formation Oculomotor neuron diversification mechanisms of the layers of the mammalian cerebral cortex (Rakic, 1974). In an A striking finding in the current work is that Phox2a forced influential series of transplantation studies, McConnell and expression is able to drive the production of both visceral and colleagues have demonstrated that the neocortical inside-out somatic motoneurons broadly in ventral midbrain. The molecular patterning is not a passive process, in which cells simply migrate mechanisms underlying the diversification of these motoneuron past earlier-born cells before settling (Frantz and McConnell, 1996; subtypes are unknown (Song and Pfaff, 2009). One possibility is that McConnell, 1991). Rather, cortical progenitor cells acquire specific the ectopic cells take advantage of endogenous signaling systems, layer fate identity before they leave the ventricular zone of neuronal such as the NOTCH pathway, that mediate binary cell fate decisions progenitors. These transplantation experiments were conducted with (Mizuguchi et al., 2006; Peng et al., 2007). A second possible cortex donor tissue transplanted to cortex host tissue. Consequently, mechanism for somatic and visceral motoneuron diversification is the execution of the laminar fate program was tested within native the action of cell-autonomous temporal specification factors cerebral cortex, where the cues used by transplanted cells could be (Maurange et al., 2008; Zhu et al., 2006). A requirement for both layer-intrinsic signals or molecules (McConnell, 1991). In our these mechanisms is that they be resident throughout the territory Phox2a-misexpression experiments, we have been able to drive competent for OMC induction or that they be triggered by Phox2a ectopic production of isolated early-born (visceral motor) and late- misexpression. A third possibility is that PHOX2A levels themselves born (somatic motor) neurons in lateral midbrain, that is, in directly specify visceral and somatic motoneuron fates. In vivo neuroepithelium without a native OMC template. Thus, the ectopic plasmid electroporation does not tightly control the gene dose singletons must employ intrinsic mechanisms, such as counting cells delivered, and it is likely that different transgenic cells will express or measuring distances, as they migrate to their appropriate depths, different levels of exogenous . In studies of hematopoietic or must draw on general cues that are deployed throughout ventral lineages, varying transcription factor levels have been found to

midbrain. A particularly attractive cueing mechanism would be a regulate cell fate specification differentially (DeKoter and Singh, DEVELOPMENT 1212 RESEARCH ARTICLE Development 137 (7)

2000). A similar scenario for PHOX2A in OMC cell fate Agarwala, S., Sanders, T. A. and Ragsdale, C. W. (2001). Sonic Hedgehog determination would be that low (or high) levels of PHOX2A in the control of size and shape in midbrain pattern formation. Science 291, 2147- 2150. earliest progenitors drive visceral motoneuron production, and that Bayly, R. D., Ngo, M., Aglyamova, G. V. and Agarwala, S. (2007). Regulation progressive accumulation (or depletion) of PHOX2A protein leads of ventral midbrain patterning by Hedgehog signaling. Development 134, 2115- to somatic motoneuron production. Testing this PHOX2A dose 2124. Bosley, T. M., Oystreck, D. T., Robertson, R. L., al Awad, A., Abu-Amero, K. hypothesis is likely to entail the development of stem cell culture and Engle, E. C. (2006). Neurological features of congenital fibrosis of the methods that permit tight regulation of PHOX2A protein delivery. 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. PHOX2A is a primary regulator of OMC and Cohen, J. (2007). Boundary cap cells constrain spinal motor neuron somal migration at motor exit points by a semaphorin-plexin mechanism. Neural Dev. nucleogenesis 2, 21. The extensive work on the specification of spinal motoneurons and Cheng, L., Chen, C. L., Luo, P., Tan, M., Qiu, M., Johnson, R. and Ma, Q. hindbrain serotonin cells has emphasized the combinatorial nature (2003). Lmx1b, Pet-1, and Nkx2.2 coordinately specify serotonergic neurotransmitter phenotype. J. Neurosci. 23, 9961-9967. of transcription factor regulation of neuronal identity (Cheng et al., Chilton, J. K. and Guthrie, S. (2004). Development of oculomotor axon 2003; Jessell, 2000; Novitch et al., 2001; Pattyn et al., 2004). The projections in the chick embryo. J. Comp. Neurol. 472, 308-317. properties of PHOX2A in midbrain development, however, fit better Coppola, E., Pattyn, A., Guthrie, S. C., Goridis, C. and Studer, M. (2005). Reciprocal gene replacements reveal unique functions for Phox2 genes during with non-combinatorial models of cell type specification, such as neural differentiation. EMBO J. 24, 4392-4403. those emerging from hematopoietic system studies, in which the DeKoter, R. P. and Singh, H. (2000). Regulation of B lymphocyte and networks of cell type control can be analyzed, at least in some cases, macrophage development by graded expression of PU.1. Science 288, 1439- 1441. as hierarchies of primary and secondary regulators (Orkin, 2000; Diaz, C., Puelles, L., Marin, F. and Glover, J. C. (1998). The relationship between Singh, 2007). In this analysis, a regulator of a cell fate can be called rhombomeres and vestibular neuron populations as assessed in quail-chicken primary if it (1) is required for that cell fate, (2) induces secondary chimeras. Dev. Biol. 202, 14-28. Dubreuil, V., Hirsch, M., Pattyn, A., Brunet, J. and Goridis, C. (2000). The regulators, such as other transcription factors, that are also required Phox2b transcription factor coordinately regulates neuronal cell cycle exit and for that cell fate, (3) is activated at the time and site of the cell fate identity. Development 127, 5191-5201. decision, and (4) is by itself sufficient to produce the cell fate. Our 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 data, together with the extensive published work of others, establish neurogenesis. Development 129, 5241-5253. PHOX2A is a primary regulator of oculomotor cell fates. First, Evinger, C. (1988). Extraocular motor nuclei: location, morphology and afferents. genetic studies have demonstrated that PHOX2A is required for the In Neuroanatomy of the Oculomotor System (ed. J. A. Buttner-Ennever), pp. 81- production of midbrain motoneurons (Guo et al., 1999; Nakano et 117. Amsterdam: Elsevier. Frantz, G. D. and McConnell, S. K. (1996). Restriction of late cerebral cortical al., 2001; Pattyn et al., 1997). Second, the present study and those of progenitors to an upper-layer fate. Neuron 17, 55-61. others have shown that Phox2a/b induce the expression of secondary Gamlin, P. D. and Reiner, A. (1991). The Edinger-Westphal nucleus: sources of regulators of OMC cell fate, such as the transcription factor ISL1, input influencing accommodation, pupilloconstriction, and choroidal blood flow. J. Comp. Neurol. 306, 425-438. which is required for all motoneuron development (Dubreuil et al., Gamlin, P. D., Reiner, A. and Karten, H. J. (1982). Substance P-containing 2000; Pfaff et al., 1996). Third, BrdU birthdating and gene neurons of the avian suprachiasmatic nucleus project directly to the nucleus of expression schedules in the chick (this study) and mouse (Pattyn et Edinger-Westphal. Proc. Natl. Acad. Sci. USA 79, 3891-3895. Gamlin, P. D., Reiner, A., Erichsen, J. T., Karten, H. 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