GATA Proteins Identify a Novel Ventral Interneuron Subclass in the Developing Chick Spinal Cord

Developmental Biology 249, 30–43 (2002) doi:10.1006/dbio.2002.0754 GATA Proteins Identify a Novel Ventral Interneuron Subclass in the Developing Chick Spinal Cord

Asanka Karunaratne,*,1,3 Murray Hargrave,† Alisa Poh,†,2 and Toshiya Yamada† *Department of Biochemistry, and †Institute for Molecular Bioscience and ARC Special Research Centre for Functional and Applied Genomics, The University of Queensland, Brisbane, Queensland 4072, Australia

Members of the GATA transcription factor gene family have been implicated in a variety of developmental processes, including that of the vertebrate central nervous system. However, the role of GATA proteins in spinal cord development remains unresolved. In this study, we investigated the expression and function of two GATA proteins, GATA2 and GATA3, in the developing chick spinal cord. We show that both proteins are expressed by a distinct subpopulation of ventral interneurons that share the same dorsoventral position as CHX10-positive V2 interneurons. However, no coexpression is observed between the two GATA proteins and CHX10. By in vivo notochord grafting and cyclopamine treatment, we demonstrate that the spatially restricted pattern of GATA3 expression is regulated, at least in part, by the signaling molecule Sonic hedgehog. In addition, we further show that Sonic hedgehog induces GATA3 expression in a dose-dependent manner. Using in ovo electroporations, we also demonstrate that GATA2 is upstream of GATA3 in the same epigenetic cascade and that GATA3 is capable of inducing GATA2 expression in vivo. Furthermore, the ectopically expressed GATA proteins can repress differentiation of other ventral cell fates, but not the development of progenitor populations identiﬁed by PAX protein expression. Taken together, our ﬁndings strongly suggest an important role for GATA2 and GATA3 proteins in the establishment of a distinct ventral interneuron subpopulation in the developing chick spinal cord. © 2002 Elsevier Science (USA) Key Words: GATA2; GATA3; spinal cord; interneurons; patterning; Sonic hedgehog; CNS.

INTRODUCTION generated by midline structures such as the notochord and the floor plate (Briscoe et al., 2001; Ericson et al., 1997; Formation of the vertebrate central nervous system Yamada et al., 1991, 1993). This graded inductive signal (CNS) involves coordinate functioning of many different initially establishes progenitor cell domains at distinct molecular mechanisms. Our current understanding of this dorsoventral positions along the neural tube (Briscoe et al., process, in particular of cell-type specification, is derived 2000; Mansouri and Gruss, 1998; Muhr et al., 2001). These from analysis of transcription factor gene expression in the progenitor domains, which are characterized by the expres- developing embryonic spinal cord. Recent investigations sion of specific homeodomain proteins (Briscoe et al., 2000; have revealed that the primary determinant of cell fate in Ericson et al., 1997; Mansouri and Gruss, 1998; Sander et the ventral spinal cord is the graded Sonic hedgehog signal, al., 2000), give rise to distinct cell populations, which express a multitude of other transcription factors (Ericson et al., 1997; Moran-Rivard et al., 2001; Pierani et al., 2001; 1 ϩ To whom correspondence should be addressed. Fax: 1 858 457 Tsuchida et al., 1994; Zhou et al., 2000). While this model 4765. E-mail: [email protected]. 2 Present address: Department of Cell and Developmental Biol- adequately describes cell-type specification for many neu- ogy, Cornell University Weill Medical College, New York, NY. rons within the ventral spinal cord, recent studies have 3 Present address: The Salk Institute, 10010 N. Toney Pines Rd., described some alternative mechanisms involved in ventral La Jolla, CA 92037. patterning. For example, retinoid signals from postmitotic,

FIG. 1. Expression of GATA3 and other ventral cell type-speciﬁc markers in stage 20 and 24 spinal cord sections. Expression of GATA3 (A), GATA2 (B), and CHX10 (C) in the lateral border of a stage 20 spinal cord. At the same stage, domains of EN1 (D) and ISL1 (E) expression appeared dorsal or ventral to the GATA3-expressing cells, respectively. (F) Note, by stage 24, the expression domain of GATA3 expanded medially with increasing cell numbers. (G) This pattern of expression was also maintained by the GATA2 and (H) CHX10 proteins. (I) EN1-positive V1 interneurons positioned dorsal to the GATA3 expression domain. (J) The expanded pool of motor neurons immediately ventral to the GATA3-expressing cells. d, dorsal; v, ventral; m, medial; l, lateral. Scale, 100 ␮m.

early born motor neurons have been found to influence the (Ericson et al., 1997; Zhou et al., 2000). The fourth and most development of later born motor neurons (Sockanathan and ventrally located neural population expresses Nkx2.2 and is Jessell, 1998). In a different study, some ventral interneuron termed V3 interneurons (Briscoe et al., 2000; Ericson et al., fates were specified by retinoid signaling in the absence of 1997). All four classes of interneurons are regulated by the Sonic hedgehog (Pierani et al., 1999). These observations graded Sonic hedgehog signal and thus occupy distinct clearly demonstrate the existence of other patterning pro- dorsoventral positions in the developing spinal cord. cesses that augment Sonic hedgehog signaling. While transcription factor gene expression allows the Previous studies have extensively investigated the Sonic classification of distinct ventral interneuron subtypes, the hedgehog-mediated specification of motor neuron subtypes current understanding of molecular cascades that establish characterized by the combinatorial expression of LIM- these cell fates is limited. For instance, the protein EN1, homeodomain genes (Tsuchida et al., 1994). More recent which is expressed by the V1 interneurons, is not involved investigations have further identified transcription factors in decisions regarding the fate of this neural population involved in the intermediary steps of motor neuron speci- (Matise and Joyner, 1997). Instead, it is required for the fication (Arber et al., 1999; Mizuguchi et al., 2001; Tanabe postmitotic differentiation of the V1 interneurons. In addi- et al., 1998; Thaler et al., 1999). However, information tion, a physiological analysis has also revealed that EN1- regarding the molecular specification of ventral interneu- expressing cells constitute a heterogeneous population of rons has been less forthcoming. ventral interneurons (Wenner et al., 2000). A number of previous studies defined four distinct classes A recent study, however, demonstrated that Evx1, ex- of postmitotic interneurons based on the combinatorial pressed by the V0 interneurons, is required for the correct patterns of transcription factor gene expression. The first differentiation of the V0 interneuron population (Moran- class, termed V0, is characterized by the expression of Evx1, Rivard et al., 2001). In this study, the lack of Evx1 caused and it is the most dorsally positioned ventral interneuron many V0 neurons to acquire V1 neural characteristics, and population (Moran-Rivard et al., 2001). The second class, overexpression of EVX repressed EN1 expression and V1 positioned ventral to the V0 interneurons, is called V1 and interneuron development. Similarly, it has also been sug- is identified by Engrailed1 (En1) expression (Ericson et al., gested that GATA2, a member of the GATA Zn-finger 1997; Matise and Joyner, 1997). The third class, which is transcription factor gene family, is required for the genera- characterized by the expression of Chx10, GATA2, and tion of V2 interneurons (Zhou et al., 2000). This study Lim3 is termed V2, and it is located ventral to the V1 further suggested that the GATA2 protein might also be an interneurons and immediately dorsal to the motor neurons upstream regulator of CHX10 expression.

GATA3 is another member of the GATA transcription factor gene family which has been implicated in various developmental processes. Previous investigations have demonstrated important, nonredundant roles for GATA3 in the development of the haematopoietic and the central nervous systems (Hendriks et al., 1999; Nawijn et al., 2001; Pandolfi et al., 1995). Even within the CNS, GATA3 has been shown to play critical roles in the development of different neural populations. For example, in the embryonic midbrain, expression of GATA3 correlates with the development of the optic tectum (Kornhauser et al., 1994), and in the developing hindbrain, loss of GATA3 affects differentiation of the vestibuloacoustic efferent neurons and migra- tion of the facial branchiomotor neurons (Pata et al., 1999). Furthermore, in adult caudal Ralphe nuclei, GATA3 has been found to regulate the development of serotonergic neurons (van Doorninck et al., 1999). Taken together, these observations strongly suggest vital roles for GATA3 in the specification of different neural subtypes within the developing CNS. Given the importance of GATA3 in CNS development, we sought to investigate its role in cell-type specification within the developing chick spinal cord. In this study, we show that a distinct population of postmitotic ventral interneurons express the GATA3 protein and that these cells also express GATA2. Furthermore, this interneuron population does not express other previously characterized molecular markers, including CHX10. We also demonstrate that the domain of GATA3 expression is determined by the Sonic hedgehog concentration gradient and that GATA3 is located downstream of GATA2, in the same epigenetic cascade. We further show that GATA3 could also induce GATA2 in vivo, which suggests the existence of a positive- feedback mechanism. Finally, we show that ectopic induction of GATA2 or -3 can disrupt gene expression in other developing ventral interneurons. Taken together, these findings indicate roles for GATA2 and GATA3 in the postmitotic determination of a distinct ventral interneuron subtype in the developing chick spinal cord.

two proteins was detected. (C, D) GATA3 (red) and CHX10 (green), respectively, at stages 20 and 24 of development. While the two cell populations appeared tightly intermingled, there was no coexpression of the two proteins. (E, F) Thoracic sections stained for GATA2 (red) and CHX10 (green) at stages 20 and 24, respectively. As with GATA3, the two populations of cells appeared to be tightly intermingled. (G) Expression of GATA3 (red) and NKX6.1 (green) in FIG. 2. GATA3 identiﬁes a novel subclass of ventral interneu- a stage 25 chick spinal cord. While no GATA3 is detected in rons. (A) Comparison of GATA3 (red) and ISL1 (green) expression in NKX6.1-expressing cells, GATA3-positive interneurons appear to a stage 24 thoracic spinal cord. Cells expressing GATA3 were arise from the dorsal most boundary of the NKX6.1-expressing positioned immediately dorsal to the ISL1-positive motor neurons progenitor population. (H) A stage 24 spinal cord stained for and no coexpression of the two proteins was observed in any of the GATA3 (red) and tubulin (green). All cells stained for GATA3 also stages examined. (B) GATA3 (red) and EN1 (green) expression in a stained positive for tubulin (arrows). d, dorsal; v, ventral; m, stage 25 thoracic spinal cord. Once again, no coexpression of the medial; l, lateral. Scale, (A–G) 30 ␮m; and (H) 10 ␮m.

FIG. 3. Sequential expression of GATA2 and GATA3 in the developing spinal cord. (A) Coexpression of GATA2 (green) and GATA3 (red) at stage 22. Note the abundance of GATA2-positive nuclei towards the medial spinal cord (arrows). (B) Increased coexpression of the two GATA proteins by stages 26–27. While many GATA2-positive cells also express GATA3 at this stage (yellow nuclei), a few medially located younger cells still only express the GATA2 protein (arrows). (C) Level of GATA2 and GATA3 coexpression during early spinal cord development. Despite an almost 100% coexpression at stage 27, a very small number of younger, more medially located GATA2-positive cells do not express GATA3, indicating a sequential pattern of GATA gene expression. (D) A schematic representation of the “proposed” cell-type distribution in the ventral spinal cord by stage 24, as identified by transcription factor expression. According to this proposed scheme, GATA2, GATA3, and CHX10 share the same dorsoventral position within the neural tube, but are expressed by two distinct populations of interneurons. d, dorsal; v, ventral; m, medial; l, lateral. Scale, 35 ␮m. FIG. 4. Induction of GATA3 expression by notochord-derived signals. In vivo notochord grafts were performed to assess the role of notochord-derived signals in the specification of GATA3-positive ventral interneurons. (A) The induction of an ectopic floor plate (fЈ) adjacent to a dorsally placed notochord (nЈ) detected by Sonic hedgehog expression. (B) Ectopic induction of ISLET1-positive motor neurons (arrows) by the grafted notochord. (C) Induction of ectopic GATA3 (red)- and CHX10 (green)-positive cells in the dorsal neural tube by the grafted notochord (nЈ). In addition to being induced at a consistent distance from the ectopic floor plate, similar to that of the ventral spinal cord, both GATA3- and CHX10-positive cells also maintained their close intermingled positions even in the ectopic expression domain (arrows) (D). n, notochord; f, floor plate. Scale, (A–C) 100 ␮m; and (D) 40 ␮m.

FIG. 5. Repression of GATA3 induction by inhibition of the Sonic hedgehog pathway. The role of Sonic hedgehog in GATA3-positive interneurons speciﬁcation was investigated by blocking the Sonic hedgehog signal cascade using cyclopamine. (A–E) Serial sections through the lumbar spinal cord of a stage 23 chick embryo treated with cyclopamine. While the expression of GATA3 (A) and CHX10 (B) proteins is completely repressed, a small number of EN1 (C)- and ISLET1 (D)-expressing cells can be detected at the medial region of the ventral spinal cord (arrows in C and D). No ﬂoor plate was detected at all axial levels as shown by the absence of Sonic hedgehog expression (E, J, O). (F–J) Serial sections through the thoracic spinal cord of the same embryo. While no GATA3 expression was detected at this axial level (F), a small number of CHX10-positive cells (G) were detected in the ventral most region of the spinal cord. Compared with the lumbar level, an increased number of EN1 (H) and ISL1 (I) cells were also detected at the ventral spinal cord. (K–O) Serial sections through cervical spinal cord of the aforementioned embryo showing expression of GATA3 (K), CHX10 (L), EN1 (M), and ISL1 (N) in the ventral region. While a larger number of nuclei are detected, the domain of expression appears considerably more ventral than that of a normal spinal cord. Scale, 200 ␮m.

MATERIALS AND METHODS type-specific antibodies: mouse anti-GATA3 (Santa Cruz, sc-268), mouse anti-GATA2 (Santa Cruz, sc-269), mouse anti-Engrailed-1 Collection of embryos. Fertilized white leghorn chicken eggs [4G11; Developmental Study Hybridoma Bank (DSHB), University (Ingham Enterprise, Brisbane) were incubated in a humidified, of Iowa], Enhb-1 (a gift of Dr. Alexandra Joyner, New York forced-draft incubator until they reached appropriate stages of University, NY), rabbit anti-CHX10 (polyclonal antibody, a gift of development (Hamburger and Hamilton, 1951). Dr. Thomas Jessell, Columbia University, NY), mouse anti-ISLET- Immunohistochemistry. Cryosectioning and antibody labeling 1/2 (4D5 and 2D6; DSHB, University of Iowa), mouse anti-Sonic of frozen tissue sections was performed as previously described hedgehog (5E1; DSHB, University of Iowa), rabbit anti-NKX6.1 (Yamada et al., 1991). Briefly, the embryos were collected in (polyclonal antibody; a gift of Dr. Martyn Goulding, The Salk ice-cold L-15 culture medium (GIBCO, Life Technology), fixed in Institute for Biological Studies, CA), and mouse anti-Tubulin anti- 4% paraformaldehyde in 10 mM phosphate-buffered saline (PBS), body TUJ1. The primary antibodies were detected by using species- and saturated with 30% sucrose in PBS overnight. Serial sections of specific secondary antibodies conjugated with Cy3 (Jackson Labo- 14-␮m thickness were then cut and labeled with following cell ratories) or BODIPY (Molecular Probes). The antibody labeling was

examined on an Olympus AX70 compound fluorescent microscope mg/ml in 10 mM PBS with 10 mg/ml of HBC. The stock solution and a BioRad MRC 1024 confocal microscope. (10 ␮L) was injected into stage 10 chick embryos with repeated Sequential immunostaining of frozen tissue sections. Possible daily injections up to stages 24–25. The cyclopamine-treated coexpression of GATA3 and GATA2, GATA2/3 and LIM3, or embryos were then fixed and processed as described. Changes to GATA3 and Tubulin proteins was investigated by the sequential the cell-type specification were determined by labeling with anti- labeling of these proteins using two mouse monoclonal antibodies. bodies as described above. First, the sections were incubated with the mouse anti-GATA3 or anti-GATA2 antibody followed by either the BODIPY or Cy3- conjugated secondary antibodies (as described above). After label- RESULTS ing was completed, the sections were thoroughly rinsed with PBS and blocked with 10% heat-inactivated goat serum. The sections Expression of GATA3 in the developing chick spinal were then incubated with either the mouse anti-GATA2, anti- cord. To determine the identity of GATA3-positive cells LIM3, or anti-Tubulin monoclonal antibody, followed by either within the developing chick spinal cord, we compared the Cy3- or BODIPY-conjugated secondary labels. Since we did not spatial and temporal expression of the GATA3 protein with observe any detectable cross-reactivity between the first and sec- other known ventral cell type-specific markers. ond antibody labeling when the primary antibody of the second Expression of the GATA3 protein was first detected round of labeling was omitted, we confirmed that this sequential between stages 20 and 21 in a small number of cells located labeling method could distinguish between two antigens expressed at the lateral border of the ventral spinal cord (Fig. 1A). The in the same cell. GATA3-positive cells were positioned at the dorsal border In vivo notochord grafting. In vivo grafting of ectopic notochord was performed as previously described (Yamada et al., 1991). of the motor column (Fig. 1E), similar to that of GATA2- In brief, a small fragment of notochord was isolated from a stage 10 and CHX10-expressing V2 interneurons (Figs. 1B and 1C) donor embryo by using Dispase (1 mg/ml; Roche Diagnostics, cat. and ventral to the EN1-positive V1 interneurons (Fig. 1D). # 241750) in L-15 culture medium. The notochord fragment was By stage 24, the number of GATA3-positive cells markedly then grafted either lateral or dorsal to the midline position of the increased and spread mediolaterally as the spinal cord neural tube at the caudal end of the embryo. Following the grafting, increased in size (Fig. 1F). The relative position of the the operated embryo was allowed to grow for a further 72–80hat GATA3-positive cells remained dorsomedial to the motor 37°C. The embryo was then fixed, sectioned, and immunostained column (Fig. 1J) and was still very similar to those of as described above. GATA2- and CHX10-expressing V2 interneurons (Figs. 1G In vitro neural plate explant assay. Neural plate explant and 1H). At this stage, cells expressing GATA3 were clearly culture assays were performed as described by Yamada et al. (1993). located more ventral to those of the EN1-positive V1 The intermediate neural plates were dissected from the caudal neural tubes of stage 10 chick embryos and embedded in a drop of interneurons (Fig. 1I). While this study mainly focused on collagen gel. The explants were then allowed to grow in the the lumbar region of the spinal cord, it is possible that presence or absence of defined concentrations of purified recombi- variations in cell-type distribution exist at different rostral– nant, N-terminal Sonic hedgehog protein (corresponding to amino caudal levels along the neuraxis. acid residues 1–198, batch 5066; a gift of Drs. Thomas Jessell and GATA3 is expressed by a novel class of ventral inter- Susan Morton, Columbia University, NY). After 80 h in culture, neurons in the developing spinal cord. Analysis of spinal the explants were fixed in 4% PFA in PBS at 4°C for 1–2 h, rinsed cord serial sections suggested that GATA3-positive cells with ice-cold PBS, and immunostained with the cell type-specific might also express CHX10 and GATA2, but not other marker antibodies as described above. The number of labeled ventral cell type-specific markers. We therefore examined nuclei was counted from 6 to 12 explants in 3–4 different experi- this possibility at the single cell level by colabeling GATA3 ments for each data point. with EN1, CHX10, GATA2, and ISL1/2, respectively. Con- In ovo electroporation of chick neural tube. In ovo electropo- focal microscopy was used to resolve the observed expres- rations of the developing chick neural tubes were performed according to a published protocol (Nakamura and Funahashi, 2001). sion patterns. Full-length chick GATA3 and -2 cDNA were cloned into the We found that GATA3 does not coexpress with EN1, eukaryotic expression vector pcDNA3 (Invitrogen) containing an CHX10, or ISL1/2 in any of the stages examined (Figs. internal ribosome entry site (IRES) followed by a green fluorescent 2A–2F). In contrast, significant coexpression between protein (GFP) gene, a 1676-bp fragment from pSKeG (nucleotide GATA3 and GATA2 was detected in the same population positions 669 to 2345; a gift from Dr. Brian Key, University of of ventral interneurons (Figs. 3A and 3B). During early Queensland). The purified plasmid DNAs were resuspended in development, cells which expressed both GATA3 and sterile PBS (10 mM) at concentrations of 2–5 ␮g/␮l. Following GATA2 were located toward the lateral half of the spinal electroporation, the embryos were allowed to grow at 37.5°C for a cord, whereas those which only expressed GATA2 were further 48–72 h. Changes in the cell pattern in the spinal cord were located in the medial half (Fig. 3A). As development pro- analyzed by labeling with the cell type-specific markers as de- gressed, the number of double-labeled cells increased with scribed above. In ovo cyclopamine treatment of chick embryos. In ovo cyclo- only a small number of “young” cells located at the medial pamine treatment of chick embryos was performed according to most of region of the spinal cord expressing just the GATA2 the published methods with a modification (Incardona et al., 1998). protein (Figs. 3B–3D). This suggests a sequential pattern of The cyclopamine was kindly provided by Dr. William Garfield GATA2 and GATA3 expression where only the more ma- (USDA). Cyclopamine was dissolved at the concentration of 1 ture, laterally located cells express both proteins. These

expressing cell types (Ericson et al., 1997), expression of GATA2 and GATA3 was also compared with that of LIM3. No expression of LIM3 was detected in any of the cells expressing GATA2 or GATA3 at stage 25 (data not shown). At all stages examined, GATA3-positive cells were not observed within the mitotically active ventricular zone (data not shown). Consistent with this observation, no coexpression was detected between GATA3 and NKX6.1 (Fig. 2G), which together with IRX3 defines the p2 progenitor domain (Briscoe et al., 2000). The cells expressing GATA3 were also restricted to the dorsal-most border of the NKX6.1 expression domain, suggesting the p2 progenitor population as the likely source of GATA3-expressing cell fates. Furthermore, all cells that expressed GATA3 also expressed ␤-tubulin, a general marker for differentiated neurons (Fig. 2H). Taken together, these observations suggest that GATA3-positive cells of the ventral spinal cord represent a distinct population of postmitotic ventral interneurons. Induction of ectopic GATA3-positive interneurons by in vivo notochord grafts. The restricted pattern of GATA3 expression implicates inductive signals from ventral midline structures in the generation of GATA3-positive interneurons. Further, as GATA3-positive and CHX10-positive cells are in close proximity in the developing spinal cord, we reasoned that precursors of both populations would likely receive similar, if not identical, inductive signals. This raises the possibility that the development of these two fates requires an additional mechanism. We therefore sought to determine whether induction GATA3 and CHX10 in an ectopic region of the spinal cord would result in exclusive or coexpression of the two factors. To test these hypotheses, we performed an in vivo notochord graft, where a small fragment of notochord was placed at an ectopic position along the dorsoventral axis of neural tube (Yamada et al., 1993). Subsequent changes to cell-type distribution was monitored by immunostaining using cell FIG. 6. In vitro induction of GATA3 by a SHH-N concentration type-specific antibodies. gradient. (A, i–iv) Gradual induction of GATA3 (red) and CHX10 The notochord-graft induced floor plate, motor neuron, (green) in neural plate explants by increasing concentrations of and GATA3-positive cells at ectopic locations within the SHH-N. (B) Quantitative analysis of ventral cell-type induction in neural tube (Figs. 4A–4C). Moreover, the relative positions response to SHH-N gradients in neural plate explants. At lower SHH-N concentrations, all the ventral markers examined show of these cells were similar to that of endogenous cell fates in little, if any, expression. However, at subsequently higher concen- that ectopic floor plate cells were induced immediately trations, the dorsal-most ventral marker, EN1, shows a significant adjacent to the grafted notochord, with motor neurons induction. Other ventral markers examined show only a limited being generated next to the induced floor plate (Figs. 4A and level of induction at this concentration. With a further increase in 4B). The GATA3- and CHX10-positive interneurons also SHH-N concentration, a downregulation of EN1 expression is appeared further away from the ectopic floor plate in a observed. This coincides with the up regulation of GATA3, manner reminiscent of the endogenous cell populations CHX10, and ISL1 expression. These observations are consistent (Figs. 4C and 4D). Importantly, though induced at ectopic with the proposed model of ventral cell-type specification by the positions, the GATA3- and CHX10-positive cells main- graded Sonic hedgehog signal. tained the close intermixed distribution observed in the ventral neural tube (Fig. 4D). These observations suggest that an inductive signal from the notochord initiated the observations were in disagreement with a previous finding generation of GATA3-positive interneurons, and the sepa- that suggested coexpression of CHX10 and GATA2 in the ration of this fate from that of CHX10-expressing cells was same population of ventral interneurons (Zhou et al., 2000). intrinsic to this induction and not dependent on a local, Since the expression of CHX10 identifies a subset of LIM3- ventral cue.

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FIG. 7. Effects of GATA protein mis-expression on ventral patterning of the spinal cord. (A–C) Ectopic induction of the GATA3 protein by mis-expressed GATA2. (A) Expression of GFP identifying the ectopic GATA2-expressing cells. (B) Cells expressing the ectopic GATA3 protein along the entire dorsoventral axis. The arrow marks endogenous GATA3 expression in the nonelectroporated side. (D–F) Repression of ISL1-positive motor neurons by the ectopically expressed GATA2 protein. Regions with strong GFP staining show a significant reduction in ISL1 expression (F). (G) Changes to ventral cell-type specification after GATA2 electroporation plotted as a factor of normal expression. All markers except GATA3 were repressed following GATA2 electroporation, with EN1- and CHX10-positive interneurons being the most affected. (H–J) Induction of ectopic GATA2 protein by mis-expressed GATA3. (H) Expression of the GATA3 protein as identified by GFP. (I, J) Ectopically induced GATA2 protein in cells mis-expressing GATA3. (K–M) Repression of ISL1-positive motor neurons by ectopic GATA3 expression. As with GATA2, regions of the highest GFP concentrations show the most significant reduction in motor neuron induction (M). (N) Changes to ventral cell-type specification following GATA3 electroporation. The ectopically expressed GATA3 protein repressed the induction of other known ventral cell types, particularly that of EN1- and CHX10-positive interneurons (N). The level of repression shown by GATA3 mis-expression was strikingly similar to that of the

ectopically expressed GATA2 (G). Scale, 100 ␮m. 37 38 Karunaratne et al.

In vivo induction of GATA3-positive ventral interneu- CHX10-positive cells showed substantial overlap in the rons by Sonic hedgehog gradient. Previous studies have SHH-N concentrations required for their induction (Fig. demonstrated that distinct classes of ventral neurons are 6B). These observations strongly suggest that, in the devel- induced by a gradient of Sonic hedgehog along the dorso- oping spinal cord, Sonic hedgehog regulates induction of ventral axis of the neural tube (Ericson et al., 1997). There- GATA3-positive interneurons in a dose-dependent manner. fore, to test whether Sonic hedgehog is both necessary and Regulation of GATA3 expression and cross-talk be- sufficient to induce GATA3 expression, we first blocked tween GATA3 and GATA2 in the developing spinal cord. Sonic hedgehog signaling using cyclopamine, a specific The sequential pattern of GATA2 and GATA3 expression Sonic hedgehog signal cascade inhibitor (Incardona et al., in developing spinal interneurons suggests that expression 1998), and then used recombinant Sonic hedgehog protein of GATA3 may be controlled by GATA2. Evidence from to induce GATA3 expression in neural plate explants. two previous studies supports this hypothesis. First, When cyclopamine was applied to stage 10 chick em- GATA2 knockout mice lose GATA3 mRNA expression in bryos in vivo, the differentiation of floor plate cells, motor the developing spinal cord and brain (Nardelli et al., 1999), neurons, and CHX10- and GATA3-positive interneurons and second, the 5Ј-upstream region of the chick GATA3 was completely inhibited despite strong expression of Sonic genomic sequence contains putative GATA protein-binding hedgehog in the notochord (Figs. 5A–5E). We also observed sites (Ishihara et al., 1995). As the GATA2 genomic se- a small number of EN1-positive cells at the ventral midline quence in certain species also contains putative GATA- position (Fig. 5C). This phenotype is very similar to that of binding sites (Fleenor et al., 1996), it is possible that the spinal cord of Sonic hedgehog knockout mice (Liting- GATA2 is also regulated by another GATA factor during tung and Chiang, 2000), and may also represent EN1- interneuron development, possibly GATA3. We therefore positive interneurons generated via the Sonic hedgehog- tested these hypotheses by ectopically expressing the independent retinoid-signaling pathway (Pierani et al., GATA2 and GATA3 proteins using in ovo electroporations 1999). In a slightly more rostral segment of the same (Nakamura and Funahashi, 2001). When GATA2 was ec- embryo, we also detected some ISL1/2-expressing motor topically expressed in the developing neural tube, signifi- neurons and CHX10-positive interneurons, but no GATA3- cant numbers of ectopic GATA3-positive cells were ob- expressing cells were observed (Figs. 5F–5J). At the ventral served along the electroporated neural tube (Figs. 7A–7C). midline position of even further rostral segments, a signifi- Conversely, ectopic expression of GATA3 also induced cant number of motor neurons and EN1-, CHX10-, and ectopic GATA2 protein expression (Figs. 7H–7J). However, GATA3-expressing interneurons were detected (Figs. 5K– induction of GATA3 by the GATA2 protein appeared 5O). As neurons of the rostral spinal cord generally develop slightly more efficient than the induction of GATA2 by the earlier than those of caudal regions, presumably these cells GATA3 protein (Figs. 7G and 7N). These results, together received a fate-inducing signal of Sonic hedgehog before the with the expression analysis, strongly suggested that administration of cyclopamine. Therefore, these observa- GATA3 resides downstream of GATA2 in the epigenetic tions clearly suggest that Sonic hedgehog is indeed neces- cascade and that, in turn, GATA2 expression can be posi- sary for the induction of the GATA3-positive ventral inter- tively regulated by GATA3. neurons in the developing spinal cord. GATA proteins influence specification of ventral inter- By using a chick neural plate explant culture assay neurons in the developing spinal cord. To determine system (Yamada et al., 1993), we investigated whether whether GATA3 and GATA2 are involved in spinal inter- Sonic hedgehog protein alone was sufficient to induce neuron specification, we again ectopically expressed each GATA3-positive interneurons. Different concentrations of protein by performing in ovo electroporations. When the the recombinant Sonic hedgehog protein were applied to GATA3 protein was mis-expressed, it repressed the expres- intermediate neural plate explants from stage 10 chick sion of molecular markers indicative of other ventral neural embryos. After approximately 80 h in culture, the explants subtypes (Fig. 7N). Similarly, ectopic GATA2 expression were fixed and immunostained with GATA3-, EN1-, also effectively repressed the differentiation of other ventral CHX10-, and ISL1/2-specific antibodies. cell fates (Fig. 7G). The repression of EN1- and CHX10- No GATA3-positive cells were induced in the control positive interneurons by ectopic GATA3 and GATA2 ex- (0 nM) explant culture (Fig. 6Ai). However, we observed a pression was particularly severe, while repression of ISL1/ dose-dependent induction of GATA3 at subsequently 2-expressing motor neurons was less in comparison (Figs. higher concentrations of SHH-N (Fig. 6B). Expression of 7G and 7N). Taken together, these results suggest that GATA3 was also induced at a distinct dose range relative to GATA3 and GATA2 could function in the specification of the EN1-positive V1 neural subtype (Fig. 6B). For example, interneuron subtypes in the developing spinal cord. EN1-positive cells were strongly induced at a lower concen- GATA3 and GATA2 act downstream of progenitor cell tration of SHH-N (0.4 nM) than that required for the specification in the developing spinal cord. Both PAX6 induction of GATA3-positive interneurons (Fig. 6B). At and PAX7, which are expressed in the ventricular zone, higher concentrations of SHH-N (4.0 nM), expression of have been implicated in the establishment of progenitor EN1 was greatly reduced and strong expression of GATA3 domains within the developing spinal cord in response to was detected (Fig. 6B). In contrast, both GATA3- and midline signaling (Ericson et al., 1997; Mansouri and Gruss,

1998). As these proteins are the earliest markers of neural tions are likely to carry different functional properties, as development, we investigated the effect of GATA3 overex- specified by their transcription factor gene expression. pression on spinal cord PAX6 and PAX7 expression. The While expression of transcription factors can define dis- ectopically expressed GATA proteins did not significantly crete neural subtypes, very little is known of the actual alter expression of PAX6 or PAX7 in the developing spinal anatomical and physiological properties of these cells in the cord (Fig. 8). These observations suggest that ectopic expres- context of functional spinal circuits. A number of recent sion of GATA proteins have no effect on the progenitor studies have identified some anatomical features of the cells, and thus, are presumably involved in the early differ- EN1- and EVX1-expressing spinal interneurons (Moran- entiation of this interneuron population. Rivard et al., 2001; Saueressig et al., 1999; Wenner et al., 2000). For example, EN1-expressing neurons project their axons ipsilaterally and target somatic motor neurons of the DISCUSSION ventral spinal cord (Saueressig et al., 1999). Notably, a majority of these EN1-expressing cells have been also Spatially restricted expression of GATA3 and GATA2 identified as inhibitory interneurons (Wenner et al., 2000). defines a novel class of postmitotic ventral interneurons. Therefore, an obvious question to be addressed is the exact In this study, we examined the expression pattern of the morphology of GATA3- and GATA2-expressing interneu- Zn-finger protein GATA3 in the developing chick spinal rons. In this study, we did not investigate the axon projec- cord. Our analysis revealed that GATA3 was sequentially tions of either GATA3- or GATA2-positive cells of the expressed after GATA2 in a novel population of ventral developing spinal cord. However, a previous investigation interneurons. Despite being expressed at the same dorso- on brain stem development suggested a role for GATA3 in ventral position as CHX10, no coexpression of GATA2/3 serotonergic raphe neuron and olivocochlear neuron devel- was detected with any of the previously identified ventral opment (van Doorninck et al., 1999). Thus, it appears that, interneuron or motor neuron-specific markers. Indeed, in the developing spinal cord, GATA3 specifies a popula- while the CHX10-positive and GATA2/3-positive cell tion of neurons yet again distinct in functionality from populations were intermixed, they remained mutually ex- other GATA3-expressing cells of the CNS. clusive. This observation is in disagreement with two Sonic hedgehog regulates the spatially restricted pat- previous studies that suggested GATA2 (and therefore tern of GATA3 expression. Spinal cord expression of GATA3) was expressed in motor neuron precursor cells or GATA3 was restricted to a narrow domain along the the CHX10-expressing V2 interneurons (Pandolfi et al., dorsoventral axis of the neural tube. Therefore, we hypoth- 1995; Zhou et al., 2000). The difference between these esized that this expression was likely to be regulated by the observations could be attributed to a difference in the signaling molecule Sonic hedgehog. The induction of domain of GATA2/3 mRNA expression and GATA2/3 GATA3-positive cells by ectopic notochord grafts and the protein expression. It is possible that CHX10-positive cells inhibition of GATA3 expression by cyclopamine treatment express GATA2/3 mRNA but do not translate it, perhaps revealed that Sonic hedgehog is indeed both necessary and indicating expression control at the level of translation. An sufficient to induce GATA3 expression. Furthermore, we alternative explanation is a difference in resolution of the also demonstrated that SHH regulates the expression of histological techniques used for analysis. The previous GATA3 in a dose-dependent manner. This was in agree- studies used either in situ hybridization or cytoplasmic ment with previous findings that suggested the concentra- lacZ reporter expression, while we used GATA3-, GATA2-, tion of SHH along the D-V axis determines the fate of and CHX10-specific antibodies to detect proteins localized ventral cell types during early spinal cord development in cell nuclei. This enabled us to use confocal microscopy (Ericson et al., 1997). and unequivocally resolve differences in protein expression However, cell-type specification based on the SHH gradi- at the single cell level. We have also recently examined ent alone cannot explain generation of distinct neural GATA3 and CHX10 protein expression in the developing subtypes at similar, or overlapping, D-V positions. The mouse spinal cord and have found them to be expressed in expression domains of GATA3 and CHX10 exemplify this exclusive populations (Smith et al., 2002). Therefore, based point. Both genes share identical expression domains, yet on protein expression, we propose to designate the CHX10- two distinct populations of ventral interneurons express positive neurons of the ventral spinal cord as V2a inter- them. This suggests the involvement of additional mecha- neurons, and the GATA2/GATA3-positive neurons as V2b nisms in the specification of ventral neural subtypes. Cre- interneurons. dence to hypothesis comes from a previous study that A previous study divided neurons of the motor pool into implicates signaling from motor neurons in development of different subclasses based on the combinatorial expression the EN1-positive V1 interneurons (Pfaff et al., 1996). Fur- on LIM-homeodomain genes (Tsuchida et al., 1994). Impor- thermore, it has been proposed that temporal regulation tantly, though located within the same motor pool, each may be important in ventral cell-fate specification in re- subclass innervates specific peripheral targets (Tsuchida et sponse to SHH (Ericson et al., 1997). There is evidence that al., 1994). Similarly, while intermixed within the same progenitors of floor plate cells, motor neurons, and some dorsoventral location, V2a and V2b interneuron popula- ventral interneurons remain competent to appropriate SHH

FIG. 8. Ectopic expression of GATA3 does not affect PAX6 or PAX7 protein expression in progenitor cells. (A) Expression of ectopic GATA3 as shown by the presence of GFP. (B) PAX6 expression on a GATA3 electroporated spinal cord shown in (A). No signiﬁcant change to PAX6 expression is detected in the electroporated side of the spinal cord (B, C). (D–F) Expression of PAX7 (E) in a GATA3 electroporated spinal cord. PAX7 expression was also unchanged in the electroporated side in comparison with the normal side (E, F).

signals for a differing periods of time (Ericson et al., 1997; not supported by our observations as expression of GATA2 Poh et al., 2002). Given that CHX10, but not GATA3, is and CHX10 appear to commence at the same time (see Figs. expressed in the cylopamine-treated embryos at the tho- 1B, 1C, and 2E). Another possibility is that a single precur- racic level (Figs. 5F and 5G), it is conceivable that the sor subtype (such as NKX6.1- and IRX3-expressing) gives mechanism responsible for the separation of the V2a and rise to both V2a and V2b populations. In this model, V2a V2b fates includes a temporal component. neurons do not at any stage express GATA2, but instead Furthermore, it was reported that the loss of GATA2 require the “local” presence of V2b neurons for their activity led to a significant reduction in CHX10 expression survival (Fig. 9B). This may explain the specific loss of (Zhou et al., 2000). The same study also indicated that the CHX10 expression in the spinal cord of GATA2 knockout loss of GATA2 appeared to have no effect on the differen- mice (Zhou et al., 2000). A third possibility is the genera- tiation other ventral cell types. Based on this observation, it tion of these two interneuron populations from two inde- is conceivable that CHX10-expressing cells are generated pendent progenitor domains (Fig. 9C). However, this would from a GATA2-positive lineage, where expression of require the presence of an intrinsic mechanism that could GATA2 is downregulated prior to CHX10 expression (Fig. generate an alternative precursor population in the same 9A). If so, the expression of GATA2 may identify a popula- dorsoventral position as those that give rise to the V2a tion of precursor cells that gives rise to two different ventral interneurons. interneuron subtypes: the GATA2/3-negative, CHX10- Molecular cascade of interneurons specification by tran- positive V2a interneurons and the GATA2/3-positive, scription factors. While it is widely accepted that induc- CHX10-negative V2b interneurons. This model, however, is tive signals from midline structures determine cell-type

FIG. 9. Proposed models for V2a and V2b interneuron generation by progenitor cell domains. (A) Generation of V2a and V2b interneurons by a population of precursors that express the GATA2 protein. This model requires the cessation of GATA2 expression in cells that give rise to CHX10-positive V2a interneurons. As both GATA2 and CHX10 appear to be expressed simultaneously, it is unlikely that V2a and V2b interneurons are generated by this mechanism. (B) A single progenitor population that does not express GATA2 independently gives rise to CHX10-positive V2a interneurons and GATA2- and GATA3-positive V2b interneurons. Once generated, the V2b interneurons provide “support” for the survival of V2a interneurons (green arrows). (C) Generation of V2a and V2b interneurons from two independent progenitor domains. This model assumes the existence of two distinct progenitor domains within the same dorsoventral position of the spinal cord. These progenitors subsequently give rise to the V2a and V2b interneuron subpopulations. However, current evidence does not support the existence of two distinct progenitor populations within the same dorso-ventral position of the spinal cord.

specification in the ventral spinal cord, the subsequent GATA2 is indeed upstream of GATA3, and when taken molecular cascades that generate these individual cell types together with previous findings that GATA2 is necessary remains largely unknown. for the induction GATA3 expression. Moreover, we also Previous studies on CNS GATA expression suggested observed a repression of other ventral cell fates by the GATA2 and GATA3 are in the same epigenetic pathway ectopically expressed GATA2 protein. This suggests that (Kornhauser et al., 1994). Our expression analysis also when ectopically expressed, GATA2 can change the “pre- revealed a sequential pattern of GATA expression, where programmed” fate of other ventral interneurons. Similar expression of GATA2 preceded that of GATA3. It has also parallels have also been observed between EVX1 and EVX2 been reported that there is a lack of GATA3 gene expression in V0 interneuron development (Moran-Rivard et al., 2001). in the CNS of GATA2 mutant mice (Nardelli et al., 1999). In this study, it was demonstrated that EVX1 expression It is not clear, however, whether this is due to a specificor preceded EVX2, and that EVX1 was required for EVX2 general effect as a large proportion of GATA2Ϫ/Ϫ mice expression (Moran-Rivard et al., 2001). In addition, it was apparently die at 10.5 days postcoitum (Nardelli et al., further shown that EVX1 overexpression led to repression 1999; Tsai et al., 1994), shortly after the onset of GATA3 of V1 interneuron fates (Moran-Rivard et al., 2001). expression (Nardelli et al., 1999; Smith et al., 2002). A We also examined the effects of ectopic GATA3 expres- specific effect is supported by the observation that, al- sion on cells of the ventral spinal cord. As with GATA2, though there was distortion of the spinal cord of GATA2Ϫ/Ϫ ectopic expression of GATA3 resulted in the repression of mutant embryos at 10.5 days postcoitum, a number of other ventral cell types, with ventral interneurons being the ventral cell types were evident (Zhou et al., 2000). most effected. The relative level of ventral interneuron We sought to investigate whether GATA2 and GATA3 repression was comparable with that of ectopic GATA2 were in the same epigenetic cascade by using in ovo expression, an observation consistent with GATA3 being a electroporations. Our results clearly demonstrated that downstream factor of GATA2 in the epigenetic cascade.

However, we found that GATA3 could also induce ectopic controls progenitor cell identity and neuronal fate in response to GATA2 expression. This suggests the existence of a graded Shh signaling. Cell 90, 169–180. positive-feedback pathway, where once expressed, GATA3 Fleenor, D. E., Langdon, S. D., deCastro, C. M., and Kaufman, R. E. can either induce or maintain GATA2 expression in vivo. (1996). Comparison of human and Xenopus GATA-2 promoters. The similar abilities of GATA2 and GATA3 to influence Gene 179, 219–223. Hamburger, V., and Hamilton, H. L. (1951). A series of normal stages gene expression may also reflect a partial functional redun- in the development of the chick embryo. J. of Morph. 88, 49–92. dancy between these two factors in the developing chick Hendriks, R. W., Nawijn, M. C., Engel, J. D., van Doorninck, H., spinal cord. Grosveld, F., and Karis, A. (1999). Expression of the transcription Our study also revealed that ectopic expression of the factor GATA-3 is required for the development of the earliest T GATA3 protein had no significant effect on the PAX6 and cell progenitors and correlates with stages of cellular prolifera- PAX7 expression domains. Previous studies have impli- tion in the thymus. Eur. J. Immunol. 29, 1912–1918. cated both PAX6 and PAX7 in the establishment of progeni- Incardona, J. P., Gaffield, W., Kapur, R. P., and Roelink, H. (1998). tor cells in the developing neural tube (Ericson et al., 1997; The teratogenic Veratrum alkaloid cyclopamine inhibits sonic Mansouri and Gruss, 1998). This suggests that GATA3 is hedgehog signal transduction. Development 125, 3553–3562. involved in the postmitotic differentiation of a unique Ishihara, H., Engel, J. D., and Yamamoto, M. (1995). Structure and ventral interneuron subtype during development of the regulation of the chicken GATA-3 gene. J. Biochem. (Tokyo) 117, ventral spinal cord. 499–508. Kornhauser, J. M., Leonard, M. W., Yamamoto, M., LaVail, J. H., The findings of this study point to important roles for Mayo, K. E., and Engel, J. D. (1994). Temporal and spatial changes GATA2 and GATA3 proteins in spinal cord interneuron in GATA transcription factor expression are coincident with specification. Furthermore, they also indicate a possible development of the chicken optic tectum. Brain Res. Mol. Brain paradigm shift in the mechanisms governing ventral inter- Res. 23, 100–110. neuron specification. While previous investigations as- Litingtung, Y., and Chiang, C. (2000). Specification of ventral signed motor neurons into different subclasses based on neuron types is mediated by an antagonistic interaction between combinatorial expression of transcription factor genes Shh and Gli3. Nat. Neurosci. 3, 979–985. (Tsuchida et al., 1994), this is the first study to further Mansouri, A., and Gruss, P. (1998). Pax3 and Pax7 are expressed in subdivide a ventral interneuron population using differen- commissural neurons and restrict ventral neuronal identity in tial expression of transcription factor genes. the spinal cord. Mech. Dev. 78, 171–178. Matise, M. P., and Joyner, A. L. (1997). Expression patterns of developmental control genes in normal and Engrailed-1 mutant mouse spinal cord reveal early diversity in developing interneu- ACKNOWLEDGMENTS rons. J. Neurosci. 17, 7805–7816. Mizuguchi, R., Sugimori, M., Takebayashi, H., Kosako, H., Nagao, We thank Dr. Tom Jessell and Dr. Susan Morton for gifts of M., Yoshida, S., Nabeshima, Y., Shimamura, K., and Nakafuku, SHH-N recombinant protein and antibodies, Dr. Martyn Goulding M. (2001). Combinatorial roles of olig2 and neurogenin2 in the for the NKX6.1 antibody, and Dr. Brian Key for expression vector coordinated induction of pan-neuronal and subtype-specific constructs. We also thank Dr. Peter Koopman for critically reading properties of motoneurons. Neuron 31, 757–771. the manuscript. This work was supported by grants from the Moran-Rivard, L., Kagawa, T., Saueressig, H., Gross, M. K., Burrill, Australian National Health and Medical Research Council. A.K. J., and Goulding, M. (2001). Evx1 is a postmitotic determinant of was supported by an Australian Post-Graduate Award. Infrastruc- v0 interneuron identity in the spinal cord. Neuron 29, 385–399. ture support was provided in part by the ARC Special Research Muhr, J., Andersson, E., Persson, M., Jessell, T. M., and Ericson, J. Centre for Functional and Applied Genomics. This article is (2001). Groucho-mediated transcriptional repression establishes dedicated to the memory of Toshiya Yamada (October 15, 1960– progenitor cell pattern and neuronal fate in the ventral neural May 12, 2001). tube. Cell 104, 861–873. Nakamura, H., and Funahashi, J. (2001). Introduction of DNA into chick embryos by in ovo electroporation. Methods 24, 43–48. REFERENCES Nardelli, J., Thiesson, D., Fujiwara, Y., Tsai, F. Y., and Orkin, S. H. (1999). Expression and genetic interaction of transcription factors Arber, S., Han, B., Mendelsohn, M., Smith, M., Jessell, T. M., and GATA-2 and GATA-3 during development of the mouse central Sockanathan, S. (1999). Requirement for the homeobox gene Hb9 nervous system. Dev. Biol. 210, 305–321. in the consolidation of motor neuron identity. Neuron 23, Nawijn, M. C., Dingjan, G. M., Ferreira, R., Lambrecht, B. N., Karis, 659–674. A., Grosveld, F., Savelkoul, H., and Hendriks, R. W. (2001). Briscoe, J., Chen, Y., Jessell, T. M., and Struhl, G. (2001). A Enforced expression of GATA-3 in transgenic mice inhibits Th1 hedgehog-insensitive form of patched provides evidence for di- differentiation and induces the formation of a T1/ST2-expressing rect long-range morphogen activity of sonic hedgehog in the Th2- committed T cell compartment in vivo. J. Immunol. 167, neural tube. Mol. Cell 7, 1279–1291. 724–732. Briscoe, J., Pierani, A., Jessell, T. M., and Ericson, J. (2000). A Pandolfi, P. P., Roth, M. E., Karis, A., Leonard, M. W., Dzierzak, E., homeodomain protein code specifies progenitor cell identity and Grosveld, F. G., Engel, J. D., and Lindenbaum, M. H. (1995). neuronal fate in the ventral neural tube. Cell 101, 435–445. Targeted disruption of the GATA3 gene causes severe abnormali- Ericson, J., Rashbass, P., Schedl, A., Brenner-Morton, S., Kawakami, ties in the nervous system and in fetal liver haematopoiesis. Nat. A., van Heyningen, V., Jessell, T. M., and Briscoe, J. (1997). Pax6 Genet. 11, 40–44.

Pata, I., Studer, M., van Doorninck, J. H., Briscoe, J., Kuuse, S., Thaler, J., Harrison, K., Sharma, K., Lettieri, K., Kehrl, J., and Pfaff, Engel, J. D., Grosveld, F., and Karis, A. (1999). The transcription S. L. (1999). Active suppression of interneuron programs within factor GATA3 is a downstream effector of Hoxb1 specification in developing motor neurons revealed by analysis of homeodomain rhombomere 4. Development 126, 5523–5531. factor HB9. Neuron 23, 675–687. Pfaff, S. L., Mendelsohn, M., Stewart, C. L., Edlund, T., and Jessell, Tsai, F. Y., Keller, G., Kuo, F. C., Weiss, M., Chen, J., Rosenblatt, T. M. (1996). Requirement for LIM homeobox gene Isl1 in motor M., Alt, F. W., and Orkin, S. H. (1994). An early haematopoietic neuron generation reveals a motor neuron-dependent step in defect in mice lacking the transcription factor GATA-2. Nature interneuron differentiation. Cell 84, 309–320. 371, 221–226. Pierani, A., Brenner-Morton, S., Chiang, C., and Jessell, T. M. Tsuchida, T., Ensini, M., Morton, S. B., Baldassare, M., Edlund, T., (1999). A sonic hedgehog-independent, retinoid-activated path- Jessell, T. M., and Pfaff, S. L. (1994). Topographic organization of way of neurogenesis in the ventral spinal cord. Cell 97, 903–915. embryonic motor neurons defined by expression of LIM ho- Pierani, A., Moran-Rivard, L., Sunshine, M. J., Littman, D. R., meobox genes. Cell 79, 957–970. Goulding, M., and Jessell, T. M. (2001). Control of interneuron van Doorninck, J. H., van Der Wees, J., Karis, A., Goedknegt, E., fate in the developing spinal cord by the progenitor homeodo- Engel, J. D., Coesmans, M., Rutteman, M., Grosveld, F., and Dez main protein Dbx1. Neuron 29, 367–384. Eeuw, C. I. (1999). GATA-3 is involved in the development of Poh, A., Karunaratne, A., Kolle, G., Huang, N., Smith, E., Starkey, serotonergic neurons in the caudal raphe nuclei. J. Neurosci. J., Wen, D., Wilson, I., Yamada, T., and Hargrave, M. (2002). (Online) 19, RC12. Patterning of the vertebrate ventral spinal cord. Int. J. Dev. Biol. Wenner, P., O’Donovan, M. J., and Matise, M. P. (2000). Topograph- ical and physiological characterization of interneurons that ex- 46, 597–608. press engrailed-1 in the embryonic chick spinal cord. J. Neuro- Sander, M., Paydar, S., Ericson, J., Briscoe, J., Berber, E., German, physiol. 84, 2651–2657. M., Jessell, T. M., and Rubenstein, J. L. (2000). Ventral neural Yamada, T., Pfaff, S. L., Edlund, T., and Jessell, T. M. (1993). patterning by Nkx homeobox genes: Nkx6.1 controls somatic Control of cell pattern in the neural tube: Motor neuron induc- motor neuron and ventral interneuron fates. Genes Dev. 14, tion by diffusible factors from notochord and floor plate. Cell 73, 2134–2139. 673–686. Saueressig, H., Burrill, J., and Goulding, M. (1999). Engrailed-1 and Yamada, T., Placzek, M., Tanaka, H., Dodd, J., and Jessell, T. M. netrin-1 regulate axon pathfinding by association interneurons (1991). Control of cell pattern in the developing nervous system: that project to motor neurons. Development 126, 4201–4212. Polarizing activity of the floor plate and notochord. Cell 64, Smith, E., Hargrave, M., Yamada, T., Begley, C. G., and Little, M 635–647. (2002). Co-expression of SCL and GATA3 in the V2 interneurons Zhou, Y., Yamamoto, M., and Engel, J. D. (2000). GATA2 is of the developing mouse spinal cord. Dev. Dyn. 224, 231–237. required for the generation of V2 interneurons. Development Sockanathan, S., and Jessell, T. M. (1998). Motor neuron-derived 127, 3829–3838. retinoid signaling specifies the subtype identity of spinal motor neurons. Cell 94, 503–514. Received for publication November 12, 2001 Tanabe, Y., William, C., and Jessell, T. M. (1998). Specification of Revised May 28, 2002 motor neuron identity by the MNR2 homeodomain protein. Cell Accepted May 28, 2002 95, 67–80. Published online August 7, 2002