Heightened activation of embryonic megakaryocytes causes aneurysms in the developing brain of mice lacking podoplanin.

During early embryonic development in mammals, including humans and mice, megakaryocytes (Mks) first originate from primitive hematopoiesis in the yolk sac. These embryonic Mks (eMks) circulate in the vasculature with unclear function. Herein, we report that podoplanin (PDPN), the ligand of C-type lectin-like receptor (CLEC-2) on Mks/platelets, is temporarily expressed in neural tissue during midgestation in mice. Loss of PDPN or CLEC-2 resulted in aneurysms and spontaneous hemorrhage, specifically in the lower diencephalon during midgestation. Surprisingly, more eMks/platelets had enhanced granule release and localized to the lower diencephalon in mutant mouse embryos than in wild-type littermates before hemorrhage. We found that PDPN counteracted the collagen-1-induced secretion of angiopoietin-1 from fetal Mks, which coincided with enhanced TIE-2 activation in aneurysm-like sprouts of PDPN-deficient embryos. Blocking platelet activation prevented the PDPN-deficient embryo from developing vascular defects. Our data reveal a new role for PDPN in regulating eMk function during midgestation.

Zebrafish Cdx1b modulates epithalamic asymmetry by regulating ndr2 and lft1 expression.

Nodal signaling is essential for mesoderm and endoderm formation, as well as neural plate induction and establishment of left-right asymmetry. However, the mechanisms controlling expression of Nodal pathway genes in these contexts are not fully known. Previously, we showed that Cdx1b induces expression of downstream Nodal signaling factors during early endoderm formation. In this study, we show that Cdx1b also regulates epithalamic asymmetry in zebrafish embryos by modulating expression of ndr2 and lft1. We first knocked down cdx1b with translation-blocking and splicing-blocking morpholinos (MOs). Most embryos injected with translation-blocking MOs showed absent ndr2, lft1 and pitx2c expression in the left dorsal diencephalon during segmentation and pharyngula stages accompanied by aberrant parapineal migration and habenular laterality at 72 â€‹h post fertilization (hpf). These defects were less frequent in embryos injected with splicing-blocking MO. To confirm the morphant phenotype, we next generated both zygotic (Z)cdx1b-/- and maternal zygotic (MZ)cdx1b-/- mutants by CRISPR-Cas9 mutagenesis. Expression of ndr2, lft1 and pitx2c was absent in the left dorsal diencephalon of a high proportion of MZcdx1b-/- mutants; however, aberrant dorsal diencephalic pitx2c expression patterns were observed at low frequency in Zcdx1b-/- mutant embryos. Correspondingly, dysregulated parapineal migration and habenular laterality were also observed in MZcdx1b-/- mutant embryos at 72 hpf. On the other hand, Kupffer's vesicle cilia length and number, expression pattern of spaw in the lateral plate mesoderm and pitx2c in the gut as well as left-right patterning of various visceral organs were not altered in MZcdx1b-/- mutants compared to wild-type embryos. Chromatin immunoprecipitation revealed that Cdx1b directly regulates ndr2 and lft1 expression. Furthermore, injection of cdx1b-vivo MO1 but not cdx1b-vivo 4 â€‹mm MO1 in the forebrain ventricle at 18 hpf significantly downregulated lft1 expression in the left dorsal diencephalon at 23-24 â€‹s stages. Together, our results suggest that Cdx1b regulates transcription of ndr2 and lft1 to maintain proper Nodal activity in the dorsal diencephalon and epithalamic asymmetry in zebrafish embryos.

Apoptosis is involved in maintaining the character of the midbrain and the diencephalon roof plate after neural tube closure.

Apoptosis, a major form of programmed cell death, is massively observed in neural plate border and subsequently in the roof plate (RP). While deficiency of apoptosis often results in brain malformations including exencephaly and hydrocephalus, the impact of apoptosis on RP formation and maintenance remains unclear. Here we described that mouse embryos deficient in Apaf1, a gene crucial for the intrinsic apoptotic pathway, in C57BL/6 genetic background exhibited narrow and discontinuous expression of RP marker genes in the midline of the midbrain and the diencephalon. Instead, cells positive for the neuroectodermal gene SOX1 ectopically accumulated in the midline. A lineage-tracing experiment suggests that these ectopic SOX1-positive cells began to accumulate in the midline of apoptosis-deficient embryos after E9.5. These embryos further displayed malformation of the subcommissural organ, which has been discussed in the etiology of hydrocephalus. Thus, the apoptosis machinery prevents ectopic emergence of SOX1-positive cells in the midbrain and the diencephalon RP, and helps in maintaining the character of the RP in the diencephalon and midbrain, thereby ensuring proper brain development.

gdnf affects early diencephalic dopaminergic neuron development through regulation of differentiation-associated transcription factors in zebrafish.

Glial cell line-derived neurotrophic factor (GDNF) has been reported to enhance dopaminergic neuron survival and differentiation in vitro and in vivo, although those results are still being debated. Glial cell line-derived neurotrophic factor (gdnf) is highly conserved in zebrafish and plays a role in enteric nervous system function. However, little is known about gdnf function in the teleost brain. Here, we employed clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 to impede gdnf function in the maintenance of dopaminergic neuron development. Genotyping of gdnf crispants revealed successful deletions of the coding region with various mutant band sizes and down-regulation of gdnf transcripts at 1, 3 and 7 day(s) post fertilization. Notably, ~20% reduction in ventral diencephalic dopaminergic neuron numbers in clusters 8 and 13 was observed in the gdnf-deficient crispants. In addition, gdnf depletion caused a modest reduction in dopaminergic neurogenesis as determined by 5-ethynyl-2'-deoxyuridine pulse chase assay. These deleterious effects could be partly attributed to deregulation of dopaminergic neuron fate specification-related transcription factors (otp,lmx1b,shha,and ngn1) in both crispants and established homozygous mutants with whole mount in-situ hybridization (WISH) on gdnf mutants showing reduced otpb and lmx1b.1 expression in the ventral diencephalon. Interestingly, locomotor function of crispants was only impacted at 7 dpf, but not earlier. Lastly, as expected, gdnf deficiency heightened crispants vulnerability to 1-methyl-4-phenylpyridinium toxic insult. Our results suggest conservation of teleost gdnf brain function with mammals and revealed the interactions between gdnf and transcription factors in dopaminergic neuron differentiation.

Fgf10+ progenitors give rise to the chick hypothalamus by rostral and caudal growth and differentiation.

Classical descriptions of the hypothalamus divide it into three rostro-caudal domains but little is known about their embryonic origins. To investigate this, we performed targeted fate-mapping, molecular characterisation and cell cycle analyses in the embryonic chick. Presumptive hypothalamic cells derive from the rostral diencephalic ventral midline, lie above the prechordal mesendoderm and express Fgf10Fgf10+ progenitors undergo anisotropic growth: those displaced rostrally differentiate into anterior cells, then those displaced caudally differentiate into mammillary cells. A stable population of Fgf10+ progenitors is retained within the tuberal domain; a subset of these gives rise to the tuberal infundibulum - the precursor of the posterior pituitary. Pharmacological approaches reveal that Shh signalling promotes the growth and differentiation of anterior progenitors, and also orchestrates the development of the infundibulum and Rathke's pouch - the precursor of the anterior pituitary. Together, our studies identify a hypothalamic progenitor population defined by Fgf10 and highlight a role for Shh signalling in the integrated development of the hypothalamus and pituitary.

VEGF-A and neuropilin 1 (NRP1) shape axon projections in the developing CNS via dual roles in neurons and blood vessels.

Visual information is relayed from the eye to the brain via retinal ganglion cell (RGC) axons. Mice lacking NRP1 or NRP1-binding VEGF-A isoforms have defective RGC axon organisation alongside brain vascular defects. It is not known whether axonal defects are caused exclusively by defective VEGF-A signalling in RGCs or are exacerbated by abnormal vascular morphology. Targeted NRP1 ablation in RGCs with a Brn3bCre knock-in allele reduced axonal midline crossing at the optic chiasm and optic tract fasciculation. In contrast, Tie2-Cre-mediated endothelial NRP1 ablation induced axon exclusion zones in the optic tracts without impairing axon crossing. Similar defects were observed in Vegfa120/120 and Vegfa188/188 mice, which have vascular defects as a result of their expression of single VEGF-A isoforms. Ectopic midline vascularisation in endothelial Nrp1 and Vegfa188/188 mutants caused additional axonal exclusion zones within the chiasm. As in vitro and in vivo assays demonstrated that vessels do not repel axons, abnormally large or ectopically positioned vessels are likely to present physical obstacles to axon growth. We conclude that proper axonal wiring during brain development depends on the precise molecular control of neurovascular co-patterning.

Six3 dosage mediates the pathogenesis of holoprosencephaly.

Holoprosencephaly (HPE) is defined as the incomplete separation of the two cerebral hemispheres. The pathology of HPE is variable and, based on the severity of the defect, HPE is divided into alobar, semilobar, and lobar. Using a novel hypomorphic Six3 allele, we demonstrate in mice that variability in Six3 dosage results in different HPE phenotypes. Furthermore, we show that whereas the semilobar phenotype results from severe downregulation of Shh expression in the rostral diencephalon ventral midline, the alobar phenotype is caused by downregulation of Foxg1 expression in the anterior neural ectoderm. Consistent with these results, in vivo activation of the Shh signaling pathway rescued the semilobar phenotype but not the alobar phenotype. Our findings show that variations in Six3 dosage result in different forms of HPE.

Diencephalic Size Is Restricted by a Novel Interplay Between GCN5 Acetyltransferase Activity and Retinoic Acid Signaling.

Diencephalic defects underlie an array of neurological diseases. Previous studies have suggested that retinoic acid (RA) signaling is involved in diencephalic development at late stages of embryonic development, but its roles and mechanisms of action during early neural development are still unclear. Here we demonstrate that mice lacking enzymatic activity of the acetyltransferase GCN5 ((Gcn5hat/hat )), which were previously characterized with respect to their exencephalic phenotype, exhibit significant diencephalic expansion, decreased diencephalic RA signaling, and increased diencephalic WNT and SHH signaling. Using a variety of molecular biology techniques in both cultured neuroepithelial cells treated with a GCN5 inhibitor and forebrain tissue from (Gcn5hat/hat ) embryos, we demonstrate that GCN5, RARα/γ, and the poorly characterized protein TACC1 form a complex in the nucleus that binds specific retinoic acid response elements in the absence of RA. Furthermore, RA triggers GCN5-mediated acetylation of TACC1, which results in dissociation of TACC1 from retinoic acid response elements and leads to transcriptional activation of RA target genes. Intriguingly, RA signaling defects caused by in vitro inhibition of GCN5 can be rescued through RA-dependent mechanisms that require RARß. Last, we demonstrate that the diencephalic expansion and transcriptional defects seen in (Gcn5hat/hat ) mutants can be rescued with gestational RA supplementation, supporting a direct link between GCN5, TACC1, and RA signaling in the developing diencephalon. Together, our studies identify a novel, nonhistone substrate for GCN5 whose modification regulates a previously undescribed, tissue-specific mechanism of RA signaling that is required to restrict diencephalic size during early forebrain development.SIGNIFICANCE STATEMENT Changes in diencephalic size and shape, as well as SNPs associated with retinoic acid (RA) signaling-associated genes, have been linked to neuropsychiatric disorders. However, the mechanisms that regulate diencephalic morphogenesis and the involvement of RA signaling in this process are poorly understood. Here we demonstrate a novel role of the acetyltransferase GCN5 in a previously undescribed mechanism of RA signaling in the developing forebrain that is required to maintain the appropriate size of the diencephalon. Together, our experiments identify a novel nonhistone substrate of GCN5, highlight an essential role for both GCN5 and RA signaling in early diencephalic development, and elucidate a novel molecular regulatory mechanism for RA signaling that is specific to the developing forebrain.

Tcf7l2 plays crucial roles in forebrain development through regulation of thalamic and habenular neuron identity and connectivity.

The thalamus acts as a central integrator for processing and relaying sensory and motor information to and from the cerebral cortex, and the habenula plays pivotal roles in emotive decision making by modulating dopaminergic and serotonergic circuits. These neural compartments are derived from a common developmental progenitor domain, called prosomere 2, in the caudal forebrain. Thalamic and habenular neurons exhibit distinct molecular profile, neurochemical identity, and axonal circuitry. However, the mechanisms of how their progenitors in prosomere 2 give rise to these two populations of neurons and contribute to the forebrain circuitry remains unclear. In this study, we discovered a previously unrecognized role for Tcf7l2, a transcription factor known as the canonical Wnt nuclear effector and diabetes risk-conferring gene, in establishing neuronal identity and circuits of the caudal forebrain. Using genetic and chemical axon tracers, we showed that efferent axons of the thalamus, known as the thalamocortical axons (TCAs), failed to elongate normally and strayed from their normal course to inappropriate locations in the absence of Tcf7l2. Further experiments with thalamic explants revealed that the pathfinding defects of Tcf7l2-deficient TCAs were associated at least in part with downregulation of guidance receptors Robo1 and Robo2 expression. Moreover, the fasciculus retroflexus, the main habenular output tract, was missing in embryos lacking Tcf7l2. These axonal defects may result from dysregulation of Nrp2 guidance receptor. Strikingly, loss of Tcf7l2 caused a post-mitotic identity switch between thalamic and habenular neurons. Despite normal acquisition of progenitor identity in prosomere 2, Tcf7l2-deficient thalamic neurons adopted a molecular profile of a neighboring forebrain derivative, the habenula. Conversely, habenular neurons failed to maintain their normal post-mitotic neuronal identity and acquired a subset of thalamic neuronal features in the absence of Tcf7l2. Our findings suggest a unique role for Tcf7l2 in generating distinct neuronal phenotypes from homogeneous progenitor population, and provide a better understanding of the mechanism underlying neuronal specification, differentiation, and connectivity of the developing caudal forebrain.

The COUP-TFII/Neuropilin-2 is a molecular switch steering diencephalon-derived GABAergic neurons in the developing mouse brain.

The preoptic area (POa) of the rostral diencephalon supplies the neocortex and the amygdala with GABAergic neurons in the developing mouse brain. However, the molecular mechanisms that determine the pathway and destinations of POa-derived neurons have not yet been identified. Here we show that Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII)-induced expression of Neuropilin-2 (Nrp2) and its down-regulation control the destination of POa-derived GABAergic neurons. Initially, a majority of the POa-derived migrating neurons express COUP-TFII and form a caudal migratory stream toward the caudal subpallium. When a subpopulation of cells steers toward the neocortex, they exhibit decreased expression of COUP-TFII and Nrp2. The present findings show that suppression of COUP-TFII/Nrp2 changed the destination of the cells into the neocortex, whereas overexpression of COUP-TFII/Nrp2 caused cells to end up in the medial part of the amygdala. Taken together, these results reveal that COUP-TFII/Nrp2 is a molecular switch determining the pathway and destination of migrating GABAergic neurons born in the POa.

Hedgehog receptor function during craniofacial development.

The Hedgehog signalling pathway plays a fundamental role in orchestrating normal craniofacial development in vertebrates. In particular, Sonic hedgehog (Shh) is produced in three key domains during the early formation of the head; neuroectoderm of the ventral forebrain, facial ectoderm and the pharyngeal endoderm; with signal transduction evident in both ectodermal and mesenchymal tissue compartments. Shh signalling from the prechordal plate and ventral midline of the diencephalon is required for appropriate division of the eyefield and forebrain, with mutation in a number of pathway components associated with Holoprosencephaly, a clinically heterogeneous developmental defect characterized by a failure of the early forebrain vesicle to divide into distinct halves. In addition, signalling from the pharyngeal endoderm and facial ectoderm plays an essential role during development of the face, influencing cranial neural crest cells that migrate into the early facial processes. In recent years, the complexity of Shh signalling has been highlighted by the identification of multiple novel proteins that are involved in regulating both the release and reception of this protein. Here, we review the contributions of Shh signalling during early craniofacial development, focusing on Hedgehog receptor function and describing the consequences of disruption for inherited anomalies of this region in both mouse models and human populations.

Eph/Ephrin signalling maintains eye field segregation from adjacent neural plate territories during forebrain morphogenesis.

During forebrain morphogenesis, there is extensive reorganisation of the cells destined to form the eyes, telencephalon and diencephalon. Little is known about the molecular mechanisms that regulate region-specific behaviours and that maintain the coherence of cell populations undergoing specific morphogenetic processes. In this study, we show that the activity of the Eph/Ephrin signalling pathway maintains segregation between the prospective eyes and adjacent regions of the anterior neural plate during the early stages of forebrain morphogenesis in zebrafish. Several Ephrins and Ephs are expressed in complementary domains in the prospective forebrain and combinatorial abrogation of their activity results in incomplete segregation of the eyes and telencephalon and in defective evagination of the optic vesicles. Conversely, expression of exogenous Ephs or Ephrins in regions of the prospective forebrain where they are not usually expressed changes the adhesion properties of the cells, resulting in segregation to the wrong domain without changing their regional fate. The failure of eye morphogenesis in rx3 mutants is accompanied by a loss of complementary expression of Ephs and Ephrins, suggesting that this pathway is activated downstream of the regional fate specification machinery to establish boundaries between domains undergoing different programmes of morphogenesis.

Inhibition of Sox2-dependent activation of Shh in the ventral diencephalon by Tbx3 is required for formation of the neurohypophysis.

Tbx2 and Tbx3 are two highly related members of the T-box transcription factor gene family that regulate patterning and differentiation of a number of tissue rudiments in the mouse. Both genes are partially co-expressed in the ventral diencephalon and the infundibulum; however, a functional requirement in murine pituitary development has not been reported. Here, we show by genetic lineage tracing that Tbx2(+) cells constitute the precursor population of the neurohypophysis. However, Tbx2 is dispensable for neurohypophysis development as revealed by normal formation of this organ in Tbx2-deficient mice. By contrast, loss of Tbx3 from the ventral diencephalon results in a failure to establish the Tbx2(+) domain in this region, and a lack of evagination of the infundibulum and formation of the neurohypophysis. Rathke's pouch is severely hypoplastic, exhibits defects in dorsoventral patterning, and degenerates after E12.5. In Tbx3-deficient embryos, the ventral diencephalon is hyperproliferative and displays an abnormal cellular architecture, probably resulting from a failure to repress transcription of Shh. We further show that Tbx3 and Tbx2 repress Shh by sequestering the SRY box-containing transcription factor Sox2 away from a Shh forebrain enhancer (SBE2), thus preventing its activation. These data suggest that Tbx3 is required in the ventral diencephalon to establish a Shh(-) domain to allow formation of the infundibulum.

Conditional cell ablation via diphtheria toxin reveals distinct requirements for the basal plate in the regional identity of diencephalic subpopulations.

The mammalian diencephalon is the caudal derivative of the embryonic forebrain. Early events in diencephalic regionalization include its subdivision along the dorsoventral and anteroposterior axes. The prosomeric model by Puelles and Rubenstein (1993) suggests that the alar plate of the posterior diencephalon is partitioned into three different prosomeres (designated p1-p3), which develop into the pretectum, thalamus, and prethalamus, respectively. Here, we report the developmental consequences of genetic ablation of cell populations from the diencephalic basal plate. The strategy for conditionally regulated cell ablation is based on the targeted expression of the diphtheria toxin gene (DTA) to the diencephalic basal plate via tamoxifen- induced, Cre-mediated recombination of the ROSA(DTA) allele. We show that activation of DTA leads to specific cell loss in the basal plate of the posterior diencephalon, and disrupted early regionalization of distinct alar territories. In the basal plate-deficient embryos, the p1 alar plate exhibited reduced expression of subtype-specific markers in the pretectum, whereas p2 alar plate failed to further subdivide into two discrete thalamic subpopulations. We also show that these defects lead to abnormal nuclear organization at later developmental stages. Our data have implications for increased understanding of the interactive roles between discrete diencephalic compartments.

Transcriptional regulatory mechanisms underlying the GABAergic neuron fate in different diencephalic prosomeres.

Diverse mechanisms regulate development of GABAergic neurons in different regions of the central nervous system. We have addressed the roles of a proneural gene, Ascl1, and a postmitotic selector gene, Gata2, in the differentiation of GABAergic neuron subpopulations in three diencephalic prosomeres: prethalamus (P3), thalamus (P2) and pretectum (P1). Although the different proliferative progenitor populations of GABAergic neurons commonly express Ascl1, they have distinct requirements for it in promotion of cell-cycle exit and GABAergic neuron identity. Subsequently, Gata2 is activated as postmitotic GABAergic precursors are born. In P1, Gata2 regulates the neurotransmitter identity by promoting GABAergic and inhibiting glutamatergic neuron differentiation. Interestingly, Gata2 defines instead the subtype of GABAergic neurons in the rostral thalamus (pTh-R), which is a subpopulation of P2. Without Gata2, the GABAergic precursors born in the pTh-R fail to activate subtype-specific markers, but start to express genes typical of GABAergic precursors in the neighbouring P3 domain. Thus, our results demonstrate diverse mechanisms regulating differentiation of GABAergic neuron subpopulations and suggest a role for Gata2 as a selector gene of both GABAergic neuron neurotransmitter and prosomere subtype identities in the developing diencephalon. Our results demonstrate for the first time that neuronal identities between distinct prosomeres can still be transformed in postmitotic neuronal precursors.

Cell-autonomous FGF signaling regulates anteroposterior patterning and neuronal differentiation in the mesodiencephalic dopaminergic progenitor domain.

The structure and projection patterns of adult mesodiencephalic dopaminergic (DA) neurons are one of the best characterized systems in the vertebrate brain. However, the early organization and development of these nuclei remain poorly understood. The induction of midbrain DA neurons requires sonic hedgehog (Shh) from the floor plate and fibroblast growth factor 8 (FGF8) from the isthmic organizer, but the way in which FGF8 regulates DA neuron development is unclear. We show that, during early embryogenesis, mesodiencephalic neurons consist of two distinct populations: a diencephalic domain, which is probably independent of isthmic FGFs; and a midbrain domain, which is dependent on FGFs. Within these domains, DA progenitors and precursors use partly different genetic programs. Furthermore, the diencephalic DA domain forms a distinct cell population, which also contains non-DA Pou4f1(+) cells. FGF signaling operates in proliferative midbrain DA progenitors, but is absent in postmitotic DA precursors. The loss of FGFR1/2-mediated signaling results in a maturation failure of the midbrain DA neurons and altered patterning of the midbrain floor. In FGFR mutants, the DA domain adopts characteristics that are typical for embryonic diencephalon, including the presence of Pou4f1(+) cells among TH(+) cells, and downregulation of genes typical of midbrain DA precursors. Finally, analyses of chimeric embryos indicate that FGF signaling regulates the development of the ventral midbrain cell autonomously.

Spatial and temporal requirements for sonic hedgehog in the regulation of thalamic interneuron identity.

In caudal regions of the diencephalon, sonic hedgehog (Shh) is expressed in the ventral midline of prosomeres 1-3 (p1-p3), which underlie the pretectum, thalamus and prethalamus, respectively. Shh is also expressed in the zona limitans intrathalamica (zli), a dorsally projecting spike that forms at the p2-p3 boundary. The presence of two Shh signaling centers in the thalamus has made it difficult to determine the specific roles of either one in regional patterning and neuronal fate specification. To investigate the requirement of Shh from a focal source of expression in the ventral midline of the diencephalon, we used a newly generated mouse line carrying a targeted deletion of the 525 bp intronic sequence mediating Shh brain enhancer-1 (SBE1) activity. In SBE1 mutant mice, Shh transcription was initiated but not maintained in the ventral midline of the rostral midbrain and caudal diencephalon, yet expression in the zli was unaffected. In the absence of ventral midline Shh, rostral thalamic progenitors (pTH-R) adopted the molecular profile of a more caudal thalamic subtype (pTH-C). Surprisingly, despite their early mis-specification, neurons derived from the pTH-R domain continued to migrate to their proper thalamic nucleus, extended axons along their normal trajectory and expressed some, but not all, of their terminal differentiation markers. Our results, and those of others, suggest a model whereby Shh signaling from distinct spatial and temporal domains in the diencephalon exhibits unique and overlapping functions in the development of discrete classes of thalamic interneurons.

Retinoic acid-dependent and -independent gene-regulatory pathways of Pitx3 in meso-diencephalic dopaminergic neurons.

Development of meso-diencephalic dopamine (mdDA) neurons requires the combined actions of the orphan nuclear receptor Nurr1 and the paired-like homeobox transcription factor Pitx3. Whereas all mdDA neurons require Nurr1 for expression of Th and survival, dependence on Pitx3 is displayed only by the mdDA subpopulation that will form the substantia nigra (SNc). Previously, we have demonstrated that Pitx3(-/-) embryos lack the expression of the retinoic acid (RA)-generating enzyme Ahd2, which is normally selectively expressed in the Pitx3-dependent DA neurons of the SNc. Restoring RA signaling in Pitx3(-/-) embryos revealed a selective dependence of SNc neurons on the presence of RA for differentiation into Th-positive neurons and maintenance throughout embryonic development. Whereas these data are suggestive of an important developmental role for RA in neurons of the SNc, it remained unclear whether other Nurr1 and Pitx3 target genes depend on RA signaling in a manner similar to Th. In the search for genes that were affected in Pitx3-deficient mdDA neurons and restored upon embryonic RA treatment, we provide evidence that Delta-like 1, D2R (Drd2) and Th are regulated by Pitx3 and RA signaling, which influences the mdDA terminal differentiated phenotype. Furthermore, we show that regulation of Ahd2-mediated RA signaling represents only one aspect of the Pitx3 downstream cascade, as Vmat2, Dat, Ahd2 (Aldh1a1), En1, En2 and Cck were unaffected by RA treatment and are (subset) specifically modulated by Pitx3. In conclusion, our data reveal several RA-dependent and -independent aspects of the Pitx3-regulated gene cascade, suggesting that Pitx3 acts on multiple levels in the molecular subset-specification of mdDA neurons.

Barhl2 limits growth of the diencephalic primordium through Caspase3 inhibition of beta-catenin activation.

Little is known about the respective contributions of cell proliferation and cell death to the control of vertebrate forebrain growth. The homeodomain protein barhl2 is expressed in the diencephalons of Xenopus, zebrafish, and mouse embryos, and we previously showed that Barhl2 overexpression in Xenopus neuroepithelial cells induces Caspase3-dependent apoptosis. Here, barhl2 is shown to act as a brake on diencephalic proliferation through an unconventional function of Caspase3. Depletion of Barhl2 or Caspase3 causes an increase in diencephalic cell number, a disruption of the neuroepithelium architecture, and an increase in Wnt activity. Surprisingly, these changes are not caused by decreased apoptosis but instead, are because of an increase in the amount and activation of ß-catenin, which stimulates excessive neuroepithelial cell proliferation and induces defects in ß-catenin intracellular localization and an up-regulation of axin2 and cyclinD1, two downstream targets of ß-catenin/T-cell factor/lymphoïd enhancer factor signaling. Using two different sets of complementation experiments, we showed that, in the developing diencephalon, Caspase3 acts downstream of Barhl2 in limiting neuroepithelial cell proliferation by inhibiting ß-catenin activation. Our data argue that Bar homeodomain proteins share a conserved function as cell type-specific regulators of Caspase3 activities.

Chemoattractant axon guidance cues regulate de novo axon trajectories in the embryonic forebrain of zebrafish.

The development of axon tracts in the early vertebrate brain is controlled by combinations of soluble, membrane-bound and extracellular matrix molecules. How these multiple and sometimes conflicting guidance cues are integrated in order to establish stereotypical pathways remains to be determined. We show here that when interactions between the chemoattractive signal Netrin1a and its receptor Dcc are suppressed using a loss-of-function approach, a novel axon trajectory emerges in the dorsal diencephalon. Axons arising from a subpopulation of telencephalic neurons failed to project rostrally into the anterior commissure in the absence of either Netrin1a or Dcc. Instead these axons inappropriately exited the telencephalon and ectopically coursed caudally into virgin neuroepithelium. This response was highly specific since loss-of-function of Netrin1b, a paralogue of Netrin1a, generated a distinct phenotype in the rostral brain. These results show that a subpopulation of telencephalic neurons, when freed from long-range chemoattraction mediated by Netrin1a-Dcc interactions, follow alternative instructive cues that lead to creation of an ectopic axon bundle in the diencephalon. This work provides insight into how integration of multiple guidance signals defines the initial scaffold of axon tracts in the embryonic vertebrate forebrain.