RESUMEN
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.
Asunto(s)
Encéfalo/irrigación sanguínea , Aneurisma Intracraneal/etiología , Megacariocitos/patología , Glicoproteínas de Membrana/deficiencia , Aneurisma Roto/embriología , Aneurisma Roto/etiología , Angiopoyetina 1/metabolismo , Animales , Encéfalo/embriología , Células Cultivadas , Hemorragia Cerebral/embriología , Hemorragia Cerebral/etiología , Colágeno/farmacología , Diencéfalo/irrigación sanguínea , Diencéfalo/embriología , Regulación del Desarrollo de la Expresión Génica , Edad Gestacional , Aneurisma Intracraneal/embriología , Aneurisma Intracraneal/genética , Aneurisma Intracraneal/patología , Lectinas Tipo C/deficiencia , Lectinas Tipo C/genética , Lectinas Tipo C/fisiología , Megacariocitos/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Noqueados , Neovascularización Patológica/genética , Neovascularización Patológica/fisiopatología , Neovascularización Fisiológica/fisiología , Activación Plaquetaria , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Receptor TIE-2/metabolismoRESUMEN
The prethalamic eminence (PThE), a diencephalic caudal neighbor of the telencephalon and alar hypothalamus, is frequently described in mammals and birds as a transient embryonic structure, undetectable in the adult brain. Based on descriptive developmental analysis of Tbr1 gene brain expression in chick embryos, we previously reported that three migratory cellular streams exit the PThE rostralward, targeting multiple sites in the hypothalamus, subpallium and septocommissural area, where eminential cells form distinct nuclei or disperse populations. These conclusions needed experimental corroboration. In this work, we used the homotopic quail-chick chimeric grafting procedure at stages HH10/HH11 to demonstrate by fate-mapping the three predicted tangential migration streams. Some chimeric brains were processed for Tbr1 in situ hybridization, for correlation with our previous approach. Evidence supporting all three postulated migration streams is presented. The results suggested a slight heterochrony among the juxtapeduncular (first), the peripeduncular (next), and the eminentio-septal (last) streams, each of which followed differential routes. A possible effect of such heterochrony on the differential selection of medial to lateral habenular hodologic targets by the migrated neurons is discussed.
Asunto(s)
Hipotálamo/embriología , Neuronas/citología , Codorniz/embriología , Telencéfalo/metabolismo , Animales , Diferenciación Celular/fisiología , Movimiento Celular/fisiología , Embrión de Pollo , Pollos , Diencéfalo/embriologíaRESUMEN
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 âh 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 âmm MO1 in the forebrain ventricle at 18 hpf significantly downregulated lft1 expression in the left dorsal diencephalon at 23-24 âs 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.
Asunto(s)
Tipificación del Cuerpo/genética , Epitálamo/embriología , Proteínas de Homeodominio/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Factores de Determinación Derecha-Izquierda/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Animales , Movimiento Celular , Diencéfalo/embriología , Diencéfalo/metabolismo , Embrión no Mamífero/metabolismo , Epitálamo/metabolismo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Habénula/embriología , Corazón/embriología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Factores de Determinación Derecha-Izquierda/metabolismo , Proteína Nodal/metabolismo , Glándula Pineal/citología , Glándula Pineal/embriología , Unión Proteica , Transducción de Señal , Pez Cebra/metabolismoRESUMEN
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.
Asunto(s)
Diferenciación Celular/fisiología , Diencéfalo/embriología , Diencéfalo/metabolismo , Neuronas Dopaminérgicas/metabolismo , Factor Neurotrófico Derivado de la Línea Celular Glial/deficiencia , Factores de Transcripción/deficiencia , Proteínas de Pez Cebra/deficiencia , Animales , Animales Modificados Genéticamente , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Factores de Transcripción/genética , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
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.
Asunto(s)
Apoptosis , Diencéfalo/embriología , Mesencéfalo/embriología , Tubo Neural/embriología , Animales , Factor Apoptótico 1 Activador de Proteasas/genética , Factor Apoptótico 1 Activador de Proteasas/metabolismo , Ratones , Ratones Transgénicos , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismoRESUMEN
Promoting the regeneration or survival of retinal ganglion cells (RGCs) is one focus of regenerative medicine. Homeobox Barhl transcription factors might be instrumental in these processes. In mammals, only barhl2 is expressed in the retina and is required for both subtype identity acquisition of amacrine cells and for the survival of RGCs downstream of Atoh7, a transcription factor necessary for RGC genesis. The underlying mechanisms of this dual role of Barhl2 in mammals have remained elusive. Whole genome duplication in the teleost lineage generated the barhl1a and barhl2 paralogues. In the Zebrafish retina, Barhl2 functions as a determinant of subsets of amacrine cells lineally related to RGCs independently of Atoh7. In contrast, barhl1a expression depends on Atoh7 but its expression dynamics and function have not been studied. Here we describe for the first time a Barhl1a reporter line in vivo showing that barhl1a turns on exclusively in subsets of RGCs and their post-mitotic precursors. We also show transient expression of barhl1a:GFP in diencephalic neurons extending their axonal projections as part of the post-optic commissure, at the time of optic chiasm formation. This work sets the ground for future studies on RGC subtype identity, axonal projections and genetic specification of Barhl1a-positive RGCs and commissural neurons.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/biosíntesis , Proteínas del Tejido Nervioso/biosíntesis , Quiasma Óptico/embriología , Células Ganglionares de la Retina/metabolismo , Proteínas de Pez Cebra/biosíntesis , Pez Cebra/genética , Células Amacrinas/metabolismo , Animales , Axones/ultraestructura , Linaje de la Célula , Proteínas de Unión al ADN/fisiología , Diencéfalo/citología , Diencéfalo/embriología , Duplicación de Gen , Genes Reporteros , Proteínas de Homeodominio/genética , Microscopía Intravital , Microscopía Fluorescente , Células-Madre Neurales/metabolismo , Neuronas/metabolismo , Quiasma Óptico/citología , Células Ganglionares de la Retina/clasificación , Pez Cebra/embriología , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/fisiologíaRESUMEN
Neuronal migration is essential for constructing functional neural networks. Two posterior septal (PS) nuclei, the triangular septal nucleus and bed nuclei of the anterior commissure, are involved in fear and anxiety. During development, glutamatergic PS neurons undergo long-distance rostrodorsal migration from the thalamic eminence (TE) of the diencephalon, then settle in the caudalmost telencephalon. However, the developmental behavior of PS neurons and the guidance structures facilitating their migration remain unknown. We previously demonstrated the migration of PS neurons along the fornix, a major efferent pathway from the hippocampal formation. Here, we show that the postcommissural fornix is essential for PS neuron migration which is largely confined to its axonal tract, which grows in the opposite direction as PS neuron migration. Fornical axons reach the TE prior to initiation of PS neuron rostrodorsal migration. Ectopic expression of Semaphorin 3 A in the dorsomedial cortex resulted in defective fornix formation. Furthermore, loss of the postcommissural fornix stalled PS neuron migration resulting in abnormal accumulation near their origin. This suggests that PS neurons utilize the postcommissural fornix as a permissive corridor during migration beyond the diencephalic-telencephalic boundary. This axonal support is essential for the functional organization of the heterogeneous septal nuclear complex.
Asunto(s)
Movimiento Celular , Diencéfalo/citología , Hipocampo/fisiología , Neuronas/citología , Telencéfalo/citología , Animales , Diencéfalo/embriología , Electroporación , Femenino , Hipocampo/citología , Hipocampo/embriología , Ratones , Embarazo , Semaforina-3A/metabolismo , Telencéfalo/embriologíaRESUMEN
MARCH5 is a RING finger E3 ligase involved in mitochondrial integrity, cellular protein homeostasis, and the regulation of mitochondrial fusion and fission. To determine the function of MARCH5 during development, we assessed transcript expression in zebrafish embryos. We found that march5 transcripts were of maternal origin and evenly distributed at the 1-cell stage, except for the mid-blastula transition, with expression predominantly in the developing central nervous system at later stages of embryogenesis. Overexpression of march5 impaired convergent extension movement during gastrulation, resulting in reduced patterning along the dorsoventral axis and alterations in the ventral cell types. Overexpression and knockdown of march5 disrupted the organization of the developing telencephalon and diencephalon. Lastly, we found that the transcription of march5 was tightly regulated by the transcriptional regulators CHOP, C/EBPα, Staf, Znf143a, and Znf76. These results demonstrate the essential role of March5 in the development of zebrafish embryos.
Asunto(s)
Sistema Nervioso Central/fisiología , Diencéfalo/embriología , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Telencéfalo/embriología , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Desarrollo Embrionario , Técnicas de Silenciamiento del Gen , Células HEK293 , Homeostasis , Humanos , Proteínas de la Membrana/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación , Proteínas de Pez Cebra/genéticaRESUMEN
Recent evidence demonstrates that the pulvinar nuclei play a critical role in shaping the connectivity and function of the multiple cortical areas they connect. Surprisingly, however, little is known about the development of this area, the largest corpus of the thalamic nuclei, which go on to occupy 40% of the adult thalamus in the human. It was proposed that the nonhuman primate and the human pulvinar develop according to very different processes, with a greatly reduced neurogenic period in nonhuman primate compared to human and divergent origins. In the marmoset monkey, we demonstrate that neurons populating the pulvinar are generated throughout gestation, suggesting that this aspect of development is more similar to the human than first predicted. While we were able to confirm the diencephalic source of pulvinar neurons, we provide new evidence contesting the presence of an additional niche in the telencephalon. Finally, our study defines new molecular markers that will simplify future investigations in the development and evolution of the pulvinar.
Asunto(s)
Callithrix/fisiología , Pulvinar/crecimiento & desarrollo , Acetilcolinesterasa/metabolismo , Animales , Animales Recién Nacidos , Proliferación Celular , Diencéfalo/embriología , Diencéfalo/crecimiento & desarrollo , Femenino , Regulación de la Expresión Génica , Inmunohistoquímica , Neurogénesis/fisiología , Neuronas/fisiología , Embarazo , Pulvinar/citología , Pulvinar/embriología , Tercer Ventrículo/citología , Tercer Ventrículo/embriología , Vías Visuales/fisiologíaRESUMEN
The pituitary organizer is a domain within the ventral diencephalon that expresses Bmp4, Fgf8, and Fgf10, which induce the formation of the pituitary precursor, Rathke's pouch, from the oral ectoderm. The WNT signaling pathway regulates this pituitary organizer such that loss of Wnt5a leads to an expansion of the pituitary organizer and an enlargement of Rathke's pouch. WNT signaling is classified into canonical signaling, which is mediated by ß-CATENIN, and noncanonical signaling, which operates independently of ß-CATENIN. WNT5A is typically classified as a noncanonical WNT; however, other WNT family members are expressed in the ventral diencephalon and nuclear localized ß-CATENIN is observed in the ventral diencephalon. Therefore, we sought to determine whether canonical WNT signaling is necessary for regulation of pituitary organizer function. Using a conditional loss-of-function approach, we deleted ß-catenin within the mouse ventral diencephalon. Mutant embryos have a smaller Rathke's pouch, resulting from a reduced pituitary organizer, especially Fgf8. This result suggests that canonical WNT signaling promotes pituitary organizer function, instead of inhibiting it. To test this hypothesis, we stimulated canonical WNT signaling in the ventral diencephalon using an inducible gain-of-function allele of ß-catenin and found that stimulating canonical WNT signaling expands the domain of Fgf8 and results in a dysmorphic Rathke's pouch. These results demonstrate that canonical WNT signaling in the ventral diencephalon is necessary for proper expression of pituitary organizer genes and suggests that a balance of both canonical and noncanonical WNT signaling is necessary to ensure proper formation of Rathke's pouch.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/genética , Hipófisis/embriología , Vía de Señalización Wnt/genética , beta Catenina/genética , Animales , Proteína Morfogenética Ósea 4/metabolismo , Diencéfalo/embriología , Diencéfalo/metabolismo , Diencéfalo/patología , Factor 10 de Crecimiento de Fibroblastos/metabolismo , Factor 8 de Crecimiento de Fibroblastos/metabolismo , Hibridación in Situ , Etiquetado Corte-Fin in Situ , Ratones , Mutación , Hipófisis/metabolismo , Hipófisis/patología , Proteína Wnt-5a/genética , Proteína Wnt-5a/metabolismo , beta Catenina/metabolismoRESUMEN
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.
Asunto(s)
Tipificación del Cuerpo , Diferenciación Celular , Factor 10 de Crecimiento de Fibroblastos/metabolismo , Hipotálamo/citología , Hipotálamo/embriología , Células Madre/citología , Animales , Anisotropía , Proliferación Celular , Embrión de Pollo , Pollos , Diencéfalo/embriología , Endodermo/embriología , Proteínas Hedgehog/metabolismo , Mesodermo/embriología , Modelos Biológicos , Sistemas Neurosecretores/metabolismo , Transducción de Señal , Somitos/embriología , Somitos/metabolismo , Células Madre/metabolismo , Regulación hacia ArribaRESUMEN
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.
Asunto(s)
Axones/metabolismo , Vasos Sanguíneos/embriología , Vasos Sanguíneos/metabolismo , Sistema Nervioso Central/embriología , Sistema Nervioso Central/metabolismo , Neuropilina-1/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Tipificación del Cuerpo , Diencéfalo/embriología , Diencéfalo/metabolismo , Células Endoteliales/metabolismo , Técnicas de Silenciamiento del Gen , Proteínas de Homeodominio/metabolismo , Ratones Endogámicos C57BL , Mutación/genética , Neovascularización Fisiológica , Quiasma Óptico/embriología , Quiasma Óptico/metabolismo , Células Ganglionares de la Retina/metabolismo , Factor de Transcripción Brn-3B/metabolismo , Vías Visuales/metabolismoRESUMEN
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.
Asunto(s)
Habénula/citología , Habénula/embriología , Red Nerviosa/metabolismo , Neuronas/metabolismo , Tálamo/citología , Tálamo/embriología , Proteína 2 Similar al Factor de Transcripción 7/metabolismo , Animales , Orientación del Axón , Axones/metabolismo , Biomarcadores/metabolismo , Tipificación del Cuerpo , Diencéfalo/embriología , Diencéfalo/metabolismo , Proteínas de Homeodominio/metabolismo , Ratones , Mitosis , Mutación/genética , Unión Proteica , Células Madre/metabolismo , Transcripción GenéticaRESUMEN
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.
Asunto(s)
Diencéfalo/anatomía & histología , Diencéfalo/metabolismo , Transducción de Señal/fisiología , Tretinoina/metabolismo , Factores de Transcripción p300-CBP/metabolismo , Animales , Diencéfalo/embriología , Activación Enzimática , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Tamaño de los Órganos/fisiologíaRESUMEN
The diencephalon is the primary relay network transmitting sensory information to the anterior forebrain. During development, distinct progenitor domains in the diencephalon give rise to the pretectum (p1), the thalamus and epithalamus (p2), and the prethalamus (p3), respectively. Shh plays a significant role in establishing the progenitor domains. However, the upstream events influencing the expression of Shh are largely unknown. Here, we show that Barhl2 homeobox gene is expressed in the p1 and p2 progenitor domains and the in zona limitans intrathalamica (ZLI) and regulates the acquisition of identity of progenitor cells in the developing diencephalon. Targeted deletion of Barhl2 results in the ablation of Shh expression in the dorsal portion of ZLI and causes thalamic p2 progenitors to take the fate of p1 progenitors and form pretectal neurons. Moreover, loss of Barhl2 leads to the absence of thalamocortical axon projections, the loss of habenular afferents and efferents, and a gross diminution of the pineal gland. Thus, by acting upstream of Shh signaling pathway, Barhl2 plays a crucial role in patterning the progenitor domains and establishing the positional identities of progenitor cells in the diencephalon.
Asunto(s)
Tipificación del Cuerpo , Diencéfalo/embriología , Diencéfalo/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Axones/metabolismo , Biomarcadores/metabolismo , Corteza Cerebral/embriología , Corteza Cerebral/metabolismo , Regulación del Desarrollo de la Expresión Génica , Ratones , Tálamo/embriología , Tálamo/metabolismoRESUMEN
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.
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Cerebro/embriología , Proteínas del Ojo/genética , Haploinsuficiencia/genética , Holoprosencefalia/genética , Proteínas de Homeodominio/genética , Proteínas del Tejido Nervioso/genética , Animales , Línea Celular , Cerebro/anomalías , Diencéfalo/embriología , Diencéfalo/metabolismo , Ectodermo/metabolismo , Factores de Transcripción Forkhead/biosíntesis , Células HEK293 , Proteínas Hedgehog/biosíntesis , Proteínas Hedgehog/metabolismo , Holoprosencefalia/patología , Humanos , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/biosíntesis , Transducción de Señal/fisiología , Proteína Homeobox SIX3RESUMEN
BACKGROUND: Cerebrospinal fluid (CSF) contained within the brain ventricles contacts neuroepithelial progenitor cells during brain development. Dynamic properties of CSF movement may limit locally produced factors to specific regions of the developing brain. However, there is no study of in vivo CSF dynamics between ventricles in the embryonic brain. We address CSF movement using the zebrafish larva, during the major period of developmental neurogenesis. METHODS: CSF movement was monitored at two stages of zebrafish development: early larva [pharyngula stage; 27-30 h post-fertilization (hpf)] and late larva (hatching period; 51-54 hpf) using photoactivatable Kaede protein to calculate average maximum CSF velocity between ventricles. Potential roles for heartbeat in early CSF movement were investigated using tnnt2a mutant fish (tnnt2a (-/-)) and chemical [2,3 butanedione monoxime (BDM)] treatment. Cilia motility was monitored at these stages using the Tg(ßact:Arl13b-GFP) transgenic fish line. RESULTS: In wild-type early larva there is net CSF movement from the telencephalon to the combined diencephalic/mesencephalic superventricle. This movement directionality reverses at late larval stage. CSF moves directionally from diencephalic to rhombencephalic ventricles at both stages examined, with minimal movement from rhombencephalon to diencephalon. Directional movement is partially dependent on heartbeat, as indicated in assays of tnnt2a (-/-) fish and after BDM treatment. Brain cilia are immotile at the early larval stage. CONCLUSION: These data demonstrate directional movement of the embryonic CSF in the zebrafish model during the major period of developmental neurogenesis. A key conclusion is that CSF moves preferentially from the diencephalic into the rhombencephalic ventricle. In addition, the direction of CSF movement between telencephalic and diencephalic ventricles reverses between the early and late larval stages. CSF movement is partially dependent on heartbeat. At early larval stage, the absence of motile cilia indicates that cilia likely do not direct CSF movement. These data suggest that CSF components may be compartmentalized and could contribute to specialization of the early brain. In addition, CSF movement may also provide directional mechanical signaling.
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Ventrículos Cerebrales/embriología , Ventrículos Cerebrales/fisiología , Líquido Cefalorraquídeo/metabolismo , Pez Cebra/embriología , Pez Cebra/fisiología , Animales , Animales Modificados Genéticamente , Cilios/fisiología , Diencéfalo/embriología , Diencéfalo/fisiología , Corazón/embriología , Corazón/fisiología , Hidrodinámica , Microscopía Confocal , Movimiento , Rombencéfalo/embriología , Telencéfalo/embriología , Telencéfalo/fisiología , Troponina T/genética , Troponina T/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
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.
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Anomalías Craneofaciales/embriología , Proteínas Hedgehog/fisiología , Desarrollo Maxilofacial/fisiología , Receptores Patched/fisiología , Transducción de Señal , Animales , Movimiento Celular , Cilios/fisiología , Ciliopatías/embriología , Ciliopatías/genética , Ciliopatías/fisiopatología , Anomalías Craneofaciales/genética , Anomalías Craneofaciales/fisiopatología , Diencéfalo/embriología , Modelos Animales de Enfermedad , Ectodermo/embriología , Endodermo/embriología , Cara/anomalías , Cara/embriología , Regulación del Desarrollo de la Expresión Génica , Holoprosencefalia/embriología , Holoprosencefalia/genética , Holoprosencefalia/fisiopatología , Humanos , Desarrollo Maxilofacial/genética , Proteínas de la Membrana/fisiología , Cresta Neural/citología , Cresta Neural/embriología , Receptores Patched/genética , Transducción de Señal/genética , Cráneo/anomalías , Cráneo/embriologíaRESUMEN
The diencephalon is a complex brain area that derives from the caudal region of the prosencephalon. This structure is divided into four longitudinal neuroepithelial zones: roof, alar, basal and floor plates, which constitute its dorso-ventral (DV) columnar domains. Morphogenetic differences between alar and basal plates in the prosencephalon and mesencephalon contribute to the characteristic expansion of alar plate derivatives in the brain and the formation of the cephalic flexure. Although differential histogenesis among DV regions seems to be relevant in understanding structural and functional complexity of the brain, most of our knowledge about DV regionalization comes from the spinal cord development. Therefore, it seems of interest to study the molecular mechanisms that govern DV patterning in the diencephalon, the brain region where strong differences in size and complexity between alar and basal derivatives are evident in all vertebrates. Different morphogenetic signals, which induce specific progenitors fate to the neighboring epithelium, are involved in the spinal cord DV patterning. To study if Wnt1, one of these signaling molecules, has a role for the establishment of the diencephalic longitudinal domains, we carried out gain- and loss-of-function experiments, using mice and chick embryos. Our results demonstrated functional differences in the molecular mechanisms downstream of Wnt1 function in the diencephalon, in relation to the spinal cord. We further demonstrated that Bmp4 signal induces Wnt1 expression in the diencephalon, unraveling a new molecular regulatory code downstream of primary dorsalizing signals to control ventral regionalization in the diencephalon.
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Tipificación del Cuerpo , Diencéfalo/embriología , Diencéfalo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Vía de Señalización Wnt , Proteína Wnt1/metabolismo , Animales , Proteína Morfogenética Ósea 4/metabolismo , Pollos , Glicosiltransferasas/metabolismo , Proteínas Hedgehog , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/metabolismo , Proteína Wnt1/genética , Proteína Gli3 con Dedos de ZincRESUMEN
EphA7 is a key molecule in regulating the development of the dien- and mesencephalon. To get insight into the mechanism of how EphA7 gene expression is regulated during the dorsal specification of the dien- and mesencephalon, we investigated the cis-acting regulatory sequence driving EphA7 to the dorsal midline of the dien- and mesencephalon. Transgenic LacZ reporter analysis, using overlapping EphA7 BACs, was used to narrow down the dorsal midline-specific enhancer, revealing the 25.3 kb genomic region as the enhancer candidate. Strikingly, this genomic DNA was located far downstream of the EphA7 transcription start site, +302.6 kb to +327.9 kb. Further enhancer mapping, using comparative genomic analysis and transgenic methods, showed that the 187 bp genomic DNA alone, approximately 305 kb downstream of the EphA7 transcription start site, was sufficient to act as the dorsal midline-specific enhancer of EphA7. Importantly, our results indicate that the 187 bp dorsal midline-specific enhancer is critically regulated by homeobox transcription factors during the development of the dien- and mesencephalon.