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Seemingly simple behaviors such as swatting a mosquito or glancing at a signpost involve the precise coordination of multiple body parts. Neural control of coordinated movements is widely thought to entail transforming a desired overall displacement into displacements for each body part. Here we reveal a different logic implemented in the mouse gaze system. Stimulating superior colliculus (SC) elicits head movements with stereotyped displacements but eye movements with stereotyped endpoints. This is achieved by individual SC neurons whose branched axons innervate modules in medulla and pons that drive head movements with stereotyped displacements and eye movements with stereotyped endpoints, respectively. Thus, single neurons specify a mixture of endpoints and displacements for different body parts, not overall displacement, with displacements for different body parts computed at distinct anatomical stages. Our study establishes an approach for unraveling motor hierarchies and identifies a logic for coordinating movements and the resulting pose.
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Fixação Ocular , Movimentos Sacádicos , Animais , Camundongos , Movimentos Oculares , Neurônios/fisiologia , Colículos Superiores/fisiologia , Rombencéfalo , Movimentos da Cabeça/fisiologiaRESUMO
Posterior fossa A (PFA) ependymomas are lethal malignancies of the hindbrain in infants and toddlers. Lacking highly recurrent somatic mutations, PFA ependymomas are proposed to be epigenetically driven tumors for which model systems are lacking. Here we demonstrate that PFA ependymomas are maintained under hypoxia, associated with restricted availability of specific metabolites to diminish histone methylation, and increase histone demethylation and acetylation at histone 3 lysine 27 (H3K27). PFA ependymomas initiate from a cell lineage in the first trimester of human development that resides in restricted oxygen. Unlike other ependymomas, transient exposure of PFA cells to ambient oxygen induces irreversible cellular toxicity. PFA tumors exhibit a low basal level of H3K27me3, and, paradoxically, inhibition of H3K27 methylation specifically disrupts PFA tumor growth. Targeting metabolism and/or the epigenome presents a unique opportunity for rational therapy for infants with PFA ependymoma.
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Ependimoma/genética , Ependimoma/metabolismo , Epigenoma/genética , Neoplasias Infratentoriais/genética , Neoplasias Infratentoriais/metabolismo , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Linhagem Celular , Proliferação de Células/genética , Metilação de DNA/genética , Epigenômica/métodos , Histonas/genética , Histonas/metabolismo , Humanos , Lactente , Lisina/genética , Lisina/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Mutação/genéticaRESUMO
The genetic approach, based on the study of inherited forms of deafness, has proven to be particularly effective for deciphering the molecular mechanisms underlying the development of the peripheral auditory system, the cochlea and its afferent auditory neurons, and how this system extracts the physical parameters of sound. Although this genetic dissection has provided little information about the central auditory system, scattered data suggest that some genes may have a critical role in both the peripheral and central auditory systems. Here, we review the genes controlling the development and function of the peripheral and central auditory systems, focusing on those with demonstrated intrinsic roles in both systems and highlighting the current underappreciation of these genes. Their encoded products are diverse, from transcription factors to ion channels, as are their roles in the central auditory system, mostly evaluated in brainstem nuclei. We examine the ontogenetic and evolutionary mechanisms that may underlie their expression at different sites.
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Vias Auditivas/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Genes , Neurogênese/genética , Animais , Vias Auditivas/crescimento & desenvolvimento , Evolução Biológica , Cóclea/embriologia , Cóclea/crescimento & desenvolvimento , Cóclea/fisiologia , Ontologia Genética , Células Ciliadas Auditivas/citologia , Células Ciliadas Auditivas/fisiologia , Transtornos da Audição/genética , Humanos , Canais Iônicos/genética , Canais Iônicos/fisiologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Rombencéfalo/embriologia , Rombencéfalo/crescimento & desenvolvimento , Rombencéfalo/fisiologia , Células Receptoras Sensoriais/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologiaRESUMO
The interplay between neural progenitors and stem cells (NPSCs), and their extracellular matrix (ECM) is a crucial regulatory mechanism that determines their behavior. Nonetheless, how the ECM dictates the state of NPSCs remains elusive. The hindbrain is valuable to examine this relationship, as cells in the ventricular surface of hindbrain boundaries (HBs), which arise between any two neighboring rhombomeres, express the NPSC marker Sox2, while being surrounded with the membrane-bound ECM molecule chondroitin sulphate proteoglycan (CSPG), in chick and mouse embryos. CSPG expression was used to isolate HB Sox2+ cells for RNA-sequencing, revealing their distinguished molecular properties as typical NPSCs, which express known and newly identified genes relating to stem cells, cancer, the matrisome and cell cycle. In contrast, the CSPG- non-HB cells, displayed clear neural-differentiation transcriptome. To address whether CSPG is significant for hindbrain development, its expression was manipulated in vivo and in vitro. CSPG manipulations shifted the stem versus differentiation state of HB cells, evident by their behavior and altered gene expression. These results provide further understanding of the uniqueness of hindbrain boundaries as repetitive pools of NPSCs in-between the rapidly growing rhombomeres, which rely on their microenvironment to maintain their undifferentiated state during development.
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Células-Tronco Neurais , Proteoglicanas , Camundongos , Animais , Proteoglicanas/metabolismo , Sulfatos de Condroitina , Proteoglicanas de Sulfatos de Condroitina , Matriz Extracelular/metabolismo , Rombencéfalo/metabolismo , Células-Tronco Neurais/metabolismoRESUMO
Developing tissues are sequentially patterned by extracellular signals that are turned on and off at specific times. In the zebrafish hindbrain, fibroblast growth factor (Fgf) signalling has different roles at different developmental stages: in the early hindbrain, transient Fgf3 and Fgf8 signalling from rhombomere 4 is required for correct segmentation, whereas later, neuronal Fgf20 expression confines neurogenesis to specific spatial domains within each rhombomere. How the switch between these two signalling regimes is coordinated is not known. We present evidence that the Zbtb16 transcription factor is required for this transition to happen in an orderly fashion. Zbtb16 expression is high in the early anterior hindbrain, then gradually upregulated posteriorly and confined to neural progenitors. In mutants lacking functional Zbtb16, fgf3 expression fails to be downregulated and persists until a late stage, resulting in excess and more widespread Fgf signalling during neurogenesis. Accordingly, the spatial pattern of neurogenesis is disrupted in Zbtb16 mutants. Our results reveal how the distinct stage-specific roles of Fgf signalling are coordinated in the zebrafish hindbrain.
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Neurogênese , Peixe-Zebra , Animais , Neurogênese/genética , Rombencéfalo , Transdução de Sinais/genética , Fatores de Transcrição/genética , Peixe-Zebra/genéticaRESUMO
In the developing hindbrain, facial branchiomotor (FBM) neurons migrate caudally from rhombomere 4 (r4) to r6 to establish the circuit that drives jaw movements. Although the mechanisms regulating initiation of FBM neuron migration are well defined, those regulating directionality are not. In mutants lacking the Wnt/planar cell polarity (PCP) component Celsr1, many FBM neurons inappropriately migrate rostrally into r3. We hypothesized that Celsr1 normally blocks inappropriate rostral migration of FBM neurons by suppressing chemoattraction towards Wnt5a in r3 and successfully tested this model. First, FBM neurons in Celsr1; Wnt5a double mutant embryos never migrated rostrally, indicating that inappropriate rostral migration in Celsr1 mutants results from Wnt5a-mediated chemoattraction, which is suppressed in wild-type embryos. Second, FBM neurons migrated rostrally toward Wnt5a-coated beads placed in r3 of wild-type hindbrain explants, suggesting that excess Wnt5a chemoattractant can overcome endogenous Celsr1-mediated suppression. Third, rostral migration of FBM neurons was greatly enhanced in Celsr1 mutants overexpressing Wnt5a in r3. These results reveal a novel role for a Wnt/PCP component in regulating neuronal migration through suppression of chemoattraction.
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Regulação da Expressão Gênica no Desenvolvimento , Neurônios Motores , Neurônios Motores/fisiologia , Rombencéfalo , Polaridade Celular , Movimento Celular/genéticaRESUMO
BACKGROUND: The brain and spinal cord formation is initiated in the earliest stages of mammalian pregnancy in a highly organized process known as neurulation. Environmental or genetic interferences can impair neurulation, resulting in clinically significant birth defects known collectively as neural tube defects. The Fuz gene encodes a subunit of the CPLANE complex, a macromolecular planar polarity effector required for ciliogenesis. Ablation of Fuz in mouse embryos results in exencephaly and spina bifida, including dysmorphic craniofacial structures due to defective cilia formation and impaired Sonic Hedgehog signaling. RESULTS: We demonstrate that knocking Fuz out during embryonic mouse development results in a hypoplastic hindbrain phenotype, displaying abnormal rhombomeres with reduced length and width. This phenotype is associated with persistent reduction of ventral neuroepithelial stiffness in a notochord adjacent area at the level of the rhombomere 5. The formation of cranial and paravertebral ganglia is also impaired in these embryos. CONCLUSIONS: This study reveals that hypoplastic hindbrain development, identified by abnormal rhombomere morphology and persistent loss of ventral neuroepithelial stiffness, precedes exencephaly in Fuz ablated murine mutants, indicating that the gene Fuz has a critical function sustaining normal neural tube development and neuronal differentiation.
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During early development, the hindbrain is sub-divided into rhombomeres that underlie the organisation of neurons and adjacent craniofacial tissues. A gene regulatory network of signals and transcription factors establish and pattern segments with a distinct anteroposterior identity. Initially, the borders of segmental gene expression are imprecise, but then become sharply defined, and specialised boundary cells form. In this Review, we summarise key aspects of the conserved regulatory cascade that underlies the formation of hindbrain segments. We describe how the pattern is sharpened and stabilised through the dynamic regulation of cell identity, acting in parallel with cell segregation. Finally, we discuss evidence that boundary cells have roles in local patterning, and act as a site of neurogenesis within the hindbrain.
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Padronização Corporal/fisiologia , Rombencéfalo/crescimento & desenvolvimento , Rombencéfalo/fisiologia , Vertebrados/crescimento & desenvolvimento , Vertebrados/fisiologia , Animais , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Redes Reguladoras de Genes/fisiologia , HumanosRESUMO
OBJECTIVE: Compared with postnatal repair, prenatal myelomeningocele (MMC) repair is associated with improved motor function and decreased need for cerebrospinal fluid (CSF) diversion. It is unknown how prenatal surgery alters neuroanatomic structures identifiable on magnetic resonance imaging (MRI). The purpose of this study was to use MRI to compare neurodevelopmental anatomy in patients undergoing fetal MMC repair compared with those undergoing postnatal repair. METHODS: This was a retrospective review of neonates who underwent prenatal or postnatal MMC repair at our institution between 2016 and 2021. Imaging data, including prenatal ultrasound and pre- and postnatal MRI examinations, if available, were retrieved. We analyzed anatomical findings characteristically seen on MRI of the neuroaxis in patients with MMC and compared imaging findings between patients with prenatal vs postnatal MMC repair. RESULTS: The study population included 61 patients who underwent surgical repair for MMC during the 6-year study period, of whom 25 underwent prenatal repair and 36 postnatal repair. CSF diversion was required in 24% of the prenatally repaired cohort vs 67% of the postnatally repaired cohort (P = 0.001). On postnatal MRI, a syrinx was present in 13% of the prenatally repaired cohort vs 42% in the postnatally repaired cohort (P = 0.02). Postnatal corpus callosal (CC) morphology was abnormal in 54% of the prenatally repaired cohort vs 53% of the postnatally repaired cohort (P = 0.92), while falx morphology was normal in 92% of the prenatally repaired cohort vs 34% of the postnatally repaired cohort (P < 0.001). On postnatal MRI, patients in the prenatally repaired cohort had a shorter tentorium-to-foramen-magnum distance compared with those in the postnatally repaired cohort (mean, 18.43 mm vs 22.42 mm; P = 0.01), a larger foramen magnum diameter (mean, 22.87 mm vs 18.94 mm; P < 0.001) and a smaller degree of hindbrain herniation (mean, 1.53 mm vs 8.72 mm; P < 0.001). The cerebral aqueduct was patent in 79% of the prenatally repaired cohort vs 100% of the postnatally repaired cohort on postnatal MRI (P = 0.008). Between the two cohorts, at postnatal MRI there was no significant difference in the presence of gray-matter heterotopia, presence of the septum pellucidum or size of the massa intermedia. CONCLUSIONS: We report variations in developmental neuroanatomy in patients with MMC, including rates of CC dysgenesis, gray-matter heterotopia and additional cranial and spinal MRI findings. Compared to postnatal surgery, prenatal surgery is associated with changes to infratentorial anatomy, with minimal effect on supratentorial brain development. This information will be useful in counseling parents affected by fetal MMC and in understanding how prenatal repair of MMC affects brain development. © 2024 International Society of Ultrasound in Obstetrics and Gynecology.
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BACKGROUND: Chiari malformation type 1 (CM1) is a congenital hindbrain malformation characterized by herniation of the cerebellar tonsils below the foramen magnum. The term Chiari type 1.5 is used when herniation of the brainstem under the McRae line and anomalies of the craniovertebral junction are also present. These conditions are associated with several symptoms and signs, including headache, neck pain, and spinal cord syndrome. For symptomatic patients, surgical decompression is recommended. When radiographic indicators of craniovertebral junction (CVJ) instability or symptoms related to ventral brainstem compression are present, CVJ fixation should also be considered. CASE DESCRIPTION: We report the case of a 13-year-old girl who presented with severe tetraparesis after posterior decompression for Chiari malformation type 1.5, followed 5 days later by partial C2 laminectomy. Several months after the initial surgery, she underwent two fixations, first without and then with intraoperative cervical traction, leading to significant neurological improvement. DISCUSSION AND CONCLUSION: This case report underscores the importance of meticulous radiological analysis before CM surgery. For CM 1.5 patients with basilar invagination, CVJ fixation is recommended, and C2 laminectomy should be avoided. In the event of significant clinical deterioration due to nonadherence to these guidelines, our findings highlight the importance of traction with increased extension before fixation, even years after initial destabilizing surgery.
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Malformação de Arnold-Chiari , Descompressão Cirúrgica , Quadriplegia , Tração , Humanos , Feminino , Malformação de Arnold-Chiari/cirurgia , Malformação de Arnold-Chiari/complicações , Malformação de Arnold-Chiari/diagnóstico por imagem , Adolescente , Descompressão Cirúrgica/métodos , Descompressão Cirúrgica/efeitos adversos , Quadriplegia/etiologia , Quadriplegia/cirurgia , Tração/efeitos adversos , Tração/métodos , Resultado do TratamentoRESUMO
BACKGROUND: Chiari malformation type III (CM III), a rare hindbrain anomaly, often presents with various concurrent anomalies. This paper reports a unique case of CM III associated with Klippel-Feil syndrome (KFS), a condition previously unreported in Saudi Arabia and documented in only one other case globally in Turkey. This study aims to share insights into the unusual association between CM III and KFS, considering their close embryological development and involvement in the craniocervical junction. METHODOLOGY: The study presents a case of a 2.5-year-old female diagnosed with CM III and KFS. Diagnostic tools such as ultrasound, CT scans, MRI, and physical examinations were used to confirm the patient's condition. Surgical interventions, including decompression and encephalocele repair, were performed. RESULTS: Successful surgical interventions, including encephalocele repair and duraplasty, were carried out. Follow-up visits indicated a stable condition, marked improvement in lower limb strength, and the patient's ability to walk with assistance. CT follow-up affirmed a satisfactory surgical outcome. CONCLUSION: This case study illustrates the potential for an optimistic prognosis in CM III, even when accompanied by complex conditions such as KFS, through early diagnosis and intervention. It underscores the significance of antenatal screening for effective care planning and calls for further research and publications due to the rarity of this association. These findings contribute to our understanding of CM III and its related conditions, emphasizing the need for open-minded consideration of potential embryological associations.
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Malformação de Arnold-Chiari , Síndrome de Klippel-Feil , Gravidez , Humanos , Feminino , Pré-Escolar , Síndrome de Klippel-Feil/complicações , Síndrome de Klippel-Feil/diagnóstico por imagem , Síndrome de Klippel-Feil/cirurgia , Encefalocele , Malformação de Arnold-Chiari/complicações , Malformação de Arnold-Chiari/diagnóstico por imagem , Malformação de Arnold-Chiari/cirurgia , Tomografia Computadorizada por Raios X , Imageamento por Ressonância MagnéticaRESUMO
Gap closure is a common morphogenetic process. In mammals, failure to close the embryonic hindbrain neuropore (HNP) gap causes fatal anencephaly. We observed that surface ectoderm cells surrounding the mouse HNP assemble high-tension actomyosin purse strings at their leading edge and establish the initial contacts across the embryonic midline. Fibronectin and laminin are present, and tensin 1 accumulates in focal adhesion-like puncta at this leading edge. The HNP gap closes asymmetrically, faster from its rostral than caudal end, while maintaining an elongated aspect ratio. Cell-based physical modeling identifies two closure mechanisms sufficient to account for tissue-level HNP closure dynamics: purse-string contraction and directional cell motion implemented through active crawling. Combining both closure mechanisms hastens gap closure and produces a constant rate of gap shortening. Purse-string contraction reduces, whereas crawling increases gap aspect ratio, and their combination maintains it. Closure rate asymmetry can be explained by asymmetric embryo tissue geometry, namely a narrower rostral gap apex, whereas biomechanical tension inferred from laser ablation is equivalent at the gaps' rostral and caudal closure points. At the cellular level, the physical model predicts rearrangements of cells at the HNP rostral and caudal extremes as the gap shortens. These behaviors are reproducibly live imaged in mouse embryos. Thus, mammalian embryos coordinate cellular- and tissue-level mechanics to achieve this critical gap closure event.
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Embrião de Mamíferos/metabolismo , Crista Neural/metabolismo , Tubo Neural/metabolismo , Rombencéfalo/metabolismo , Anencefalia/embriologia , Anencefalia/genética , Anencefalia/metabolismo , Animais , Caderinas/metabolismo , Embrião de Mamíferos/embriologia , Feminino , Fibronectinas/metabolismo , Laminina/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal/métodos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Crista Neural/embriologia , Tubo Neural/embriologia , Rombencéfalo/embriologia , Imagem com Lapso de Tempo/métodosRESUMO
Though initially discovered as a proto-oncogene in virally induced mouse mammary tumors, FGF3 is primarily active in prenatal stages, where it is found at various sites at specific times. FGF3 is crucial during development, as its roles include tail formation, inner ear development and hindbrain induction and patterning. FGF3 expression and function are highly conserved in vertebrates, while it also interacts with other FGFs in various developmental processes. Intriguingly, while it is classified as a classical paracrine signaling factor, murine FGF3 was uniquely found to also act in an intracrine manner, depending on alternative translation initiation sites. Corresponding with its conserved role in inner ear morphogenesis, mutations in FGF3 in humans are associated with LAMM syndrome, a disorder that include hearing loss and inner ear malformations. While recent studies indicate of some FGF3 presence in post-natal stages, emerging evidences of its upregulation in various human tumors and cariogenic processes in mouse models, highlights the importance of its close regulation in adult tissues. Altogether, the broad and dynamic expression pattern and regulation of FGF3 in embryonic and adult tissues together with its link to congenital malformations and cancer, calls for further discoveries of its diverse roles in health and disease.
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INTRODUCTION: Reduced middle cerebral artery resistance indices (MCA-RI) in fetuses with spina bifida (fSB) are commonly observed. Compression of neuronal pathways in the brainstem due to hindbrain herniation (HH) and disturbed cerebrospinal fluid circulation likely cause an imbalance of the autonomic nervous system. This may increase systemic vasoconstriction and compensatory increase cerebral vasodilation (like brain sparing). The aim of this study was to systematically analyze all fetal MCA-RI before and after fSB repair and to compare their correlation with the presence and postsurgical resolution of HH. METHODS: 173 patients were included. Standardized ultrasound examinations including MCA and umbilical artery (UA) Doppler as well as assessment of HH presence and regression were performed. Fetuses with MCA-RI <5th percentile (P) before fetal surgery were compared to the group with normal MCA-RI and correlated to the presence of HH before and its regression after fSB repair. RESULTS: 30% (49/161) fetuses showed RI's <5th P before fSB repair. All fetuses had normal UA-RI. 99.4% of fetuses (160/161) showed normal of MCA-RI before delivery. Normalization occurred within a mean of 1.3 ± 1.2 weeks. HH regression was observed in 97% in the group with normal MCA-RI and in 96% in the group with MCA-RI <5th P before surgery (p = 0.59). Time lapse to HH regression after fSB repair was 1.8 ± 1.7 and 1.9 ± 1.6 weeks, respectively. CONCLUSION: In fetuses with MCA-RIs <5 P before fSB repair, a parallel timely course of MCA-RI normalization and HH regression was noted. To suggest common pathogenic factor(s), more studies are needed. However, normalization of the fetal cerebral circulation could be a further benefit of fSB repair.
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Activity in the dorsal vagal complex (DVC) is essential to gastric motility regulation. We and others have previously shown that this activity is greatly influenced by local GABAergic signaling, primarily because of somatostatin (SST)-expressing GABAergic neurons. To further understand the network dynamics associated with gastric motility control in the DVC, we focused on another neuron prominently distributed in this complex, neuropeptide-Y (NPY) neurons. However, the effect of these neurons on gastric motility remains unknown. Here, we investigate the anatomic and functional characteristics of the NPY neurons in the nucleus tractus solitarius (NTS) and their interactions with SST neurons using transgenic mice of both sexes. We sought to determine whether NPY neurons influence the activity of gastric-projecting neurons, synaptically interact with SST neurons, and affect end-organ function. Our results using combined neuroanatomy and optogenetic in vitro and in vivo show that NPY neurons are part of the gastric vagal circuit as they are trans-synaptically labeled by a viral tracer from the gastric antrum, are primarily excitatory as optogenetic activation of these neurons evoke EPSCs in gastric-antrum-projecting neurons, are functionally coupled to each other and reciprocally connected to SST neurons, whose stimulation has a potent inhibitory effect on the action potential firing of the NPY neurons, and affect gastric tone and motility as reflected by their robust optogenetic response in vivo. These findings indicate that interacting NPY and SST neurons are integral to the network that controls vagal transmission to the stomach.SIGNIFICANCE STATEMENT The brainstem neurons in the dorsal nuclear complex are essential for regulating vagus nerve activity that affects the stomach via tone and motility. Two distinct nonoverlapping populations of predominantly excitatory NPY neurons and predominantly inhibitory SST neurons form reciprocal connections with each other in the NTS and with premotor neurons in the dorsal motor nucleus of the vagus to control gastric mechanics. Light activation and inhibition of NTS NPY neurons increased and decreased gastric motility, respectively, whereas both activation and inhibition of NTS SST neurons enhanced gastric motility.
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Tronco Encefálico , Estômago , Animais , Tronco Encefálico/fisiologia , Feminino , Neurônios GABAérgicos/fisiologia , Masculino , Camundongos , Neuropeptídeo Y/farmacologia , Ratos , Ratos Sprague-Dawley , Núcleo Solitário/fisiologia , Estômago/inervação , Nervo Vago/fisiologiaRESUMO
The process of morphogenesis carefully crafts cells into complex organ structures which allows them to perform their unique functions. During brain development, the neuroepithelial tissue must go through apical and basal folding which is mediated through the instruction of both intrinsic and extrinsic factors. While much is known about apical folding, the mechanisms that regulate basal folding are less understood. Using the highly conserved zebrafish midbrain-hindbrain boundary (MHB) as an epithelial tissue model, we have identified the basement membrane protein laminin-111 as a key extrinsic factor in basal tissue folding. Laminin-111 is a highly conserved, heterotrimeric protein that lines the basal surface of the neuroepithelium. Laminin-111 is comprised of one alpha, one beta, and one gamma chain encoded by the genes lama1, lamb1, and lamc1, respectively. Human mutations in individual laminin-111 genes result in disparate disease phenotypes; therefore, we hypothesized that each laminin gene would have a distinctive role in tissue morphogenesis. Using zebrafish mutants for laminin-111 genes, we found that each laminin chain has a unique impact on basal folding. We found that lamc1 is the most critical gene for MHB morphogenesis, followed by lama1, and finally lamb1a. This hierarchy was discovered via three-dimensional single cell shape analysis, localization of myosin regulatory light chain (MRLC), and with analysis of MHB tissue folding later in development. These findings are essential for development of novel techniques in tissue engineering and to elucidate differences in human diseases due to specific chain mutations.
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Laminina , Peixe-Zebra , Animais , Humanos , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Laminina/genética , Laminina/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Morfogênese/genética , Epitélio/metabolismoRESUMO
Segmentation of the vertebrate hindbrain leads to the formation of rhombomeres, each with a distinct anteroposterior identity. Specialised boundary cells form at segment borders that act as a source or regulator of neuronal differentiation. In zebrafish, there is spatial patterning of neurogenesis in which non-neurogenic zones form at boundaries and segment centres, in part mediated by Fgf20 signalling. To further understand the control of neurogenesis, we have carried out single cell RNA sequencing of the zebrafish hindbrain at three different stages of patterning. Analyses of the data reveal known and novel markers of distinct hindbrain segments, of cell types along the dorsoventral axis, and of the transition of progenitors to neuronal differentiation. We find major shifts in the transcriptome of progenitors and of differentiating cells between the different stages analysed. Supervised clustering with markers of boundary cells and segment centres, together with RNA-seq analysis of Fgf-regulated genes, has revealed new candidate regulators of cell differentiation in the hindbrain. These data provide a valuable resource for functional investigations of the patterning of neurogenesis and the transition of progenitors to neuronal differentiation.
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Padronização Corporal/genética , Rombencéfalo/embriologia , Rombencéfalo/metabolismo , Transcriptoma/fisiologia , Peixe-Zebra , Animais , Animais Geneticamente Modificados , Atlas como Assunto , Diferenciação Celular/genética , Embrião não Mamífero , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurogênese/genética , Neurônios/citologia , Neurônios/fisiologia , Análise de Célula Única/métodos , Distribuição Tecidual , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
The formation and maintenance of sharp boundaries between groups of cells play a vital role during embryonic development as they serve to compartmentalize cells with similar fates. Some of these boundaries also act as organizers, with the ability to induce specific cell fates and morphogenesis in the surrounding cells. The midbrain-hindbrain boundary (MHB) is such an organizer: it acts as a lineage restriction boundary to prevent the intermingling of cells with different developmental fates. However, the mechanisms underlying the lineage restriction process remain unclear. Here, using novel fluorescent knock-in reporters, live imaging, Cre/lox-mediated lineage tracing, atomic force microscopy-based cell adhesion assays and mutant analysis, we analyze the process of lineage restriction at the MHB and provide mechanistic details. Specifically, we show that lineage restriction occurs by the end of gastrulation, and that the subsequent formation of sharp gene expression boundaries in the developing MHB occur through complementary mechanisms, i.e. cell-fate plasticity and cell sorting. Furthermore, we show that cell sorting at the MHB involves differential adhesion among midbrain and hindbrain cells that is mediated by N-cadherin and Eph-ephrin signaling.
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Adesão Celular/fisiologia , Mesencéfalo/metabolismo , Rombencéfalo/metabolismo , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados/crescimento & desenvolvimento , Animais Geneticamente Modificados/metabolismo , Sistemas CRISPR-Cas/genética , Caderinas/genética , Caderinas/metabolismo , Linhagem da Célula , Embrião não Mamífero/metabolismo , Efrinas/antagonistas & inibidores , Efrinas/genética , Efrinas/metabolismo , Gastrulação , Edição de Genes , Mesencéfalo/patologia , Microscopia de Força Atômica , Microscopia de Fluorescência , Morfolinos/metabolismo , Fatores de Transcrição Otx/genética , Fatores de Transcrição Otx/metabolismo , Rombencéfalo/patologia , Transdução de Sinais , Imagem com Lapso de Tempo , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
The choroid plexus (ChP) is a secretory tissue that produces cerebrospinal fluid (CSF) secreted into the ventricular system. It is a monolayer of secretory, multiciliated epithelial cells derived from neuroepithelial progenitors and overlying a stroma of mesenchymal cells of mesodermal origin. Zfp423, which encodes a Kruppel-type zinc-finger transcription factor essential for cerebellar development and mutated in rare cases of cerebellar vermis hypoplasia/Joubert syndrome and other ciliopathies, is expressed in the hindbrain roof plate, from which the IV ventricle ChP arises, and, later, in mesenchymal cells, which give rise to the stroma and leptomeninges. Mouse Zfp423 mutants display a marked reduction of the hindbrain ChP (hChP), which: (1) fails to express established markers of its secretory function and genes implicated in its development and maintenance (Lmx1a and Otx2); (2) shows a perturbed expression of signaling pathways previously unexplored in hChP patterning (Wnt3); and (3) displays a lack of multiciliated epithelial cells and a profound dysregulation of master genes of multiciliogenesis (Gmnc). Our results propose that Zfp423 is a master gene and one of the earliest known determinants of hChP development.
Assuntos
Plexo Corióideo/embriologia , Proteínas de Ligação a DNA/metabolismo , Rombencéfalo/embriologia , Fatores de Transcrição/metabolismo , Animais , Plexo Corióideo/citologia , Proteínas de Ligação a DNA/genética , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Camundongos , Camundongos Mutantes , Fatores de Transcrição Otx/genética , Fatores de Transcrição Otx/metabolismo , Rombencéfalo/citologia , Fatores de Transcrição/genética , Proteína Wnt3/genética , Proteína Wnt3/metabolismoRESUMO
Hox genes play key roles in the anterior-posterior (AP) specification of all 3 germ layers during different developmental stages. It is only partially understood how they function in widely different developmental contexts, particularly with regards to extracellular signaling, and to what extent their function can be harnessed to guide cell specification in vitro. Here, we addressed the role of Hoxb1 in 2 distinct developmental contexts; in mouse embryonic stem cells (mES)-derived neuromesodermal progenitors (NMPs) and hindbrain neural progenitors. We found that Hoxb1 promotes NMP survival through the upregulation of Fgf8, Fgf17, and other components of Fgf signaling as well as the repression of components of the apoptotic pathway. Additionally, it upregulates other anterior Hox genes suggesting that it plays an active role in the early steps of AP specification. In neural progenitors, Hoxb1 synergizes with shh to repress anterior and dorsal neural markers, promote the expression of ventral neural markers and direct the specification of facial branchiomotorneuron (FBM)-like progenitors. Hoxb1 and shh synergize in regulating the expression of diverse signals and signaling molecules, including the Ret tyrosine kinase receptor. Finally, Hoxb1 synergizes with exogenous Glial cell line-derived neurotrophic factor (GDNF) to strengthen Ret expression and further promote the generation of FBM-like progenitors. Facial branchiomotorneuron-like progenitors survived for at least 6 months and differentiated into postmitotic neurons after orthotopic transplantation near the facial nucleus of adult mice. These results suggested that the patterning activity of Hox genes in combination with downstream signaling molecules can be harnessed for the generation of defined neural populations and transplantations with implications for neurodegenerative diseases.