RESUMEN
The neural crest is regionalized along the anteroposterior axis, as demonstrated by foundational lineage-tracing experiments that showed the restricted developmental potential of neural crest cells originating in the head. Here, we explore how recent studies of experimental embryology, genetic circuits and stem cell differentiation have shaped our understanding of the mechanisms that establish axial-specific populations of neural crest cells. Additionally, we evaluate how comparative, anatomical and genomic approaches have informed our current understanding of the evolution of the neural crest and its contribution to the vertebrate body.
Asunto(s)
Tipificación del Cuerpo , Cabeza/embriología , Cresta Neural/embriología , Cola (estructura animal)/embriología , Animales , Tipificación del Cuerpo/genética , Diferenciación Celular/genética , Redes Reguladoras de Genes , Cresta Neural/citologíaRESUMEN
During vertebrate embryonic development complex morphogenetic events drive the formation of internal organs associated with the developing digestive tract. The foregut organs derive from hepatopancreatic precursor cells that originate bilaterally within the endoderm monolayer, and subsequently converge toward the midline where they coalesce to produce the gut tube from which the liver and pancreas form. The progenitor cells of these internal organs are influenced by the lateral plate mesoderm (LPM), which helps direct them towards their specific fates. However, it is not completely understood how the bilateral organ precursors move toward the embryonic midline and ultimately coalesce to form functional organs. Here we demonstrate that the zebrafish homeobox gene hoxb5b regulates morphogenesis of the foregut endoderm at the midline. At early segmentation stages, hoxb5b is expressed in the LPM adjacent to the developing foregut endoderm. By 24 hpf hoxb5b is expressed directly in the endoderm cells of the developing gut tube. When Hoxb5b function is disrupted, either by morpholino knockdown or sgRNA/Cas9 somatic disruption, the process of foregut morphogenesis is disrupted, resulting in a bifurcated foregut. By contrast, knockdown of the paralogous hoxb5a gene does not alter gut morphology. Further analysis has indicated that Hoxb5b knockdown specimens produce endocrine pancreas cell types, but liver cells are absent. Finally, cell transplantation experiments revealed that Hoxb5b function in the endoderm is not needed for proper coalescence of the foregut at the midline. Together, our findings imply that midline morphogenesis of foregut endoderm is guided by a hoxb5b-mediated mechanism that functions extrinsically, likely within the LPM. Loss of hoxb5b function prevents normal coalescence of endoderm cells at the midline and thus disrupts gut morphogenesis.
Asunto(s)
Tipificación del Cuerpo , Embrión no Mamífero/embriología , Endodermo/embriología , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Proteínas de Homeodominio/genética , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
The neural crest (NC) is a transient multipotent cell population that migrates extensively to produce a remarkable array of vertebrate cell types. NC cell specification progresses in an anterior to posterior fashion, resulting in distinct, axial-restricted subpopulations. The anterior-most, cranial, population of NC is specified as gastrulation concludes and neurulation begins, while more posterior populations become specified as the body elongates. The mechanisms that govern development of the more posterior NC cells remain incompletely understood. Here, we report a key role for zebrafish Cdx4, a homeodomain transcription factor, in the development of posterior NC cells. We demonstrate that cdx4 is expressed in trunk NC cell progenitors, directly binds NC cell-specific enhancers in the NC GRN, and regulates expression of the key NC development gene foxd3 in the posterior body. Moreover, cdx4 mutants show disruptions to the segmental pattern of trunk NC cell migration due to loss of normal leader/follower cell dynamics. Finally, using cell transplantation to generate chimeric specimens, we show that Cdx4 does not function in the paraxial mesoderm-the environment adjacent to which crest migrates-to influence migratory behaviors. We conclude that cdx4 plays a critical, and likely tissue autonomous, role in the establishment of trunk NC migratory behaviors. Together, our results indicate that cdx4 functions as an early NC specifier gene in the posterior body of zebrafish embryos.
Asunto(s)
Proteínas de Homeodominio/genética , Cresta Neural/metabolismo , Factores de Transcripción/genética , Animales , Tipificación del Cuerpo/genética , Diferenciación Celular/genética , Movimiento Celular/genética , Factores de Transcripción Forkhead/metabolismo , Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas de Homeodominio/metabolismo , Morfogénesis/genética , Placa Neural/metabolismo , Tubo Neural/metabolismo , Neurulación/genética , Factores de Transcripción/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismoRESUMEN
Our understanding of the neural crest, a key vertebrate innovation, is built upon studies of multiple model organisms. Early research on neural crest cells (NCCs) was dominated by analyses of accessible amphibian and avian embryos, with mouse genetics providing complementary insights in more recent years. The zebrafish model is a relative newcomer to the field, yet it offers unparalleled advantages for the study of NCCs. Specifically, zebrafish provide powerful genetic and transgenic tools, coupled with rapidly developing transparent embryos that are ideal for high-resolution real-time imaging of the dynamic process of neural crest development. While the broad principles of neural crest development are largely conserved across vertebrate species, there are critical differences in anatomy, morphogenesis, and genetics that must be considered before information from one model is extrapolated to another. Here, our goal is to provide the reader with a helpful primer specific to neural crest development in the zebrafish model. We focus largely on the earliest events-specification, delamination, and migration-discussing what is known about zebrafish NCC development and how it differs from NCC development in non-teleost species, as well as highlighting current gaps in knowledge.
Asunto(s)
Cresta Neural/embriología , Cresta Neural/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismo , Animales , Movimiento Celular/genética , Movimiento Celular/fisiología , Transición Epitelial-Mesenquimal/genética , Transición Epitelial-Mesenquimal/fisiología , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
The neural crest-a key innovation of the vertebrates-gives rise to diverse cell types including melanocytes, neurons and glia of the peripheral nervous system, and chondrocytes of the jaw and skull. Proper development of the cephalic region is dependent on the tightly-regulated specification and migration of cranial neural crest cells (NCCs). The core PCP proteins Frizzled and Disheveled have previously been implicated in NCC migration. Here we investigate the functions of the core PCP proteins Prickle1a and Prickle1b in zebrafish cranial NCC development. Using analysis of pk1a and pk1b mutant embryos, we uncover similar roles for both genes in facilitating cranial NCC migration. Disruption of either gene causes pre-migratory NCCs to cluster together at the dorsal aspect of the neural tube, where they adopt aberrant polarity and movement. Critically, in investigating Pk1-deficient cells that fail to migrate ventrolaterally, we have also uncovered roles for pk1a and pk1b in the epithelial-to-mesenchymal transition (EMT) of pre-migratory NCCs that precedes their collective migration to the periphery. Normally, during EMT, pre-migratory NCCs transition from a neuroepithelial to a bleb-based and subsequently, mesenchymal morphology capable of directed migration. When either Pk1a or Pk1b is disrupted, NCCs continue to perform blebbing behaviors characteristic of pre-migratory cells over extended time periods, indicating a block in a key transition during EMT. Although some Pk1-deficient NCCs transition successfully to mesenchymal, migratory morphologies, they fail to separate from neighboring NCCs. Additionally, Pk1b-deficient NCCs show elevated levels of E-Cadherin and reduced levels of N-Cadherin, suggesting that Prickle1 molecules regulate Cadherin levels to ensure the completion of EMT and the commencement of cranial NCC migration. We conclude that Pk1 plays crucial roles in cranial NCCs both during EMT and migration. These roles are dependent on the regulation of E-Cad and N-Cad.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Movimiento Celular/fisiología , Transición Epitelial-Mesenquimal/fisiología , Proteínas con Dominio LIM/metabolismo , Cresta Neural/embriología , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Cadherinas/genética , Cadherinas/metabolismo , Técnicas de Silenciamiento del Gen , Proteínas con Dominio LIM/genética , Cresta Neural/citología , Tubo Neural/citología , Tubo Neural/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
KEY POINTS: Chronic alcohol consumption causes a spectrum of liver diseases, but the pathogenic mechanisms driving the onset and progression of disease are not clearly defined. We show that chronic alcohol feeding sensitizes rat hepatocytes to Ca2+ -mobilizing hormones resulting in a leftward shift in the concentration-response relationship and the transition from oscillatory to more sustained and prolonged Ca2+ increases. Our data demonstrate that alcohol-dependent adaptation in the Ca2+ signalling pathway occurs at the level of hormone-induced inositol 1,4,5 trisphosphate (IP3 ) production and does not involve changes in the sensitivity of the IP3 receptor or size of internal Ca2+ stores. We suggest that prolonged and aberrant hormone-evoked Ca2+ increases may stimulate the production of mitochondrial reactive oxygen species and contribute to alcohol-induced hepatocyte injury. ABSTRACT: 'Adaptive' responses of the liver to chronic alcohol consumption may underlie the development of cell and tissue injury. Alcohol administration can perturb multiple signalling pathways including phosphoinositide-dependent cytosolic calcium ([Ca2+ ]i ) increases, which can adversely affect mitochondrial Ca2+ levels, reactive oxygen species production and energy metabolism. Our data indicate that chronic alcohol feeding induces a leftward shift in the dose-response for Ca2+ -mobilizing hormones resulting in more sustained and prolonged [Ca2+ ]i increases in both cultured hepatocytes and hepatocytes within the intact perfused liver. Ca2+ increases were initiated at lower hormone concentrations, and intercellular calcium wave propagation rates were faster in alcoholics compared to controls. Acute alcohol treatment (25 mm) completely inhibited hormone-induced calcium increases in control livers, but not after chronic alcohol-feeding, suggesting desensitization to the inhibitory actions of ethanol. Hormone-induced inositol 1,4,5 trisphosphate (IP3 ) accumulation and phospholipase C (PLC) activity were significantly potentiated in hepatocytes from alcohol-fed rats compared to controls. Removal of extracellular calcium, or chelation of intracellular calcium did not normalize the differences in hormone-stimulated PLC activity, indicating calcium-dependent PLCs are not upregulated by alcohol. We propose that the liver 'adapts' to chronic alcohol exposure by increasing hormone-dependent IP3 formation, leading to aberrant calcium increases, which may contribute to hepatocyte injury.
Asunto(s)
Consumo de Bebidas Alcohólicas/metabolismo , Alcoholismo/metabolismo , Señalización del Calcio , Hepatocitos/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Fosfolipasas de Tipo C/metabolismo , Animales , Calcio/metabolismo , Hepatocitos/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratas Sprague-Dawley , Vasopresinas/farmacologíaRESUMEN
The vertebrate brain arises from the complex organization of millions of neurons. Neurogenesis encompasses not only cell fate specification from neural stem cells, but also the terminal molecular and morphological maturation of neurons at correct positions within the brain. RE1-silencing transcription factor (Rest) is expressed in non-neural tissues and neuronal progenitors where it inhibits the terminal maturation of neurons by repressing hundreds of neuron-specific genes. Here we show that Rest repression of maturation is intimately linked with the migratory capability of zebrafish facial branchiomotor neurons (FBMNs), which undergo a characteristic tangential migration from hindbrain rhombomere (r) 4 to r6/r7 during development. We establish that FBMN migration is increasingly disrupted as Rest is depleted in zebrafish rest mutant embryos, such that around two-thirds of FBMNs fail to complete migration in mutants depleted of both maternal and zygotic Rest. Although Rest is broadly expressed, we show that de-repression or activation of Rest target genes only within FBMNs is sufficient to disrupt their migration. We demonstrate that this migration defect is due to precocious maturation of FBMNs, based on both morphological and molecular criteria. We further show that the Rest target gene and alternative splicing factor srrm4 is a key downstream regulator of maturation; Srrm4 knockdown partially restores the ability of FBMNs to migrate in rest mutants while preventing their precocious morphological maturation. Rest must localize to the nucleus to repress its targets, and its subcellular localization is highly regulated: we show that targeting Rest specifically to FBMN nuclei rescues FBMN migration in Rest-deficient embryos. We conclude that Rest functions in FBMN nuclei to inhibit maturation until the neurons complete their migration.
Asunto(s)
Nervio Facial/metabolismo , Neuronas Motoras/metabolismo , Células-Madre Neurales/metabolismo , Neurogénesis/genética , Proteínas Represoras/genética , Animales , Animales Modificados Genéticamente , Movimiento Celular/fisiología , Núcleo Celular/genética , Nervio Facial/citología , Técnicas de Silenciamiento del Gen , Morfolinos/genética , Proteínas del Tejido Nervioso/genética , Proteínas Represoras/biosíntesis , Rombencéfalo/embriología , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
During development a network of transcription factors functions to differentiate foregut cells into pancreatic endocrine cells. Differentiation of appropriate numbers of each hormone-expressing endocrine cell type is essential for the normal development of the pancreas and ultimately for effective maintenance of blood glucose levels. A fuller understanding of the details of endocrine cell differentiation may contribute to development of cell replacement therapies to treat diabetes. In this study, by using morpholino and gRNA/Cas9 mediated knockdown we establish that differential levels of the basic-helix loop helix (bHLH) transcription factor Neurod are required for the differentiation of distinct endocrine cell types in developing zebrafish. While Neurod plays a role in the differentiation of all endocrine cells, we find that differentiation of glucagon-expressing alpha cells is disrupted by a minor reduction in Neurod levels, whereas differentiation of insulin-expressing beta cells is less sensitive to Neurod depletion. The endocrine cells that arise during embryonic stages to produce the primary islet, and those that arise subsequently during larval stages from the intra-pancreatic duct (IPD) to ultimately contribute to the secondary islets, show similar dependence on differential Neurod levels. Intriguingly, Neurod-deficiency triggers premature formation of endocrine precursors from the IPD during early larval stages. However, the Neurod-deficient endocrine precursors fail to differentiate appropriately, and the larvae are unable to maintain normal glucose levels. In summary, differential levels of Neurod are required to generate endocrine pancreas subtypes from precursors during both embryonic and larval stages, and Neurod function is in turn critical to endocrine function.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Regulación del Desarrollo de la Expresión Génica , Células Secretoras de Glucagón/citología , Células Secretoras de Insulina/citología , Islotes Pancreáticos/embriología , Proteínas del Tejido Nervioso/fisiología , Pez Cebra/embriología , Animales , Benzazepinas/química , Diferenciación Celular , Linaje de la Célula , Células Endocrinas/citología , Glucagón/metabolismo , Glucosa/química , Proteínas Fluorescentes Verdes/química , Insulina/metabolismo , Mutagénesis , Oligonucleótidos Antisentido/química , Florizina/química , ARN Mensajero/metabolismoRESUMEN
Appropriate localization of neurons within the brain is a crucial component of the establishment of neural circuitry. In the zebrafish hindbrain, the facial branchiomotor neurons (FBMNs) undergo a chain-like tangential migration from their birthplace in rhombomere (r) 4 to their final destination in r6/r7. Here, we report that ablation of either the cell body or the trailing axon of the leading FBMN, or 'pioneer' neuron, blocks the migration of follower FBMNs into r5. This demonstrates that the pioneer neuron and its axon are crucial to the early migration of FBMNs. Later migration from r5 to r6 is not dependent on pioneer neurons but on the medial longitudinal fasciculus (MLF), a bundle of axons lying ventral to the FBMNs. We find that MLF axons enter r5 only after the pioneer neuron has led several followers into this region; the MLF is then contacted by projections from the FBMNs. The interactions between FBMNs and the MLF are important for migration from r5 to r6, as blocking MLF axons from entering the hindbrain can stall FBMN migration in r5. Finally, we have found that the adhesion molecule Cdh2 (N-cadherin) is important for interactions between the MLF and FBMNs, as well as for interactions between the trailing axon of the pioneer neuron and follower FBMNs. Interestingly, migration of pioneer neurons is independent of both the MLF and Cdh2, suggesting pioneer migration relies on independent cues.
Asunto(s)
Axones/fisiología , Movimiento Celular/fisiología , Nervio Facial/citología , Neuronas Motoras/fisiología , Rombencéfalo/embriología , Pez Cebra/embriología , Animales , Inmunohistoquímica , Microscopía Confocal , Modelos Neurológicos , Morfolinos/genéticaRESUMEN
The transcriptional repressor Rest (Nrsf) recruits chromatin-modifying complexes to RE1 'silencer elements', which are associated with hundreds of neural genes. However, the requirement for Rest-mediated transcriptional regulation of embryonic development and cell fate is poorly understood. Conflicting views of the role of Rest in controlling cell fate have emerged from recent studies. To address these controversies, we examined the developmental requirement for Rest in zebrafish using zinc-finger nuclease-mediated gene targeting. We discovered that germ layer specification progresses normally in rest mutants despite derepression of target genes during embryogenesis. This analysis provides the first evidence that maternal rest is essential for repression of target genes during blastula stages. Surprisingly, neurogenesis proceeds largely normally in rest mutants, although abnormalities are observed within the nervous system, including defects in oligodendrocyte precursor cell development and a partial loss of facial branchiomotor neuron migration. Mutants progress normally through embryogenesis but many die as larvae (after 12 days). However, some homozygotes reach adulthood and are viable. We utilized an RE1/NRSE transgenic reporter system to dynamically monitor Rest activity. This analysis revealed that Rest is required to repress gene expression in mesodermal derivatives including muscle and notochord, as well as within the nervous system. Finally, we demonstrated that Rest is required for long-term repression of target genes in non-neural tissues in adult zebrafish. Our results point to a broad role for Rest in fine-tuning neural gene expression, rather than as a widespread regulator of neurogenesis or cell fate.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Neurogénesis , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Pez Cebra/crecimiento & desarrollo , Pez Cebra/genética , Animales , Movimiento Celular , Transcripción Genética , Pez Cebra/embriología , Pez Cebra/metabolismoRESUMEN
The facial branchiomotor neurons (FBMNs) undergo a characteristic tangential migration in the vertebrate hindbrain. We previously used a morpholino knockdown approach to reveal that zebrafish prickle1b (pk1b) is required for this migration. Here we report that FBMN migration is also blocked in a pk1b mutant with a disruption in the consensus farnesylation motif. We confirmed that this lipid modification is required during FBMN migration by disrupting the function of farnesyl biosynthetic enzymes. Furthermore, farnesylation of a tagged Pk1b is required for its nuclear localization. Using a unique rescue approach, we have demonstrated that Pk1b nuclear localization and farnesylation are required during FBMN migration. Our data suggest that Pk1b acts at least partially independently of core planar cell polarity molecules at the plasma membrane, and might instead be acting at the nucleus. We also found that the neuronal transcriptional silencer REST is necessary for FBMN migration, and we provide evidence that interaction between Pk1b and REST is required during this process. Finally, we demonstrate that REST protein, which is normally localized in the nuclei of migrating FBMNs, is depleted from the nuclei of Pk1b-deficient neurons. We conclude that farnesylation-dependent nuclear localization of Pk1b is required to regulate REST localization and thus FBMN migration.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Movimiento Celular , Núcleo Celular/metabolismo , ADN Complementario/genética , Farnesiltransferasa/metabolismo , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Hidroximetilglutaril-CoA Reductasas/metabolismo , Proteínas con Dominio LIM , Modelos Biológicos , Datos de Secuencia Molecular , Neuronas Motoras/citología , Neuronas Motoras/metabolismo , Mutación , Neurogénesis , Prenilación de Proteína , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal , Pez Cebra/genética , Proteínas de Pez Cebra/antagonistas & inhibidores , Proteínas de Pez Cebra/genéticaRESUMEN
The vertebrate endocrine pancreas has the crucial function of maintaining blood sugar homeostasis. This role is dependent upon the development and maintenance of pancreatic islets comprising appropriate ratios of hormone-producing cells. In all vertebrate models studied, an initial precursor population of Pdx1-expressing endoderm cells gives rise to separate endocrine and exocrine cell lineages. Within the endocrine progenitor pool a variety of transcription factors influence cell fate decisions, such that hormone-producing differentiated cell types ultimately arise, including the insulin-producing beta cells and the antagonistically acting glucagon-producing alpha cells. In previous work, we established that the development of all pancreatic lineages requires retinoic acid (RA) signaling. We have used the zebrafish to uncover genes that function downstream of RA signaling, and here we identify mnx1 (hb9) as an RA-regulated endoderm transcription factor-encoding gene. By combining manipulation of gene function, cell transplantation approaches and transgenic reporter analysis we establish that Mnx1 functions downstream of RA within the endoderm to control cell fate decisions in the endocrine pancreas progenitor lineage. We confirm that Mnx1-deficient zebrafish lack beta cells, and, importantly, we make the novel observation that they concomitantly gain alpha cells. In Mnx1-deficient embryos, precursor cells that are normally destined to differentiate as beta cells instead take on an alpha cell fate. Our findings suggest that Mnx1 functions to promote beta and suppress alpha cell fates.
Asunto(s)
Diferenciación Celular/fisiología , Islotes Pancreáticos/embriología , Organogénesis/fisiología , Factores de Transcripción/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Animales Modificados Genéticamente , Linaje de la Célula , Endodermo/citología , Endodermo/fisiología , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Humanos , Islotes Pancreáticos/citología , Islotes Pancreáticos/crecimiento & desarrollo , Transducción de Señal , Células Madre/citología , Células Madre/fisiología , Factores de Transcripción/genética , Tretinoina/metabolismo , Pez Cebra/anatomía & histología , Pez Cebra/crecimiento & desarrollo , Proteínas de Pez Cebra/genéticaRESUMEN
BACKGROUND: The vertebrate nuclear receptor subfamily 2, group f (nr2f) genes encode orphan receptors that have the capacity to act as negative regulators of retinoic acid (RA) signaling. RESULTS: We describe embryonic and larval expression of four of the six zebrafish nr2f genes, nr2f1a, nr2f1b, nr2f2, and nr2f5. These genes show highly regulated patterns of expression within the central nervous system, including in the developing hindbrain, as well as in the mesoderm and endoderm. We also investigated the role of RA and fibroblast growth factor (Fgf) signaling in regulating early nr2f gene expression. RA is not required for nr2f expression in the hindbrain; however, exogenous RA can repress this expression. Conversely, we find that RA positively regulates nr2f1a expression in trunk endoderm and mesoderm. Fgf signaling is not required for nr2f expression onset in the hindbrain; however, it may play a role in maintaining rhombomere-specific expression. CONCLUSIONS: We report detailed expression analysis of four nr2f genes in all three germ layers. The onset of nr2f expression in the hindbrain does not require RA or Fgf signals. Our finding that RA positively regulates nr2f1a expression in the trunk supports the possibility that Nr2fs function in a negative feedback loop to modulate RA signaling in this region.
Asunto(s)
Factores de Transcripción COUP/metabolismo , Sistema Nervioso Central/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Transducción de Señal/fisiología , Tretinoina/metabolismo , Pez Cebra/embriología , Animales , Factores de Transcripción COUP/genética , Cartilla de ADN/genética , Perfilación de la Expresión Génica , Hibridación in SituRESUMEN
The control of organ size and position relies, at least in part, upon appropriate regulation of the signals that specify organ progenitor fields. Pancreatic cell fates are specified by retinoic acid (RA), and proper size and localization of the pancreatic field are dependent on tight control of RA signaling. Here we show that the RA-degrading Cyp26 enzymes play a critical role in defining the normal anterior limit of the pancreatic field. Disruption of Cyp26 function causes a dramatic expansion of pancreatic cell types toward the anterior of the embryo. The cyp26a1 gene is expressed in the anterior trunk endoderm at developmental stages when RA is signaling to specify pancreas, and analysis of cyp26a1/giraffe (gir) mutant zebrafish embryos confirms that cyp26a1 plays the primary role in setting the anterior limit of the pancreas. Analysis of the gir mutants further reveals that cyp26b1 and cyp26c1 function redundantly to partially compensate for loss of Cyp26a1 function. We used cell transplantation to determine that Cyp26a1 functions directly in endoderm to modulate RA signaling and limit the pancreatic field. Taken together with our finding that endodermal expression of cyp26 genes is subject to positive regulation by RA, our data reveal a feedback loop within the endoderm. Such feedback can maintain consistent levels of RA signaling, despite environmental fluctuations in RA concentration, thus ensuring a consistent size and location of the pancreatic field.
Asunto(s)
Sistema Enzimático del Citocromo P-450/fisiología , Endodermo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Páncreas/metabolismo , Animales , Linaje de la Célula , Trasplante de Células , Hibridación in Situ , Modelos Biológicos , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Páncreas/citología , Páncreas/embriología , Ácido Retinoico 4-Hidroxilasa , Transducción de Señal , Pez Cebra , Proteínas de Pez CebraRESUMEN
Coordination of cell proliferation and migration is fundamental for life, and its dysregulation has catastrophic consequences, such as cancer. How cell cycle progression affects migration, and vice versa, remains largely unknown. We address these questions by combining in silico modelling and in vivo experimentation in the zebrafish trunk neural crest (TNC). TNC migrate collectively, forming chains with a leader cell directing the movement of trailing followers. We show that the acquisition of migratory identity is autonomously controlled by Notch signalling in TNC. High Notch activity defines leaders, while low Notch determines followers. Moreover, cell cycle progression is required for TNC migration and is regulated by Notch. Cells with low Notch activity stay longer in G1 and become followers, while leaders with high Notch activity quickly undergo G1/S transition and remain in S-phase longer. In conclusion, TNC migratory identities are defined through the interaction of Notch signalling and cell cycle progression.
Asunto(s)
Cresta Neural , Pez Cebra , Animales , División Celular , Movimiento Celular/fisiología , Transducción de Señal , Pez Cebra/fisiologíaRESUMEN
Development of the vertebrate pancreas is a complex stepwise process comprising regionalization, cell differentiation, and morphogenesis. Studies in zebrafish are contributing to an emerging picture of pancreas development in which extrinsic signaling molecules influence intrinsic transcriptional programs to allow ultimate differentiation of specific pancreatic cell types. Zebrafish experiments have revealed roles for several signaling molecules in aspects of this process; for example our own work has shown that retinoic acid signals specify the pre-pancreatic endoderm. Time-lapse imaging of live zebrafish embryos has started to provide detailed information about early pancreas morphogenesis. In addition to modeling embryonic development, the zebrafish has recently been used as a model for pancreas regeneration studies. Here, we review the significant progress in these areas and consider the future potential of zebrafish as a diabetes research model.
Asunto(s)
Diabetes Mellitus/embriología , Modelos Animales , Páncreas/embriología , Páncreas/metabolismo , Pez Cebra/embriología , Animales , Linaje de la Célula , Páncreas/citología , RegeneraciónRESUMEN
The facial branchiomotor neurons undergo a characteristic tangential migration in the vertebrate hindbrain. Several signaling mechanisms have been implicated in this process, including the non-canonical Wnt/planar cell polarity (PCP) pathway. However, the role of this signaling pathway in controlling the dynamics of these neurons is unclear. Here, we describe the cellular dynamics of the facial neurons as they migrate, focusing on the speed and direction of migration, extension of protrusions, cell shape, and orientation. Furthermore, we show that the PET/LIM domain protein Prickle1b (Pk1b) is required for several aspects of these migratory behaviors, including cell orientation. However, we find that centrosome localization is not significantly affected by disruption of Pk1b function, suggesting that polarization of the neurons is not completely lost. Together, our data suggest that Pk1b function may be required to integrate the multiple migratory cues received by the neurons into polarization instructions for proper posterior movement.