RESUMEN
Efficient and accurate DNA replication is particularly critical in stem and progenitor cells for successful proliferation and survival. The replisome, an amalgam of protein complexes, is responsible for binding potential origins of replication, unwinding the double helix, and then synthesizing complimentary strands of DNA. According to current models, the initial steps of DNA unwinding and opening are facilitated by the CMG complex, which is composed of a GINS heterotetramer that connects Cdc45 with the mini-chromosome maintenance (Mcm) helicase. In this work, we provide evidence that in the absence of GINS function DNA replication is cell autonomously impaired, and we also show that gins1 and gins2 mutants exhibit elevated levels of apoptosis restricted to actively proliferating regions of the central nervous system (CNS). Intriguingly, our results also suggest that the rapid cell cycles during early embryonic development in zebrafish may not require the function of the canonical GINS complex as neither zygotic Gins1 nor Gins2 isoforms seem to be present during these stages.
RESUMEN
The vertebrate eye originates from the eye field, a domain of cells specified by a small number of transcription factors. In this study, we show that Tcf7l1a is one such transcription factor that acts cell-autonomously to specify the eye field in zebrafish. Despite the much-reduced eye field in tcf7l1a mutants, these fish develop normal eyes revealing a striking ability of the eye to recover from a severe early phenotype. This robustness is not mediated through genetic compensation at neural plate stage; instead, the smaller optic vesicle of tcf7l1a mutants shows delayed neurogenesis and continues to grow until it achieves approximately normal size. Although the developing eye is robust to the lack of Tcf7l1a function, it is sensitised to the effects of additional mutations. In support of this, a forward genetic screen identified mutations in hesx1, cct5 and gdf6a, which give synthetically enhanced eye specification or growth phenotypes when in combination with the tcf7l1a mutation.
Asunto(s)
Ojo/crecimiento & desarrollo , Morfogénesis , Proteína 1 Similar al Factor de Transcripción 7/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/crecimiento & desarrollo , Animales , Proliferación Celular , Embrión no Mamífero/metabolismo , Ojo/patología , Femenino , Regulación del Desarrollo de la Expresión Génica , Sitios Genéticos , Cinética , Masculino , Mutación/genética , Placa Neural/embriología , Neurogénesis , Penetrancia , Fenotipo , Prosencéfalo/embriología , Proteína 1 Similar al Factor de Transcripción 7/genética , Regulación hacia Arriba/genética , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Cigoto/metabolismoRESUMEN
Maintaining neurogenesis in growing tissues requires a tight balance between progenitor cell proliferation and differentiation. In the zebrafish retina, neuronal differentiation proceeds in two stages with embryonic retinal progenitor cells (RPCs) of the central retina accounting for the first rounds of differentiation, and stem cells from the ciliary marginal zone (CMZ) being responsible for late neurogenesis and growth of the eye. In this study, we analyse two mutants with small eyes that display defects during both early and late phases of retinal neurogenesis. These mutants carry lesions in gdf6a, a gene encoding a BMP family member previously implicated in dorsoventral patterning of the eye. We show that gdf6a mutant eyes exhibit expanded retinoic acid (RA) signalling and demonstrate that exogenous activation of this pathway in wild-type eyes inhibits retinal growth, generating small eyes with a reduced CMZ and fewer proliferating progenitors, similar to gdf6a mutants. We provide evidence that RA regulates the timing of RPC differentiation by promoting cell cycle exit. Furthermore, reducing RA signalling in gdf6a mutants re-establishes appropriate timing of embryonic retinal neurogenesis and restores putative stem and progenitor cell populations in the CMZ. Together, our results support a model in which dorsally expressed gdf6a limits RA pathway activity to control the transition from proliferation to differentiation in the growing eye.
Asunto(s)
Factor 6 de Diferenciación de Crecimiento/genética , Neurogénesis/genética , Retina/embriología , Tretinoina/metabolismo , Proteínas de Pez Cebra/genética , Pez Cebra/embriología , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Ciclo Celular/genética , Proliferación Celular , Embrión no Mamífero/embriología , Neurogénesis/fisiología , Transducción de Señal/genética , Células Madre/citologíaRESUMEN
BACKGROUND: Although left-right asymmetries are common features of nervous systems, their developmental bases are largely unknown. In the zebrafish epithalamus, dorsal habenular neurons adopt medial (dHbm) and lateral (dHbl) subnuclear character at very different frequencies on the left and right sides. The left-sided parapineal promotes the elaboration of dHbl character in the left habenula, albeit by an unknown mechanism. Likewise, the genetic pathways acting within habenular neurons to control their asymmetric differentiated character are unknown. RESULTS: In a forward genetic screen for mutations that result in loss of habenular asymmetry, we identified two mutant alleles of tcf7l2, a gene that encodes a transcriptional regulator of Wnt signaling. In tcf7l2 mutants, most neurons on both sides differentiate with dHbl identity. Consequently, the habenulae develop symmetrically, with both sides adopting a pronounced leftward character. Tcf7l2 acts cell automously in nascent equipotential neurons, and on the right side, it promotes dHbm and suppresses dHbl differentiation. On the left, the parapineal prevents this Tcf7l2-dependent process, thereby promoting dHbl differentiation. CONCLUSIONS: Tcf7l2 is essential for lateralized fate selection by habenular neurons that can differentiate along two alternative pathways, thereby leading to major neural circuit asymmetries.
Asunto(s)
Diferenciación Celular , Habénula/embriología , Neuronas/fisiología , Proteína 2 Similar al Factor de Transcripción 7/genética , Proteínas de Pez Cebra/genética , Pez Cebra/embriología , Animales , Embrión no Mamífero/embriología , Embrión no Mamífero/fisiología , Regulación de la Expresión Génica , Habénula/citología , Neuronas/citología , Transducción de Señal , Proteína 2 Similar al Factor de Transcripción 7/metabolismo , Pez Cebra/fisiología , Proteínas de Pez Cebra/metabolismoRESUMEN
During tissue morphogenesis and differentiation, cells must self-renew while contemporaneously generating daughters that contribute to the growing tissue. How tissues achieve this precise balance between proliferation and differentiation is, in most instances, poorly understood. This is in part due to the difficulties in dissociating the mechanisms that underlie tissue patterning from those that regulate proliferation. In the migrating posterior lateral line primordium (PLLP), proliferation is predominantly localised to the leading zone. As cells emerge from this zone, they periodically organise into rosettes that subsequently dissociate from the primordium and differentiate as neuromasts. Despite this reiterative loss of cells, the primordium maintains its size through regenerative cell proliferation until it reaches the tail. In this study, we identify a null mutation in the Wnt-pathway transcription factor Lef1 and show that its activity is required to maintain proliferation in the progenitor pool of cells that sustains the PLLP as it undergoes migration, morphogenesis and differentiation. In absence of Lef1, the leading zone becomes depleted of cells during its migration leading to the collapse of the primordium into a couple of terminal neuromasts. We show that this behaviour resembles the process by which the PLLP normally ends its migration, suggesting that suppression of Wnt signalling is required for termination of neuromast production in the tail. Our data support a model in which Lef1 sustains proliferation of leading zone progenitors, maintaining the primordium size and defining neuromast deposition rate.
Asunto(s)
Proliferación Celular , Homeostasis/genética , Sistema de la Línea Lateral/embriología , Factores de Transcripción/fisiología , Proteínas Wnt/fisiología , Proteínas de Pez Cebra/fisiología , beta Catenina/fisiología , Aletas de Animales/embriología , Aletas de Animales/crecimiento & desarrollo , Aletas de Animales/metabolismo , Animales , Animales Modificados Genéticamente , Tipificación del Cuerpo/genética , Diferenciación Celular/genética , Embrión no Mamífero , Homeostasis/fisiología , Sistema de la Línea Lateral/metabolismo , Masculino , Morfogénesis/genética , Morfogénesis/fisiología , Mutación/fisiología , Transducción de Señal/genética , Transducción de Señal/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismo , Pez Cebra/fisiología , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
In fish and amphibians, the dorsal axis is specified by the asymmetric localization of maternally provided components of the Wnt signalling pathway. Gore et al. suggest that the Nodal signal Squint (Sqt) is required as a maternally provided dorsal determinant in zebrafish. Here we test their proposal and show that the maternal activities of sqt and the related Nodal gene cyclops (cyc) are not required for dorsoventral patterning.
Asunto(s)
Tipificación del Cuerpo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Embrión no Mamífero/metabolismo , Femenino , Péptidos y Proteínas de Señalización Intracelular/genética , Modelos Biológicos , Madres , Ligandos de Señalización Nodal , Ovario/metabolismo , Óvulo/metabolismo , Empalme del ARN , Reproducibilidad de los Resultados , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genéticaRESUMEN
Bone morphogenetic proteins (BMPs) are key mediators of dorsoventral patterning in vertebrates and are required for the induction of ventral fates in fish and frogs. A widely accepted model of dorsoventral patterning postulates that a morphogenetic BMP activity gradient patterns cell fates along the dorsoventral axis. Recent work in zebrafish suggests that the role of BMP signaling changes over time, with BMPs required for global dorsoventral patterning during early gastrulation and for tail patterning during late gastrulation and early somitogenesis. Key questions remain about the late phase, including which BMP ligands are required and how the functions of BMPs differ during the early and late gastrula stages. In a screen for dominant enhancers of mutations in the homeobox genes vox and vent, which function in parallel to bmp signaling, we identified an insertion mutation in bmp4. We then performed a reverse genetic screen to isolate a null allele of bmp4. We report the characterization of these two alleles and demonstrate that BMP4 is required during the later phase of BMP signaling for the specification of ventroposterior cell fates. Our results indicate that different bmp genes are essential at different stages. In addition, we present genetic evidence supporting a role for a morphogenetic BMP gradient in establishing mesodermal fates during the later phase of BMP signaling.
Asunto(s)
Alelos , Proteínas Morfogenéticas Óseas/genética , Gastrulación/genética , Regulación del Desarrollo de la Expresión Génica , Pez Cebra/embriología , Animales , Tipificación del Cuerpo/genética , Proteína Morfogenética Ósea 2 , Proteína Morfogenética Ósea 4 , Proteína Morfogenética Ósea 7 , Proteínas Morfogenéticas Óseas/metabolismo , Diferenciación Celular/genética , Mesodermo/embriología , Transducción de Señal , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
In this study, we elucidate the roles of the winged-helix transcription factor Foxa2 in ventral CNS development in zebrafish. Through cloning of monorail (mol), which we find encodes the transcription factor Foxa2, and phenotypic analysis of mol-/- embryos, we show that floorplate is induced in the absence of Foxa2 function but fails to further differentiate. In mol-/- mutants, expression of Foxa and Hh family genes is not maintained in floorplate cells and lateral expansion of the floorplate fails to occur. Our results suggest that this is due to defects both in the regulation of Hh activity in medial floorplate cells as well as cell-autonomous requirements for Foxa2 in the prospective laterally positioned floorplate cells themselves. Foxa2 is also required for induction and/or patterning of several distinct cell types in the ventral CNS. Serotonergic neurones of the raphenucleus and the trochlear motor nucleus are absent in mol-/- embryos, and oculomotor and facial motoneurones ectopically occupy ventral CNS midline positions in the midbrain and hindbrain. There is also a severe reduction of prospective oligodendrocytes in the midbrain and hindbrain. Finally, in the absence of Foxa2, at least two likely Hh pathway target genes are ectopically expressed in more dorsal regions of the midbrain and hindbrain ventricular neuroepithelium, raising the possibility that Foxa2 activity may normally be required to limit the range of action of secreted Hh proteins.
Asunto(s)
Sistema Nervioso Central/embriología , Inducción Embrionaria/fisiología , Neuronas Motoras/citología , Oligodendroglía/citología , Factores de Transcripción/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Sistema Nervioso Central/citología , Sistema Nervioso Central/fisiología , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Factores de Transcripción Forkhead , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas Hedgehog , Neuronas Motoras/metabolismo , Mutación/genética , Oligodendroglía/metabolismo , Núcleos del Rafe/citología , Núcleos del Rafe/embriología , Núcleos del Rafe/metabolismo , Serotonina/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Nervio Troclear/citología , Nervio Troclear/embriología , Nervio Troclear/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genéticaRESUMEN
Retinal ganglion cell (RGC) axons are topographically ordered in the optic tract according to their retinal origin. In zebrafish dackel (dak) and boxer (box) mutants, some dorsal RGC axons missort in the optic tract but innervate the tectum topographically. Molecular cloning reveals that dak and box encode ext2 and extl3, glycosyltransferases implicated in heparan sulfate (HS) biosynthesis. Both genes are required for HS synthesis, as shown by biochemical and immunohistochemical analysis, and are expressed maternally and then ubiquitously, likely playing permissive roles. Missorting in box can be rescued by overexpression of extl3. dak;box double mutants show synthetic pathfinding phenotypes that phenocopy robo2 mutants, suggesting that Robo2 function requires HS in vivo; however, tract sorting does not require Robo function, since it is normal in robo2 null mutants. This genetic evidence that heparan sulfate proteoglycan function is required for optic tract sorting provides clues to begin understanding the underlying molecular mechanisms.
Asunto(s)
Axones/metabolismo , Proteoglicanos de Heparán Sulfato/biosíntesis , N-Acetilglucosaminiltransferasas/fisiología , Vías Visuales/metabolismo , Proteínas de Pez Cebra/fisiología , Animales , Proteoglicanos de Heparán Sulfato/genética , Datos de Secuencia Molecular , N-Acetilglucosaminiltransferasas/biosíntesis , N-Acetilglucosaminiltransferasas/genética , Células Ganglionares de la Retina/metabolismo , Vías Visuales/embriología , Pez CebraRESUMEN
Large-scale genetic screens in zebrafish have identified thousands of mutations in hundreds of essential genes. The genetic mapping of these mutations is necessary to link DNA sequences to the gene functions defined by mutant phenotypes. Here, we report two advances that will accelerate the mapping of zebrafish mutations: (1) The construction of a first generation single nucleotide polymorphism (SNP) map of the zebrafish genome comprising 2035 SNPs and 178 small insertions/deletions, and (2) the development of a method for mapping mutations in which hundreds of SNPs can be scored in parallel with an oligonucleotide microarray. We have demonstrated the utility of the microarray technique in crosses with haploid and diploid embryos by mapping two known mutations to their previously identified locations. We have also used this approach to localize four previously unmapped mutations. We expect that mapping with SNPs and oligonucleotide microarrays will accelerate the molecular analysis of zebrafish mutations.