Zebrafish mnx1 controls cell fate choice in the developing endocrine pancreas.

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.

Chondrogenic and gliogenic subpopulations of neural crest play distinct roles during the assembly of epibranchial ganglia.

In vertebrates, the sensory neurons of the epibranchial (EB) ganglia transmit somatosensory signals from the periphery to the CNS. These ganglia are formed during embryogenesis by the convergence and condensation of two distinct populations of precursors: placode-derived neuroblasts and neural crest- (NC) derived glial precursors. In addition to the gliogenic crest, chondrogenic NC migrates into the pharyngeal arches, which lie in close proximity to the EB placodes and ganglia. Here, we examine the respective roles of these two distinct NC-derived populations during development of the EB ganglia using zebrafish morphant and mutants that lack one or both of these NC populations. Our analyses of mutant and morphant zebrafish that exhibit deficiencies in chondrogenic NC at early stages reveal a distinct requirement for this NC subpopulation during early EB ganglion assembly and segmentation. Furthermore, restoration of wildtype chondrogenic NC in one of these mutants, prdm1a, is sufficient to restore ganglion formation, indicating a specific requirement of the chondrogenic NC for EB ganglia assembly. By contrast, analysis of the sox10 mutant, which lacks gliogenic NC, reveals that the initial assembly of ganglia is not affected. However, during later stages of development, EB ganglia are dispersed in the sox10 mutant, suggesting that glia are required to maintain normal EB ganglion morphology. These results highlight novel roles for two subpopulations of NC cells in the formation and maintenance of EB ganglia: chondrogenic NC promotes the early-stage formation of the developing EB ganglia while glial NC is required for the late-stage maintenance of ganglion morphology.

Lef1 is required for progenitor cell identity in the zebrafish lateral line primordium.

The zebrafish posterior lateral line (pLL) is a sensory system that comprises clusters of mechanosensory organs called neuromasts (NMs) that are stereotypically positioned along the surface of the trunk. The NMs are deposited by a migrating pLL primordium, which is organized into polarized rosettes (proto-NMs). During migration, mature proto-NMs are deposited from the trailing part of the primordium, while progenitor cells in the leading part give rise to new proto-NMs. Wnt signaling is active in the leading zone of the primordium and global Wnt inactivation leads to dramatic disorganization of the primordium and a loss of proto-NM formation. However, the exact cellular events that are regulated by the Wnt pathway are not known. We identified a mutant strain, lef1(nl2), that contains a lesion in the Wnt effector gene lef1. lef1(nl2) mutants lack posterior NMs and live imaging reveals that rosette renewal fails during later stages of migration. Surprisingly, the overall primordium patterning, as assayed by the expression of various markers, appears unaltered in lef1(nl2) mutants. Lineage tracing and mosaic analyses revealed that the leading cells (presumptive progenitors) move out of the primordium and are incorporated into NMs; this results in a decrease in the number of proliferating progenitor cells and eventual primordium disorganization. We concluded that Lef1 function is not required for initial primordium organization or migration, but is necessary for proto-NM renewal during later stages of pLL formation. These findings revealed a novel role for the Wnt signaling pathway during mechanosensory organ formation in zebrafish.

Zebrafish dorsal root ganglia neural precursor cells adopt a glial fate in the absence of neurogenin1.

The proneural transcription factor neurogenin 1 (neurog1) has been shown to be a key regulator of dorsal root ganglion (DRG) neuron development. Here we use a novel transgenic zebrafish line to demonstrate that the neural crest population that gives rise to DRG neurons becomes fate restricted to a neuronal/glial precursor before the onset of neurog1 function. We generated a stable transgenic zebrafish line that carries a modified bacterial artificial chromosome that expresses green fluorescent protein (GFP) under the control of the neurog1 promoter [Tg(neurog1:EGFP)]. In contrast to previously described neurog1 transgenic lines, Tg(neurog1:EGFP) expresses GFP in DRG neuronal precursors cells as they migrate ventrally and after their initial differentiation as neurons. Using this line, we are able to track the fate of DRG neuronal precursor cells during their specification. When Neurog1 function is blocked, either by neurog1 morpholino antisense oligonucleotide injection or in neurog1 mutants, GFP expression initiates in neural crest cells, although they fail to form DRG neurons. Rather, these cells take on a glial-like morphology, retain proliferative capacity, and express glial markers and become associated with the ventral motor root. These results suggest that, within the zebrafish neural crest, there is a fate-restricted lineage that is limited to form either sensory neurons or glia in the developing DRG. Neurog1 acts as the key factor in this lineage to direct the formation of sensory neurons.

Specification of epibranchial placodes in zebrafish.

In all vertebrates, the neurogenic placodes are transient ectodermal thickenings that give rise to sensory neurons of the cranial ganglia. Epibranchial (EB) placodes generate neurons of the distal facial, glossopharyngeal and vagal ganglia, which convey sensation from the viscera, including pharyngeal endoderm structures, to the CNS. Recent studies have implicated signals from pharyngeal endoderm in the initiation of neurogenesis from EB placodes; however, the signals underlying the formation of placodes are unknown. Here, we show that zebrafish embryos mutant for fgf3 and fgf8 do not express early EB placode markers, including foxi1 and pax2a. Mosaic analysis demonstrates that placodal cells must directly receive Fgf signals during a specific crucial period of development. Transplantation experiments and mutant analysis reveal that cephalic mesoderm is the source of Fgf signals. Finally, both Fgf3 and Fgf8 are sufficient to induce foxi1-positive placodal precursors in wild-type as well as Fgf3-plus Fgf8-depleted embryos. We propose a model in which mesoderm-derived Fgf3 and Fgf8 signals establish both the EB placodes and the development of the pharyngeal endoderm, the subsequent interaction of which promotes neurogenesis. The coordinated interplay between craniofacial tissues would thus assure proper spatial and temporal interactions in the shaping of the vertebrate head.

Sustained Bmp signaling is essential for cloaca development in zebrafish.

Bone morphogenetic protein (Bmp) signaling has long been known to be important for the early development of the ventral mesoderm, including blood, vasculature and kidney cells. Although Bmp genes are continually expressed in the ventral cells throughout gastrulation and somitogenesis, previous studies in zebrafish have not addressed how the role of Bmp signaling changes over time to regulate ventral mesoderm development. Here, we describe the use of a transgenic inducible dominant-negative Bmp receptor line to examine the temporal roles of Bmp signaling in ventral mesoderm patterning. Surprisingly, we find that Bmp signaling from the mid-gastrula stage through early somitogenesis is important for excluding blood and vascular precursors from the extreme ventral mesoderm, and we show that this domain is normally required for development of the cloaca (the common gut and urogenital opening). Using a novel assay for cloacal function, we find that larvae with reduced mid-gastrula Bmp signaling cannot properly excrete waste. We show that the cloacal defects result from alterations in the morphogenesis of the cloaca and from changes in the expression of genes marking the excretory system. Finally, we show that HrT, a T-box transcription factor, is a Bmp-regulated gene that has an essential function in cloacal development. We conclude that sustained Bmp signaling plays an important role in specification of the zebrafish cloaca by maintaining the fate of extreme ventral cells during the course of gastrulation and early somitogenesis. Furthermore, our data suggest that alterations in Bmp signaling are one possible cause of anorectal malformations during human embryogenesis.

Endoderm-derived Fgf3 is necessary and sufficient for inducing neurogenesis in the epibranchial placodes in zebrafish.

In vertebrates, epibranchial placodes are transient ectodermal thickenings that contribute sensory neurons to the epibranchial ganglia. These ganglia innervate internal organs and transmit information on heart rate, blood pressure and visceral distension from the periphery to the central nervous system. Despite their importance, the molecular mechanisms that govern the induction and neurogenesis of the epibranchial placodes are only now being elucidated. In this study, we demonstrate that endoderm is required for neurogenesis of the zebrafish epibranchial placodes. Mosaic analyses confirm that endoderm is the source of the neurogenic signal. Using a morpholino knockdown approach, we find that fgf3 is required for the majority of placode cells to undergo neurogenesis. Tissue transplants demonstrate that fgf3 activity is specifically required in the endodermal pouches. Furthermore, ectopic fgf3 expression is sufficient for inducing phox2a-positive neurons in wild-type and endoderm-deficient embryos. Surprisingly, ectodermal foxi1 expression, a marker for the epibranchial placode precursors, is present in both endoderm-deficient embryos and fgf3 morphants, indicating that neither endoderm nor Fgf3 is required for initial placode induction. Based on these findings, we propose a model for epibranchial placode development in which Fgf3 is a major endodermal determinant required for epibranchial placode neurogenesis.