Apoptosis and proliferation in developing, mature, and regressing epibranchial placodes

Epibranchial placodes and rhombencephalic neural crest provide precursor cells for the geniculate, petrosal, and nodose ganglia. In chick embryos and in Tupaia belangeri, apoptosis in rhombomeres 3 and 5 helps to select premigratory precursor cells and to segregate crest cell streams derived from the even-numbered rhombomeres. Much less is known about the patterns and functions of apoptosis in epibranchial placodes. We found that, in Tupaia belangeri, combined anlagen of the otic placode and epibranchial placode 1 transiently share a primordial low grade thickening with post-otic epibranchial placodes. Three-dimensional reconstructions reveal complementary, spatially, and temporally regulated apoptotic and proliferative events that demarcate the otic placode and epibranchial placode 1, and help to individualize three pairs of epibranchial placodes in a rostrocaudal sequence. Later, rostrocaudal waves of proliferation and apoptosis extend from dorsal to ventral parts of the placodes, paralleled by the dorsoventral progression of precursor cell delamination. These findings suggest a role for apoptosis during the process of neuroblast generation in the epibranchial placodes. Finally, apoptosis eliminates remnants of the placodes in the presence of late invading macrophages.     

Epibranchial and otic placodes are induced by a common Fgf signal, but their subsequent development is independent

The epibranchial placodes are cranial, ectodermal thickenings that give rise to sensory neurons of the peripheral nervous system. Despite their importance in the developing animal, the signals responsible for their induction remain unknown. Using the placodal marker, sox3, we have shown that the same Fgf signaling required for otic vesicle development is required for the development of the epibranchial placodes. Loss of both Fgf3 and Fgf8 is sufficient to block placode development. We further show that epibranchial sox3 expression is unaffected in mutants in which no otic placode forms, where dlx3b and dlx4b are knocked down, or deleted along with sox9a. However, the forkhead factor, Foxi1, is required for both otic and epibranchial placode development. Thus, both the otic and epibranchial placodes form in a common region of ectoderm under the influence of Fgfs, but these two structures subsequently develop independently. Although previous studies have investigated the signals that trigger neurogenesis from the epibranchial placodes, this represents the first demonstration of the signaling events that underlie the formation of the placodes themselves, and therefore, the process that determines which ectodermal cells will adopt a neural fate.     

Molecular analysis of neurogenic placode development in a basal ray-finned fish

Neurogenic placodes are transient, thickened patches of embryonic vertebrate head ectoderm that give rise to the paired peripheral sense organs and most neurons in cranial sensory ganglia. We present the first analysis of gene expression during neurogenic placode development in a basal actinopterygian (ray-finned fish), the North American paddlefish (Polyodon spathula). Pax3 expression in the profundal placode confirms its homology with the ophthalmic trigeminal placode of amniotes. We report the conservation of expression of Pax2 and Pax8 in the otic and/or epibranchial placodes, Phox2b in epibranchial placode-derived neurons, Sox3 during epibranchial and lateral line placode development, and NeuroD in developing cranial sensory ganglia. We identify Sox3 as a novel marker for developing fields of electrosensory ampullary organs and for ampullary organs themselves. Sox3 is also the first molecular marker for actinopterygian ampullary organs. This is consistent with, though does not prove, a lateral line placode origin for actinopterygian ampullary organs.     

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.     

Pax2/8 proteins coordinate sequential induction of otic and epibranchial placodes through differential regulation of foxi1, sox3 and fgf24

Vertebrate cranial placodes contribute vitally to development of sensory structures of the head. Amongst posterior placodes, the otic placode forms the inner ear whereas nearby epibranchial placodes produce sensory ganglia within branchial clefts. Though diverse in fate, these placodes show striking similarities in their early regulation. In zebrafish, both are initiated by localized Fgf signaling plus the ubiquitous competence factor Foxi1, and both express pax8 and sox3 in response. It has been suggested that Fgf initially induces a common otic/epibranchial field, which later subdivides in response to other signals. However, we find that otic and epibranchial placodes form at different times and by distinct mechanisms. Initially, Fgf from surrounding tissues induces otic expression of pax8 and sox3, which cooperate synergistically to establish otic fate. Subsequently, pax8 works with related genes pax2a/pax2b to downregulate otic expression of foxi1, a necessary step for further otic development. Additionally, pax2/8 activate otic expression of fgf24, which induces epibranchial expression of sox3. Knockdown of fgf24 or sox3 causes severe epibranchial deficiencies but has little effect on otic development. These findings clarify the roles of pax8 and sox3 and support a model whereby the otic placode forms first and induces epibranchial placodes through an Fgf-relay.     

Sox3 expression defines a common primordium for the epibranchial placodes in chick

The epibranchial placodes are ectodermal thickenings that generate sensory neurons of the distal ganglia of the branchial nerves. Although significant advances in our understanding of neurogenesis from the placodes have recently been made, the events prior to the onset of neurogenesis remain unclear. We found that chick Sox3 (cSox3) shows a highly dynamic pattern of expression before becoming confined to the final placodes: one pre-otic (geniculate) and three post-otic (one petrosal and two nodose) placodes. A fate-mapping study using lipophilic dyes revealed that all post-otic placodes arise within a single broad cSox3-positive domain, where cSox3 expression and epithelial thickness will be retained only in much smaller final neurogenic placodes. The data presented here suggest that post-otic placodes are remnants of a common primordium defined as a discrete domain of cSox3 expression.     

Integrin-α5 coordinates assembly of posterior cranial placodes in zebrafish and enhances Fgf-dependent regulation of otic/epibranchial cells

Vertebrate sensory organs develop in part from cranial placodes, a series of ectodermal thickenings that coalesce from a common domain of preplacodal ectoderm. Mechanisms coordinating morphogenesis and differentiation of discrete placodes are still poorly understood. We have investigated whether placodal assembly in zebrafish requires Integrin- α5 (itga5), an extracellular matrix receptor initially expressed throughout the preplacodal ectoderm. Morpholino knockdown of itga5 had no detectable effect on anterior placodes (pituitary, nasal and lens), but posterior placodes developed abnormally, resulting in disorganization of trigeminal and epibranchial ganglia and reduction of the otic vesicle. Cell motion analysis in GFP-transgenic embryos showed that cell migration in itga5 morphants was highly erratic and unfocused, impairing convergence and blocking successive recruitment of new cells into these placodes. Further studies revealed genetic interactions between itga5 and Fgf signaling. First, itga5 morphants showed changes in gene expression mimicking modest reduction in Fgf signaling. Second, itga5 morphants showed elevated apoptosis in the otic/epibranchial domain, which was rescued by misexpression of Fgf8. Third, knockdown of the Fgf effector erm had no effect by itself but strongly enhanced defects in itga5 morphants. Finally, proper regulation of itga5 requires dlx3b/4b and pax8, which are themselves regulated by Fgf. These findings support a model in which itga5 coordinates cell migration into posterior placodes and augments Fgf signaling required for patterning of these tissues and cell survival in otic/epibranchial placodes.     

The zebrafish forkhead transcription factor Foxi1 specifies epibranchial placode-derived sensory neurons

Vertebrate epibranchial placodes give rise to visceral sensory neurons that transmit vital information such as heart rate, blood pressure and visceral distension. Despite the pivotal roles they play, the molecular program underlying their development is not well understood. Here we report that the zebrafish mutation no soul, in which epibranchial placodes are defective, disrupts the fork headrelated, winged helix domain-containing protein Foxi1. Foxi1 is expressed in lateral placodal progenitor cells. In the absence of foxi1 activity, progenitor cells fail to express the basic helix-loop-helix gene neurogenin that is essential for the formation of neuronal precursors, and the paired homeodomain containing gene phox2a that is essential for neuronal differentiation and maintenance. Consequently, increased cell death is detected indicating that the placodal progenitor cells take on an apoptotic pathway. Furthermore, ectopic expression of foxi1 is sufficient to induce phox2a-positive and neurogenin-positive cells. Taken together, these findings suggest that Foxi1 is an important determination factor for epibranchial placodal progenitor cells to acquire both neuronal fate and subtype visceral sensory identity.     

Expression of Fibroblast growth factor 19 (Fgf19) during chicken embryogenesis and eye development, compared with Fgf15 expression in the mouse

The normal development of eyes relies on proper signaling through Fibroblast growth factor (FGF) receptors, but the source and identity of cognate ligands have remained largely unknown. We have found that Fgf19 is expressed in the developing chicken retina. In situ hybridization discloses dynamic expression patterns for Fgf19 in the optic vesicle, lens primordia and retinal horizontal cells. Overall expression pattern of Fgf19 during chicken embryogenesis was also examined: Fgf19 is expressed in the regions associated with cranial placodes induction, boundary regions of rhombomeres, somites, specific groups of neural cells in midbrain, hindbrain, and those derived from epibranchial placodes, and the apical ectodermal ridge of limb buds. Expression pattern of the Fgf19-orthologous gene Fgf15 was further examined in the mouse developing eye. Fgf15 is expressed in the optic vesicle, a subset of progenitor cells of neural retina, and emerging ganglion and amacrine cells during retinogenesis.     

PRDC regulates placode neurogenesis in chick by modulating BMP signalling

The epibranchial placodes generate the neurons of the geniculate, petrosal, and nodose cranial sensory ganglia. Previously, it has been shown that bone morphogenetic proteins (BMPs) are involved in the formation of these structures. However, it has been unclear as to whether BMP signalling has an ongoing function in directing the later development of the epibranchial placodes, and how this signalling is regulated. Here, we demonstrate that BMPs maintain placodal neurogenesis and that their activity is modulated by a member of the Cerberus/Dan family of BMP antagonists, Protein Related to Dan and Cerberus (PRDC). We find that Bmp4 is expressed in the epibranchial placodes while Bmp7 and PRDC are expressed in the pharyngeal pouches. The timing and regional expression of these three genes suggest that BMP7 is involved in inducing placode neurogenesis and BMP4 in maintaining it and that BMP activity is modulated by PRDC. To investigate this hypothesis, we have performed both gain- and loss- of-function experiments with PRDC and find that it can modulate the BMP signals that induce epibranchial neurogenesis: a gain of PRDC function results in a loss of Bmp4 and hence placode neurogenesis is inhibited; conversely, a loss of PRDC function induces ectopic Bmp4 and an expansion of placode neurogenesis. This modulation is therefore necessary for the number and positioning of the epibranchial neurons.     

Requirements for endoderm and BMP signaling in sensory neurogenesis in zebrafish

Cranial sensory neurons largely derive from neurogenic placodes (epibranchial and dorsolateral), which are ectodermal thickenings that form the sensory ganglia associated with cranial nerves, but the molecular mechanisms of placodal development are unclear. Here, we show that the pharyngeal endoderm induces epibranchial neurogenesis in zebrafish, and that BMP signaling plays a crucial role in this process. Using a her5:egfp transgenic line to follow endodermal movements in living embryos, we show that contact between pharyngeal pouches and the surface ectoderm coincides with the onset of neurogenesis in epibranchial placodes. By genetic ablation and reintroduction of endoderm by cell transplantation, we show that these contacts promote neurogenesis. Using a genetic interference approach we further identify bmp2b and bmp5 as crucial components of the endodermal signals that induce epibranchial neurogenesis. Dorsolateral placodes (trigeminal, auditory, vestibular, lateral line) develop independently of the endoderm and BMP signaling, suggesting that these two sets of placodes are under separate genetic control. Our results show that the endoderm regulates the differentiation of cranial sensory ganglia, which coordinates the cranial nerves with the segments that they innervate.     

Modulation of zebrafish pitx3 expression in the primordia of the pituitary, lens, olfactory epithelium and cranial ganglia by hedgehog and nodal signaling

In this article we report the isolation of a novel zebrafish gene, pitx3, which plays an important role in the formation of several placode-derived structures. In wildtype embryos, pitx3 is first expressed in a crescent-shaped area in the anterior end of the embryo. At later stages, the primordia of the anterior pituitary, the lens, the olfactory sensory epithelium, and cranial ganglia express this gene. Pitx3 is not expressed in the more posterior preplacodal region that gives rise to the epibranchial, otic, and lateral line placodes. The dynamics of pitx3 in the anterior region of wildtype embryos suggests that pitx3 expression marks a common step in the formation of the pituitary, lens, olfactory placode as well as the trigeminal placode. Analysis of pitx3 expression in mutants lacking the hedgehog or nodal function demonstrates the differential dependence of pitx3 expression in these structures on nodal and hedgehog signaling. While the lens and trigeminal placodes express pitx3 in the absence of hedgehog and nodal signaling, there is no expression of pitx3 in the anteriormost ectoderm adjacent to the neural plate from which the anterior pituitary would derive. In mutants with impaired hedgehog signaling, the lens placode frequently extends into more anterior ventral regions of the embryo.     

Hypobranchial placodes in<Emphasis Type="Italic"> Xenopus laevis</Emphasis> give rise to hypobranchial ganglia,a novel type of cranial ganglia

Recently, a novel type of neurogenic placode was described in anurans. These hypobranchial placodes were recognized as ectodermal thickenings situated ventral to the second and third pharyngeal pouch that give rise to neurons of unknown fate. Here, the development of hypobranchial placodes in Xenopus laevis is described in more detail using in situ hybridization and immunohistochemistry for various placodal ( Six1, Eya1) and neurogenic ( NGNR-1, NeuroD, Delta-1, Hu, acetylated tubulin) markers. Moreover, the fate of hypobranchial placodes was determined by analyzing tadpoles that had received orthotopic grafts of ventral branchial arch ectoderm at embryonic stages from donor embryos injected with the lineage tracer green fluorescent protein. The neurogenic epibranchial and hypobranchial placodes are shown to develop in certain subregions of a broader branchial placodal area as defined by Six1 and Eya1 expression, viz., adjacent to the dorsal and ventral tip of the pharyngeal pouches, respectively. Grafting experiments show that each of the two hypobranchial placodes gives rise to a small and previously undescribed hypobranchial ganglion (identified by its immunoreactivity for the neuron-specific Hu protein) of unknown function located in the ventral branchial arch region. No contributions of hypobranchial placodes to any other ganglia (including cardiac ganglia and the ganglia of branchiomeric nerves located dorsal to pharyngeal pouches) were found.