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.     

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.     

Signaling mechanisms controlling cranial placode neurogenesis and delamination

The neurogenic cranial placodes are a unique transient epithelial niche of neural progenitor cells that give rise to multiple derivatives of the peripheral nervous system, particularly, the sensory neurons. Placode neurogenesis occurs throughout an extended period of time with epithelial cells continually recruited as neural progenitor cells. Sensory neuron development in the trigeminal, epibranchial, otic, and olfactory placodes coincides with detachment of these neuroblasts from the encompassing epithelial sheet, leading to delamination and ingression into the mesenchyme where they continue to differentiate as neurons. Multiple signaling pathways are known to direct placodal development. This review defines the signaling pathways working at the finite spatiotemporal period when neuronal selection within the placodes occurs, and neuroblasts concomitantly delaminate from the epithelium. Examining neurogenesis and delamination after initial placodal patterning and specification has revealed a common trend throughout the neurogenic placodes, which suggests that both activated FGF and attenuated Notch signaling activities are required for neurogenesis and changes in epithelial cell adhesion leading to delamination. We also address the varying roles of other pathways such as the Wnt and BMP signaling families during sensory neurogenesis and neuroblast delamination in the differing placodes.     

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.     

Delamination of cells from neurogenic placodes does not involve an epithelial-to-mesenchymal transition

Neurogenic placodes are specialized regions of embryonic ectoderm that generate the majority of the neurons of the cranial sensory ganglia. Here we examine in chick the mechanism underlying the delamination of cells from the epibranchial placodal ectoderm. We show that the placodal epithelium has a distinctive morphology, reflecting a change in cell shape, and is associated with a breach in the underlying basal lamina. Placodal cell delamination is distinct from neural crest cell delamination. In particular, exit of neuroblasts from the epithelium is not associated with the expression of Snail/Snail2 or of the Rho family GTPases required for the epithelial-to-mesenchymal transition seen in neural crest cell delamination. Indeed, cells leaving the placodes do not assume a mesenchymal morphology but migrate from the epithelium as neuronal cells. We further show that the placodal epithelium has a pseudostratified appearance. Examination of proliferation shows that the placodal epithelium is mitotically quiescent, with few phosphohistone H3-positive cells being identified. Where division does occur within the epithelium it is restricted to the apical surface. The neurogenic placodes thus represent specialized ectodermal niches that generate neuroblasts over a protracted period.     

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.     

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.     

Molecular and tissue interactions governing induction of cranial ectodermal placodes

Whereas neural crest cells are the source of the peripheral nervous system in the trunk of vertebrates, the “ectodermal placodes,” together with neural crest, form the peripheral nervous system of the head. Cranial ectodermal placodes are thickenings in the ectoderm that subsequently ingress or invaginate to make important contributions to cranial ganglia, including epibranchial and trigeminal ganglia, and sensory structures, the ear, nose, lens, and adenohypophysis. Recent studies have uncovered a number of molecular signals mediating induction and differentiation of placodal cells. Here, we described recent advances in understanding the tissue interactions and signals underlying induction and neurogenesis of placodes, with emphasis on the trigeminal and epibranchial. Important roles of Fibroblast Growth Factors, Platelet Derived Growth Factors, Sonic Hedgehog, TGFβ superfamily members, and Wnts are discussed.     

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.     

Chicken HOXA3 gene: its expression pattern and role in branchial nerve precursor cell migration

In vertebrates, the proximal and distal sensory ganglia of the branchial nerves are derived from neural crest cells (NCCs) and placodes, respectively. We previously reported that in Hoxa3 knockout mouse embryos, NCCs and placode-derived cells of the glossopharyngeal nerve were defective in their migration. In this report, to determine the cell-type origin for this Hoxa3 knockout phenotype, we blocked the expression of the gene with antisense morpholino oligonucleotides (MO) specifically in either NCCs/neural tube or placodal cells of chicken embryos. Our results showed that HOXA3 function was required for the migration of the epibranchial placode-derived cells and that HOXA3 regulated this cell migration in both NCCs/neural tube and placodal cells. We also report that the expression pattern of chicken HOXA3 was slightly different from that of mouse Hoxa3.     

Lateral line, otic and epibranchial placodes: developmental and evolutionary links?

Two embryonic cell populations, the neural crest and cranial ectodermal placodes, between them give rise to many of the unique characters of vertebrates. Neurogenic placode derivatives are vital for sensing both external and internal stimuli. In this speculative review, we discuss potential developmental and evolutionary relationships between two placode series that are usually considered to be entirely independent: lateral line placodes, which form the mechanosensory and electroreceptive hair cells of the anamniote lateral line system as well as their afferent neurons, and epibranchial placodes (geniculate, petrosal and nodose), which form Phox2b(+) visceral sensory neurons with input from both the external and internal environment. We illustrate their development using molecular data we recently obtained in shark embryos, and we describe their derivatives, including the possible geniculate placode origin of a mechanosensory sense organ associated with the first pharyngeal pouch/cleft (the anamniote spiracular organ/amniote paratympanic organ). We discuss how both lateral line and epibranchial placodes can be related in different ways to the otic placode (which forms the inner ear and its afferent neurons), and how both are important for protective somatic reflexes. Finally, we put forward a highly speculative proposal about the original function of the cells whose evolutionary descendants today include the derivatives of the lateral line, otic and epibranchial placodes, namely that they produced sensory receptors and neurons for Phox2b-dependent protective reflex circuits. We hope this review will stimulate both debate and a fresh look at possible developmental and evolutionary relationships between these seemingly disparate and independent placodes.     

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.