Placode Induction Research Articles

Hydrocortisone treatment as a tool to study conjunctival placode induction.

Conjunctival placodes are a series of placodes that develop into the conjunctival (scleral) papillae and ultimately induce a series of scleral ossicles in the eyes of many vertebrates. This study establishes a hydrocortisone injection procedure (incl. dosage) that consistently inhibits all conjunctival papillae in the embryonic chicken eye. The effects of this hydrocortisone treatment on apoptosis, vasculature, and placode-related gene expression were assessed. Hydrocortisone treatment does not increase apoptotic cell death or have a major effect on the ciliary artery or vascular plexus in the eye. β-catenin and Eda expression levels were not significantly altered following hydrocortisone treatment, despite the absence of conjunctival papillae. Notably, Fgf20 expression was significantly reduced following hydrocortisone treatment, and the distribution of β-catenin was altered. Our study showed that conjunctival papillae induction begins as early as HH27.5 (E5.5). Hydrocortisone treatment reduces Fgf20 expression independently of β-catenin and Eda and may instead affect other members of the Wnt/β-catenin or Eda/Edar pathways, or it may affect the ability of morphogens to diffuse through the extracellular matrix. This study contributes to a growing profile of gene expression data during placode development and enhances our understanding of how some vertebrate eyes develop these fascinating bones.

Inactivation of Fgf3 and Fgf4 within the Fgf3/Fgf4/Fgf15 gene cluster reveals their redundant requirement for mouse inner ear induction and embryonic survival.

Fibroblast growth factors (Fgfs) are required for survival and organ formation during embryogenesis. Fgfs often execute their functions redundantly. Previous analysis of Fgf3 mutants revealed effects on inner ear formation and embryonic survival with incomplete penetrance. Here, we show that presence of a neomycin resistance gene (neo) replacing the Fgf3 coding region leads to reduced survival during embryogenesis and an increased penetrance of inner ear defects. Fgf3neo/neo mutants showed reduced expression of Fgf4, which is positioned in close proximity to the Fgf3 locus in the mouse genome. Conditional inactivation of Fgf4 during inner ear development on a Fgf3 null background using Fgf3/4 cis mice revealed a redundant requirement between these Fgfs during otic placode induction. In contrast, inactivation of Fgf3 and Fgf4 in the pharyngeal region where both Fgfs are also co-expressed using a Foxg1-Cre driver did not affect development of the pharyngeal arches. However, these mutants showed reduced perinatal survival. These results highlight the importance of Fgf signaling during development. In particular, different members of the Fgf family act redundantly to guarantee inner ear formation and embryonic survival.

The Importance Of Understanding The Micro‐Environment Enabling Tissue Induction: A Case Study Of The Scleral Ossicle System

Developmental induction is the cornerstone of many developmental processes, with research most often focused on identifying the inductive factors and/or tissues. Here, we examine the microenvironment that enables the inductive events that occur during development of the scleral ossicles. The scleral ossicle system of avians is composed of a series of 13-16 flat neural-crest derived overlapping bones situated in the sclera of the eye. The skeletogenic condensations, preceding these bones, develop over a two day period and are induced by placode-derived conjunctival epithelial papillae. These placodes develop in a fascinating spatiotemporal pattern that is conserved across avians and reptiles. There is a gap in our understanding of the ontogenetic growth of these condensations and the factors that are involved in inducing the scleral ossicle system. Using a combination of histological analyses, qPCR and in situ hybridization gene expression analyses, we provide insight into the tissue characteristics of this system. We show that while the condensations increase in size, their relative depth remains constant, indicating that the distance inducing morphogens need to diffuse is constant over the two day period of induction despite scleral width increasing. Our gene expression analyses of the early inductive phases reveals a complex network of interacting factors, that are also implicated in epithelial placode induction in other systems. Specifically, several members of each of the following key signaling pathways - β-catenin, FGF, BMP, EDA and HH - are expressed during placode formation. Correlation analyses reveal two distinct gene interaction modules providing evidence of subgroups of genes with unique spatiotemporal expression characteristics. Additionally, through careful embryonic manipulations, we created knock-out phenotypes for both the placodes and the skeletogenic condensations. These phenotypes may be the result of vascular disruption and/or matrix disruption and demonstrates the importance of each in enabling inductive mechanisms. The mesenchymal matrix is denser in these knock-outs, suggesting that diffusible morphogens cannot reach their destinations deeper in the tissue. Collectively these results underscore the importance of understanding the microenvironmental context that supports morphogen-based tissue induction.

Fgf8 genetic labeling reveals the early specification of vestibular hair cell type in mouse utricle.

FGF8 signaling plays diverse roles in inner ear development, acting at multiple stages from otic placode induction to cellular differentiation in the organ of Corti. As a secreted morphogen with diverse functions, Fgf8 expression is likely to be spatially restricted and temporally dynamic throughout inner ear development. We evaluated these characteristics using genetic labeling mediated by Fgf8mcm gene-targeted mice and determined that Fgf8 expression is a specific and early marker of Type-I vestibular hair cell identity. Fgf8mcm expression initiates at E11.5 in the future striolar region of the utricle, labeling hair cells following EdU birthdating, and demonstrates that sub-type identity is determined shortly after terminal mitosis. This early fate specification is not apparent using markers or morphological criteria that are not present before birth in the mouse. Although analyses of Fgf8 conditional knockout mice did not reveal developmental phenotypes, the restricted pattern of Fgf8 expression suggests that functionally redundant FGF ligands may contribute to vestibular hair cell differentiation and supports a developmental model in which Type-I and Type-II hair cells develop in parallel rather than from an intermediate precursor.

Spatial and temporal inhibition of FGFR2b ligands reveals continuous requirements and novel targets in mouse inner ear morphogenesis.

Morphogenesis of the inner ear epithelium requires coordinated deployment of several signaling pathways, and disruptions cause abnormalities of hearing and/or balance. The FGFR2b ligands FGF3 and FGF10 are expressed throughout otic development and are required individually for normal morphogenesis, but their prior and redundant roles in otic placode induction complicates investigation of subsequent combinatorial functions in morphogenesis. To interrogate these roles and identify new effectors of FGF3 and FGF10 signaling at the earliest stages of otic morphogenesis, we used conditional gene ablation after otic placode induction, and temporal inhibition of signaling with a secreted, dominant-negative FGFR2b ectodomain. We show that both ligands are required continuously after otocyst formation for maintenance of otic neuroblasts and for patterning and proliferation of the epithelium, leading to normal morphogenesis of both the cochlear and vestibular domains. Furthermore, the first genome-wide identification of proximal targets of FGFR2b signaling in the early otocyst reveals novel candidate genes for inner ear development and function.

Counter-rotational cell flows drive morphological and cell fate asymmetries in mammalian hair follicles.

Organ morphogenesis is a complex process coordinated by cell specification, epithelial-mesenchymal interactions, and tissue polarity. A striking example is the pattern of regularly spaced, globally aligned mammalian hair follicles, which emerges through epidermal-dermal signaling and planar polarized morphogenesis. Here, using live-imaging, we discover that developing hair follicles polarize through dramatic cell rearrangements organized in a counter-rotational pattern of cell flows. Upon hair placode induction, Shh signaling specifies a radial pattern of progenitor fates that, together with planar cell polarity (PCP), induce counter-rotational rearrangements through myosin and ROCK-dependent polarized neighbor exchanges. Importantly, these cell rearrangements also establish cell fate asymmetry by repositioning radial progenitors along the anterior-posterior axis. These movements concurrently displace associated mesenchymal cells, which then signal asymmetrically to maintain polarized cell fates. Our results demonstrate how spatial patterning and tissue polarity generate an unexpected collective cell behavior that in turn, establishes both morphological and cell fate asymmetry.

Sox2 and sox3 cooperate to regulate otic/epibranchial placode induction in zebrafish

Expression of sox3 is one of the earliest markers of Fgf-dependent otic/epibranchial placode induction. We report here that sox2 is also expressed in the early otic/epibranchial placode in zebrafish. To address functions of sox2 and sox3, we generated knockouts and heat shock-inducible transgenes. Mutant analysis, and low-level misexpression, showed that sox2 and sox3 act redundantly to establish a full complement of otic/epibranchial cells. Disruption of pax8, another early regulator, caused similar placodal deficiencies to sox3 mutants or pax8-sox3 double mutants, suggesting that sox3 and pax8 operate in the same pathway. High-level misexpression of sox2 or sox3 during early stages cell-autonomously blocked placode induction, whereas misexpression several hours later could not reverse placodal differentiation. In an assay for ectopic placode-induction, we previously showed that misexpression of fgf8 induces a high level of ectopic sox3, but not pax8. Partial knockdown of sox3 significantly enhanced ectopic induction of pax8, whereas full knockdown of sox3 inhibited this process. Together these findings show that sox2 and sox3 are together required for proper otic induction, but the level of expression must be tightly regulated to avoid suppression of differentiation and maintenance of pluripotency.

Neural induction by the node and placode induction by head mesoderm share an initial state resembling neural plate border and ES cells

Around the time of gastrulation in higher vertebrate embryos, inductive interactions direct cells to form central nervous system (neural plate) or sensory placodes. Grafts of different tissues into the periphery of a chicken embryo elicit different responses: Hensen's node induces a neural plate whereas the head mesoderm induces placodes. How different are these processes? Transcriptome analysis in time course reveals that both processes start by induction of a common set of genes, which later diverge. These genes are remarkably similar to those induced by an extraembryonic tissue, the hypoblast, and are normally expressed in the pregastrulation stage epiblast. Explants of this epiblast grown in the absence of further signals develop as neural plate border derivatives and eventually express lens markers. We designate this state as "preborder"; its transcriptome resembles embryonic stem cells. Finally, using sequential transplantation experiments, we show that the node, head mesoderm, and hypoblast are interchangeable to begin any of these inductions while the final outcome depends on the tissue emitting the later signals.

Retinoic acid is required and Fgf, Wnt, and Bmp signaling inhibit posterior lateral line placode induction in zebrafish

The lateral line system is a mechanosensory systems present in aquatic animals. The anterior and posterior lateral lines develop from anterior and posterior lateral line placodes (aLLp and pLLp), respectively. Although signaling molecules required for the induction of other cranial placodes have been well studied, the molecular mechanisms underlying formation of the lateral line placodes are unknown. In this study we tested the requirement of multiple signaling pathways, such as Wnt, Bmp Fgf, and Retinoic Acid for aLLp and pLLp induction. We determined that aLLp specification requires Fgf signaling, whilst pLLp specification requires retinoic acid which inhibits Fgf signaling. pLLp induction is also independent of Wnt and Bmp activities, even though these pathways limit the boundaries of the pLLp. This is the first report that the aLLp and pLLp depend on different inductive mechanisms and that pLLp induction requires the inhibition of Fgf, Wnt and Bmp signaling.

Cell fate specification in the lingual epithelium is controlled by antagonistic activities of Sonic hedgehog and retinoic acid

The interaction between signaling pathways is a central question in the study of organogenesis. Using the developing murine tongue as a model, we uncovered unknown relationships between Sonic hedgehog (SHH) and retinoic acid (RA) signaling. Genetic loss of SHH signaling leads to enhanced RA activity subsequent to loss of SHH-dependent expression of Cyp26a1 and Cyp26c1. This causes a cell identity switch, prompting the epithelium of the tongue to form heterotopic minor salivary glands and to overproduce oversized taste buds. At developmental stages during which Wnt10b expression normally ceases and Shh becomes confined to taste bud cells, loss of SHH inputs causes the lingual epithelium to undergo an ectopic and anachronic expression of Shh and Wnt10b in the basal layer, specifying de novo taste placode induction. Surprisingly, in the absence of SHH signaling, lingual epithelial cells adopted a Merkel cell fate, but this was not caused by enhanced RA signaling. We show that RA promotes, whereas SHH, acting strictly within the lingual epithelium, inhibits taste placode and lingual gland formation by thwarting RA activity. These findings reveal key functions for SHH and RA in cell fate specification in the lingual epithelium and aid in deciphering the molecular mechanisms that assign cell identity.

Epithelial cell behaviours during neurosensory organ formation.

Perception of the environment in vertebrates relies on a variety of neurosensory mini-organs. These organs develop via a multi-step process that includes placode induction, cell differentiation, patterning and innervation. Ultimately, cells derived from one or more different tissues assemble to form a specific mini-organ that exhibits a particular structure and function. The initial building blocks of these organs are epithelial cells that undergo rearrangements and interact with neighbouring tissues, such as neural crest-derived mesenchymal cells and sensory neurons, to construct a functional sensory organ. In recent years, advances in in vivo imaging methods have allowed direct observation of these epithelial cells, showing that they can be displaced within the epithelium itself via several modes. This Review focuses on the diversity of epithelial cell behaviours that are involved in the formation of small neurosensory organs, using the examples of dental placodes, hair follicles, taste buds, lung neuroendocrine cells and zebrafish lateral line neuromasts to highlight both well-established and newly described modes of epithelial cell motility.

The molecular basis of craniofacial placode development.

The sensory organs of the vertebrate head originate from simple ectodermal structures known as cranial placodes. All cranial placodes derive from a common domain adjacent to the neural plate, the preplacodal region, which is induced at the border of neural and non-neural ectoderm during gastrulation. Induction and specification of the preplacodal region is regulated by the fibroblast growth factor, bone morphogenetic protein, WNT, and retinoic acid signaling pathways, and characterized by expression of the EYA and SIX family of transcriptional regulators. Once the preplacodal region is specified, different combinations of local signaling molecules and placode-specific transcription factors, including competence factors, promote the induction of individual cranial placodes along the neural axis of the head region. In this review, we summarize the steps of cranial placode development and discuss the roles of the main signaling molecules and transcription factors that regulate these steps during placode induction, specification, and development. For further resources related to this article, please visit the WIREs website.

The mouse Foxi3 transcription factor is necessary for the development of posterior placodes

The inner ear develops from the otic placode, one of the cranial placodes that arise from a region of ectoderm adjacent to the anterior neural plate called the pre-placodal domain. We have identified a Forkhead family transcription factor, Foxi3, that is expressed in the pre-placodal domain and down-regulated when the otic placode is induced. We now show that Foxi3 mutant mice do not form otic placodes as evidenced by expression changes in early molecular markers and the lack of thickened placodal ectoderm, an otic cup or otocyst. Some preplacodal genes downstream of Foxi3-Gata3, Six1 and Eya1-are not expressed in the ectoderm of Foxi3 mutant mice, and the ectoderm exhibits signs of increased apoptosis. We also show that Fgf signals from the hindbrain and cranial mesoderm, which are necessary for otic placode induction, are received by pre-placodal ectoderm in Foxi3 mutants, but do not initiate otic induction. Finally, we show that the epibranchial placodes that develop in close proximity to the otic placode and the mandibular division of the trigeminal ganglion are missing in Foxi3 mutants. Our data suggest that Foxi3 is necessary to prime pre-placodal ectoderm for the correct interpretation of inductive signals for the otic and epibranchial placodes.

Cooperative and independent functions of FGF and Wnt signaling during early inner ear development.

BackgroundIn multiple vertebrate organisms, including chick, Xenopus, and zebrafish, Fibroblast Growth Factor (FGF) and Wnt signaling cooperate during formation of the otic placode. However, in the mouse, although FGF signaling induces Wnt8a expression during induction of the otic placode, it is unclear whether these two signaling pathways functionally cooperate. Sprouty (Spry) genes encode intracellular antagonists of receptor tyrosine kinase signaling, including FGF signaling. We previously demonstrated that the Sprouty1 (Spry1) and Sprouty2 (Spry2) genes antagonize FGF signaling during induction of the otic placode. Here, we investigate cross talk between FGF/SPRY and Wnt signaling during otic placode induction and assess whether these two signaling pathways functionally cooperate during early inner ear development in the mouse.MethodsEmbryos were generated carrying combinations of a Spry1 null allele, Spry2 null allele, β-catenin null allele, or a Wnt reporter transgene. Otic phenotypes were assessed by in situ hybridization, semi-quantitative reverse transcriptase PCR, immunohistochemistry, and morphometric analysis of sectioned tissue.ResultsComparison of Spry1, Spry2, and Wnt reporter expression in pre-otic and otic placode cells indicates that FGF signaling precedes and is active in more cells than Wnt signaling. We provide in vivo evidence that FGF signaling activates the Wnt signaling pathway upstream of TCF/Lef transcriptional activation. FGF regulation of Wnt signaling is functional, since early inner ear defects in Spry1 and Spry2 compound mutant embryos can be genetically rescued by reducing the activity of the Wnt signaling pathway. Interestingly, we find that although the entire otic placode increases in size in Spry1 and Spry2 compound mutant embryos, the size of the Wnt-reporter-positive domain does not increase to the same extent as the Wnt-reporter-negative domain.ConclusionsThis study provides genetic evidence that FGF and Wnt signaling cooperate during early inner ear development in the mouse. Furthermore, our data suggest that although specification of the otic placode may be globally regulated by FGF signaling, otic specification of cells in which both FGF and Wnt signaling are active may be more tightly regulated.Electronic supplementary materialThe online version of this article (doi:10.1186/s12861-015-0083-8) contains supplementary material, which is available to authorized users.

Sox9 regulates development of neural crest and otic placode in a time- and dose-dependent fashion

Neural crest and placodes share a number of important features, pointing to a possible common evolutionary origin. They both arise from the neural plate border, which is the boundary between the non-neural ectoderm and neural plate. The transcription factor Sox9 has been implicated in neural crest and otic placode induction in several species. To investigate the differential regulation of neural crest and otic placode induction by Sox9, a gain of function assay was performed using a hormone-inducible version of the Sox9 construct at different doses and time periods. Sox9 was expressed in both neural crest and otic placode cell populations in the same stage embryos by in situ hybridization. Using a gain of function approach, increased expression of neural crest marker (Snail2) and otic placode marker (Pax8) in Sox9-overexpressed embryos was observed. Higher dose of Sox9 reduced or eliminated both neural crest and placode cells in the embryos. Interestingly, otic placodes cells were more strongly affected as compared to neural crest cells. So, optimal dosage and timing of Sox9 expression are important for the development of the neural crest and otic placode. The development of the neural crest and otic placode are affected by Sox9 in a time- and dose-dependent manner.

Fgf10 is required for specification of non-sensory regions of the cochlear epithelium

The vertebrate inner ear is a morphologically complex sensory organ comprised of two compartments, the dorsal vestibular apparatus and the ventral cochlear duct, required for motion and sound detection, respectively. Fgf10, in addition to Fgf3, is necessary for the earliest stage of otic placode induction, but continued expression of Fgf10 in the developing otic epithelium, including the prosensory domain and later in Kolliker׳s organ, suggests additional roles for this gene during morphogenesis of the labyrinth. While loss of Fgf10 was implicated previously in semicircular canal agenesis, we show that Fgf10−/+ embryos also exhibit a reduction or absence of the posterior semicircular canal, revealing a dosage-sensitive requirement for FGF10 in vestibular development. In addition, we show that Fgf10−/− embryos have previously unappreciated defects of cochlear morphogenesis, including a somewhat shortened duct, and, surprisingly, a substantially narrower duct. The mutant cochlear epithelium lacks Reissner׳s membrane and a large portion of the outer sulcus–two non-contiguous, non-sensory domains. Marker gene analyses revealed effects on Reissner׳s membrane as early as E12.5-E13.5 and on the outer sulcus by E15.5, stages when Fgf10 is expressed in close proximity to Fgfr2b, but these effects were not accompanied by changes in epithelial cell proliferation or death. These data indicate a dual role for Fgf10 in cochlear development: to regulate outgrowth of the duct and subsequently as a bidirectional signal that sequentially specifies Reissner׳s membrane and outer sulcus non-sensory domains. These findings may help to explain the hearing loss sometimes observed in LADD syndrome subjects with FGF10 mutations.

CD133 Expression Correlates with Membrane Beta-Catenin and E-Cadherin Loss from Human Hair Follicle Placodes during Morphogenesis

Genetic studies suggest that the major events of human hair follicle development are similar to those in mice, but detailed analyses of this process are lacking. In mice, hair follicle placode ‘budding’ is initiated by invagination of Wnt-induced epithelium into the underlying mesenchyme. Modification of adherens junctions is clearly required for budding. Snail-mediated downregulation of adherens junction component E-cadherin is important for placode budding in mice. Beta-catenin, another adherens junction component, has been more difficult to study due to its essential functions in Wnt signaling, a prerequisite for hair follicle placode induction. Here, we show that a subset of human invaginating hair placode cells expresses the stem cell marker CD133 during early morphogenesis. CD133 associates with membrane beta-catenin in early placodes and its continued expression correlates with loss of beta-catenin and E-cadherin from the cell membrane at a time when E-cadherin transcriptional repressors Snail and Slug are not implicated. Stabilization of CD133 via anti-CD133 antibody treatment of human fetal scalp explants depresses beta-catenin and E-cadherin membrane localization. We discuss this unique correlation and suggest a hypothetical model whereby CD133 promotes morphogenesis in early hair follicle placodes through the localized removal of membrane beta-catenin proteins and subsequent adherens junction dissolution.

RA and FGF signalling are required in the zebrafish otic vesicle to pattern and maintain ventral otic identities.

During development of the zebrafish inner ear, regional patterning in the ventral half of the otic vesicle establishes zones of gene expression that correspond to neurogenic, sensory and non-neural cell fates. FGF and Retinoic acid (RA) signalling from surrounding tissues are known to have an early role in otic placode induction and otic axial patterning, but how external signalling cues are translated into intrinsic patterning during otic vesicle (OV) stages is not yet understood. FGF and RA signalling pathway members are expressed in and around the OV, suggesting important roles in later patterning or maintenance events. We have analysed the temporal requirement of FGF and RA signalling for otic development at stages after initial anteroposterior patterning has occurred. We show that high level FGF signalling acts to restrict sensory fates, whereas low levels favour sensory hair cell development; in addition, FGF is both required and sufficient to promote the expression of the non-neural marker otx1b in the OV. RA signalling has opposite roles: it promotes sensory fates, and restricts otx1b expression and the development of non-neural fates. This is surprisingly different from the earlier requirement for RA signalling in specification of non-neural fates via tbx1 expression, and highlights the shift in regulation that takes place between otic placode and vesicle stages in zebrafish. Both FGF and RA signalling are required for the development of the otic neurogenic domain and the generation of otic neuroblasts. In addition, our results indicate that FGF and RA signalling act in a feedback loop in the anterior OV, crucial for pattern refinement.

Vertebrate neurogenic placode development: historical highlights that have shaped our current understanding.

With the flood of published research encountered today, it is important to occasionally reflect upon how we arrived at our current understanding in a particular scientific discipline, thereby positioning new discoveries into proper context with long-established models. This historical review highlights some of the important scientific contributions in the field of neurogenic placode development. By viewing cumulatively the rich historical data, we can more fully appreciate and apply what has been accomplished. Early descriptive work in fish and experimental approaches in amphibians and chick yielded important conceptual models of placode induction and cellular differentiation. Current efforts to discover genes and their molecular functions continue to expand our understanding of the placodes. Carefully considering the body of work may improve current models and help focus modern experimental design.

Foxi3 is necessary for the induction of the chick otic placode in response to FGF signaling

Vertebrate cranial sensory organs are derived from region at the border of the anterior neural plate called the pre-placodal region (PPR). The otic placode, the anlagen of the inner ear, is induced from PPR ectoderm by FGF signaling. We have previously shown that competence of embryonic ectoderm to respond to FGF signaling during otic placode induction correlates with the expression of PPR genes, but the molecular basis of this competence is poorly understood. Here, we characterize the function of a transcription factor, Foxi3 that is expressed at very early stages in the non-neural ectoderm and later in the PPR of chick embryos. Ablation experiments showed that the underlying hypoblast is necessary for the initiation of Foxi3 expression. Mis-expression of Foxi3 was sufficient to induce markers of non-neural ectoderm such as Dlx5, and the PPR such as Six1 and Eya2. Electroporation of Dlx5, or Six1 together with Eya1 also induced Foxi3, suggesting direct or indirect positive regulation between non-neural ectoderm genes and PPR genes. Knockdown of Foxi3 in chick embryos prevented the induction of otic placode markers, and was able to prevent competent cranial ectoderm from expressing otic markers in response to FGF2. In contrast, Foxi3 expression alone was not sufficient to confer competence to respond to FGF on embryonic ectoderm. Our analysis of PPR and FGF-responsive genes after Foxi3 knockdown at gastrula stages suggests it is not necessary for the expression of PPR genes at these stages, nor for the transduction of FGF signals. The early expression but late requirement for Foxi3 in ear induction suggests it may have some of the properties associated with pioneer transcription factors.