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Cranial placodes are transient thickenings of the vertebrate embryonic head ectoderm that will give rise to sensory (olfactory, lens, and otic) and non-sensory (hypophyseal) components of the peripheral nervous system (PNS). In most vertebrate embryos, these four sensory placodes undergo invagination. Epithelial invagination is a morphological process in which flat cell sheets transform into three-dimensional structures, like an epithelial pit/cup. The process of invagination is crucial during development as it plays an important role for the formation of the lens, inner ear, nasal cavity, and adenohypophysis. Using the chick as the model system the following questions were addressed. What signals are involved in placode invagination? Is there any common regulatory molecular mechanism for all sensory placode invagination, or is it controlled by unique molecular codes for each individual placode? Are placode invagination and acquisition of placode-specific identities two independent developmental processes or coupled together? To address this we used in vivo assays like electroporation and whole embryo culture. Our in vivo results provide evidence that RhoA and F-actin rearrangements, apical constriction, cell elongation and epithelial invagination are regulated by a common BMP (Bone morphogenetic protein) dependent molecular mechanism. In addition, our results show that epithelial invagination and acquisition of placode-specific identities are two independent developmental processes. BMP signals have been shown to be essential for lens development and patterning of the retina. However, the spatial and temporal requirement of BMP activity during early events of lens development has remained elusive. Moreover, when and how retinal cells are specified, and whether the lens plays any role for the early development of the retina is not completely known. To address these questions, we have used gain- and loss-of-function analyses in chick explant and intact embryo assays. Here, we show that during lens development BMP activity is both required and sufficient to induce the lens specific marker, L-Maf. After the L-Maf upregulation the cells are no longer dependent on BMP signaling for the next step of fiber cell differentiation, which is characterized by up-regulation of δ-crystallin expression. Regarding the specification of retinal cells our results provide evidence that at blastula stages, BMP signals inhibit the acquisition of eye-field character. Furthermore, from optic vesicle stages, BMP signals emanating from the lens are essential for maintaining eye-field identity, inhibiting telencephalic character and inducing neural retina cells.

Vertebrates have evolved a unique way to sense their environment: placodallyderived sense organs. These sensory structures emerge from a crescent-shaped domain, the preplacodal domain, which surrounds the anterior neural plate and generates the paired sense organs as well as the cranial ganglia. For decades, embryologists have attempted to determine the tissue interactions required for induction of various placodal tissues. More recently, technological advances have allowed investigators to ask probing questions about the molecular nature of placodal development. In this dissertation I largely focus on development of the otic placode. I utilize loss-of-function techniques available in the zebrafish model system to demonstrate that two members of the fibroblast growth factors family of secreted ligands, Fgf3 and Fgf8, are redundantly required for otic placode induction. I go on to show that these factors are expressed in periotic tissues from the beginning of gastrulation. These findings are consistent with a model where Fgf3 and Fgf8 signal to preotic tissue to induce otic-specific gene expression. This model does not address other potential inducers in otic induction. A study using chick explant cultures suggests that a member of the Wnt family of secreted ligands also has a role in otic induction. I therefore test the relative roles of Wnt and Fgf in otic placode induction. The results demonstrate that Wnt functions primarily to correctly position the Fgf expression domain and that it is these Fgf factors which are directly received by future otic cells. Lastly, I examine the function of the muscle segment homeobox (msx) gene family expressed in the preplacodal domain. This study demonstrates that Msx proteins refine the boundary between the preplacodal domain and the neural plate. Further, msx genes function in the differentiation and survival of posterior placodal tissues (including the otic field), neural crest and dorsal neural cell types. Loss of Msx function results in precocious cell death and morphogenesis defects which may reflect perturbed BMP signaling.

The inner ear is a complex sensory organ of interconnected chambers, each with a sensory epithelium comprised of hair cells and support cells for detection of sound and motion. This dissertation focuses on the development and regulation of sensory epithelia in zebrafish and utilizes loss of function, gain of function and laser ablation techniques. Hair cells and support cells develop from an equivalence group specified by proneural genes encoding bHLH transcription factors. The vertebrate Atoh1 bHLH transciption factor is a potential candidate for this role. However, data in mouse has led some researchers to conclude it does not have a proneural activity, but, rather, is involved in later stages of hair cell differentiation. In addition, the factors regulating Atoh1 are mostly unknown. We address these issues in zebrafish and show that the zebrafish homologs atoh1a and atoh1b are required during two developmental phases, first in the preotic placode and later in the otic vesicle. They interact with the Notch pathway and are necessary and sufficient for specification of sensory epithelia. Our data confirm atoh1 genes have proneural function. We also go on to show Atoh1 works in a complex network of factors, Pax2/5/8, Sox2, Fgf and Notch. Misexpression of atoh1 alters axial patterning and leads to expanded sensory epithelia, which is enhanced by misexpression of either fgf8 or sox2. Lastly, we examine the role of sox2 in sensory epithelia development and regeneration. Sox2 has been implicated in maintainence of pluripotent stem cells as well as cell differentiation. In the inner ear, Sox2 is initially expressed in the prosensory domain and is required for its formation. Eventually, Sox2 is downregulated in hair cells and maintained in support cells; however, its later role has not been determined. We show that in the zebrafish inner ear, sox2 is expressed after sensory epithelium development has begun and, like in mouse, expression is down regulated in hair cells and maintained in support cells. Our data demonstrate a role for sox2 in maintenance of hair cells and in transdifferentation of support cells into hair cells after laser ablation. Additionally, sox2 is regulated by Aoth1a/1b, Fgf, and Notch.

The vertebrate inner ear is a complex structure responsible for hearing and balance. The inner ear houses sensory epithelia composed of mechanosensory hair cells and non-sensory support cells. Hair cells synapse with neurons of the VIIIth cranial ganglion, the statoacoustic ganglion (SAG), and transmit sensory information to the hindbrain. This dissertation focuses on the development and regulation of both sensory and neuronal cell populations. The sensory epithelium is established by the basic helixloop- helix transcription factor Atoh1. Misexpression of atoh1a in zebrafish results in induction of ectopic sensory epithelia albeit in limited regions of the inner ear. We show that sensory competence of the inner ear can be enhanced by co-activation of fgf8/3 or sox2, genes that normally act in concert with atoh1a. The developing sensory epithelia express several factors that regulate differentiation and maintenance of hair cells. We show that pax5 is differentially expressed in the anterior utricular macula (sensory epithelium). Knockdown of pax5 function results in utricular hair cell death and subsequent loss of vestibular (balance) but not auditory (hearing) defects. SAG neurons are formed normally in these embryos but show disorganized dendrites in the utricle following loss of hair cells. Lastly, we examine the development of SAG. SAG precursors (neuroblasts) are formed in the floor of the ear by another basic helix-loophelix transcription factor neurogenin1 (neurog1). We show that Fgf emanating from the utricular macula specifies neuroblasts, that later delaminate from the otic floor and undergo a phase of proliferation. Neuroblasts then differentiate into bipolar neurons that extend processes to hair cells and targets in the hindbrain. We show evidence that differentiating neurons express fgf5 and regulate further development of the SAG. As more differentiated neurons accumulate, increasing level of Fgf terminates the phase of neuroblast specification. Later on, elevated Fgf stabilizes the transit-amplifying phase and inhibits terminal differentiation. Thus, Fgf signaling regulates SAG development at various stages to ensure that proper number of neurons is generated.

Vertebrates have evolved a unique way to sense their environment: placodallyderived sense organs. These sensory structures emerge from a crescent-shaped domain, the preplacodal domain, which surrounds the anterior neural plate and generates the paired sense organs as well as the cranial ganglia. For decades, embryologists have attempted to determine the tissue interactions required for induction of various placodal tissues. More recently, technological advances have allowed investigators to ask probing questions about the molecular nature of placodal development. In this dissertation I largely focus on development of the otic placode. I utilize loss-of-function techniques available in the zebrafish model system to demonstrate that two members of the fibroblast growth factors family of secreted ligands, Fgf3 and Fgf8, are redundantly required for otic placode induction. I go on to show that these factors are expressed in periotic tissues from the beginning of gastrulation. These findings are consistent with a model where Fgf3 and Fgf8 signal to preotic tissue to induce otic-specific gene expression. This model does not address other potential inducers in otic induction. A study using chick explant cultures suggests that a member of the Wnt family of secreted ligands also has a role in otic induction. I therefore test the relative roles of Wnt and Fgf in otic placode induction. The results demonstrate that Wnt functions primarily to correctly position the Fgf expression domain and that it is these Fgf factors which are directly received by future otic cells. Lastly, I examine the function of the muscle segment homeobox (msx) gene family expressed in the preplacodal domain. This study demonstrates that Msx proteins refine the boundary between the preplacodal domain and the neural plate. Further, msx genes function in the differentiation and survival of posterior placodal tissues (including the otic field), neural crest and dorsal neural cell types. Loss of Msx function results in precocious cell death and morphogenesis defects which may reflect perturbed BMP signaling.

Preplacodal ectoderm (PPE) is a contigous horse-shoe shaped domain that enwraps the anterior neural plate towards the end of gastrulation and eventally resolves into a number of focal epithelial thickenings called placodes. These placodes together with Neural Crest (NC), contributes to the peripheral nervous system in vertebrates. PPE and NC arise at the neural-non neural interface by distinct mechanisms during development. However, a general idea in the field was that a Bmp signaling gradient specifies different ectodermal fates: high Bmp levels specify epidermis, intermediate levels PPE and NC and no Bmp signaling is required for neural fate specification. We showed that while NC responds to intermediate levels of Bmp signals, PPE is specified by a distinct mechanism that involves a two step model for PPE specification. In the first step, Bmp is positively required to activate four competence factors, tfap2a, tfap2c, foxi1 and gata3 throughout the ventral ectoderm and renders this domain competent to respond to inductive factors. In the second step, inductive factors Fgf and Bmp antagonists act to completely block all Bmp signaling to specify PPE at neural-non neural interface. These Bmp-activated competence factors do not need Bmp for subsequent maintenance because they positively cross-regulate and autoregulate each other’s expression forming a gene regulatory network. This network is sufficient to rescue both PPE and NC in the complete absence of Bmp. The subsequent resolution of PPE into discerte placodal thickenings was hypothesized to involve localized migration of placodal progenitors and one of the molecules that could play an important role during cell migration was extracellular matrix binding molecule, integrin alpha 5 (itga5) because it was expressed at the right time and place. Knockdown of itga5 results in disorganised trigeminal, epibranchial ganglia and smaller otic placodes. Tracing the cell trajectories of placodal progenitors revealed that cells failed to migrate directionally. Additionally, we observed elevated levels of cell death in itga5 morphants which could be rescued by overexpression of Fgf ligands suggesting that Itga5 and Fgf pathways cooperate during placodal development. All together, this dissertation reveals novel genetic mechanisms that regulate placodal development from late-blastula to mid-somitogenesis stages.

In vertebrates, the otic placode forms inner ear and epibranchial placodes produce sensory ganglia within branchial clefts. Fibroblast growth factor (FGF) family of protein ligands from the surrounding tissues are responsible for otic and epibranchial placode induction. Members of pax2/5/8 family of transcription factors function as mediators during otic induction. To understand the temporal and spatial requirements of Fgf and their interaction with pax2/8 for otic induction, we used heat shock inducible transgenic lines of zebrafish to misexpress fgf3/8 and pax2a/8 under the control of hsp70 promoter. Loss of function studies were done to examine the functions of pax2/8 genes in regulating otic and epibranchial development. We show that global transient activation of hs:fgf3 or hs:fgf8 at mid-late gastrula stages (7-8 hpf) severely impairs otic induction, in part by disrupting formation of the principal signaling centers in the hindbrain. Additionally, mosaic studies show that high-level misexpression blocks otic fate cell-autonomously, whereas low to moderate levels promote otic development. At later stages high-level Fgf misexpression, both globally and locally does not inhibit otic fate, but rather causes a dramatic expansion of endogenous otic domains. Misexpression of hs:pax2a or hs:pax8 also expands endogenous otic domains but is not sufficient to bypass the requirement for Fgf signaling. Co-misexpression of Fgf with pax2a or pax8 leads to production of ectopic otic tissue in a broad range of cranial ectoderm. These data show that changes in timing, distribution and level of Fgf signaling and its downstream effectors influences otic induction. We show that otic and epibranchial placodes are induced 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 along with pax2a/pax2b downregulate foxi1 expression in otic cells, which is necessary for further otic development. Additionally, pax2/8 activate otic expression of fgf24, which induces epibranchial expression of sox3. Blocking functions of fgf24 or sox3 causes severe epibranchial deficiencies but has little effect on otic development. These results support the model whereby the otic placode forms first and induces epibranchial placodes through pax2/8-dependent Fgf24 signaling.

Indiana University-Purdue University Indianapolis (IUPUI) / Loss of the finite cochlear hair cells of the inner ear results in sensorineural deafness. Human cochlear hair cells do not regenerate, and there is no cure for deafness. Our laboratory has established a three-dimensional culture system for deriving functional sensory hair cells from human pluripotent stem cells. A major limitation of this approach is that derived hair cells exhibit a morphological and gene expression phenotype reflective of native vestibular hair cells. Previous studies have shown that establishment of localized domains of gene expression along the dorso-ventral axis of the developing otic vesicle is necessary for proper morphogenesis of both auditory and vestibular inner ear structures. Sonic hedgehog (SHH) signaling has been shown to play a key role in specification of the ventral otic vesicle and subsequent cochlear development. Here, SHH treatment was pursued as a potential strategy for inducing a patterning phenotype permissive to cochlear induction in vitro. Single-cell RNAsequencing analysis revealed that while treatment with the SHH pathway agonist Purmorphamine reduced expression of markers for the vestibular-yielding dorsal otic vesicle, upregulation of ventral otic marker genes was modest. More strikingly, the number of otic progenitors exhibiting a neuroprogenitor phenotype increased in response to Purmorphamine treatment. These results suggest that SHH pathway modulation in early-stage inner ear organoids may bias their differentiation toward a neural lineage at the expense of an epithelial lineage. The present study is the first to evaluate the patterning phenotype of human stem cell derived otic progenitors, and sheds light on the transcriptomic profile at this critical point of inner ear development. This study may also cultivate future efforts to derive cochlear cell types as well as inner ear neural cell types from human pluripotent stem cells, and contribute to the establishment of a more complete in vitro model of inner ear development. / 2021-08-21

La surdité neurosensorielle est définie par une atteinte de l’oreille interne, il résulte principalement d’une perte de cellules ciliées (CC). Chez les mammifères, ce processus est malheureusement irréversible. Le développement de la thérapie cellulaire a fait naître de nouveaux espoirs pour le traitement des surdités neurosensorielles. Les cellules souches d’origine embryonnaire ou adulte seraient capables de se différencier in vitro en progéniteurs otiques et de restaurer partiellement les fonctions auditives in vivo après transplantation. Cependant, les protocoles de différenciation in vitro des CC à partir de cellules souches sont insatisfaisants, et les signaux qui contrôlent ce phénomène restent mal connus. Ainsi, l’objectif de ce travail de thèse était d’étudier in vitro la différenciation des CC à partir de cellules souches pluripotentes induites humaines (hiPSC). Nous nous sommes intéressés à deux voies de signalisation majeures impliquées dans le développement de l’oreille interne in vivo, la voie Notch et la voie Wnt. Dans une première partie, nous avons montré que l’inhibition tardive de la voie Notch favorise la différenciation des hiPSC en CC. Dans une seconde partie, nous avons étudié le rôle de la voie Wnt dans la différenciation des hiPSC en cellules otiques. Nos résultats indiquent que l'inhibition de la voie Wnt durant la première phase d’induction favorise l'expression des marqueurs de la placode otique et initie la spécification des CC.Les travaux présentés dans cette thèse améliorent ainsi les protocoles de différenciation des hiPSC et suggèrent que ce type de cellules serait parfaitement adapté pour traiter les surdités neurosensorielles. / Neurosensory hearing loss is associated to inner ear disorders and degeneration of hair cells (HCs). Unfortunately, this process is irreversible in mammals. Currently, no curative treatment allows these cells to regenerate. For this reason, the development of cell therapy arose new hopes for the treatment of neurosensory hearing loss. Stem cells, either of embryonic or adult origin, seem able to differentiate in vitro into otic progenitors and to partially restore auditory functions in vivo. However, current protocols for in vitro differentiation of stem cells into HCs are unsatisfactory, and the signals that control this phenomenon remain poorly understood. Thus, the objective of this thesis was to study in vitro HC differentiation from human induced pluripotent stem cells (hiPSCs). We were particularly interested in two major signaling pathways involved in vivo in inner ear development, the Notch and Wnt signaling pathways.In a first part, we demonstrated that Notch inhibition during late otic differentiation enhances hiPSC differentiation into hair cell-like cells. In a second part, we studied the role of the Wnt signaling pathway during otic induction and HC specification. Our results indicate that Wnt inhibition during early otic induction promotes the expression of otic placode markers and initiate HC specification. The work presented here thus propose improved protocols to obtain HCs from hiPSCs, and suggest that this cell type is perfectly adapted for the treatment of neurosensory hearing loss.