Cranial placodes are ectodermal regions that contribute extensively to the vertebrate peripheral nervous system. The development of the ophthalmic trigeminal (opV) placode, which gives rise only to sensory neurons of the ophthalmic lobe of the trigeminal ganglion, is a well-studied model of sensory neuron development. While key differentiation processes have been characterized at the tissue and cellular levels, the molecules governing opV placode development have not been well described. This study identifies the canonical Wnt signaling pathway as a regulator of opV trigeminal placode development. Introducing dominant-negative TCF and dominant-active β-catenin expression constructs by in ovo electroporation, we have manipulated the canonical Wnt pathway within the opV placode domain and surrounding ectoderm of chick embryos. Inhibition of canonical Wnt signaling results in the failure of targeted cells to express or maintain Pax3 protein, the earliest known specific molecular marker of opV placode cells. Misexpression of dominant-active β-catenin as an activator of canonical Wnt signaling, however, is not sufficient to promote the opV placode cell fate. We conclude that canonical Wnt signaling is necessary for normal opV placode development, and propose that other molecular cues are required in addition to Wnt signaling to promote cells to an opV placode fate. Strategies for manipulating the Wnt pathway at the level of ligand and receptor are also reviewed. Because it is clear that Wnt signaling is not acting alone in early development of the opV placode, we have also begun to investigate additional signaling pathways, such as FGFs, that may be involved in these developmental processes.

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.

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.

No description available.

In vertebrate embryos, the ophthalmic trigeminal (opV) placode contributes sensory neurons to the trigeminal ganglion during development of the peripheral nervous system. FGFR4 is expressed transiently in the trigeminal placode as cells undergo an epithelial-mesenchymal transition and begin to migrate toward the condensing ganglion. Because of the restricted spatiotemporal expression of FGFR4 in the opV placode, it was hypothesized that FGFR4 is necessary for the process of epithelial-mesenchymal transition in the opV placode. Using electroporation, an FGFR4 inhibitory gene construct was introduced into 6—10 somite stage chick embryos. This secreted inhibitory form of the FGFR4 gene blocked endogenous FGFR4 signaling, which resulted in trigeminal placode cells remaining in the ectoderm that would have normally begun migration into the mesenchyme. These results show that FGFR4 is involved in trigeminal placode cell delamination.

The vertebrate nervous system is a highly complex and intriguing structure with diverse functions. To understand the functional nervous system, we first have to be aware of how it is assembled during development. In this thesis the mechanism of early diversification and regionalisation necessary for subsequent formation of part of the nervous system, namely the telencephalon and the placodes, will be addressed. We have identified signalling molecules involved in the dorsoventral patterning of the telencephalon and we propose a mechanism for the induction and differential specification of the olfactory and lens placodes. The telencephalon is regionalised along the dorsoventral axis during development. The cells situated dorsally will give rise to the cerebral cortex while the ventral and intermediate cells are mainly progenitors for the basal ganglia. The cerebral cortex is associated with higher cognitive functions whereas the basal ganglia control movements. We provide evidence that dorsal and intermediate telencephalic cells are re-specified from cells with an intrinsic ventral character. Dorsal telencephalic cells are specified at stage 10 in chick, while the intermediate cells are specified a few hours later, at stage 14. The expression of Wnt and Fibroblast growth factors (Fgfs) coincides with the time point when the dorsal cells are specified, and we provide evidence that Wnt and FGF signals act in a sequential way to specify dorsal telencephalic cells. The retinoic acid (RA) synthesising enzyme Raldh3 is expressed in proximity to the telencephalon, and our result suggests that RA is both required and sufficient to induce intermediate telencephalic cell types. Additionally, Fgf8 is expressed in the anterior neural ridge and the ventral telencephalic cells require FGF signals that oppose RA to maintain their character. The olfactory and lens placodes contribute to the special sense organs associated with olfaction and vision, respectively. Olfactory and lens placodes are specified at gastrula stage in chick, and become spatially separated at the neural fold stage. We provide evidence that Bone morphogenetic protein (BMP) signalling is required for the induction of a pool of placodal progenitor cells. Furthermore, time of exposure to BMP signals plays a key role in the differential specification of the olfactory and lens placodes, where continued exposure to BMP signals promotes lens character at the expense of olfactory placodal cells.

The development of the nervous system is a complex process. Cell divisions, cell differentiation and signalling interactions must be tightly regulated. To comprehend the mature nervous system, we have to understand its assembly during development. Two main questions were addressed in this thesis: (1) how is the caudal part of the central nervous system specified and (2) how is the early development of the olfactory placode regulated? By using tissue and whole embryo assays in the chick, we identified signalling molecules involved in these processes and propose possible mechanisms for their function. The central nervous system is regionalized along its rostrocaudal axis during development. However, the mechanisms by which cells in the caudal part of the neuraxis acquire rostrocaudal regional identity have been unresolved. We provide evidence that at gastrula stages cells in the caudal neural plate are specified as cells of caudal spinal cord character in response to Wnt and FGF signals and that cells of rostral spinal cord and caudal hindbrain character only emerge later at neurulation stages in response to retinoic acid signalling acting on previously caudalized cells. In the hindbrain and spinal cord distinct motor neuron subtypes differentiate at precise rostrocaudal positions from progenitor cells. We provide evidence that cells in the caudal neural plate have acquired sufficient positional information to differentiate into motor neurons of the correct rostrocaudal subtype. The olfactory placode gives rise to all the structures of the peripheral olfactory system, which, in the chick consists of the olfactory nerve, the sensory epithelium, where the olfactory sensory neurons (OSN) are located and the respiratory epithelium, that produces the mucus. Several studies have addressed the role of signalling cues in the specification of OSNs but much less is known about the regulation of sensory versus respiratory patterning and the events controlling early neurogenesis in the developing olfactory placode. We show that by stage 14 the olfactory placode is specified to give rise to both cells of sensory and respiratory epithelial character. Moreover, cells of respiratory epithelial character require BMP signalling, whereas cells of sensory epithelial character require FGF signalling. We suggest a mechanism in which FGF and BMP signals act in an opposing manner to regulate olfactory versus respiratory epithelial cell fate decision. BMP signalling has also been implicated in the regulation of neurogenesis in the sensory epithelium, and we show that BMP signals are required for the generation of OSNs, because in the absence of BMP signalling cells in the sensory epithelium do not mature. Independently, we also analyzed the role of Notch signalling during early olfactory development both in vitro and in vivo and provide evidence that active Notch signalling is required to prevent cells in the olfactory placode from premature differentiation.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第22283号 / 理博第4597号 / 新制||理||1659(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)准教授 佐藤 ゆたか, 教授 高橋 淑子, 准教授 秋山 秋梅 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

Wnt signaling has been shown to have several roles in the development of sensory neurons, particularly in the ophthalmic portion of the trigeminal nerve. Many of these studies have relied on the conclusion that Wnt is necessary but not sufficient for the induction and maintenance of the neural precursor cells that develop in the ophthalmic placode. Wnt had been inhibited in the ophthalmic placode using a dominant negative t-cell factor (TCF) and resulted in the loss of Pax3 expression (indicative of undifferentiated placode cells) in all targeted cells, suggesting a loss of specification/commitment of these cells to the sensory neuron fate. This study aimed to build on that conclusion by identifying the source of Wnt signaling that allowed for the maintenance of these placode cells. To investigate this, chick embryo ex ovo cultures were used and treated with small molecule chemical Wnt inhibitors to globally knock out Wnt signaling. The embryos were then sectioned and stained for cell markers of undifferentiated placode and differentiated neural cells (Pax3 and Islet1, respectively). Also used was a conditional knockout of Porcn, a gene critical to post-transcriptional modification of the Wnt ligand, using Wnt1-cre as a driver; this allowed for the knockout of Wnt secretion from the dorsal neural tube as well as neural crest cells. The data showed a decrease in placode cell differentiation but did not indicate a necessity for Wnt in maintenance of the ophthalmic placode cells—there was no loss of Pax3 expressing cells in the ectoderm. This suggested that maintenance of the ophthalmic placode could be through alternate pathways. Data is also presented describing how loss of Porcn in Wnt1 expressing cells impacts craniofacial development, where the mouse mutant used in this study displayed the absence and underdevelopment of cranial neural crest structures.