Embryonic Origin of the Olfactory Sensory System: Fate Map, Lineage Analysis and Specification of the Avian Olfactory Placode

Citation

Bhattacharyya, Sujata (2005) Embryonic Origin of the Olfactory Sensory System: Fate Map, Lineage Analysis and Specification of the Avian Olfactory Placode. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/hftc-6c39. https://resolver.caltech.edu/CaltechETD:etd-05282005-205447

Abstract

Coordinating the generation of myriad cell types within the developing nervous system is an exquisite and intensely studied puzzle. The entire vertebrate peripheral nervous system derives from two multipotential cell types in the embryo: neural crest and placodes. Neurogenic placodes are focal ectodermal thickenings present in stereotypic positions in the head. Their derivatives are responsible for much of our sensory perceptions in the craniofacial region. The olfactory placode which gives rise to the olfactory epithelium mediates our sense of smell. Its derivatives include the regenerating olfactory sensory neurons, gonadotropin releasing hormone neurons, olfactory ensheathing glial cells and the basal and supporting cells. While we have some clues to the molecular mechanisms driving its differentiation into the various cell types mentioned above, little is known about the source and induction of the olfactory placode precursor cells in the early embryo.

To trace definitively the origin of the olfactory placode precursor cells, we generated a fate-map and compared it with patterns of gene expression in the region of the chick olfactory placode. To this end, small populations of cells in Hamburger-Hamilton (HH) stage 6 to stage 10 chick embryos were labeled with DiI and DiO and their derivatives were analyzed two days later. At head-fold stages, olfactory placode precursor cells are spread out over a broad domain and intermingle with lens, epidermal and neural precursors. As the neural folds close, the precursors appear to converge anteriorly within the ectoderm. The lens and nasal precursors sort out from each other around HH stage 8, at which time, Pax-6 is differentially upregulated in the region fated to form the lens while Dlx-5 expression is enhanced in the anterior area where the nasal precursors accumulate. To further study the cell movements that lead to the eventual formation of the olfactory placode, I performed confocal time-lapse analysis. The cell rearrangements that I observed are consistent with two possible outcomes: 1.specified lens and nasal precursors have differential adhesive properties and hence sort out or 2.unspecified placodal precursors differentiate according to the environment in which they are positioned by stochastic movements. To distinguish between these possibilities and to clarify whether the precursors are multipotent before they segregate, I have undertaken single cell lineage analysis. Surprisingly, I find no evidence for a shared olfactory and lens placode lineage from single precursors even at early neurula stages, prior to their sorting out from each other in order to contribute to one or the other placode. This raises an interesting question: does the fate of these cells motivate their migration to a certain region of the embryo?

In the next part of my thesis, I have sought to answer a fundamental question in developmental biology, which is how are organs generated in precise and reproducible locations within the body. I have attempted to answer this question in the context of the olfactory sensory system. To understand how and when the nasal structure is first induced, I decided to delineate the tissue that is competent to form the olfactory placode and to determine the spatiotemporal localization of the inducing signals. In general, either one of these two parameters is strictly delimited such that the induced structure arises only in a distinct position. In order to define the extent of competence within the ectoderm to form the nasal placode, I have grafted quail ectoderm from different axial levels to the chick anterior neural fold at stage 8. Cranial and trunk level ectoderm are capable of responding to the inducing signals; they express PAX6 and subsequently form the olfactory placode. However, hindbrain and trunk level ectoderm lose this competence rapidly; by stage 10 neither tissue can express PAX6. This suggests that either the inducing signals are localized anteriorly at early stages or that later signals further refine the olfactory placode-forming region. The presumptive olfactory placode ectoderm is defined by co-expression of several markers. Therefore, I have also analyzed these grafts for DLX3 expression and find a similar trend in loss of competence as seen with PAX6. A prerequisite for studying the induction of a particular fate in a tissue is determining the time at which it is still unspecified i.e. the tissue does not express markers exclusive to its fate when removed from its original context in the embryo and placed in a neutral environment in vitro. I examined the specification of the presumptive olfactory placode ectoderm to express PAX6 and DLX3 and form neurons by culturing this tissue at various stages in three-dimensional collagen gel matrices. Presumptive olfactory placode ectoderm is specified to express PAX6 and DLX3 between stages 8-10. Neuronal specification as assayed by expression of the post-mitotic neuronal marker, Hu, begins around stage 14. This implies that the ectoderm has seen signals that will direct its fate even before it is morphologically visible as a placode. I have also determined the time at which the presumptive olfactory placode ectoderm is irreversibly committed to its fate by grafting this tissue at different stages to the lateral plate ectoderm at the level of the most recently formed somites in the stage 8/9 chick embryo. This occurs by stage 14 as assayed by expression of PAX6 and DLX3, concomitant with a visible thickening of the placode.

The next step is to determine the molecular nature of the inducing signals. Such embryological manipulations in combination with fate-mapping and lineage studies will hopefully afford us some insight into the basic principles by which sensory systems are assembled during development.

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