Kathleen Whitlock - US grants

The olfactory organ, which gives rise to both the olfactory sensory neurons and a group of neuroendocrine cells containing gonadotropin- releasing hormone (GnRH), arises from the olfactory placode during development. GnRH is a hormone that has essential reproductive and neuromodulatory functions (Pfaff et. al., 1987) and Kallman's Syndrome in humans, characterized by anosmia and hypogonadism, has now been shown to result from the failure of the GnRH cells to migrate from the olfactory placode (Schwanzel-Fukuda et al., 1989; Legouis et al., 1991). While the development of the olfactory organ has been described after the time of olfactory placode differentiation, the early developmental events giving rise to the olfactory placode and its derivatives remain poorly understood. Two crucial unanswered questions in the field of olfactory biology will be addressed here: how does the olfactory placode differentiate and what is the lineage relationship between olfactory sensory neurons and GnRH cells? This proposal will address the question of which cellular and genetic processes control the initial development of the olfactory placode add subsequent differentiation of the GnRH cells and olfactory sensory neurons. Zebrafish will be used because of the ability to mark cells and follow them during development of live embryos. In addition, zebrafish provide a unique opportunity to screen for mutant phenotypes to identify genes important in the early development of the olfactory system and its derivatives.

The olfactory organ gives rise to the olfactory sensory neurons and is also thought to give rise to a group of neuroendocrine cells containing gonadotropin-releasing hormone (GnRH). The cell bodies of the olfactory sensory neurons lie in the olfactory epithelium of the nose and during development their axons grow from this peripherally located sensory structure into the central nervous system (CNS). These sensory axons create a pathway, the olfactory nerve, connecting the peripheral and central nervous system. The neuroendocrine cells containing GnRH use the olfactory pathway to gain access to the CNS. Improper development of the olfactory pathway will lead not only to loss of sense of smell, but also to loss of reproductive neuroendocrine function crucial for the development of vertebrate animals, as evidenced by Kallman's Syndrome in humans. This proposal will present and test several hypothesis central to questions in developmental biology: 1) Cranial neural crest plays an inductive role in olfactory placode formation, 2) Neuroendocrine cells containing GnRH arise from cranial neural crest and not the olfactory placode, 3) Olfactory neurons expressing the same olfactory receptor type are related by lineage. Zebrafish will e used to address these questions because of the ability to mark cells and follow them in live embryos, manipulate cells in live embryos, and examine mutant phenotypes resulting from genetic screens in order to understand cellular interactions crucial to the early development of the olfactory system and its derivatives.

9981445
Whitlock

The goals of this project are to measure in the zebrafish, a vertebrate model organism: (1) the number of recessive lethals (an extreme kind of harmful mutation) in the genome and (2) the rate that such harmful mutations arise. The number of lethal mutations carried in the genome will be estimated by measuring the fraction of lethal-free offspring that are produced using a new technique that makes individuals have only a single copy of the genome and so express all lethal mutations. The rate at which lethal mutations arise will be measured by making individuals in which both copies of the genome are identical (and thus carry no recessive lethals) and then measuring the number of lethals that arise in the next generation.

This study will provide the first data in any vertebrate on the genomic rate at which new harmful mutations arise. Among animals, data on the genomic rate of the new appearance of harmful mutations is available only for fruit flies. Knowledge of the rate that harmful mutations arise is essential to understanding evolutionary processes in living organisms, in managing small populations of endangered animals, as well as in assessing health risks in humans. While it is known that an average human newborn carries ~100 de novo mutations, it is not clear what fraction of them are harmful and so might contribute to miscarriage and a variety of hereditary diseases.

DESCRIPTION (provided by applicant): Our long-term goals are to understand the embryonic origin of the endocrine gonadotropin releasing hormone (GnRH) cells in the hypothalamus, and the mechanisms controlling the differentiation and migration of these cells. Kallmann Syndrome in humans, which is characterized by lack of GnRH cells in the hypothalamus and by loss of a sense of smell, has been used as evidence supporting a common origin for the GnRH cells of the hypothalamus and the olfactory sensory neurons. However, this link is likely only a consequence of the disruption in the migration of GnRH cells to the hypothalamus along the olfactory nerve. Consistent with this interpretation we have shown, in zebrafish, that mutant embryos lacking their pituitary also lack hypothalamic GnRH cells, suggesting that the GnRH cells arise from the anterior pituitary placode, not the olfactory placode as previously proposed. We will test the hypotheses that 1) the hypothalamic GnRH cells arise from precursors in the anterior pituitary placode and 2) migration of the hypothalamic GnRH cells is dependent upon anosminl and fibroblast growth factor receptor-1 gene function, 2 genes which when mutant cause Kallmann Syndrome phenotypes. We will test the first hypotheses by: labeling single cells within anterior pituitary placode domain and following the differentiation of the resulting clones of cells, and creating transgenic fish expressing Green Fluorescent Protein in the migrating GnRH cells to visualize them in living embryos. We will test the second hypothesis by: blocking the function of anosminl and fibroblast growth factor receptor-1 genes in the developing embryo using RNA interference technologies and examining effects on development of the olfactory system, anterior pituitary and hypothalamus. Understanding factors controlling the origin, differentiation and migration of the hypothalamic GnRH cells will allow us to make specific diagnoses based on developmental defects observed early in human development, thus alerting clinicians to the possibility of GnRH deficits later in life.