Forward and reverse genetics to establish novel mechanisms underlying mammalian organogenesis.

摘要

The embryonic development of vertebrate species, from a single cell to a newborn organism consisting of a diverse array of properly proportioned and patterned tissues and organs, has long fascinated biologists. The genes that direct and coordinate developmental processes in mammals have been eagerly sought, both for the insights they can provide into fundamental questions of biology, as well as for the relevance of genetic mutations to human congenital malformations and disorders. For this purpose, the mouse has proven to be a valuable and tractable model organism with which to search for and study the genes that govern mammalian development.One critical aspect of embryonic development in mammals is the process of organogenesis. The tissue types comprising each organ must be properly specified and differentiated, organized correctly, and expand according to the appropriate size of the organ. In general, organogenesis involves the assembly of progenitor cells into organ primordia that consist of mesenchymal and epithelial tissues derived from different germ layers. As the organ primordium, referred to also as the anlage, begins to form, the specification of the different cell types that will constitute the mature organ must take place. At the same time as the proper cell lineages are being specified in the organ primordium, sufficient cell proliferation must occur in order to generate a critical mass of progenitors, to allow for organ morphogenesis and expansion.In this thesis, I will describe how I investigated the processes of organogenesis during embryonic development in the mouse. Specifically, I have identified and characterized genes and genetic pathways that play critical roles in enabling the proper specification, formation, morphogenesis, and expansion of mammalian organs. Toward this end, I will describe two different approaches I have undertaken. In Chapter 1, I will discuss a forward genetic screen that led me to identify a novel mouse mutation, named Morgante, which causes severe abnormal morphogenesis and severe hypoplasia of several tissues and organs, including skeletal cartilage, skeletal muscle, and heart. I will also describe a linkage analysis I performed to identify the specific chromosomal region bearing the mutation that causes these defects, as well as efforts I carried out to establish the exact genetic lesion and gene specifically affected by this mutation. In Chapter 2, I will describe an opposite approach I followed, employing the tools of reverse genetics to dissect genetic networks governing the organogenesis of the mammalian spleen. To this end, I characterized existing mutant mouse models bearing loss-of-function mutations in specific, targeted genes, including Pbx1, p15Ink4b, and Nkx2.5. Through this analysis, I will illustrate the critical importance of the homeodomain transcription factor Pbx1 in controlling the expansion of the developing spleen, and specifying appropriate organ size. Furthermore, I will describe the identification and validation of two downstream target genes of Pbx1, through which Pbx1 exercises control over spleen organogenesis and expansion. In sum, I will show how both types of approaches, both forward and reverse genetics, can be fruitfully employed to discover and characterize novel and essential genetic mechanisms that govern the fascinating processes of organ formation and expansion during mammalian development.