| The roof plate is a dorsal midline organizing center in the central nervous system that controls specification and differentiation of adjacent neurons through secretion of the BMP and WNT signaling molecules. The LIM-homeodomain transcription factor (LIM-HD) Lmx1a is expressed in the roof plate and its progenitors at all axial levels of the CNS and is necessary and sufficient for roof plate formation in the spinal cord. Loss of Lmx1a, however, does not completely abolish roof plate formation in anterior hindbrain. A residual roof plate still forms which is sufficient to at least partially direct adjacent cerebellar cell fate specification. I hypothesize that Lmx1b, another member of the LIM-HD transcription factor family which is highly related to Lmx1a, functions redundantly with Lmx1a to form the hindbrain roof plate. Although Lmx1b null mice do not show a substantial deficiency in hindbrain roof plate formation, Lmx1a/Lmx1b double null mutant mice completely lack hindbrain roof plate. This observation indicates that both genes act in concert during hindbrain roof plate formation. Because of the requirement of Lmx1b to maintain the isthmic organizer (IsO), which is an important signaling center lying along the anterior/posterior axis at the midbrain-hindbrain junction, the adult cerebellar phenotype in Lmx1b null homozygous mutant mice cannot be observed due to perinatal lethality. To isolate the anterior/posterior patterning functions from the dorsal/ventral patterning role of Lmx1b, I used a conditional knockout strategy to specifically delete dorsal midline Lmx1b expression leaving IsO expression intact. Phenotypic analysis of single and conditional Lmx1b and compound Lmx1a:Lmx1b conditional mutants confirmed overlapping roles for Lmx1 genes in regulating hindbrain roof plate formation and growth and also revealed roles in regulating adjacent cerebellar development. My analyses provide the first evidence of overlapping function of the Lmx1 genes during embryonic CNS development. Despite their importance, little is understood regarding the regulation of the Lmx1 genes. To begin to address this question, I used LacZ reporter-tagged BACs surrounding the Lmx1a locus to interrogate the regulatory sequences in transient transgenic mouse assays. Three BACs that tile across 357kb of the Lmx1a locus showed both distinct and overlapping patterns replicating most of the endogenous expression pattern of the Lmx1a gene and localizing a potential roof plate enhancer element(s) to a 70 kb region within the large second intron of this gene. Since Lmx1a is expressed in multiple important regions within the developing CNS including the hypothalamus, ventral midbrain, and subsets of hindbrain nuclei, the research presented in this thesis serves as a platform for future studies of the Lmx1 genes in CNS development. |
