The regulation and function of the transcription factor Lhx9 during mouse limb and urogenital development.

Abstract

Developmentally important genes often have pleiotropic effects, being involved in and necessary for multiple developmental processes. An example of this is the LIM- homeobox gene, Lhx9, a transcription factor required for correct limb, brain and gonad development in mice. To understand how genes can contribute to more than one trait, how Lhx9 was used during the development of two very different tissues, limb and gonad. In mice, Lhx9 and its paralogue, Lhx2 are required for proper growth and pattering of the limb bud. Only null mutations of both genes in a single mouse strain results in defects in the growth and patterning of the proximal-distal (PD) and anterior-posterior (AP) axes of the limb. In comparison, mammalian gonads arise from a common progenitor tissue known as the urogenital ridge (UGR). Current development of the bipotential gonad is crucial for sex determination and sexual differentiation, but very little is known about how the molecular networks that shape its formation. Lhx9 is among only a handful of genes known to be required for formation of the bipotential gonad, but its broader regulatory network is poorly understood.

To begin with, the regulation of Lhx9 gene expression via known signaling molecules was investigated. These factors were fibroblast growth factor (Fgf) and sonic hedgehog (Shh), which govern limb bud growth and patterning of the axes. Fgf signaling, but not Shh, was found to be necessary for Lhx9 expression during early limb development. In addition, the expression patterns of Lhx9 isoforms in the developing limb was also determined, with only two out of three isoforms expressed at 11.5 dpc.

To investigate the gene networks that underlie bipotential gonad formation, chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq) was used. Over 1,300 putative target genes of Lhx9, including some with known roles in gonad development were uncovered, confirming Lhx9 is crucial for this process. RT-qPCR confirmed altered expression of some of these target genes in Lhx9+/- gonads. Gene ontology analyses showed that putative target genes are involved in biological processes such as angiogenesis, cell growth/ proliferation and cell signaling.