Identification of a Hoxd10‐regulated transcriptional network and combinatorial interactions with Hoxa10 during spinal cord development

Abstract

Hoxd10 is expressed in the posterior spinal cord and hindlimbs of the mouse. Hoxd10, along with other Hox transcription factors, is thought to regulate the activity of genes involved in nervous system patterning and motor neuron development, but little is known about the downstream targets regulated by this gene. cDNA microarrays were used to investigate the transcriptional network regulated by Hoxd10 in homozygous knockout animals. Sixty‐nine genes were identified with altered expression levels in mutant spinal cords. Among these were genes involved in such diverse cellular events as cellular communication, cell cycle control, development and differentiation, and neuronal survival. The expression of some of these genes was investigated using reverse transcriptase‐polymerase chain reaction (RT‐PCR) and in situ hybridization. Nine genes showed changes in expression of the same sign and similar magnitude using RT‐PCR in Hoxd10 single mutant animals, with additional changes in expression seen in Hoxa10/Hoxd10 double mutant animals. In situ hybridization studies also demonstrated changes in expression consistent with microarray results. Analysis of putative promoter regions for Hox protein binding sites suggested that some genes may be direct Hoxd10 targets, whereas others likely are regulated through intermediate steps. Using cDNA microarrays to study a single gene knockout during critical developmental stages has identified a large number of genes regulated by Hoxd10, many of which would not have been approached as candidates for Hox gene regulation based on function or expression.