Activity of a limb enhancer from the Shox2 genomic region, as assayed in a transgenic embryo.

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Our current research can be divided into three categories: 1) the function of Shox2 during limb and brain development. 2) enhancers controlling Shox gene expression 3) the function of R-spondins during limb development.

Shox2 Function

    First we are focusing on how Shox2 expression is translated into function by downstream target genes. Although many transcription factors that are required to form limb elements have been identified, we know comparatively little about how these proteins actually function. Most notably, very few of the target genes that are regulated by transcription factors during limb development have been identified. We searched for candidate Shox2 target genes by microarray analysis and we are now characterizing their expression. We are exploring the function of the most promising candidates with transgenic and gene targeting techniques. We particularly want to understand how Shox2 influences the differentiation of the chondrocytes (cartilage-producing) cells of the developing skeleton.

    We have recently discovered that Shox2 is necessary for proper innervation of the forelimb (below right) and for proper formation of the triceps muscles.  This suggests the intriguing possibility that patterning of the different tissues of the proximal limb is coordinated by the function of Shox2. We are now investigating what genes downstream of Shox2 mediate the tissue patterning in the proximal limb.

Regulation of SHOX genes

    Second we are studying how DNA elements called enhancers control the expression of the Shox genes. This analysis is important because deletions on the human chromosome near the SHOX gene can cause limb malformations without disturbing the SHOX gene itself, presumably by removing enhancer sequences. Therefore we are using transgenic mice to understand how the expression of Shox genes is controlled. By using bacterial artificial chromosomes (BACs) as our transgene constructs we can scan large regions of the genome for enhancer activity and then assess the activity of individual sequences by deleting them from the transgenes. We have recently acquired the equipment necessary for performing pronuclear injections within our own laboratory, which will be a considerable asset for increasing the throughput for these experiments.

Another quality that makes the Shox genes a particularly attractive subject for study is that Shox2 is the only short-stature gene in the mouse genome, while humans have two. The remaining mouse short-stature gene, Shox2, is an excellent subject for study since mutation of this single gene causes a severe, discrete phenotype. The evolutionary loss of the Shox gene in the rodent lineage has thus provided an efficacious tool for understanding the function of a developmental transcription factor. The lack of the Shox gene in the mouse genome is especially noteworthy since virtually all other human genes involved in patterning basic embryonic structures have a single matching ortholog in the mouse. The Shox genes present a unique opportunity to study how very similar structures (i.e. human and mouse limbs) can be patterned by different gene complements.

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Our research is (or has been) funded by these agencies: