| Summary: | The thymus develops from the ventral portion of 3<sup>rd</sup> pharyngeal pouch endoderm. At E9.5 this region is marked by expression of the transcription factor <i>Rhox4, Rhox4 </i>may therefore have a role in the developing thymic primordia. <i>Rhox4 </i>is a member of the recently described <i>Rhox </i>family of homeobox-containing genes, which are present in 3 clusters on the mouse X chromosome and are primarily expressed in reproductive and extra-embryonic tissues. The expression of <i>Rhox4 </i>in the thymus, in addition to the testis and placenta makes it unique amongst family members. Here we show that there are seven copies of <i>Rhox4 </i>present in a tandem array within the <i>Rhox </i><i>α</i><i> locus. All </i>alleles of <i>Rhox4 </i>are expressed, although preferential expression was observed and differed between tissues. In contrast to reproductive tissues, no evidence of co-linear expression of the <i>Rhox </i><i>α</i><i> </i>cluster during thymus development was found. Two other members of the <i>α</i><i> </i>cluster, <i>Rhox2 </i>and <i>Rhox3 </i>are also present in the duplicated unit, both having 8 copies. The only predicted orthologue of <i>Rhox4 </i>is present in a single copy in the rat; however, no <i>Rhox4 </i>expression was detected in the developing or postnatal rat thymus. Examination of different sub-species of mice showed that all contain multiple copies of <i>Rhox4, </i>suggesting that the duplication event arose at the time of divergence of the rat and mouse lineages. Finally, no changes in <i>Rhox4 </i>expression were detected in mice with defects in thymus organogenesis, placing it upstream or outside of established transcriptional pathways.
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