Supplemental Data Rhox: A New Homeobox Gene Cluster

Cell Type Specificity of Rhox Gene Expression in Testes To identify the cell types in the testis that express the Rhox genes, we purified testicular cell fractions enriched for Sertoli and interstitial (e.g., Leydig) cells. Although it is not possible to generate entirely pure populations of these cell types, our fractions were substantially enriched, based on the relative levels of Sertoli and Leydig cell markers. The interstitial cell fraction, compared to the whole testis, had 6and 12fold higher mRNA levels corresponding to the Leydig markers P450scc and lutenizing hormone (LH) receptor, respectively (Supplemental Table S1). The Sertoli cell fraction was 5-fold enriched for the Sertoli cell marker Gata1. This fraction was also 12-fold enriched for Rhox5, which we and others have previously shown is specifically expressed in Sertoli cells within the testis (mainly present in their nuclei; Lindsey and Wilkinson, 1996; Maiti et al., 2001; Pitman et al., 1998; Sutton et al., 1998). Most of the other Rhox genes were also expressed in Sertoli cells but three (Rhox1, 4, and 11) were also substantially enriched in the interstitial cell fraction. Rhox4 may be exclusively expressed in interstitial cells, as its expression was 12fold higher in the interstitial cell fraction than it was in whole testis. We suspect that Leydig cells are the source of Rhox4 in the interstitial-cell fraction, as it was also the only Rhox gene expressed in the Leydig cell line Tm3 (as assayed by real-time RT-PCR analysis; data not shown). Rhox4 expression was not detected in the three Sertoli cell lines that we tested: 15P-1, MSC1, and Tm4 (data not shown).