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First published online June 8, 2005
doi: 10.1242/10.1242/dev.01885

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Comparative analysis of genes downstream of the Hoxd cluster in developing digits and external genitalia

National Research Center (NCCR) `Frontiers in Genetics', Department of Zoology and Animal Biology, University of Geneva, Sciences III, Quai Ernest Ansermet 30, 1211 Geneva 4, Switzerland

View larger version (27K): [in a new window]   Fig. 1. Experimental strategy for identifying Hoxd-regulated genes in developing genitalia and forelimbs. (A) In situ hybridization of a wild-type E12.5 embryo stained with a Hoxd13 probe illustrates the tissues dissected for this study. Hoxd13 is strongly and broadly expressed in the genital bud (right) and distal forelimb (left). Genital buds and distal forelimb buds were dissected (arrows indicate cut points) from E12.5 wild-type (WT) embryos and from homozygous mutant embryos in which all nine Hoxd genes had been deleted (HoxDDel1-13). (B) Gene expression was analyzed with Affymetrix microarrays (each array is represented schematically as a circle; see Materials and methods). (C) Microarray analyses yielded two sets of initially non-overlapping differentially-expressed candidate genes from limbs and genitals. A subset of the candidates (16 genes) was validated by quantitative real-time RT-PCR analysis. (D) Expression of the 16 candidate genes was measured by real-time RT-PCR over developmental time from secondary samples collected from E11.5 to E14.5. Nine out of 14 confirmed genes also had highly significant differential expression in the other tissue on at least one day of development. (E) The transcript profiles of the 14 genes were visualized by whole-mount in situ hybridization (WISH), in several cases further validating their differential expression.

View larger version (21K): [in a new window]   Fig. 2. Hoxa11 is upregulated in genital buds when Hoxd genes are fully deleted. (A) WISH reveals an upregulation of Hoxa11 in the genital bud (arrows) of an E12.5 embryo. Upregulation is also apparent in the trunk, whereas it is not obvious in the forelimbs. Hindlimbs were removed to visualize the genitalia. (B) Real-time RT-PCR analysis confirms that Hoxa11 is expressed 2- to 3-fold higher in developing HoxDDel1-13 genitalia from E12.5 through E14.5. By contrast, Hoxa11 is only slightly increased in mutant forelimbs, to a level that is statistically significant at E11.5 only (1.35-fold). For these and all graphs in other figures: *Paired Student's t-test P<0.05; **P-value<0.01. Values are means of triplicate samples; error bars represent s.d.

View larger version (40K): [in a new window]   Fig. 3. Expression of Sgk is dependent on Hoxd gene function in genitals and forelimb buds. (A) Sgk expression appears distally in E11 wild-type limb buds. At E12.5 and E13.5, expression is prominent in the proximal portion of the interdigital zone. By contrast, Sgk expression in mutant HoxDDel1-13 limbs is largely limited to a proximal domain at E12.5, with little interdigital staining. (B) In the developing genital bud, Sgk is expressed in a distal domain of the ventral mesenchyme, adjacent to the urethral epithelium (arrow). This domain is not visible in mutant genital buds. (C) Real-time RT-PCR measurements mirror the WISH results, with highly significant (**P<0.01) differential expression in both limbs and genitals. Although the differential expression was initially detected in genitals, the highest fold change in the developmental series was measured in limbs (2.36-fold at E11.5).

View larger version (45K): [in a new window]   Fig. 4. Gfra2 is downregulated in limbs and genitals in the absence of Hoxd gene function. (A) Ventral view of Gfra2 expression in right forelimbs. Gfra2 expression is first visible at E13.5 on the ventral side of the developing autopod. This domain is only weakly stained in HoxDDel1-13 mutants. (B) Gfra2 is expressed strongly in the genital bud from the time of its emergence. By E13.5, the expression is much stronger in the distal half of the bud. In mutant genitalia, the expression is reduced and is more distally restricted at all days stained. (C) Real-time RT-PCR analysis quantifies and confirms the differences observed by WISH.

View larger version (43K): [in a new window]   Fig. 5. Analysis of candidates for downregulation (Odz4 and Epha3) or upregulation (Gdf10) by Hoxd gene products. WISH staining of E12.5 right forelimbs are shown for each gene. Lateral views of genitals (at right in each panel) are at E13.5 for Odz4 and Epha3, and E12.5 for Gdf10. Real-time RT-PCR quantification for each gene is shown as graphs. (A) The Odz4 forelimb expression domain extends more distally in HoxDDel1-13 mutants at E12.5, and expression is gained in the mutant genitalia. (B) Epha3 is expressed at a higher level in mutant forelimbs. The limb expression domain increases posteriorly (right) and distally, when compared with the wild type (left). Real-time RT-PCR measurements also indicate an increase in genital expression in the HoxDDel1-13 mutant (**P<0.01 at E13.5). By WISH, this increase is visualized as a slight broadening of the expression domain in the mutant genitals. (C) Gdf10 expression is weaker in mutant forelimbs and genital buds. Gdf10 is expressed in a broad domain in both wild-type and mutant limbs and genitals, making visualization of differences by WISH difficult, although some decrease in mutant tissues is apparent. Real-time RT-PCR quantification shows highly significant differences (**P<0.01) in both tissues.

View larger version (87K): [in a new window]   Fig. 6. Analysis of Epha3 and Sgk expression in the HoxDDel1-10 gain-of-function mutant. (A) Anterior gain of Hoxd13 expression (arrow) in an E11.5 HoxDDel1-10 right forelimb. (B) Epha3 expression is completely abolished in the region where Hoxd13 expression is gained (arrow). This observation contrasts with the increased Epha3 expression seen in HoxDDel1-13 mutant limbs (Fig. 5B), further supporting Epha3 as a candidate gene for Hoxd repression. Complete Epha3 repression was observed in 11 out of 12 HoxDDel1-10 forelimbs stained. (C) By contrast, Sgk expression is gained in the region of presumptive digit 1 (arrow) in the HoxDDel1-10 forelimb. This weakly stained domain was visible in eight out of eight mutant E11.5 forelimbs. Accordingly, Sgk expression was lost in the E11.5 HoxDDel1-13 mutant (see Fig. 3A), indicating that Sgk is upregulated by Hoxd gene expression.

View larger version (40K): [in a new window]   Fig. 7. Epha3 expression in combined Hoxa13/Hoxd mutants. WISH staining of right forelimbs from E12.5 littermates; anterior is to the left and posterior to the right. (A) Wild-type Epha3 expression pattern at E12.5. (B) Epha3 expression in transheterozygote littermate, where one copy each of Hoxa13 and the Hoxd cluster are intact. Epha3 expression is moderately gained similar to that seen when both copies of the Hoxd cluster are deleted (Fig. 5B). (C) The gain of Epha3 expression spreads through much more of the autopod, distally and posteriorly, when a single Hoxa13 is inactivated in combination with a homozygous deletion of the Hoxd cluster. (D) Epha3 expression in a double homozygote limb. Even though most of the autopod does not form in this mutant, Epha3 expression is gained to the posterior extreme of the limb bud (arrow). Because of the small size of the mutant limbs, it is useful to compare with the expression in E11.5 embryos to illustrate the gain of expression (as shown in Fig. 6B).