DEV01884 Supplementary Material

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• Supplemental Figure 1 (Adobe PDF) -

  Fig. S1. The Pod1 and Nkx3.2 transcription factors use separate pathways to regulate early spleen development. Transcripts for the spleen regulators Nkx3.2, Pod1 and Nkx2.5 were detected by in situ hybridization of frozen sagittal sections of E12-12.5 wild-type, Nkx3.2^–/– and Pod1^–/– upper abdominal organs, as indicated. (A,B) Nkx3.2 is expressed in the condensing mesenchyme of Pod1^–/– embryos, compared with wild-type littermates, although clear signs of involution of the Pod1^–/– splenic primordium start to be present at this gestational day, as previously reported (Lu et al., 2000). (C,D) Pod1 expression is unperturbed in the spleen anlage of Nkx3.2^–/– embryos, compared with wild-type littermates. (E,F) Nkx2.5 is expressed within the spleen primordium of Nkx3.2^–/– embryos compared with wild-type littermates. The splenic anlage is outlined by black dashes. L, liver; Sp, spleen; St, stomach.

• Supplemental Figure 2 (Adobe PDF) -

  Fig. S2. Recruitment of Pbx1 to the human Hox11 promoter. A region of the human Hox11 promoter (–837-419) (NCBI AF067443), which is highly conserved between mouse and human, and contains Pbx1-binding sites, was amplified by PCR analysis. In addition, a 5¢ region within the same promoter (–4630-4360) that does not contain Pbx1-binding sites was amplified as negative control. The following Hox11 primer pairs were used: 5¢-CAGGTGGCCTCAGAACCAGAGA-3¢ (–4630 s), 5¢-aCTCACAgaGCAACTGCCATGTG-3¢ (–4360 as) product size 270 bp; 5¢-AGCCAGTTGTTCATAGGCCTGT-3¢ (–837 s), 5¢-CAAGACTCGGAGTAGTTCATGAAG-3¢ (–419 as) product size 418 bp. For ChIP analysis, chromatin from K562 cells (Ishiko et al., 2004), a human erythroid cell line expressing Pbx and Hox11, was immunoprecipitated (IP) using antibodies specific for Pbx1b (Jacobs et al., 1999), Pbx1a/2/3a and Pbx2 (both from Santa Cruz Biotech, CA), or no antibody (No Ab). An anti-Gal4 antibody (Santa Cruz Biotech, CA) was used as an additional negative control. After IP with the anti-Pbx1a/2/3a antibody, only primers spanning the conserved region –837-419 amplify the intervening sequence within the Hox11 promoter. These results indicate that Pbx1a or Pbx3, or both, are recruited to the human Hox11 promoter, an,d therefore, recruitement of Pbx proteins to the Hox11 promoter occurs both in mouse and humans. Finally, it has previously been reported that activation of Hox11 in K562 human cells occurrs through a complex containing Pbx2, as detected by EMSA and transcriptional assays (Brake et al., 2002). Intriguingly, we demonstrate here that Pbx1 is recruited to the Hox11 promoter by performing ChIP experiments on chromatin derived from K562 human cells. The different techniques used might account for this discrepancy. However, we excluded the possibility that Pbx2 might be the only binding protein in the complex in mouse, by the use of both nuclear extracts and chromatin derived from an embryonic spleen cell line generated from Pbx2^-/- embryos (Selleri et al., 2004) (data not shown). Finally, in the present study, we have established that Pbx1 is essential for spleen genesis via direct regulation of Hox11, whereas mice lacking Pbx2 develop normal spleens (Selleri et al., 2004).