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. 2011 Oct 3;6(10):e25689. doi: 10.1371/journal.pone.0025689

Figure 4. Distribution of Hoxd4/miR-10b transcripts along the embryonic AP axis.

Figure 4

(A) Hoxd4 probes and transcripts. The in situ hybridization probes are shown above a diagram of the Hoxd4 genomic locus. Previously, the P2 ex 4 probe spanning exon 4 and extending partially into intron 4, detected P2 transcripts at a point posterior to the r6/7 boundary, while the P1+P2 ex 5 probe spanning exon 5 detected P1+P2 transcripts with an r6/7 boundary. The newly designed P2 ex 1-3 probe spanned the first 3 exons, and detected spliced P2 transcripts, while the P1 probe spans the 5′ UTR of P1 transcripts, a region that is spliced out in mature P2 transcripts. Exons are denoted as for Fig. 1B. The P1 5′ end maps to nucleotide 4066 downstream of the major P2 start site. The Hoxd4 P1-Cy3 probe used for RNA FISH in Fig 5D and E extends from the P1 5′ end to the 3′ end of Hoxd4 exon 5 as drawn. The grey triangle represents a hypothetical mRNA destabilizing element that is proposed to be absent from hypothetical transcript P2.3. (B,C) Expression of Hoxd4 in E9.5 mouse embryos. Both the Drosha-cleaved P1 transcript detected by the P1 probe (B) and the P2 transcript detected by the P2 ex 1-3 probe (C) show anterior expression boundaries that are posterior to the anterior limit of the full Hoxd4 expression domain at r6/7. ba1,2,3: branchial arches 1, 2 and 3; 4th v: fourth ventricle within the developing brain; neuroepi: neuroepithelium; flb: forelimb bud; opv: optic vesicle. The r6/7 boundary is approximately opposite branchial arch 3 as indicated. (D) Schematic of distribution of Hoxd4 transcripts in the E9.5 mouse embryo. The solid red plus blue shading represents the cumulative distribution of all Hoxd4 transcripts. The most anteriorly expressed transcripts (red) have an anterior expression border at the r6/7 boundary. Such an expression pattern is detected with the P1+P2 ex 5 probe [26] as well as probes spanning the homeobox-containing exon and 3′ UTR [34], [40], [45]. The region in blue represents the expression domain of those Hoxd4 transcripts detected by the more upstream probes, P2 ex 1-3, P2 ex 4 and P1 [26].