Hoxa11eGFP fluorescence in whole mount embryos. A, B: E9.5 Hoxa11eGFP heterozygous embryos showing the brightfield view (A) and Hoxa11eGFP fluorescence (B). C: Hoxa11 RNA in situ hybridization on an E10.5 wild type embryo. D: An E10.5 Hoxa11eGFP heterozygous embryo showing eGFP fluorescence. E, F: E11.5 heterozygous Hoxa11eGFP embryo showing the brightfield view (E) and eGFP fluorescence (F). G, H: An E12.5 Hoxa11eGFP heterozygous embryo with the brightfield view (G) and eGFP fluorescence (H). I: An E13.5 wild-type embryo on the green fluorescence channel has no visible fluorescence at settings used for eGFP detection. J: An E13.5 Hoxa11eGFP heterozygous embryo showing eGFP fluorescence. K, L: An E15.5 Hoxa11eGFP heterozygous embryo with the brightfield view (K) and eGFP fluorescence (L). Arrowheads indicate regions of Hoxa11eGFP expression in the limbs in each panel.

Hoxa11eGFP fluorescence in transverse cryosections of the developing kidney. A: Hoxa11eGFP fluorescence in the condensing metanephric mesenchyme (arrows) of an E10.5 heterozygous embryo. B: Hoxa11eGFP heterozygous embryo at E11.5 with fluorescing metanephric mesenchyme (arrows) being invaded by the ureteric bud (white asterisk), which does not express Hoxa11eGFP. C: E12.5 kidney showing Hoxa11eGFP localized in the condensing nephrogenic mesenchyme (arrow) surrounding the ureteric bud (white asterisk). D: Hoxa11eGFP heterozygous kidney at E13.5, E: co-stained with anti-Pax2 antibody (in red), and F: colocalizations of Hoxa11eGFP and Pax2 in this section. Co-localization of Hoxa11eGFP and Pax2 is observed in the condensing nephrogenic mesenchyme (arrow). The ureteric bud (white asterisk) and ureteric bud-derived elements show only Pax2 expression, and mesenchyme surrounding the ureter demonstrates only Hoxa11eGFP. G: In the E15.0 Hoxa11eGFP heterozygous kidney, fluorescence is detected in the condensing nephrogenic mesenchyme cells (arrow) and in mesenchyme surrounding the ureteric bud (white asterisk), which does not express the Hoxa11eGFP allele. H: PNA, which labels early collecting tubules, is largely non-overlapping with Hoxa11eGFP at E16.5. Scale bars indicate 200 microns in all panels.

Hoxa11eGFP fluorescence in the neural tube. A–C: Transverse cryosections through the sacral region of the neural tube of Hoxa11eGFP heterozygous embryos at E11.5 (A), E12.5 (B) and E13.5 (C). D: Transverse cryosection through the sacral region of the neural tube of a wild-type embryo at E13.5 photographed using the same fluorescence settings as for C. Scale bar indicates 200 microns in all panels.

Generation of Hoxa11eGFP targeted allele in mice. A: Schematic of the Hoxa11 genomic locus, Hoxa11eGFP targeting vector and Hoxa11eGFP allele. Exon 1 of Hoxa11 is replaced by eGFP followed by an SV40 T-antigen stop sequence and an ACN cassette in reverse orientation flanked by LoxP sites. A TK cassette is 5′ to the Hoxa11 genomic flanking sequence. ApaLI restriction sites are shown for the wild type allele as well as the Hoxa11eGFP targeted allele, and the sizes generated upon digest (used for Southern analysis). B: Southern analyses of Hoxa11eGFP targeted ES cells. Genomic DNA extracted from ES cells was digested with ApaLI and hybridized with either a 813 bp 5′ flanking probe or a 621 bp 3′ flanking probe. The 5′ probe detected 10.7 and 12.7 kb bands for wild type and mutant alleles, respectively. The 3′ probe detected 9.4 and 11.6 kb bands for wild type and mutant alleles, respectively. C: PCR genotyping of Hoxa11eGFP mice. The bottom 218 bp band is the wild type allele, and the top 405 bp band is the mutant Hoxa11eGFP allele. (Further details in Experimental Methods.)

Hoxa11eGFP localization in cryosections through the forelimbs at various stages of development. A: A transverse section through a Hoxa11eGFP heterozygous forelimb at E10.5. B: Transverse section of a Hoxa11eGFP heterozygous forelimb at E11.5 co-stained with antibodies to Sox9, a pre-chrondrogenic marker. C: A transverse section through a Hoxa11eGFP heterozygous forelimb at E12.5. D: A transverse section through an E13.5 wild type forelimb (no Hoxa11eGFP), using anti-Sox9 antibody staining (in red) and green channel fluorescence at the same settings as E. E: Transverse section through an Hoxa11eGFP heterozygous E13.5 forelimb co-stained with Sox9 in red. F: A transverse section through a Hoxa11eGFP heterozygous forelimb at E15.5. The scale bar in each panel represents 500 microns.

Hoxa11eGFP fluorescence in heterozygous reproductive tracts from both sexes. A, C, E and G: Dissected female reproductive tracts at E16.5 (A), P1 (C), P28 (E) and 10 months (G). Hoxa11eGFP is observed in the developing uterus (arrows) and is excluded from the ovaries. B, D, F and H: Dissected male reproductive tracts at E16.5 (B), P1 (D), P28 (F) and 10 months (H). Intense Hoxa11eGFP localization is observed in the vas deferens (arrows). K, kidney; O, ovary; T, testis.