Delineating a conserved genetic cassette promoting outgrowth of body appendages

Abstract

The acquisition of the external genitalia allowed mammals to cope with terrestrial-specific reproductive needs for internal fertilization, and thus it represents one of the most fundamental steps in evolution towards a life on land. How genitalia evolved remains obscure, and the key to understanding this process may lie in the developmental genetics that underpins the early establishment of the genital primordium, the genital tubercle (GT). Development of the GT is similar to that of the limb, which requires precise regulation from a distal signaling epithelium. However, whether outgrowth of the GT and limbs is mediated by common instructive signals remains unknown. In this study, we used comprehensive genetic approaches to interrogate the signaling cascade involved in GT formation in comparison with limb formation. We demonstrate that the FGF ligand responsible for GT development is FGF8 expressed in the cloacal endoderm. We further showed that forced Fgf8 expression can rescue limb and GT reduction in embryos deficient in WNT signaling activity. Our studies show that the regulation of Fgf8 by the canonical WNT signaling pathway is mediated in part by the transcription factor SP8. Sp8 mutants elicit appendage defects mirroring WNT and FGF mutants, and abolishing Sp8 attenuates ectopic appendage development caused by a gain-of-function β-catenin mutation. These observations indicate that a conserved WNT-SP8-FGF8 genetic cassette is employed by both appendages for promoting outgrowth, and suggest a deep homology shared by the limb and external genitalia.

Figure 1. Distal signaling epithelia in the outgrowth of body appendages.

(A) The outgrowth of the limb is instructed by a specialized ectodermal epithelium, the AER (in red), positioned at the distal edge of the limb bud expanding across the anteroposterior axis. Inset shows a cross section through the plain indicated by arrow. (B) The Fgf8-expressing GT signaling center dUE (in red) is positioned at the distal most part of the cloacal endoderm right below the ventral ectoderm (inset, coronal section through the plain indicated by arrow). (C, D) The dUE remains at the distal end of the urethral epithelium as the GT and the urethra undergo continuous proximodistal outgrowth. Endodermal cloaca was illustrated in yellow in B–D. (E–H) Fgf8 expression marks the AER (E) and dUE (F–H). bl, bladder; ls, lateral swellings; ds, dorsal swelling; ugs, urogenital sinus; ue, urethral epithelium; r, rectum; hg, hindgut; tg, tail gut; PCM, para-cloacal mesenchyme.

Figure 2. Defective GT development in GT-Fgfr1;r2-dcKO embryos.

(A–F) SEM analyses on age-matched control (A, C) and dcKO mutants (B, D) showing a retarded proximodistal outgrowth in the mutants. (E–L) Whole mount in situ hybridization on E11.5 control and dcKO mutants using probes indicated. Note the downregulation of Bmp4 and Wnt5a in the PCM (F and H), and Shh in the UE of the dcKO mutants (J). The dUE-Fgf8 expression in the dcKO (L) was comparable to the control (K). Bars represent 500 µm.

Figure 3. Appendage over-development in the R26^Fgf8-GOF mutants.

(A) A schematic diagram for R26^Fgf8 allele. (B–G) Whole mount in situ analyses on control (B, D, and F) and UE-R26^Fgf8 (C, E, and G) embryos at consecutive time points after Tm treatment. Note the upregulation of PCM-Bmp4 ( inset in C) and dUE-Fgf8 (C, arrowhead) in the UE-R26^Fgf8 embryos 8 hours after Tm administration, and a downregulation of dUE-Fgf8 expression in the UE-R26^Fgf8 mutants 16 (E, arrowhead) and 24 hours (G) after Tm treatment. The ectopic Fgf8 expression in the anterior cloacal endoderm is indicated by arrows in C and E. (H–I) SEM on E14.5 control (H) and UE-R26^Fgf8 GT (I). Note that the GT in the GOF mutant is bigger. (J–K) Skeleton staining of E18.5 AER-R26^Fgf8-GOF embryos showing excessive limb development in the mutants. The postaxial extra digit in the forelimb is indicated by an asterisk in J, and the ectopic skeleton components were indicated by arrows in J and K. The arrowhead in K indicates an enlarged calcaneus. h, humerus; r, radius; u, ulna; fm, femur; t, tibia; f, fibula. Bars represent 500 µm.

Figure 4. FGF8 rescues appendage reduction in β-catenin-LOF mutants, but not in Shh-KO mutants.

(A–C) SEM analyses revealed an absence of GT in UE-β-Cat-LOFs (B), and a distinct tubercle structure in the LOF mutant carrying R26^Fgf8 allele (C). (D–F) Histological and in situ analyses on E12.0 β-Cat-LOF;R26^Fgf8 GT showing a normal patterning (D) and Hox genes expression (E and F). (G–L) Skeleton preparation on E18.5 embryos showing an absence of autopod and radius, truncation of ulna, underdeveloped humerus in the forelimb (I), and a complete absence of all stylopod, zeugopod and autopod elements in the hindlimb of AER-β-Cat-LOF mutant (J); and a presence of autopod rudiments (arrow and inset in K) and radius with proper humerus and ulna in the forelimb (K), and a fully developed femur in the hindlimb of R26^Fgf8-rescued β-Cat-LOF mutant (L). h, humerus; r, radius; u, ulna; fm, femur; t, tibia; f, fibula. Bars represent 500 µm.

Figure 5. GT gene expression analyses on UE-β-Cat-LOF embryos with forced Fgf8 expression.

(A–L) Whole mount in situ analyses using probes indicated. Note that the LOF mutants lack cloacal endoderm/UE Fgf8 (B) and Shh (H) expression, and PCM Bmp4 (E) expression; whereas weak Fgf8 expression (C), partially restored PCM Bmp4 (F) expression, and UE Shh (I) expression can be detected in the LOF mutants with R26^Fgf8 allele. Also note the lack of Sp8 expression in LOF mutants with (L) or without (K) R26^Fgf8 allele.

Figure 6. Appendage deficiencies in Sp8 mutants.

(A–F) Whole mount Sp8 in situ showing a downregulation of Sp8 expression in the UE and the AER of corresponding β-Cat-LOF mutants (B, E), and an upregulation of Sp8 expression in the UE (C) and the ventral limb ectoderm (F) of β-Cat-GOF mutants. Note the tail expression of Sp8 remains unchanged in (E compared to D). (G, H) SEM on E15.5 embryos showing an absence of GT in the Sp8 KO mutant (H). (I, J) Fgf8 in situ showing loss of expression in E11.0 Sp8 KO mutant. (K, L) SEM on E13.5 embryos showing a mild distal reduction in the UE-Sp8-LOF (L). Insets showing whole mount Fgf8 in situ on E12.5 control (K) and UE-Sp8-LOF (L) GTs. Note the downregulation of Fgf8 in the UE-Sp8-LOF mutant (L). (M–W) Skeletal preparation of E18.5 embryos showing that AER-Sp8-LOF lacked most autopod elements in the forelimb (Q and R), and all zeugopod and autopod elements in the hindlimb (S); whereas in the R26^Fgf8-rescued LOF mutants, several digits were formed in the forelimb (T, U), and the tibia and fibula were fully developed along with some autopod rudiments in the hindlimb (V, W). h, humerus; r, radius; u, ulna; fm, femur; t, tibia; f, fibula. Bars represent 500 µm in G and H, 400 µm in K and L.

Figure 7. WNT-induced ectopic Fgf8 expression was attenuated in Sp8 conditional knockouts.

(A–D) Whole mount Fgf8 expression on E12.5 GTs from embryos with genotypes indicated. Note the gradual reduction in Fgf8 expression in correlation with Sp8 allele number from B to D. (E) Real time-RT PCR analyses on E12.5 GTs with genotypes indicated. (F–H) Whole mount Fgf8 in situ on E11.5 limbs showing expanded expression in the AER-β-Cat-GOF mutant (G), and near normal expression in the GOF embryos with both Sp8 alleles abolished (H). (I–N) The autopod phenotype of AER-β-Cat-GOF mutants with (J, M) or without Sp8 (K, N) alleles. Quantification of digit numbers is shown in O.

Figure 8. Construction and characterization of R26^Sp8 knock-in allele.

(A) A schematic diagram for R26^Sp8 allele. (B, C) Sp8 in situ on E12.5 GTs showing an upregulation of Sp8 expression in the UE-R26^Sp8 embryos (C). (D–F) SEM analysis on E15.5 embryos showing comparable GT development between control and the UE-R26^Sp8 mutant (E), and no tubercle formation in the UE-β-Cat-LOF mutant carrying R26^Sp8 allele (F). (G) Fgf8 in situ showing no Fgf8 expression in the UE-β-Cat-LOF;R26^Sp8 mutant. (H–M) Skeletal preparation showing no difference between control (H) and AER-R26^Sp8 GOF mutant (I), or between the AER-β-Cat-LOF mutants with (K, M) or without the R26^Sp8 allele (J, L). Bars represent 500 µm in D and E, 400 µm in F.