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doi: 10.1242/10.1242/dev.00530

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Loss of Bmp7 and Fgf8 signaling in Hoxa13-mutant mice causes hypospadia

¹ Shriners Hospital for Children, Portland, OR 97239, USA
² Oregon Health and Sciences University, Department of Molecular and Medical Genetics, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA

View larger version (71K): [in a new window]   Fig. 1. Characterization of hypospadiac and proliferative defects in Hoxa13GFP-homozygous mutants. (A,B) Hoxa13GFP is expressed in both the GT mesenchyme (M) as well as the urethral plate epithelium (UPE) in E12.5 littermates. Arrows denote the distal UPE (dUPE) which is thickened in homozygous mutants. (C,D) Proliferation in the GT mesenchyme of E12.5 male embryos. Proliferation was measured in the lateral shelf mesenchyme (LSM) by counting phosphohistone H3 positive cells in a 2.5x2.5 cm region (white square). Note that proliferation is maintained in the cells immediately adjacent to the UPE in heterozygous embryos (C, arrowheads), whereas mutant embryos exhibit a dramatic decrease immediately adjacent to the UPE (D, arrowheads). (E) Closure of the distal urethra and glans by proliferating mesenchyme in an E15.5 heterozygous male embryo. Arrow denotes the initial site of closure. Arrowhead indicates future site of the rectal mesenchyme condensation. (F) Hypospadia in homozygous-mutant littermates. Arrow denotes the loss of urethral closure as well as the persistence of the epidermal layer, which is not covered by the proliferating mesenchyme. Arrowhead indicates precocious condensation of rectal mesenchyme. Note that formation of the rectum (R) is precocious and more rostral in homozygous mutants (F). Scale bars: 50 µm.

View larger version (131K): [in a new window]   Fig. 2. Male embryos lacking Hoxa13 exhibit decreased programmed cell death (PCD) in the developing genital tubercle. (A) At E11.5, PCD is restricted to the dUPE (arrowhead) and proximal UPE (pUPE) in heterozygous controls. (B) PCD is reduced in the dUPE (arrowhead), but not in the pUPE of Hoxa13GFP-homozygous mutants. (C) By E12.5, PCD shifts from the dUPE to the mesenchyme (M) flanking the dUPE (arrow). (D) Homozygous mutants also exhibit a shift in PCD to the flanking mesenchyme, although the number of TUNEL-positive cells in the mutant mesenchyme is consistently reduced when compared with heterozygous controls. Scale bars: 50 µm.

View larger version (94K): [in a new window]   Fig. 3. The expression of Bmp7, its target genes, Msx1 and Msx2, and its antagonist, Nog, are affected in Hoxa13GFP-mutant embryos. (A) Expression of Bmp7 in the UPE and lateral shelf mesenchyme (LSM; black arrow) in E12.5 control embryos. Black bracket shows sites of gene expression, white portion of brackets show sites where gene expression is absent. White arrow denotes the thin layer of epidermis. (B) Absence of Bmp7 expression in pUPE of E12.5 male homozygous mutants, as well as in the later shelf mesenchyme (black arrow). Note the thickened epithelial layer (white arrow) in the distal genital tubercle. (C) Expression of Bmp7 in E14.5 male wild-type embryos. Note high levels of Bmp7 expression in the developing meatus (black arrow), as well as in the urethral epithelium which is covered by mesenchyme (black arrowhead) and the developing preputial gland (white arrowhead). (D) Malformation of the meatus is concomitant with reduced Bmp7 expression in E14.5 male homozygous mutants (black arrow). Note that Bmp7 expression is reduced in the preputial gland (white arrowhead) and absent in the epithelial chord (black arrowhead), which is not covered by mesenchyme. (E) Nog is normally expressed in the lateral shelf mesenchyme (LSM) of E11.5 wild-type embryos. Black arrowhead in E denotes position of ectopic Nog expression seen in the mesenchyme flanking pUPE in E11.5 male homozygous mutants (F). (G) In E12.5 control embryos, Nog is expressed at low levels in mesenchyme flanking the pUPE (black arrowhead); dUPE (black arrow). (H) E12.5 homozygous mutants exhibit strong ectopic Nog expression in the mesenchyme adjacent to the medial-proximal UPE (red arrowhead), as well as in a narrow band in the GT mesenchyme (black arrowhead). Nog expression was reduced in the mesenchyme flanking the dUPE in these same mutant embryos (black arrow). Inset (H) reflects Bmp4 expression in the mesenchyme flanking the medial-proximal UPE. (I) In E11.5 wild-type embryos, Msx1 expression is localized to the mesenchyme (M) flanking the entire UPE. Black bracket marks the rostral-caudal extent of Msx1 expression. (J) In E11.5 homozygous mutants, Msx1 expression is absent in the mesenchyme flanking the pUPE. Black portion of bracket represents Msx1 expression in the rostral mesenchyme. White portion of bracket represents caudal portion of mesenchyme lacking Msx1 expression. (K) In E12.5 embryos, Msx1 is predominantly expressed in the distal GT where the glans will eventually form (black arrow); in homozygous mutants (L) this is noticeably reduced in size (black arrow). (M) Msx2 is strongly expressed in the rostral mesenchyme, as well as in the UPE and sinus epithelium in E11.5 wild-type male embryos. Black bracket denotes Msx2 expression along the rostral-caudal axis of the genital tubercle. (N) Homozygous mutants also exhibit strong Msx2 expression in the UPE and mesenchyme flanking the dUPE (black portion of bracket); however, no expression could be detected in pUPE as denoted by the white portion of the bracket. (O) In E12.5 embryos, Msx2 is expressed in the developing glans mesenchyme (G) as well as in the UPE. Black bracket reflects continuous UPE expression of Msx2. (P) In E12.5 mutants, Msx2 expression is also seen in the dUPE (black portion of bracket) and glans (G), whereas no Msx2 expression is seen in the dUPE (white portion of the bracket).

View larger version (73K): [in a new window]   Fig. 4. Bmp-receptor expression is affected in Hoxa13GFP-homozygous mutants. (A,B) Low levels of Bmpr1a can be detected in both wild-type and mutant E12.5 embryos in the dUPE and GSM. (C,D) Bmpr1b expression is elevated in developing rectum (R) (black arrows) of homozygous mutant E12.5 embryos. (E,F) Bmpr2 expression is present in the UPE and GSM of both wild-type and mutant E12.5 embryos. (G,H) In E13.5 embryos, Bmpr1a expression is restricted to the distal genital tubercle and the developing preputial glands (P) in both wild-type and homozygous mutants. (I,J) Bmpr1b expression is reduced in the UPE (arrows) of E13.5 homozygous mutants when compared with wild type controls. (K,L) Bmpr2 expression appears elevated in the UPE of E13.5 homozygous mutants (arrows), whereas expression in the developing preputial glands (P) appears unaffected.

View larger version (51K): [in a new window]   Fig. 5. Blocking of Bmp7 and Bmp4 signaling recapitulates the Hoxa13GFP homozygous mutant phenotype. (A) Heterozygous male GT explant treated with control IgGs. Arrow denotes the normal formation of the meatus (M), arrowhead denotes the progression of mesenchyme closing the urethra. (B) Male GT explant treated with a Bmp4-blocking antibody (Bmp4). Arrow denotes the disruption of meatus (M) formation. Arrowhead denotes the formation of an ectopic fistula. (C) Male GT explant treated with a Bmp7-blocking antibody (Bmp7). Arrow denotes the complete absence of a meatus (M*). Arrowhead denotes poor progression of the GT mesenchyme to close the urethra. (D) TUNEL analysis of PCD in the UPE of heterozygous male GT explants treated with control IgGs. Arrowhead denotes normal levels of PCD in the dUPE. Arrow denotes normal levels of PCD in the mesenchyme flanking the UPE. (E) TUNEL analysis of PCD of a male GT explant treated with the Bmp7-blocking antibody (Bmp7). Note the reduced PCD in the dUPE (arrowhead) and mesenchyme flanking the UPE (arrow) when compared with the IgG-treated control. Scale bar: 50 µm.

View larger version (82K): [in a new window]   Fig. 6. Ectopic application of Fgf8 rescues reduced GT proliferation in Hoxa13GFP-homozygous mutants. (A) Expression of Fgf8 in the genital tubercle of E11.5 wild-type male embryos. Note that Fgf8 expression is seen in both dUPE and pUPE (black arrow) of E11.5 wild-type male embryos. Genital Shelf Mesenchyme, GSM. (B) Fgf8 expression is absent in the pUPE (arrow) of E 11.5 Hoxa13GFP-homozygous mutants. (C) Section analysis of Fgf8 expression in the urethral plate epithelium (UPE) of E11.5 wild type male embryos. Note that Fgf8 expression is present in both the proximal (arrowhead) and distal UPE. (D) Fgf8 expression is restricted to the dUPE in E11.5 homozygous mutants. Note the complete loss of Fgf8 expression in the pUPE (arrowhead). (E,G) Implantation of heparin beads (arrow) treated with BSA into the UPE of E11.5 homozygous mutants had no effect on the reduced proliferation seen in the GSM. (F,H) Implantation of beads treated with 0.1 mg/ml Fgf8b (arrow) stimulated proliferation of the GSM in age-matched homozygous mutant embryos. (I,J) Typical levels of cell proliferation in the E11.5 GT of normally developing heterozygous male embryos. Arrow denotes normal thickening of the UPE. (K,L) Cell proliferation in the GT of a E11.5 Hoxa13GFP homozygous male mutant. Arrow denotes the earliest detection of the abnormally thickened UPE. Note how Fgf8 applications alter cell proliferation in the mutant GT to resemble proliferation levels exhibited by heterozygous littermates (compare H with I), whereas mutant embryos treated with BSA maintain reduced levels of cell proliferation (compare G with K). Scale bars: 50 µm.

View larger version (95K): [in a new window]   Fig. 7. Stratification and differential cytokeratin expression in the UPE and sinus epithelium in E12.5 Hoxa13GFP male embryos. (A,B) Sonic hedgehog (Shh) expression in the UPE is unaffected by loss of Hoxa13 function. Arrows denote a shift in epithelial morphology and stratification from a multilayered cuboidal shape (A) to rounded simple epithelium (B) in homozygous mutants. (C) Cytokeratin 14 (K-14) is highly expressed in the stratified epithelium of heterozygous embryos. (D) K-14 expression is severely reduced in the UPE of Hoxa13GFP-homozygous mutants, whereas both control and homozygous-mutant embryos exhibit normal levels of cytokeratin 8/18 expression in the same epithelial tissues (E,F). Scale bars: 50 µm.

View larger version (86K): [in a new window]   Fig. 8. Capillary vessel malformations in the genital tubercle of Hoxa13 deficient embryos. (A) Capillary vessel localization detected by the vascular endothelial marker CD-31 (Pecam; red) in E13.5 heterozygous male embryos. Note the strong co-localization of Hoxa13GFP(green) with the Pecam-positive capillary endothelial cells (yellow), as well as the symmetric arrangement of capillary vessels around the UPE (arrowhead). (B) Expansion of the capillary vessels in the E13.5 homozygous mutant genital tubercle. Arrowhead denotes a typical sevenfold expansion in vessel diameter in the mutant genital tubercle. Note the lack of Hoxa13GFP co-localization (yellow) with Pecam in the capillary vessels in the mutant GT. Scale bar: 50 µm.

View larger version (97K): [in a new window]   Fig. 9. Androgen receptor (AR) expression in the developing glans and penile urethra of E15.5 Hoxa13GFP male embryos. (A) AR expression is elevated in the UPE (arrow) as well as the surface ectoderm (arrowhead) of heterozygous controls. In these same embryos, the penian bone condensation (P) is readily detected. (B) AR expression in the distal glans of a homozygous mutant. Arrow denotes hypospadiac UPE. Arrowhead denotes reduced AR expression in the surface ectoderm. Note the poor condensation of the penian bone mesenchyme (P*). (C) A more proximal cross-section through the glans. Arrowhead denotes high levels of Hoxa13GFP/AR co-localization (yellow cells) in the mesenchyme immediately adjacent the UPE. Arrow denotes high levels of AR expression in the glans ectoderm. Note the extensive condensation of mesenchyme forming the penian bone (P). (D) Hoxa13GFP/AR co-localization (yellow cells) is absent from the mesenchyme flanking the UPE in homozygous mutants and is relocated to the lateral glans mesenchyme (arrowhead). Arrow denotes reduced AR expression in the ectoderm covering the UPE. AR expression was also seen in the penian bone condensation (P), which was more disorganized than was the same site in the heterozygous control. Scale bar: 50 µm. (Below) Diagram to show planes of section through the developing glans and penile urethra (lines A,B and C,D).

View larger version (37K): [in a new window]   Fig. 10. A model for Hoxa13 function during development of the external genitalia. (A) Fgf8 and Bmp7 are co-expressed in the UPE at E11.5, whereas Hoxa13GFP is expressed in the UPE as well as the GT mesenchyme. (B,D) Hoxa13 is essential for normal expression of Fgf8 and Bmp7 in the UPE, as well as for the mesenchymal repression of Nog. In combination, these signals control cell proliferation and PCD to establish normal formation of the meatus and urethral tube closure. (C,E) In the absence of Hoxa13 function, Fgf8 and Bmp7 expression is reduced in the UPE, whereas Nog expression is ectopic, which, in combination, causes decreased cell proliferation, reduced PCD and capillary vessel enlargement, as well as defects in urethral tube closure and meatus development. Asterisks indicate sites of programmed cell deaths. Red triangle in A indicates regions of colocalized Fgf8, Bmp7 and Hoxa13 expression. The developing meatus in D is indicated by the purple symbol.