Figure 1.
Dynamic expression patterns of Six1 and Six2 during urogenital development.
Whole-mount in situ hybridization of staged embryos, using Six1- (A–F) and Six2- (G–L) specific probes, were visualized laterally (A–L), dorsally (B′–L′) and ventrally (C″–L″). (M–P). Six2 in situ hybridization was performed on a series of e11.5 sagittal sections. C, cloaca; GT, genital tubercle; ICM, intra-cloacal mesenchyme; PCM, peri-cloacal mesenchyme; dPCM, dorsal PCM; vPCM, ventral PCM; PF, preputial fold; T, tail; arrow, metanephric mesenchyme; UGS, urogenital sinus.
Figure 2.
A genetic fate map of Six2-expressing PCM progenitors.
X-gal staining (blue) of sagittal (A–D, G and H) and cross (E, F, I, J) sections from e11.75, e13.5 and e15.5 Six2GC/+;R26RLacZ double heterozygous embryos. All sections were counterstained with eosin (pink). A, anus; PG, preputial gland; see Figure 1 for more abbreviations.
Figure 3.
An inducible genetic fate map of Six2-expressing PCM progenitors.
Double Six2GCE/+;R26RLacZ pregnant females were treated with a single dose of tamoxifen at e11.5, e13.5, e14.5 and e15.5, and all embryos were collected and analyzed at e17.5 with X-gal staining (blue). (A, E, I and M) kidney sections; (B–D, F–H, J–L and N–P) urogenital sections. CB, prospective corporal body; GT, genital tubercle; P, perineum; PF, preputial fold; PG, preputial gland; U, urethra.
Figure 4.
Genital urinary and anorectal defects of Six1;Six2 compound mutants.
(A) A table of urogenital phenotypes of Six1;Six2 compound mutants. (B–G) Gross ventral views of external urogenital structures. (H–S) Hematoxylin and eosin (H&E) staining of midline sagittal sections of urogenital structures from newborn pups. A, anus; B, bladder; GT, genital tubercle; T, tail; UM, urethral meatus; UC, umbilical cord; U, urethra; V, vagina.
Figure 5.
Hypoplastic genital tubercles of Six1;Six2 compound mutants at e11.5.
Hematoxylin and eosin (H&E) staining of the serial sagittal sections of e11.5 urogenital structure. Asterisk, juxtaposition of ICM, dPCM and the cloacal membrane (CM); B, bladder; CND, common nephric duct; lPCM, lateral PCM; R, rectum; see Figure 1 for more abbreviations.
Figure 6.
Six1;Six2 compound mutant genital tubercles have aberrant apoptosis patterns.
(A–D) Comparable levels of serial of sagittal sections were stained with TUNEL for apoptotic cells (red). All sections were counter stained with DAPI (blue). (E–H) Ventral views of genital tubercles (GTs) stained with Lysotracker® for apoptotic cells (white dots). Apoptotic cells were observed in e11.5 control GTs within distal urethral plate epithelia (dUPE), proximal urethral plate epithelia (pUPE), distal mesenchymal (DM) (E). In Six1−/−;Six2+/− mutant, Lysotracker® signals were enhanced within dUPE and pUPE but not detectable within DM (F). Ectopic apoptotic cells in the lateral mesenchyme region (LM) was observed (E′, insert). At later stages, apoptotic cells were reduced in Six1−/−;Six2+/− mutants at e12.0 within DM (G and H). Ectopic cell death in LM persisted at e12.0 (arrowheads, H). (I) Schematic representations of dynamic changes of apoptosis patterns in control and mutants. CND, common nephric duct; distal mesenchymal (DM); LM, lateral mesenchyme; dUPE, distal urethral plate epithelium; pUPE; proximal urethral plate epithelium; R, rectum; see Fig. 1 for more abbreviations.
Figure 7.
Six1 and Six2 are required for proliferation of PCM progenitors.
(A–F) Phospho-histone H3 staining (p-HH3, green) of proliferating cells using a series of sagittal sections at e11.5. (G) Quantification of p-HH3 staining results.
Figure 8.
Aberrant expression of signal molecules during urogenital development in Six1;Six2 compound mutants.
(A–P) Whole mount in situ hybridization of genital tubercles using gene-specific probes of Bmp4, Bmp7, Fgf8, and Dkk1. (Q) Real time quantitative PCR analysis of gene expression levels of micro-dissected genital tubercle tissue at e11.5. Arrow, ventral distal mesenchyme; arrowhead, dorsal lateral mesenchyme; bracket, enhanced expression of Bmp7; dUPE, distal urethral plate epithelium; LM, lateral mesenchyme; PG, preputial gland.
Figure 9.
A working model: patterning of cloacal mesoderm leads to occlusion of the cloaca and outgrowth of the genital tubercle.
(A and B) Asymmetric growth and patterning along the rostrocaudal axis (A) and dorsoventral axis (B) causes occlusion and division of cloaca into urinary and digestive tracts. The process also displaces the cloacal duct (CD), remnant of the cloacal epithelium, to the surface of perineum as a thin epithelial lining. (C and D) Midline sagittal diagrams of genital tubercle at e11.5 (C) and e17.5 (D). Continuous growth of peri-cloacal mesenchyme leads to remodeling and opening of the anal canal and urethra, and of the digestive and urinary outlets, respectively. Peri-cloaca mesenchymal progenitors contribute to most, if not all, stromal tissues of genital tubercle and perineum. Asterisk, juxtaposition of ICM, dPCM and the cloacal membrane; A, anus; C, cloaca; CD, cloacal duct; CM, cloacal membrane; ICM, intro-cloacal mesenchyme; PCM; peri-cloacal mesenchyme; dPCM, dorsal PCM; vPCM, ventral PCM; Per, perineum; R, rectum; T, tail; TG, tail gut; U, urethra; UGS, urogenital sinus; UM, urethral meatus.