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Tail gut endoderm and gut/genitourinary/tail development: a new tissue-specific role for Hoxa13

Departments of Pathology and Pediatric Surgery Research, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA

View larger version (71K): [in a new window]   Fig. 1. Spatiotemporal expressions of Hoxa13 (A-E) and Hoxa13 protein (F) during posterior GGU/tail development. (A) Stage 10 [arrow indicates caudal intestinal portal (CIP)]. (B) Stage 14 (arrow indicates CIP). (C) Stage 17 (long arrows show tail tip and hindgut, arrowheads at tail gut). C1 and C2 note planes of cryosection. (C1) Hoxa13 endodermal expression in the cloaca (Cl). (D) Expression in tail and posterior hindlimb bud (red arrows) at stage 22. (E) Dissected E4 posterior gut. Expression is present in the hindgut (HG) and cloacal (CL) endoderm and cloacal mesoderm, no expression is detected in the ceca (CE) or midgut (MG). (F) Protein expression in hindgut endoderm (endo.), arrow, at E6.

View larger version (81K): [in a new window]   Fig. 2. Human hand-foot-genital (HFG) and chick HFGa13 overexpressed embryos have similar limb phenotypes. (A-D) Control (left panel) and HFGa13-infected (right panel) hindlimbs at E6. Planar sections of E6 limbs (B), uninjected control (left) HFGa13-infected hindlimb [right, arrows indicate fibula maldevelopment (fibula*) with undifferentiated mesenchymal cells]. (C) In situ hybridization of Fgf8 in sectioned hindlimb. No Fgf8 expression is detected in the HFGa13-infected hindlimb compared with the uninfected hindlimb (arrows). (D) In situ hybridization of Bapx1 in sectioned hindlimb. There is altered Bapx1 expression in the HFGa13-infected hindlimb (arrows).

View larger version (62K): [in a new window]   Fig. 3. Endodermally expressed HFGa13 causes abnormal hindgut and tail development. (A) Whole-mount in situ hybridization showing RCAS expression (arrow at malformed tail) in an E6 mutant HFGa13-infected embryo. (B) 3C2 immunohistochemistry analysis of sectioned HFGa13-infected mutant embryo shows posterior endoderm (endo.) and mesoderm (meso.) infection. (C) RCAS in situ showing absence of the mutant phenotype (arrow at normal tail) when HFGa13 infection is present only in the mesoderm, demonstrated in D using anti-Gag immunohistochemistry to show no infection in the caudal endoderm (at E6). (E) E6 survivors after electroporation of HFGa13 constructs in the posterior endodermal layer. The phenotype involves maldevelopment of the cloaca (CL*), hindgut (HG*) and tail. Allantoic internalization is present (AL*) and ceca are unaffected (CE). (F) Anti-N-flag immunostaining demonstrating expression of the tagged-HFGa13 in the endoderm of the hindgut; the mesoderm is not stained. Note that the electroporated endodermal cells appear undamaged and intact. Misexpression of HFGa13 and Hoxa13 constructs by electroporation show similar expression levels in the gut endoderm layer (data not shown).

View larger version (112K): [in a new window]   Fig. 4. Histological analysis of mutant HFGa13-infected embryos. Hematoxylin and Eosin stained transverse (A-D; E7) and longitudinal (E-H; E6) sections of mutant HFGa13-infected (A,C,E,G) and control embryos (B,D,F,H). Ourentery is present in A. HFGa13-infected embryos show cloacal stenosis (CL*; C), atresia of the hindgut (HG*) anterior to the cloaca (CL; E), allantoic internalization (AL*; E) and defects in the cloaca-associated viscera, including more distal Müllerian duct (MN duct) atresia and cystic mesonephric (MN*) maldevelopment (G). Note the correct development of the more anterior gut structures. CE, ceca; GU, genitourinary; NC, notocord.

View larger version (77K): [in a new window]   Fig. 5. HFGa13 affects expression of Fgf8, Hoxd13 and Bapx1. (A-E) Whole-mount in situ hybridization of control (left panel) and HFGa13-infected (right panel) embryos (arrows at malformed tail). (A) Fgf8 expression at E5, note absence of expression in the HFGa13 tail (red arrow) but normal expression in the hindlimb AER. (B) Bapx1 (expression) at E6. Bapx1 shows a diminished expression in the tail (red arrow) of the HFGa13-infected embryo but normal expression in the non-infected hindlimbs. (C) Wnt5a gene expression shows normal expression in the HFGa13-infected embryo at E7 (red arrow). (D) Hoxd13 expression at E6 shows strong downregulation in the tail (red arrow) of the HFGa13-infected embryo and normal expression in hindlimbs. (E) Shh expression at E4 is normal. Arrows indicate hindlimb and notochord.

View larger version (72K): [in a new window]   Fig. 6. Whole embryo explants developed in culture demonstrate the physical necessity of caudal endoderm for normal tail development. (A) Whole embryo explanted at stage 10 and grown on albumen/agarose for 48 hours, showing normal tail development and expression of Hoxa13 in tail (blue arrow). Arrowhead shows normal placement of allantois ventral to hindgut, which expresses Hoxa13. (B) Stage 10 embryo grown with caudal endoderm removed, demonstrating blunted tail without Hoxa13 expression (arrow). Arrowhead indicates hindgut dorsal to Hoxa13-expressing allantois. Location of sections shown in C,D are indicted. (C) Anterior section of caudal endoderm-less embryo as in B, showing normal endoderm (arrows) and notochord (arrowhead). (D) Hematoxylin and Eosin stained section through a caudal endoderm-less embryo that developed ourentery (large arrowhead) within hindgut lumen (short arrow). The bracket and long arrow indicate allantois. Small arrowhead indicates normal notochord. (E) Embryo cultured for 24 hours with caudal endoderm develops normal CIP (long arrow), expresses Shh in notochord (red stain, arrowhead, most of notochord deep to plane of photograph) and co-expresses Shh and CdxA in endoderm of CIP (short arrow, purple/black stain). (F) Embryo cultured for 24 hours without caudal endoderm fails to develop CIP or express Shh or CdxA in midline caudal endoderm (long arrow), both are co-expressed in right lateral caudal endoderm (short arrow, purple/black stain). Notochord is curved but expresses Shh (red stain, arrowhead). (G) Embryo after caudal endoderm removed and donor caudal endoderm transplanted, cultured for 48 hours showing tail growth (arrow). lb, hindlimb buds. (H) Embryo after caudal endoderm removed and donor anterior endoderm transplanted, cultures at 48 hours showing blunted tail (long arrow), allantois (arrowhead); AE, anterior endoderm; –CE, caudal endoderm removed; ht, heart.

View larger version (78K): [in a new window]   Fig. 7. HFGa13 interferes with the cellular functions of Hoxa13 and Hoxd13. (A) Transcriptional transactivation by wild-type Hoxa13 and HFGa13 proteins in a GAL4-fusion assay in COS-7 cells. Relative luciferase activities were normalized to the empty GAL4 DNA-binding domain expression vector. Fusion protein of the GAL4 DNA-binding domain and Hoxa13 shows transcriptional activation of the synthetic reporter. By contrast, fusion protein of the GAL4 DNA-binding domain and HFGa13 fails to activate transcription of the same promoter and is able to decrease the basal activity. Luciferase assays were performed after two independent transfections, each done in triplicate (A,B). (B) Perturbation of the transcriptional transactivation of wild-type Hoxa13 and Hoxd13 by HFGa13 in a GAL4-fusion assay in COS-7 cells. In this assay, we monitored GAL4 transcriptional activity induced by GAL4 DBD fusion proteins without or with pcDNA3-HFGa13 construct. In a same molar ratio, the HFGa13 form specifically decreases Hoxa13 and Hoxd13 transcriptional activation. (C,D) Intracellular localization of Hoxa13 and HFGa13 proteins. Immunostaining of transfected N-flag tagged Hoxa13 (C) and HFGa13 (D) constructs in COS-7 cells with specific N-flag antibody shows that both have nuclear localization. Note an additive cytoplasmic signal with the HFGa13 construct. (E-K) Hematoxylin and Eosin stained sections of E18 control (E,F) or infected (G-K) guts. (E) Normal midgut with thin and long villi. (F) Normal hindgut with flat and short villi. (G) HFGa13 mesodermally infected midgut has wild-type midgut epithelium. Hoxa13 (H) and Hoxd13 (J) mesodermally infected midgut shows hindgut-like epithelial transformation (as shown by arrows). HFGa13 midguts co-infected with either Hoxa13 (I) or Hoxd13 (K) show rescue of the epithelial hindgut phenotype. (L) Hoxd13 and HFGa13 mesodermal midgut co-infection show presence of virus (detected by 3C2-Ab, L1), and ectopic HFGa13 (detected with Hoxa13 probe, L2) and Hoxd13 (L3) co-expression, which is associated with normal epithelial phenotype.