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Functional specificity of the Hoxa13 homeobox

Yuanxiang Zhao and S. Steven Potter*

Division of Developmental Biology, Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45224, USA
* Author for correspondence (e-mail: steve.potter{at}chmcc.org )



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  		Fig. 1. Homeobox swap strategy. (A) Organization of the genes of the HoxA cluster. (B) Amino acid sequence comparison of the A11 and A13 homeodomains. Dashes represent sequence identity. (C) The targeting construct at the top consisted of the A11 gene with a substituted A13 homeodomain (HD*, black rectangle), inserted loxP flanked Neo and HSV thymidine kinase gene. Double homologous recombination, as shown, results in homeobox swap and insertion of loxP flanked Neo. Mating with transgenic Cre mice removes Neo, leaving a single 34 bp loxP. PCR genotyping used primers a-d, and Southern blot genotyping used restriction sites (P) PstI, (N) NheI and DNA segment probes (open lines and solid rectangles).

 


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  		Fig. 2. Southern blot and PCR analysis. (A) Southern blot analysis of targeted ES cell lines. The positions of external probe and internal probe used for screening targeted ES cell lines are indicated in Fig. 1c. Targeted cell lines were confirmed by two different restriction digestions (P) PstI, and (N) NheI, generating a 6.1 kb wild-type band and 4.1 kb targeted band, and 5.5 kb wild-type band and 7.3 kb targeted band, respectively. (B) PCR identification of the swapped homeobox encoding the A13 homeodomain (HD) using primers a and b (indicated in Fig. 1C). PCR amplified fragments were digested with PstI, HincII (HcII) and EcoRV (RV). Only the swapped A13 PCR fragment was cut by all three enzymes to give distinguishable smaller sized bands. (PstI, 480, 354 and 126 bp; HincII, 480, 323 and 157 bp; EcoRV, 480, 260 and 220 bp). The presence of the precise homeobox swap was confirmed by sequencing. (C) PCR identification of Cre recombination. Cre-expressing transgenic mice were used to remove the Neo gene in the targeted A11 locus. A11 specific primers c and d (indicated in Fig. 1C) did not amplify the 2 kb (loxpNeoloxp) fragment under the PCR conditions used, but gave a 317 bp fragment with loxp, or a 285 bp fragment without loxp (wild type). wt, wild type; M, pBR322 DNA-MspI marker.

 


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  		Fig. 3. The A1113hd allele provided near normal function in kidney development. (A) Gross appearances of wild type, A11-/- D11-/- and A1113hd/- D11-/- kidneys. The A11-/- D11-/- kidneys shown are among the least affected, coming from one of the two mice that survived to P30. One kidney was severely reduced in size, with the other more normal. The A1113hd/- D11-/- kidneys appeared grossly normal except for a reproducible indentation on the left kidney (arrow). (B) Kidney histology. Top and middle panels: the A1113hd/- D11-/- kidneys appeared relatively normal compared with the A11-/- D11-/- kidneys, which showed many occluded proximal tubule (P) lumens (long arrows) and severely dilated distal (D) tubules (arrowheads). Short arrows point to glomeruli. Bottom panels: the medulla layer of both the A11-/- D11-/- kidney and the A1113hd/- D11-/- kidney (not shown) was severely reduced in size and disrupted by cysts (asterisk) when compared with the wild type. C, cortex; M, medulla.

 


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  		Fig. 4. Wild-type function of A1113hd in the male reproductive tract. (A) Sperm were present in the lumens of wild-type and A1113hd/13hd D11+/+ seminiferous tubules (arrows), but not in A11-/- D11+/+ testis. (B) The A1113hd/13hd D11+/+ ductus deferens showed a wild-type morphology, while A11-/- D11+/+ mutants showed a coiled configuration resulting from an anteriorization towards epidydimis (arrows). E, epididymis; T, testis; V, vas deferens.

 


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  		Fig. 5. A1113hd hindlimb phenotype. (A) The arrowhead points to the normal tibia and fibula fusion point and broad arrow points to the calcaneus bone in wild-type hindlimb. Mice of A1113hd/+ D11+/+ or A11-/- D11+/+ genotypes showed similar more distal separation of tibia and fibula, while A1113hd/13hd D11+/+ mice showed more extreme tibia, fibula separation (thin arrows) when compared with wild type. (B) Substitution of the A11- allele by an A1113hd allele in either A11+/- D11-/- or A11+/- D11+/- mutants resulted in greater separation of tibia and fibula (arrows). (C) Ventral views of isolated autopods of hindlimbs. A1113hd/13hd D11+/+ mutants had a severely truncated calcaneus compared with wild type (arrows). A11-/- D11+/+ mutants were normal. c, calcaneus; F, fibula; T, tibia.

 


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  		Fig. 6. The A1113hd allele antagonizes A11 and D11 function in forelimb development. (A) In A1113hd/13hd D11+/+ mice, the zeugopod region was shortened to near half of normal length, resembling A11-/- D11+/- mice. R, radius; U, ulna. A central bulge was observed in the shortened radii of A1113hd/13hd D11+/+ and A11-/- D11-/- mice (arrowheads). The olecranon of the ulna, however, was truncated and replaced with a floating sesamoid bone in A11-/- D11-/- mice and was more wild type in appearance in A1113hd/- D11-/- mice (arrows). (B) Wrist region. The styloid apophyses of A1113hd/13hd D11+/+ mice were reduced and/or fused with the ulna and radius, while they were only very mildly affected in A11-/- D11+/+ mutants (thin arrows). The styloid apophyses were also reduced and/or fused in A11-/- D11-/- and A1113hd/- D11-/- mutants (thick arrows).

 


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  		Fig. 7. Histological comparison of female reproductive tracts. (A) Wild-type uterus showed single columnar epithelial cell layer (arrow), and contained endometrial glands (G) in the stromal cell layer (asterisk). (B) The A11-/- D11+/+ mutant uterus had fewer glands and a thinner stromal cell layer than wild type, but its epithelium and stromal tissue resembled the wild-type uterus morphologically. (C) The A1113hd/13hd D11+/+ mutant uterus showed an absence of glands, a multi-layered epithelium and a more fibrous stromal layer, all consistent with a posteriorization to cervix. (D) The wild type cervical canal showed a multi-layered squamous epithelium and a fibrous stromal layer with a lower cell density than seen in the uterus.

 

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© The Company of Biologists Ltd 2001