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First published online 10 December 2003
doi: 10.1242/dev.00936

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Multiple roles of Hoxa11 and Hoxd11 in the formation of the mammalian forelimb zeugopod

Howard Hughes Medical Institute, Department of Human Genetics, University of Utah School of Medicine, Salt Lake City, UT 84112, USA

View larger version (36K): [in a new window]   Fig. 1. Mutations in paralogous Hox genes cause dramatic disruptions of limb skeletal morphology. (A-D) Alcian Blue/Alizarin Red-stained limbs. Forelimbs of (A) control newborn and (B) Hoxa11/Hoxd11 double mutants; hindlimbs of (C) control and (D) Hoxa10/Hoxc10/Hoxd10 triple mutant newborn.

View larger version (82K): [in a new window]   Fig. 2. Histological analysis, cell proliferation and apoptosis in radius and ulna condensations of Hoxa11/Hoxd11 double mutant embryos. (A) Paraffin section of forelimb bud of a control E12.5 embryo stained with Hematoxylin and Eosin. (B) Section from E12.5 Hoxa11-/-Hoxd11-/- forelimb bud. Although the size of the radius condensation appears similar to control, the ulna condensation is significantly smaller. (C) Forelimb bud of control E13.5 embryo stained with Alcian Blue. (D) Double mutant E13.5 forelimb stained with Alcian Blue. (E,F) Sections of an E13.5 control (E) and (F) double mutant forelimb bud staind with Hematoxylin and Eosin. (G,H) Adjacent sections to those shown in E,F, respectively, stained with an anti-BrdU antibody. Brackets mark distal end of the ulna where more labeled cells are seen in control than in double mutant embryo section. (I-L) TUNEL analysis performed on E12.5 control (I,K) and double mutant (J,L) embryo sections. A greater number of apoptotic cells (arrowheads) are detected in double mutant embryo.

View larger version (105K): [in a new window]   Fig. 3. Expression patterns of Sox9 and type II collagen. Hematoxylin-stained sections of E12.5 (A) control and (B) double mutant embryos. Pattern of Sox9 expression in control (C) and double mutant (D) section, as revealed by radioactive in situ hybridization. (E,F) Type II collagen protein expression in E12.5 control embryo (E) and double mutant forelimb bud (F). r, radius; u, ulna; h, humerus.

View larger version (101K): [in a new window]   Fig. 4. Forelimb development is delayed in Hoxa11/Hoxd11 double mutant embryos. (A) Limb bud of E11.5 control embryo [stage 6 limb bud (Wanek et al., 1989)]. (B) Limb bud of E11.5 Hoxa11/Hoxd11 double mutant embryo (stage 5-6). (C) Stage 8 limb bud of E12.5 control embryo. (D) Limb bud of double mutant E12.5 embryo of approximately the same crown-rump length as control embryo shown in C. Limb bud appears to be at stage 6-7. (E) Stage 9 limb bud of E13.5 control embryo. (F) Limb bud of Hoxa11/Hoxd11 mutant embryo of approximately the same size as control embryo in E. Limb bud appears to be at stage 8-9, and indentations between digits are slightly deeper in control embryo (arrows in E,F).

View larger version (101K): [in a new window]   Fig. 5. Fgf8 and Fgf10 expression patterns in double mutant embryos. (A) Fgf8 expression in stage 3 limb bud of control embryo and (B) Hoxa11/Hoxd11 double mutant embryo. Fgf8 stained region is narrower in double mutant forelimb bud. (C) Fgf8 staining in control embryo with stage 4-5 forelimb buds. (D) Stage 4-5 double mutant forelimb bud hybridized with Fgf8 probe. Staining with Fgf8 probe is weaker in anterior AER of mutant embryo than in control (arrows). (E) Expression pattern of Fgf10 in E11.5 control embryo and (F) E11.5 double mutant. Arrows indicate the alteration in the staining pattern in the anterior limb bud mesenchyme.

View larger version (110K): [in a new window]   Fig. 6. Histology and type X collagen expression at E16.5. Hematoxylin and Eosin-stained sections of control (A) and double mutant (B) E16.5 embryos. Anti-type X collagen staining of control E16.5 forelimb (C) and double mutant E16.5 forelimb (D).

View larger version (68K): [in a new window]   Fig. 7. Expression patterns of cartilage differentiation markers in mid-gestation embryos. (A) Pattern of Ihh expression in E12.5 control embryo detected by whole-mount in situ hybridization. Arrow indicates Ihh expression in the radius and ulna condensations of the forelimb bud. (B) Double mutant embryo with forelimb bud at approximately the same limb bud stage as that of control embryo in A stained with Ihh probe. Note significantly lower level of Ihh expression in radius and ulna (arrow) compared with control. (C) Ihh transcription in stage 12 control radius and ulna. (D) In situ hybridization of Ihh probe to section of a double mutant stage 12 forelimb. Transcripts are abundant in digit elements, but absent from radius and ulna. (E) Pthlh transcripts are found in the perichondrium surrounding carpal elements and the distal ends of the radius and ulna in E15.5 wild-type embryos. (F) Although Pthlh expression in the carpal region is readily observed in Hoxa11/Hoxd11 double mutant embryos, expression extending proximally around the distal radius and ulna is not seen. (G) In situ hybridization of the parathyroid hormone receptor (Pthr) probe to section of a stage 10 control embryo. (H) Section of a stage 12 double mutant forelimb hybridized with the Pthr probe. Transcripts are present in the digits but undetectable in the radius or the ulna. (I) Expression pattern of Hoxd11 in a stage 9-10 wild-type forelimb detected by in situ hybridization using a 33P-labeled probe. Hoxd11 transcripts surround the ulna, with high levels at the distal end, and also surround the radius, carpals and digit condensations at this stage (data not shown). (J) Hoxa11 transcripts, detected by a 33P-labeled probe, are abundant in mesenchyme surrounding the radius and ulna.

View larger version (107K): [in a new window]   Fig. 8. Expression of Ihh and Pthr in control and double mutant newborns. (A,B) Brightfield views of control (A) and double mutant (B) newborn forelimb sections hybridized with an Ihh probe. (C) Darkfield view of control section shown in A. (D) Darkfield view of double mutant section shown in B. (E,F) Brightfield views of control (E) and double mutant (F) newborn forelimb sections hybridized with a Pthr probe. (G) Darkfield view of control section shown in E. (H) Darkfield view of double mutant section shown in F.

View larger version (74K): [in a new window]   Fig. 9. Distal growth plate abnormalities in radius and ulna of mice with three mutant alleles of Hoxa11 and Hoxd11. (A) Forelimb skeleton of control newborn (Hoxa11+/+Hoxd11+/-) stained with Alcian Blue and Alizarin Red. (B) Forelimb skeleton of Hoxa11+/-Hoxd11-/- newborn. (C) Hematoxylin and Eosin-stained section of 14-day-old control forelimb showing growth plate of the distal ulna. (D) Growth plate of distal ulna in 14-day-old Hoxa11-/- Hoxd11+/- mouse. Asterisks are adjacent to secondary ossification centers. (E) Distal growth plate of ulna in newborn (P0) control specimen. (F) Growth plate of distal ulna from a P0 Hoxa11+/- Hoxd11-/- mouse. Double-headed arrows demarcate proximodistal extent of growth plates. (G,H) Bright-field views of distal radius and ulna of Hoxa11-/- Hoxd11+/- newborn (hz, hypertrophic zone). (I) Dark-field image of G, showing hybridization with the Ihh probe. (J) Dark-field image of H, showing Pthr expression. (K) Hoxd11 expression pattern at E16.5 revealed by in situ hybridization with a 33P-labeled probe. Transcripts are detectable adjacent to the distal end of the ulna (white bracket).