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doi: 10.1242/10.1242/dev.00354

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A role for the mesenchymal T-box gene Brachyury in AER formation during limb development

Chunqiao Liu^*,, Eiichiro Nakamura, Vladimir Knezevic, Sherrie Hunter, Katherine Thompson and Susan Mackem

Laboratory of Pathology, Center for Cancer Research, NCI, NIH, Bethesda, MD 20892, USA
^* Present address: Carnegie Inst Washington, Dept Embryol, Baltimore, MD 21210, USA
Present address: 20/20 Gene Systems, Inc., Rockville, MD 20850, USA

View larger version (84K): [in a new window]   Fig. 1. Expression of T during limb development in chick. Whole-mount in situ hybridization with chick T probe. (A) T expression in lateral plate of the wing region at stage 15 (arrow), as well as weak expression in adjacent somites, and strong expression in notochord and tail bud. (B) At stage 18, T is expressed in a narrow strip of distal mesoderm underlying both wing and leg AER (arrows), as well as in somites, notochord and tail bud. (C) By stage 22, T expression is beginning to decline in wing, but is still very evident in leg bud (arrows). (D) Transverse section through wing region of a stage 15 embryo (as indicated in A) showing strong T expression in notochord (no), weak to moderate expression in wing lateral plate, somite (so) and nephric/Wolffian ducts (nd). (E,F) Transverse sections through wing (E) and leg (F) buds from a stage 18 embryo (as indicated in B), and (G) transverse section through leg bud of a stage 22 embryo (as indicated in C) showing T expression in the immediate subridge mesoderm beneath the AER (arrow) and continued weak expression in dermomyotome of somites (so) and in nephric ducts (nd). Note T expression is also seen in tail epithelial somites, at stage 22 (G). Signal development time was extended to visualize sites with weak expression (lateral plate, somites, limb bud). d, dorsal; v, ventral.

View larger version (39K): [in a new window]   Fig. 2. Tissue distribution of T protein in limb bud stage chick and mouse embryos. (A) Western analysis of T protein in dissected tissues using affinity-purified polyclonal antibody. T protein (47 kDa band) was detected in chick wing (W) and leg (L) buds and in somites (So) at stages (st) 18 and 20, as well as in wild-type (wt) E10 mouse forelimb (FL) and hindlimb (HL) buds. No T protein band was detected in head or trunk (including forelimb buds) from T-/- mouse embryos (negative control). Chick or mouse tailbud protein was loaded as a positive control. A ubiquitous 100 kDa protein was recognized by affinity-purified T antibody in every tissue evaluated. Protein loading was assessed by reprobing blots with -tubulin antibody (bottom). (B) Immunohistochemical detection of T protein in transverse hindlimb section of E10.5 mouse embryo, showing expression in notochord (no) somitic dermomyotome (so) and limb mesoderm subjacent to AER. Affinity purified antibody was exhaustively pre-adsorbed against 100 kDa proteins prior to use. d, dorsal; v, ventral.

View larger version (50K): [in a new window]   Fig. 3. Retroviral misexpression of T in prospective hindlimb causes anterior extension of the AER and early changes in Fgf10 expression. All panels show dorsal side of embryos except B, which is viewed from the top (craniad) down and D, which shows a ventral view. The anteroposterior (a-p) or dorsoventral (d-v) orientations are indicated to left. (A-C) Fgf8 expression in infected embryos harvested at stages 20 (A), 22 (B), and 24 (C). The embryo in C was also hybridized with a Shh probe. Broadening of the anterior limb bud on the infected (inf.) side compared to the contralateral control (con.) side was always accompanied by anterior extension of Fgf8 expression in AER (arrows). No ectopic Shh expression was seen in the context of AER extension (C). (D) Fgf4 expression in infected embryo at stage 21 showing expression has extended anteriorly in the AER on the infected side (arrows). (E) Fgf10 expression in infected embryos at stage 20 was stronger and more extensive in the distal subridge mesoderm on the infected side. Color development times for Fgf10 probe were kept very short to visualize mainly the distal mesoderm and prevent signal saturation. (F) Wnt5a expression was only slightly increased by stage 23 on the infected side (arrows), but showed no expression difference at earlier stages following infection (data not shown).

View larger version (52K): [in a new window]   Fig. 4. Skeletal and soft tissue phenotypes due to retroviral misexpression of T. (A-D) Embryos harvested at day 9-10 were stained for cartilage. (A) Uninfected control leg with tarso-metatarsals (tm) and digits I-IV (anterior to posterior order) indicated. Digit identity was based on the number of phalangeal elements (1-4 for I-IV respectively). Skeletal phenotypes in infected legs (B-D) included partial (C) or complete (B,D) anterior digit duplications, and occasionally thickening (arrow, C) or duplication (arrow, D) of anterior tarsometatarsals. Some infected embryos at day 9-10 also showed changes in digital soft tissues in the infected (inf.) compared to contralateral control (con.) leg (panels E,F). These included digital soft tissue broadening (E,F arrowheads), ectopic cartilage condensations (D,E asterisk), and delayed loss of interdigital webbing (E, compare arrows). E, ventral side; F, dorsal side shown.

View larger version (52K): [in a new window]   Fig. 5. WNT3a and FGF8b induce T expression in primary limb mesenchyme culture. Mesenchymal cells were dissociated from stage 19/20 chick embryo limb buds and used immediately. RNA from cells infected with Wnt3a-expressing retrovirus (Wnt3a-RV), control viral vector (RV), uninfected control (uninf.) or after treatment with either heparin sulphate (hs) alone or with recombinant FGF8b+hs (FGF-8b) were harvested at times indicated and hybridized with a chick T 3'-UTR probe (Ch-T), followed by a chick ß-actin probe to control for loading. RNA extracted from stage 18 chick tail bud was included as a positive control. Both WNT3a and FGF8b induced T expression within 18 hours and activation by FGF8b protein was detectable by 8 hours. FGF4 protein had no effect on T expression at either time (data not shown).

View larger version (85K): [in a new window]   Fig. 6. Disrupted AER maturation and reduced Fgf10 expression in T-/- embryos. Comparison of Fgf8 (A-E), Bmp4 (F-J), En1 (K-O), Wnt7a (P-S) and Fgf10 (T-X) expression in wild-type (wt, left sides) and T-/- (right sides) embryos at different stages as indicated to the left of each row by age and average somite (so) number in wild type. As indicated in the upper left corner, limb buds shown in each panel are oriented with dorsal to ventral axis (dv) from left to right, and with anterior to posterior axis (ap) from top to bottom of the panel. The small arrows highlight some of the focal gaps, irregularities, and wavy borders in pre-AER or AER zone revealed by marker expression patterns in T-/- limb buds. Note that T-/- embryos showed mottled early pre-AER expression (A,F) and later broad, weak, and irregular patterns of AER markers compared to very sharply demarcated expression in the wild type. Fgf10 expression was substantially decreased by E9.5 and later (V-X). Note some T-/- limb buds were very irregularly shaped (eg. Q,T,V). Hybridization signals at various expression sites unaffected by loss of T (e.g. craniofacial, branchial arch, gut) were similar in T-/- and wild-type embryos, serving as internal controls (data not shown).

View larger version (91K): [in a new window]   Fig. 7. Abnormal AER morphogenesis in T-/- embryos. AER progenitors were stained with affinity-purified anti-DLX (a gift from Grace Panganiban) in transverse sections of paraffin-embedded wild-type (wt, A,C,E,G) and T-/- (B,D,F,H,H') forelimb buds at ages (average somite number: so) indicated to the left of the panels. The small arrows indicate approximate extents of pre-AER or AER. Note poor and variable maturation of T-/- AER, as illustrated by H and H' which show nearby sections from a T-/- limb bud with a particularly mild phenotype. Some T-/- limb buds were highly irregular in shape (* in B; see also Fig. 6Q,T,V). To evaluate distribution of Bmp4 expression, embryos hybridized in whole-mount were transversely cryo-sectioned and wild type (wt, I,K,M) was compared to T-/- (J,L,N). Similar abnormalities in pre-AER and AER morphology were observed, with poor AER compaction during maturation (eg. M vs.N). Additionally, reduced mesodermal Bmp4 expression was evident in E9.25 T-/- limb buds (J). d, dorsal; v, ventral.

View larger version (70K): [in a new window]   Fig. 8. Selective mesodermal changes consistent with disturbed AER function in T-/- limb buds. Potential changes in limb mesoderm due to altered AER function were evaluated in T-/- embryos (right sides) compared to wild type (wt, left sides). Proliferation was assessed by immunostaining multiple transverse sections with the mitosis-specific anti-phospho-histone H3 (representative sections shown). Similar numbers of mitotic cells were present in wild-type and T-/- limb buds at both E9.5 (not shown) and at E9.75 (A). Apoptosis was evaluated by TUNEL assay at E9.5 (not shown) and E9.75 (B) using fluorescein-UTP (green) and propidium iodide nuclear counterstaining (red) and also by whole-mount Nile Blue sulfate staining at E10 (not shown). At all stages, levels of apoptosis in T-/- limb buds were minimal and similar to wild type. Lmx1b and Shh were checked to evaluate overall limb `maturity' and level of AER signaling. Lmx1b is expressed uniformly at early stages and dorsally later. Shh activation depends on AER signals. While Lmx1b expression was preserved and showed nornal dorsal restriction in T-/- limb buds at E10 (C), Shh was not expressed at all in T-/- forelimb bud by E10 (D), but was detected at levels similar to wild type in hindgut (data not shown). d, dorsal; v, ventral; a, anterior; p, posterior.

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