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doi: 10.1242/10.1242/dev.00504

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Loss of Tbx4 blocks hindlimb development and affects vascularization and fusion of the allantois

Department of Genetics and Development, College of Physicians and Surgeons, Columbia University, 701 W. 168th Street, New York, NY 10032, USA

View larger version (31K): [in a new window]   Fig. 1. Disruption of the Tbx4 gene by homologous recombination in ES cells. (A) 7.3 kb of mouse genomic DNA was used to create a targeting construct containing a loxP site inserted into intron 4, a floxed PGK-neo PGK-thymidine kinase dual selection cassette (neo tk) inserted into intron 5 and a diptheria toxin (DT) gene 5' of the homologous DNA for negative selection against random integration. The indicated region of the Tbx4 cDNA was used for in situ hybridization (in situ probe). E, EagI; X, XhoI; Xb, XbaI; K, KpnI; S, SpeI; RV, EcoRV. Solid arrowheads indicate loxP sites. Boxes indicate exons: black, untranslated regions; white, coding regions; gray, T-box domain. Labeled boxes indicate selection cassettes. Figure not drawn to scale. (B) After electroporation, G418-resistant colonies were screened for homologous recombination by Southern hybridization of EagI/XbaI genomic digests probed with the 3' external probe shown in A. (C) Homologous recombination of the 5' end of the vector, including the loxP site, was confirmed using a KpnI digest probed with the 5' internal probe shown in A. (D) Oligonucleotides a and b were used to amplify the indicated genomic region by PCR, producing a 500 bp endogenous band and a slightly larger Tbx4tm1Pa band with the loxP site insertion. Recombination of the Tbx4tm1Pa allele was achieved by mating to a cre-expressing mouse. The recombined Tbx4tm1.1Pa allele was genotyped using oligonucleotides a, b and c, to produce the same 500 bp endogenous band and a 260 bp mutant band.

View larger version (90K): [in a new window]   Fig. 4. In vitro culture of forelimb and hindlimb buds from 10.5 dpc embryos. (A) Forelimb tissue explanted at 10.5 dpc. Dashed lines indicate planes of dissection; gray area represents tissue explanted. (B,C) Forelimb buds dissected from normal and mutant embryos, respectively, immediately after explantation. (D,E) Forelimb explants from control and mutant embryos, respectively, show an increase in distance between the distal limb margins (arrowheads) and the development of a paddle-shaped autopod after 3 days in culture. (F) Explanted hindlimb tissue. (G,H) Hindlimb buds from normal and mutant embryos, respectively, immediately after dissection. (I) Control hindlimb buds also exhibit increased distance between limb margins (arrowheads) and autopod development after 3 days in culture. (J) By contrast, explants from Tbx4-mutant embryos have no visible limb structures remaining after the same time period. (K,L) Limb explants hybridized with a Tbx4 probe from outside the region deleted in the mutant. The wild type (K) expresses Tbx4 in the morphologically apparent hindlimb buds, whereas the mutant explant (L) shows only faint Tbx4 expression, and regions of Tbx4 expression show no morphological limb structure. All panels are at the same magnification.

View larger version (68K): [in a new window]   Fig. 2. Morphology of wild-type (+/+) and Tbx4-homozygous mutants (-/-). Black arrowhead indicates the allantois or umbilicus. (A) An 8-somite wild-type embryo. The allantois has fused with the chorion and ectoplacental cone (epc). (B) An 8-somite Tbx4-mutant embryo shows a stunted, unfused allantois. (C) 9.5 dpc embryos partially dissected out of the yolk sac (ys). The allantois of the wild-type embryo has formed a vascular umbilicus connecting it to the placenta (p), whereas the mutant embryo is loose in the yolk sac and the allantois has formed only a small, amorphous stump. (D) 10.5 dpc wild-type and mutant embryos dissected out of their membranes. The mutant is hemorrhagic and has only the stump of an allantois, but it is otherwise normal and shows a distinct hindlimb bud similar to that of the wild type (red arrowheads). (E) Close-up of a wild-type umbilical connection at 10.5 dpc, with large umbilical blood vessels connected to the chorionic plate (ch) of the placenta. (F) Close-up of the unfused allantoic stump of a Tbx4 mutant at the same stage, which is not connected to the placenta and shows no coherent blood vessels. (G) Section through a normal 8.25 dpc allantois. The allantois has a funnel shape, the chorionic end of which contains loose, cavitated mesenchyme and a layer of cells tightly opposed to the chorion; the base has a more compact, uniform mesenchyme. (H) Section through the base of an unfused mutant allantois, showing dense cell packing and distinctive vesicles. (I) Detail of H, showing two of the characteristic mutant double-layered vesicles and cell debris from dying cells. (J) Detail of L, showing the mutant allantois at 9.5 dpc. The irregular mesenchyme contains dense condensations that have no apparent connections between them in serial sections. (K) Section through a normal 9.5 dpc allantois and embryo. Blue arrowhead indicates a blood vessel in the allantois. (L) Section through a mutant 9.5 dpc allantois and embryo. The allantois is unfused and the chorion is out of the section plane at the top. Embryonic tissues appear normal. (M) Transverse section through a wild-type 10.5 dpc embryo, with normal lung buds (lb), hindlimb bud (hl) and umbilicus. (N) Transverse section of a 10.5 dpc mutant in a similar plane as the embryo in M, as indicated by the lung buds. All structures appear normal. (O) A more ventral section of the same embryo showing a normal heart (a, atrium; v, ventricle), a small hindlimb bud (hl) and the remnants of an allantois.

View larger version (98K): [in a new window]   Fig. 3. Growth and differentiation of wild-type (+/+) and Tbx4-mutant (-/-) allantoises. All embryos were dissected at 8.0 dpc and were at the 0- to 8-somite stages. Black arrowheads indicate the base of the allantois in all panels. (A) A summary of 8.0 dpc embryos grouped by somite number and genotype and scored for extension of the allantois as early, late or fused (see methods). Each section of the stacked bar represents the proportion of those embryos at a specific allantois stage. The number in each set is shown at the top of the bar. (B,C) Anti-phospho-Histone H3 staining showing cells undergoing mitosis. The wild-type (B) and the mutant (C) allantoises show similar numbers of mitotic cells, both in the base of the allantois and in the distal tip. (D,E) Sets of 0- to 8-somite embryos (somite number decreases to the right in each panel) TUNEL stained to indicate apoptotic cells. Although wild-type embryos (D) show few apoptotic cells, the mutant embryos (E) exhibit extensive cell death over the entire distal portion of the allantois. (F,G) Bmp4 is expressed in the base of the allantois in wild-type (F) and mutant (G) embryos. (H,I) Tbx2 is expressed in the distal two-thirds of the wild-type allantois at the 2-somite stage (H), but is extremely faint in mutant allantoises (I). (J,K) The cell adhesion molecule VCAM1 is expressed in the distal tip of the allantois in wild-type (J), but not mutant (K), embryos. (L,M) Endogenous alkaline phosphotase staining marks the PGCs, which are similar in location and number in wild-type (L) and mutant (M) embryos. (N-P) PECAM marks differentiating endothelial cells, which in the wild type appear in clumps at the 0-somite stage (N); they elongate into small vessels at the 4-somite stage (O) and resolve into a central vessel at the 8-somite stage (P). In Tbx4 mutants (N-P), normal looking clumps appear at 0 somites, whereas at 4 somites more clumps have appeared but no elongated vessels are seen. At the 8-somite stage, no central vessel is seen.

View larger version (49K): [in a new window]   Fig. 5. Expression of genes involved in limb bud initiation in wild type (+/+) and Tbx4 mutants (-/-) at 10.5 dpc. (A,B) Fgf8 expression is visible in the AER of the forelimb bud and the presumptive AER of the hindlimb bud in wild-type and Tbx4-mutant embryos. (C,D) Expression of Msx1 is present in the mesenchyme of the forelimbs and hindlimbs of both wild-type and mutant embryos. (E,F) Twist is expressed normally throughout the hindlimb mesenchyme of wild-type and mutant embryos. (G,H) Thick sections through the forelimbs (top) and hindlimbs (bottom) of 10.5 dpc embryos. Anti-dp-ERK marks regions of FGF signaling and is seen in the mesenchyme immediately underlying the AER of forelimbs, and asymmetrically in the mesenchyme of the hindlimbs in the wild-type embryo. Tbx4-mutant embryos also show similar dp-ERK staining in both sets of limbs. (I,J) Fgf10 is expressed in the distal mesenchyme of the forelimb and throughout the mesenchyme of the hindlimb bud of the wild type. In Tbx4 mutants, forelimb expression of Fgf10 is unaltered, but expression in the hindlimb appears only in the proximal mesenchyme and is absent from the distal hindlimb bud. Arrowheads indicate the distal edge of hindlimb bud.

View larger version (50K): [in a new window]   Fig. 6. Expression of limb patterning genes in the limb buds of wild-type (+/+) and Tbx4-mutant embryos (-/-) at 10.5 dpc. (A,B) Tbx5 is expressed in the forelimb bud and absent in the hindlimb bud (arrowheads) of wild-type and mutant embryos. (C,D) Ptx1 is expressed in the hindlimb bud, but not the forelimb bud of both wild-type and mutant embryos. (E,F) Msx2 marks the ventral limb ectoderm in both normal and mutant hindlimb buds. Embryos are oriented for a posterior view of the hindlimb bud, with forelimbs at the bottom of the picture and the head removed for clarity. Arrowheads mark the dorsal boundary of Msx2 expression in the hindlimb. (G,H) dHand, an upstream regulator of Shh, is expressed strictly in the posterior edge of the hindlimb bud of normal embryos, but throughout the margin of the Tbx4-mutant hindlimb bud. Small arrowheads mark anterior and posterior boundaries of the hindlimb bud. (I,J) Tbx3 is expressed in the posterior edge of both forelimb and hindlimb buds (arrowheads) in both wild-type and mutant embryos. (K) Tbx2 is expressed in both anterior and posterior edges of the forelimb bud and in the posterior edge of the wild-type hindlimb bud (arrowhead). (L) In the Tbx4 mutants, Tbx2 is normally expressed in the forelimb but absent from the posterior hindlimb.

View larger version (22K): [in a new window]   Fig. 7. A proposed model for Tbx4 function in the hindlimb. During hindlimb initiation in normal embryos, dHand is repressed in the anterior but not the posterior limb bud by the FGF-dependent action of Gli3. Simultaneously, Fgf10 signals to the overlying ectoderm to upregulate Fgf8. As the limb bud progresses, dHand repression in the anterior limb bud is maintained, while Fgf8 reciprocal signaling maintains Fgf10 in the limb mesenchyme. In the mutant hindlimb bud, failure of Tbx4-mediated FGF signaling leads to derepression of dHand in the anterior limb bud. Mesenchymal FGF signaling to the ectoderm is normal and Fgf8 is properly upregulated, but reciprocal signaling fails and Fgf10 is not maintained.