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doi: 10.1242/10.1242/dev.00473

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Tbx5 is required for forelimb bud formation and continued outgrowth

Charalampos Rallis1, Benoit G. Bruneau2, Jo Del Buono1, Christine E. Seidman3,5, J. G. Seidman3,4, Sahar Nissim3, Clifford J. Tabin3 and Malcolm P. O. Logan1,*

1 Division of Developmental Biology, National Institute for Medical Research, Mill Hill, London NW7 1AA, UK
2 Cardiovascular Research and Developmental Biology, The Hospital for Sick Children and Department of Molecular and Medical Genetics, University of Toronto, Toronto, ON, Canada
3 Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
4 Department of Genetics, Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115, USA
5 Cardiovascular Division, Howard Hughes Medical Institute, Brigham and Women's Hospital, Boston, MA 02115, USA



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  		Fig. 1. Absence of forelimb skeletal elements in newborn Tbx5lox/lox;Prx1Cre pups. (A) Tbx5lox/lox;Prx1Cre pup viewed from the side. The forelimb has failed to form although the hindlimb has developed normally. (B) Ventral view of the pup shown in A. The arrow indicates a small flap of skin, present on one side of the embryo. (C) Tbx5 expression in the developing forelimb but not the developing hindlimb of an E10.5 mouse embryo. (D) PCR analysis identifies the presence of the wild-type (wt) and conditional allele (lox) in heterozygous Tbx5lox/wt embryos, the conditional allele (lox) in the homozygous Tbx5lox/lox embryos and the lox-out deleted allele in Tbx5lox/lox;Prx1Cre limb buds. (E) Skeletal preparation of a control littermate. (F) Dorsal view of the thoracic region of the embryo in E showing the pectoral girdle and forelimb skeletal elements. (G) An outline of the skeletal preparation shown in F. The forelimb skeletal elements are highlighted in red. (H) Skeletal preparation of a Tbx5lox/lox;Prx1Cre pup. (I) Dorsal view of the thoracic region of the embryo shown in H. All the elements of the forelimb and pectoral girdle are absent. (J) An outline diagram of the skeleton shown in I. FL, forelimb; HL, hindlimb.

 


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  		Fig. 2. Absence of a forelimb bud in Tbx5lox/lox;Prx1Cre embryos. Scanning electron micrographs of (A) a control embryo and (B,C) Tbx5lox/lox;Prx1Cre embryos at E10.5. In B, no morphological limb bud or AER is present in the forelimb region (arrowed). In C, a small outgrowth of tissue from the flank is present (arrowed). (D-G) Expression analysis of limb bud markers by whole-mount RNA in situ hybridisation. (D) Fgf8 expression in the AER of the forelimb (arrowed) and hindlimb of a control embryo at E9.5. (E) Fgf8 expression in the hindlimb AER but absence in the forelimb region of a Tbx5lox/lox;Prx1Cre embryo. (F) Shh expression in the ZPA of the forelimb (arrowed) and hindlimb of a control embryo at E10.5. (G) Shh expression in the hindlimb ZPA but absence in the forelimb region of a Tbx5lox/lox;Prx1Cre embryo. (H-K) Hindlimb markers are not upregulated in Tbx5lox/lox;Prx1Cre embryos. (H) Tbx4 expression in the hindlimb but not the forelimb (arrowed) of a control embryo at E10.5. (I) Tbx4 is expressed normally in the hindlimb and is not upregulated in the forelimb region of an E10.5 Tbx5lox/lox;Prx1Cre embryo. (J) Pitx1 expression in the hindlimb but not the forelimb (arrowed) of a control embryo at E10.5. (K) Pitx1 is expressed normally in the hindlimb but is not upregulated in the forelimb region of an E10.5 Tbx5lox/lox;Prx1Cre embryo. FL, forelimb; HL, hindlimb.

 


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  		Fig. 3. Early markers of the prospective forelimb mesenchyme are absent in Tbx5lox/lox;Prx1Cre embryos. (A-F) Expression analysis by whole-mount RNA in situ hybridisation. (A) Expression of Fgf10 in the forelimb (arrowed) of a control embryo E9.5. (B) Fgf10 is not expressed in the forelimb region of a Tbx5lox/lox;Prx1Cre embryo at E9.5. (C) Dorsal view of the embryo shown in A. Fgf10 expression in the forelimb buds is indicated by an asterisk. (D) Dorsal view of the embryo shown in B. No Fgf10 expression is detected in the forelimb-forming region. (E) Expression of Pea3 in the forelimb (black arrow) and intermediate mesoderm (red arrow) of a control embryo at E9.5. (D) Pea3 is expressed in the intermediate mesoderm (red arrow) but not the forelimb region of Tbx5lox/lox;Prx1Cre embryos at E9.5 (black arrow). (G-J) Analysis of cell death in the limb. G) Whole-mount TUNEL staining of the forelimb region of a control embryo at E9.5. (H) Whole-mount TUNEL staining of the forelimb region of a Tbx5lox/lox;Prx1Cre embryo at E9.5. A zone of increased apoptotic cell death is present at the site where the forelimb would normally form (arrowed). (I) Whole-mount TUNEL staining of the forelimb region of a control embryo at E10.5. Some cell death is observed in the limb and foci of apoptotic cells are observed in the somites (arrowed). (J) Whole-mount TUNEL staining of the forelimb region of a Tbx5lox/lox;Prx1Cre embryo at E10.5. A region of increased cell death is present in the forelimb-forming region (bracketed). Foci of apoptotic cells are also observed in the somites in a similar pattern to the control embryo (arrowed).

 


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  		Fig. 4. Limb outgrowth is disrupted following misexpression of dominant-negative Tbx5 in the chick wing bud. Scanning electron micrographs of (A) control and (B) dominant-negative Tbx5 (Tbx5{Delta}) injected limb buds at stage 20 show a dramatic reduction in limb size after Tbx5{Delta} misexpression. A, anterior; P, posterior. Sections through (C) contralateral control and (D) dominant-negative Tbx5 (Tbx5{Delta}) injected limb buds at stage 20 stained with an antibody against phosphohistone H3 to identify cells undergoing mitosis (pink). Sections through (E) contralateral control and (F) dominant-negative Tbx5 (Tbx5{Delta}) injected limb buds at stage 20 stained with TUNEL reagents to identify cells undergoing apoptotic cell death (green). (G) Schematic representation of the Tbx5 full-length, truncated and En/VP16-fusion retroviral constructs (see Materials and Methods for details). (H) Transverse section in situ hybridisation analysis of chick Grg4 expression in the chick limb at stage 22.

 


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  		Fig. 5. RNA whole-mount in situ hybridisation analysis of limb buds injected with dominant-negative forms of Tbx5. (A) Dorsal view of the control, uninjected limb bud showing Fgf8, expressed in the AER, and Shh, expressed in the ZPA at stage 23. (B) Dorsal view of Tbx5{Delta}-injected limb. Fgf8 is not expressed in the anterior of the injected limb and Shh expression is not detected in the ZPA. In this example, the injection was carried out at stage 8 to achieve high-level infection. In A, the image of the contralateral uninjected control limb has been reversed to provide a clearer comparison with the respective experimental, injected limb in B. (C) Distal view of an uninjected control limb showing Fgf8 expression in the AER and Shh expression in the ZPA. (D) Distal view of the limb injected with Tbx5{Delta} virus at stage 10 and harvested at stage 23. Injection at the later stage produces lower-level infection and a weaker phenotype. Cells expressing Fgf8 are present in the anterior of the limb in a broader region of flattened ectoderm and have not formed a ridge of cells (arrow). (E) A transverse section through the anterior of the limb shown in C. The Fgf8-expressing cells (arrow) form a ridge of cells at the distal tip of the limb. (F) Transverse section through the anterior of the limb shown in D. The Fgf8-expressing cells (arrow) are present in the flattened ectoderm and do not form a ridge. The effect of Tbx5 misexpression is observed primarily in the anterior mesenchyme.

 


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  		Fig. 6. (A-F) Misexpression of dominant-negative Tbx5 leads to downregulation of anterior mesenchymal markers. (A) In the contralateral control, uninjected limb Msx is expressed in the distal mesenchyme of the limb just under the AER at stage 21. (B) In the dominant-negative Tbx5-(Tbx5en) injected limb, Msx is downregulated in the distal mesenchyme, most obviously in the anterior of the limb. (C) Lhx9 is expressed in the anterior limb mesenchyme in the control limb at stage 19 and (D) in the Tbx5en-injected limb, expression is downregulated (arrowed). (E) Hoxc4 is expressed in the anterior limb mesenchyme of a control limb (stage 20) and (F) is downregulated in the Tbx5en-injected limb. (G-J) Misexpression of dominant-active forms of Tbx5 (Tbx5vp16) leads to an anterior expansion of the limb. (G) Fgf8 expression in the uninjected control limb (stage 20). (H) After misexpression of Tbx5vp16, the domain of cells expressing Fgf8 in the AER is expanded anteriorly (arrow). (I) Expression of Lhx9 in the anterior of the control limb at stage 20. (J) After misexpression of Tbx5vp16, Lhx9 expression is expanded anteriorly (arrow). In A,C,E,G,I, the image of the contralateral uninjected control limb has been reversed to provide a clearer comparison with the respective experimental, injected limbs in B,D,F,H,J. A, anterior; P, posterior. (K) A model for Tbx5 function in the developing limb bud. Tbx5 is first expressed as limb bud initiation commences in response to axial cues. Tbx5 is acting genetically upstream of Fgf10, and is required for Fgf10 expression in the limb mesenchyme. Later, Tbx5 is also required for the positive inductive loop between Fgf8, which is expressed by cells in the AER, and Fgf10, which is expressed by cells of the distal mesenchyme.

 

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