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. 2003 Nov 1;17(21):2630-5.
doi: 10.1101/gad.274103.

mBtd is required to maintain signaling during murine limb development

Dieter Treichel  1 , Frieder Schöck, Herbert Jäckle, Peter Gruss, Ahmed Mansouri
Affiliations

mBtd is required to maintain signaling during murine limb development

Dieter Treichel et al. Genes Dev. .

Abstract

buttonhead (btd) encodes an SP1-like transcription factor required for the generation and specification of Drosophila head segments. We identified a murine btd homolog, termed mouse Btd (mBtd), which can support btd-dependent head development in transgenic fly embryos. Functional studies show that mBtd-deficient mice develop to term and die at birth. They exhibit brain malformations, posterior axial skeleton truncations, and shortened limbs. We present evidence that mBtd is required during early limb development to maintain, but not to initiate Wnt/beta-catenin-dependent FGF, Shh, and BMP-mediated signaling. The data indicate that mBtd represents a novel key player mediating proximodistal outgrowth of the limb.

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Figures

Figure 1.
Figure 1.
Cloning of mBtd and rescue experiment in Drosophila. (A) Complete amino acid sequence of the mBtd protein. Functional domains are highlighted in different colors. (B) Schematic comparison of the protein structure of the BTD, D-SP1, mBTD, BTS1, SP1, and SP4 proteins. Domains shown are as follows: zinc finger (dark green); btd domain (light green); serine/threonine-rich (red); alaninerich (yellow); glycine-rich (blue); glutamine-rich (purple). (opa) Glutamine-rich opa-elements. (C-H) mBtd rescues btd-dependent aspects of Drosophila embryo head development. (C) Dorsal view of head segments in wild-type embryos visualized by anti-Engrailed antibody staining. The intercalary (ic), mandibular (md), and maxillary (mx) stripes are visible. (D) Antennomaxillary complex of a wild-type larva. Note the maxillary sense organ (mxso) of the maxillary segment, the dorsolateral papilla (dlp) of the intercalary segment, the antennal sense organ (anso) of the antennal origin, and the dorsomedial papilla (dmp) of the ocular (oc) segment. (E) btdXG81 mutant embryo lacking the antennal, intercalary, and mandibular segments (anti-Engrailed antibody staining). (F) In btdXG81 mutant larvae, corresponding sense organs are absent. (G) btdXG81 mutant embryos bearing one copy of the btd-mBtd transgene show rescue of mandibular and intercalary segments. (H) btdXG81 mutant larva showing the corresponding sensory organs. Anterior is to the left.
Figure 2.
Figure 2.
A highly restricted expression pattern of mBtd in mouse embryogenesis. Whole-mount in situ hybridization (A,B,C,D,E,H), LacZ staining (G), and vibratome sections of whole-mount staining (30 µm; F,I) presenting the expression of mBtd at different stages of development. mBtd is detected during gastrulation in the embryonic ectoderm and primitive streak (A). Subsequently, mBtd is found in the developing neural tube, in the prospective telencephalon, midbrain-hindbrain boundary, and spinal cord (B,D,G). Expression is also obvious in the otic vesicle, in the nasal placode, and tail bud (D). In the limb bud mBtd is shown at E9.5 in the entire ectoderm (C,F) and at E10.5, E10.75, and E11.0 in the AER (E,G,H,I) and ventral ectoderm (I). In C and H, anterior is to the left. In F, dorsal and anterior are to the top. In I, cross-section is at the level of the hindlimb. Arrows and arrowheads indicate expression domains. (D) Dorsal; (AER) apical ectodermal ridge; (bd) tail bud; (lb) limb bud; (MHB) midbrain-hindbrain boundary; (np) nasal placode; (ov) otic vesicle; (V) ventral.
Figure 3.
Figure 3.
Severe truncations of mBtd-deficient limbs. Skeletal preparations at E17.5 show the severity and variability of the defects observed in the mutant limbs. Control fore- and hindlimbs are shown in A and C, respectively. Several limb truncations are presented for the fore- and hindlimbs in B and D. Some of these defects are shown, and the frequency, how often these or similar anomalies are observed. For the forelimbs: (I) 30/51; (II) 8/51; (III) 12/51; (IV) 1. For the hindlimbs (I) 20/51; (II) 7/51; (III) 24/51. The numbers label individual limb variations. Whole skeletal preparations (E17.5) of control (J) and mutant (K) embryos showing the malformations in the trunk. The expression of Fgf8 in the AER is shown at E10.5 for control (E) and mutant (F,G) whole-mount embryos. It is obvious that at this stage of development, Fgf8 expression is already abolished in the forelimb (F, five embryos tested), whereas a patchy expression domain is still recognizable in the hindlimb (G, five embryos). H and I represent scanning electron microscope of whole embryos at E12.5 from wild-type (H) and mBtd-/- embryos. The arrows in G point to the patchy expression in the AER of the hindlimbs. (f) Femur; (fi) fibula; (fl) forelimb; (hl) hindlimb; (h) humerus; (r) radius; (sc) scapula; (t) tibia; (u) ulna.
Figure 4.
Figure 4.
Analysis of the limb phenotype with molecular markers. Whole-mount in situ hybridization at E9.5 or E10.5 showing the expression pattern of molecular markers involved in limb patterning. (I,J) FGF8 expression is detected in the forelimb of E9.5 mBtd-/- embryos, as compared with E10.5 in Figure 3F. (K,L) The expression of Fgf4 is not initiated (10 embryos tested). (G,H) The ectodermal Wnt3 expression is not altered by the loss of mBtd activity (five embryos tested). (A,B) The expression domain of Lmx1b in the forelimb is extended into the ventral portion of the bud (arrows; seven embryos tested), pointing to a progressive dorsalization of the limb. (C,D) In the same embryo, the younger hindlimb still exhibits a normal dorsoventral expression pattern. Initial expression of En1 at E9.5 is normal. (E,F) The expression of Gli3 is not maintained in mBtd-/- limbs. Except for A, B, and J, rostral is to the left. Arrows indicate the expression domains in the limb.
Figure 5.
Figure 5.
Expression of molecular markers and cell death analysis in the mBtd mutant limbs. (A,B,E,F,I,J) Sonic Hedgehog, BMP, and FGF signaling is not maintained in m-Btd mutant limbs shown in whole-mount in situ at E10.5. Two other markers are also affected: Gbx2 (C,D) and Hoxd12 (G,H). In A-J, anterior is to the left. Apoptotic cell death was assayed on cross-sections of E10.5 embryos at the level of the forelimb bud by using TUNEL. (K,M) Cell death in the forelimb bud of E10.5 control and mutant embryos is shown. (M) Apoptosis is detected in the mesenchyme and ectoderm of the mBtd-deficient forelimb bud at E10.5. (K) There is little or no apoptosis in the control forelimb bud. L and N indicate the corresponding DAPI staining to K and M, respectively. In K-N, dorsal is to the left.
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