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First published online March 7, 2008
doi: 10.1242/10.1242/dev.011759

Development 135, 1377-1388 (2008)
Published by The Company of Biologists 2008
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Regulation of Dlx5 and Dlx6 gene expression by p63 is involved in EEC and SHFM congenital limb defects

Nadia Lo Iacono1,2, Stefano Mantero1,3, Anna Chiarelli2, Elvin Garcia4, Alea A. Mills4, Maria I. Morasso5, Antonio Costanzo6, Giovanni Levi7, Luisa Guerrini2,*,{dagger} and Giorgio R. Merlo1,3,*,{dagger}

1 Dulbecco Telethon Institute, Molecular Biotechnology Center, University of Torino, Via Nizza 52, Torino, 10126, Italy.
2 Department of Biomolecular Science and Biotechnology, University of Milan, Via Celoria 26, 20133 Milan, Italy.
3 CNR Istituto Tecnologie Biomediche, Segrate Milano, Italy.
4 Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA.
5 Developmental Skin Biology Unit, NIAMS, NIH, Bethesda, MD, USA.
6 Department of Dermatology, University of Rome, TorVergata, Italy.
7 Evolution des Régulations Endocriniennes CNRS, UMR5166, Muséum National d'Histoire Naturelle, Paris, France.


Figure 1
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  		Fig. 1. p63 and Dlx proteins in the embryonic limbs. (A-D) Whole-mount in situ hybridization on wild-type FLs (A,B) and HLs (C,D) of E10 embryos for Dlx5 (A,C) and Dlx6 (B,D). (D') Hybridization for Dlx6 on wild-type HLs of E11 embryos. Lateral views are shown, the signal is indicated with black arrows. Anterior is to the top. (E-H) Double immunostaining for pan-Dlx (red) and p63 (green) on sections of E10.5 HLs. The merged image is shown in G. (H) Nuclei were counterstained with DAPI. Dashed line indicates the border between the AER and the limb mesenchyme. Section plane and orientation is indicated (I). (J,K) Double immunostaining for pan-Dlx and p63 on sections of the frontonasal region of E11 embryos, counterstained with DAPI. The olfactory placode is shown, dashed boxes indicate the neural and non-neural regions. Colocalization of p63 and Dlx proteins occurs in the non-neural epithelium. Section plane is indicated (L). Dor, dorsal; Ven, ventral; Di, distal; Pr, proximal; OP, olfactory placode. Scale bar: 25 µm.

 

Figure 2
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  		Fig. 2. Expression of p63 and Dlx genes in embryonic limbs. (A) Expression of TAp63 and {Delta}Np63 (left), and of Dlx1, Dlx2, Dlx5 and Dlx6 (right) in FLs (top) and HLs (bottom), collected at E10.5 (white bars), E11.5 (grey bars) and E12.5 (black bars). Data are normalized against GAPDH. The E10.5 abundance is set at 1. (B) RT-PCR semiquantitative analysis of each TAp63 ({alpha}, β, {gamma}; left) and {Delta}Np63 ({alpha}, β, {gamma}; right) isoform mRNAs in FLs and HLs at the indicated embryonic ages. For each mRNA, 29 or 38 amplification cycles are shown (indicated on the right). GAPDH mRNA is used as a reference. As a positive control (C+), the cloned cDNA of each isoform was used (right-most lanes). (C) Western blot analysis on total protein extracts from HaCaT cells (expressing {Delta}Np63{alpha}) and FLs collected at E10.5 and E12.5, showing the {Delta}Np63{alpha} protein (black arrowhead). Expression sharply increases between E10.5 and E12.5. β-actin is used for control.

 

Figure 3
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  		Fig. 3. Regulation of the Dlx5 and Dlx6 promoters by p63. (A) Left histograms: co-transfection of mDlx5luc and mDlx6luc reporters (300 ng) with 50 (white bars), 100 (grey bars) or 300 ng (black bars) of TAp63 and {Delta}Np63 expression plasmids. Data are reported as fold of activation relative to basal expression (reporter vector alone, set=1). Right histograms: mutated {Delta}Np63{alpha} proteins EEC R279H and C306R, and SHFM-IV K193E and K194E, show reduced activity on the Dlx5 and Dlx6 promoters. The AEC L518F mutation did not affect p63 activity. Standard deviation is indicated. (B) Expression of DLX1, DLX2, DLX5 and DLX6 analysed by RT-PCR in H1299 cells induced to express {Delta}Np63{alpha}. White bars, no induction; black bars, induced cells (20 µM Dox). p63 induction was confirmed by western blot. (C) p63 is bound in vivo to the Dlx5 and DLX6 promoters. Specific enrichment was detected in two regions of the Dlx5 promoter, R1 (-1200/-800) and R2 (-500/-100), and in one region of the DLX6 promoter, R2 (-500/-100). Input is shown on the left. Amplification of the I{kappa}Ka and of the {Delta}Np63 promoters is shown as a positive control. (D,E) Deletions of the mDlx5luc (D) and mDlx6luc (E) promoters. The position of the predicted p53 sites and the X5-R1, X5-R2, X6-R1 and X6-R2 regions are indicated. Luciferase activity is shown on the right.

 

Figure 4
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  		Fig. 4. Expression of Dlx genes in p63 mutant limbs. (A,A') Lateral view of p63+/EEC (A) and p63EEC/EEC (A') E11 embryos. Note the dysmorphology of both FLs and HLs in the homozygous embryo (black arrows). (B-E) Immunostaining for E-cadherin on sections of p63+/EEC (B,D) and p63EEC/EEC (C,E) E10.5 FL (B,C) and HL (D,E), to examine stratification of the AER (white arrows). Section plane as in Fig. 1. Scale bar: 25 µm. White arrows indicate the dorsal and ventral border of the AER. (F-K) Whole-mount in situ hybridization for Dlx5 on p63+/EEC (F,H,J) and p63EEC/EEC (G,I,K) E10 embryos, showing the pharyngeal arches (F,G), the FLs (H,I) and the HLs (J,K). Signal is reduced in the AER of p63EEC/EEC limbs (black arrows in H-K), but not in the p63EEC/EEC pharyngeal arches (white arrows in F,G). (L-O) Whole-mount in situ hybridization for Dlx5 on FLs (L,M) and HLs (N,O) of E9.5 p63+/- (L,N) and p63-/- (M,O) embryos, in lateral view. The signal on the pre-AER is indicated (black arrows). Anterior is to the right. (P-W) In situ hybridization for Dlx2 (P-S) and Dlx5 (T-W) on sections of WT, p63+/EEC; p63EEC/EEC and p63-/- E10.5 HLs. The AER is indicated with black arrows. Asterisks indicate absence of hybridization signal. (X-Z) Immunostaining for pan-Dlx on sections of wild-type, p63EEC/EEC and p63-/- E10.5 HLs.

 

Figure 5
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  		Fig. 5. Expression of p63 and Dactylyn in Dlx5;Dlx6 DKO limbs. (A,B) Morphology of E11 wild-type (A) and Dlx5;Dlx6 DKO (B) HLs. Section plane is shown in A. (C-F) Immunostaining for p63 on sections of normal (C,E) and Dlx5;Dlx6 DKO (D,F) FLs (top panels) and HLs (bottom panels) at E11. Nuclei were counterstained with DAPI. The AER is indicated with white arrows. The dorsoventral orientation is indicated. (G) Dissection of the medial (Me) sector from wild-type and Dlx5;Dlx6 DKO limbs. (H) TAp63, {Delta}Np63 and Dactylyn mRNA abundance in the Me sector of wild-type (white bars) and Dlx5;Dlx6 DKO (black bars) FLs and HLs, by RealTime qPCR. Wild-type samples are set=1. Scale bar: 25 µm.

 

Figure 6
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  		Fig. 6. Limb phenotypes of p63EEC/Dlx combined mutant mice. Skeletal staining of neonatal FL (A-K) and HL (L-V). Wild type is shown in A and L. For the p63+/EEC limbs, both normal (common phenotype, B,M) and mild ectrodactyly (rare phenotype, C,N) are shown. Dlx5;Dlx6 DKO limbs are shown in H,T. Note that the defects observed in p63+/EEC (rare) and Dlx5;Dlx6 DKO mice only affect the distal-most portion of the central phalanges. (F,G,R,S) FL and HL of Dlx5+/-;Dlx6+/- and Dlx5-/-;Dlx6+/- mice. (J,K,U,V) FL and HL of p63+/EEC;Dlx5-/-;Dlx6+/- mice. Note the severe HL defects: missing fingers (black asterisks), bent and fused distal phalanges (black arrows), and altered proximal elements (U,V). Note the defects in the FLs, observed in two out of three cases (K), whereas one was normal (J). FLs and HLs of p63EEC/EEC animals are also shown for comparison (D,E and P,Q). Digit numbers are indicated (A,L).

 

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© The Company of Biologists Ltd 2008