The apical ectodermal ridge of the mouse model of ectrodactyly Dlx5;Dlx6−/− shows altered stratification and cell polarity, which are restored by exogenous Wnt5a ligand

Human Molecular Genetics, 2016, Vol. 25, No. 4 740–754 doi: 10.1093/hmg/ddv514 Advance Access Publication Date: 18 December 2015 Original Article ORIGINAL ARTICLE The apical ectodermal ridge of the mouse model of Daniele Conte1, Giulia Garaffo1, Nadia Lo Iacono2, Stefano Mantero2, Stefano Piccolo3, Michelangelo Cordenonsi3, David Perez-Morga4, Valeria Orecchia1, Valeria Poli1 and Giorgio R. Merlo1,* 1 Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy, 2Human Genome Department, Istituto Tecnologie Biomediche, CNR Milano, Italy, 3Department of Molecular Medicine, University of Padova, Padova, Italy and 4Laboratoire de Parasitologie Moléculaire, IBMM-DBM, Université Libre de Bruxelles, B-6041 Gosselies, Belgium *To whom correspondence should be addressed at: Department of Molecular Biotechnology and Health Sciences, Via Nizza 52, 10126 Torino, Italy. Tel: +39 0116706449; Fax: +39 0116706432; Email: Abstract The congenital malformation split hand/foot (SHFM) is characterized by missing central fingers and dysmorphology or fusion of the remaining ones. Type-1 SHFM is linked to deletions/rearrangements of the DLX5–DLX6 locus and point mutations in the DLX5 gene. The ectrodactyly phenotype is reproduced in mice by the double knockout (DKO) of Dlx5 and Dlx6. During limb development, the apical ectodermal ridge (AER) is a key-signaling center responsible for early proximal–distal growth and patterning. In Dlx5;6 DKO hindlimbs, the central wedge of the AER loses multilayered organization and shows down-regulation of FGF8 and Dlx2. In search for the mechanism, we examined the non-canonical Wnt signaling, considering that Dwnt-5 is a target of distalless in Drosophila and the knockout of Wnt5, Ryk, Ror2 and Vangl2 in the mouse causes severe limb malformations. We found that in Dlx5;6 DKO limbs, the AER expresses lower levels of Wnt5a, shows scattered β-catenin responsive cells and altered basolateral and planar cell polarity (PCP). The addition of Wnt5a to cultured embryonic limbs restored the expression of AER markers and its stratification. Conversely, the inhibition of the PCP molecule c-jun N-terminal kinase caused a loss of AER marker expression. In vitro, the addition of Wnt5a on mixed primary cultures of embryonic ectoderm and mesenchyme was able to confer re-polarization. We conclude that the Dlx-related ectrodactyly defect is associated with the loss of basoapical and PCP, due to reduced Wnt5a expression and that the restoration of the Wnt5a level is sufficient to partially reverts AER misorganization and dysmorphology. Received: November 4, 2015. Revised and Accepted: December 10, 2015 © The Author 2015. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact 740 ectrodactyly Dlx5;Dlx6 −/− shows altered stratification and cell polarity, which are restored by exogenous Wnt5a ligand Human Molecular Genetics, 2016, Vol. 25, No. 4 Introduction cellular level the role of Wnt5a is not fully understood; recent findings indicate that it takes part in developmental processes such as oriented cell migration and division (32), establishment of PCP and convergent extension (CE). Since the AER forms via CE (33–35), we raised the hypothesis that a Wnt5a-activated PCP pathway is downstream of Dlx5 and Dlx6 and its perturbation causes altered AER organization and function, resulting in ectrodactyly. Notably, Wnt5a has been shown to be a transcriptional target of Dlx5 in the brain (36). In this work, we sought to identify novel targets of Dlx5, examine the function of Wnt5a and clarify the cellular pathogenesis of the AER misfunction in the Dlx5;6 DKO model of ectrodactyly. The results show that the Dlx-caused ectrodactyly is associated with the loss of Wnt5a expression in the central AER of limb buds, which results in altered basoapical and PCP. Exposure of Dlx5;6 DKO mutant limbs to exogenous Wnt5a rescues AER stratification and expression of specific markers. Results Histology of the AER in Dlx5;6 DKO limbs The central sector of the AER of Dlx5;6 DKO limbs, at early embryonic ages (E10.5–E11.5) has been previously shown to be defective in the expression of molecules such as Fgf8,Msx2 and Bmp4 (11,12,37). In addition, whole-mount in situ hybridization with a probe detecting the Dlx2 mRNA showed the same altered expression pattern (Fig. 1A). At slightly later ages (E12.5) a dysmorphology affecting the central sector of the Dlx5;6 DKO hindlimbs is clearly visible (Fig. 1B). Defective AER often shows altered stratification. Indeed we have previously reported a reduced stratification of the central wedge of the AER cells in Dlx5;6 DKO limbs (14). We also showed that p63, a marker of ectodermal stem/progenitor cells required for skin stratification (38), is still expressed in AER and ectoderm cells of Dlx5;6 mutant limbs, although at reduced level. Thus, we decided to further examine histological parameters of organization and integrity of the AER cells in the developing limbs. We focused on the embryonic ages E10.5–E11.5, e.g. prior to the onset of ectrodactyly and prior to overt changes in the expression of AER markers. We also examined the limbs at the age E12.5, when morphological and molecular defects first appear. Limbs were sectioned parallel to the Do–Ve plane (scheme in Fig. 1D). We initially immunostained for E-cadherin and ZO-1 to examine basoapical polarity. E-cadherin is a general epithelial marker present mostly at the basolateral membrane of epithelial cells, while ZO-1 stains the mostly apical lateral membrane. The AER of the Dlx5;6 DKO hindlimbs appeared normal in lateral sectors; however, in the central sector the AER appeared composed of a less stratified epithelium. E-cadherin staining showed a normal expression on the lateral cell borders, a reduction of the basal expression and the appearance of apical expression (Fig. 1C). ZO-1 staining revealed the converse alteration: its expression was reduced in the apical position and appeared in the basal layer, absent in the normal limb control (Fig. 1C). We confirmed the altered basoapical polarity defect by staining for GM130, which recognizes the Golgi organelle. This is generally located in a supra-nuclear position in epithelial cells. Combining immunostaining for GM130 and DAPI staining (for nuclei), we determined the overall orientation of AER cells relative to the Do–Ve and Pr–Di directions. In normal limbs we observed that nearly all the AER cells showed a basoapical orientation parallel to the Pr–Di orientation and parallel to the Do–Ve plane, with the GM130 fluorescent staining always apical with respect to the nuclei. In Ectrodactyly, or split-hand/foo (...truncated)