Mammalian-specific ectodermal enhancers control the expression of Hoxc genes in developing nails and hair follicles Marc Fernandez-Guerreroa, Nayuta Yakushiji-Kaminatsuib,1, Lucille Lopez-Delisleb, Sofía Zdrala, Fabrice Darbellayb,2, Rocío Perez-Gomeza, Christopher Chase Boltb, Manuel A. Sanchez-Martinc, Denis Dubouleb,d,e,3, and Marian A. Rosa,f,3 aInstituto de Biomedicina y Biotecnología de Cantabria, Consejo Superior de Investigaciones Científicas–Universidad de Cantabria–Sociedad para el Desarrollo de Cantabria, 39011 Santander, Spain; bSchool of Life Sciences, Federal Institute of Technology, Lausanne, 1015 Lausanne, Switzerland; cDepartment of Medicine, University of Salamanca, 37007 Salamanca, Spain; dDepartment of Genetics and Evolution, University of Geneva, 1211 Geneva 4, Switzerland; eCollège de France, Paris, France; and fDepartamento de Anatomía y Biología Celular, Facultad de Medicina, Universidad de Cantabria, 39011 Santander, Spain Contributed by Denis Duboule, October 6, 2020 (sent for review June 1, 2020; reviewed by Nadav Ahituv and Robert Hill) Vertebrate Hox genes are critical for the establishment of structures and thus to show less functional redundancy. Examples of this during the development of the main body axis. Subsequently, they situation may be found either in the function of some Hoxa genes play important roles either in organizing secondary axial structures in uterine physiology (9–11) or in the apparent specialization of such as the appendages, or during homeostasis in postnatal stages some Hoxc genes for epidermal derivatives (12, 13), while other and adulthood. Here, we set up to analyze their elusive function in clusters apparently do not play any detectable function there. the ectodermal compartment, using the mouse limb bud as a model. Indeed, while the single deletion of the HoxC gene cluster did We report that the HoxC gene cluster was co-opted to be transcribed not reveal any gross phenotype (14), several studies have sug- in the distal limb ectoderm, where it is activated following the rule of gested that Hoxc genes are involved in the development of ec- temporal colinearity. These ectodermal cells subsequently produce todermal organs (12, 15). The fact that such organs usually various keratinized organs such as nails or claws. Accordingly, dele- become fully developed after birth explains why the full HoxC DEVELOPMENTAL BIOLOGY HoxC tion of the cluster led to mice lacking nails (anonychia), a con- cluster deletion, which is lethal at birth because of respiratory dition stronger than the previously reported loss of function of problems (14), did not unmask these functional contributions. Hoxc13, which is the causative gene of the ectodermal dysplasia 9 However, both expression and inactivation studies have revealed (ECTD9) in human patients. We further identified two mammalian- potential contributions, in particular at late fetal stages as well as specific ectodermal enhancers located upstream of the HoxC gene during postnatal development and even adulthood (12, 16, 17). cluster, which together regulate Hoxc gene expression in the hair and nail ectodermal organs. Deletion of these regulatory elements alone or in combination revealed a strong quantitative component in Significance the regulation of Hoxc genes in the ectoderm, suggesting that these two enhancers may have evolved along with the mammalian taxon In this study, we report a unique and necessary function for the to provide the level of HOXC proteins necessary for the full devel- HoxC gene cluster in the development of some ectodermal opment of hair and nail. organs, as illustrated both by the hair and nail phenotype displayed by mice lacking the Hoxc13 function and by the Hox genes | enhancers | nails | hair follicles | transcription congenital anonychia (absence of nails) in full HoxC cluster mutants. We show that Hoxc genes are activated in a colinear n most bilateria, genes members of the Hox family of tran- manner in the embryonic limb ectoderm and are subsequently scription factors are important for the proper organization of transcribed in developing nails and hairs. We identify two I HoxC structures along the main body axis, during early development. mammalian-specific enhancers located upstream of the On top of this major task, vertebrate Hox genes are also neces- cluster, which display an exclusive ectodermal specificity. In- sary for the morphogenesis of a variety of secondary structures dividual or combined enhancer deletions suggest that they act in combination to raise the transcription level of several Hoxc such as the appendages or the external genitalia (1). In amniotes, genes during hair and nail development. Hox genes are organized in four clusters (HoxA, B, C, and D), all of them being involved in the development of the trunk axis. Author contributions: N.Y.-K., D.D., and M.A.R. designed research; M.F.-G., N.Y.-K., S.Z., Subsequently however, subgroups of gene clusters display some F.D., R.P.-G., M.A.S.-M., D.D., and M.A.R. performed research; C.C.B. contributed new specificities for particular tissues or structures. For example, reagents/analytic tools; M.F.-G., N.Y.-K., L.L.-D., D.D., and M.A.R. analyzed data; and both the HoxA and HoxD clusters are essential for the organi- L.L.-D., D.D., and M.A.R. wrote the paper. zation of the tetrapod limb morphology (2–7), while the deletion Reviewers: N.A., University of California San Francisco Medical Center; and R.H., The of both HoxA and HoxB clusters leads to severe cardiac mal- University of Edinburgh. formations, not detected in any of the single deletion mutants. The authors declare no competing interest. Likewise, deletions of both the HoxA and HoxC clusters induced This open access article is distributed under Creative Commons Attribution-NonCommercial- NoDerivatives License 4.0 (CC BY-NC-ND). a complete renal aplasia, which was not detected with either 1Present address: Laboratory for Developmental Genetics, RIKEN Center for Integrative deletion alone (8). Medical Sciences, Yokohama, Kanagawa 230-0045, Japan. Such a functional redundancy between Hox clusters can be 2Present address: Environmental Genomics and Systems Biology Division, Lawrence Ber- explained by their patterns of duplication during the evolution of keley Laboratories, Berkeley, CA 94720. vertebrates, with particular functional traits being conserved in 3To whom correspondence may be addressed. Email: [email protected] or rosm@ all or some gene clusters after duplication (8). In this context, unican.es. Hox gene function associated with recent vertebrate features, This article contains supporting information online at https://www.pnas.org/lookup/suppl/ i.e., features that must have appeared late, after the two rounds doi:10.1073/pnas.2011078117/-/DCSupplemental. of duplication, can be expected to involve single gene clusters First published November 16, 2020. www.pnas.org/cgi/doi/10.1073/pnas.2011078117 PNAS | December 1, 2020 | vol. 117 | no. 48 | 30509–30519 Downloaded by guest on September 29, 2021 In such instances, some Hoxc genes display complex patterns of good approximation of the progress zone (24–26). As a conse- expression in relation to the differentiation of epidermal organs quence, we microdissected a 150-μm-thick band of the distal limb such as the hairs and nails (12, 15, 18, 19), two ectodermal de- bud, and the mesoderm and ectoderm components were sepa- rivatives that form through epithelial–mesenchymal interactions rated by mild trypsin digestion and processed separately (SI between the surface ectoderm and the underlying mesoderm, the Appendix, Fig. S1A and Materials and Methods). Two biological latter being responsible for the development of site-specific skin replicates were collected per stage, and each pair of ectoderm derivatives (17, 20). and mesoderm replicates was obtained from the same batch The morphogenesis of both hairs and nails begins with the of embryos. induction of ectodermal thickenings or placodes by a subjacent Because a few mesoderm cells could occasionally remain at- dermal condensate (21). Once the placode forms, signaling tached to the ectoderm during dissection, potentially contami- events between the ectodermal and mesodermal components nating the ectodermal samples, we evaluated the purity of drive proliferation of the ectoderm to produce the hair peg or samples by analyzing the expression of genes known to be the nail matrix and eventually lead to a mature hair follicle (HF) expressed exclusively in the ectoderm or mesoderm components or nail organ. Both the morphogenetic steps and the signaling of limb buds. Fgf8, Sp6, Trp63, Perp, Krt14, Sp8, and Wnt7a were molecules involved are remarkably similar during the develop- selected as ectoderm specific genes, whereas Prrx1, Tbx5, Hoxa9, ment of these two integumentary structures (reviewed in refs. 22 Hoxd10, Fgf10, Pecam1, and Cdh5 were selected as mesoderm and 23), and there is evidence suggesting that Hoxc genes may specific genes (1, 27–31). A heatmap visualization of the tran- participate in these processes. This is mostly supported by the scription profiles of these genes across samples showed that the inactivation of Hoxc13, which induced specific hair and nail de- separation of the ectoderm and the mesoderm was clean, with a fects in adult homozygous mutant animals (12, 13). These mice negligible level of contamination between both limb components showed long and fragile nails; they lacked vibrissae at birth and (SI Appendix, Fig. S1B), except in replicate 1 of the E11.5 ec-