Limb development - SHFM
Dlx genes are expressed on the tip of growing appendages of all animal species, including the mammalian limb buds. In human, the DLX5-DLX6 cluster is deleted or translocated in the congenital malformation SHFM type I. We previously published that the combined Dlx5+Dlx6 knockout mice reproduce the SHFM phenotype, indicating that the human orthologs DLX5 and DLX6 are the disease genes for SHFM-I. This finding has opened the way to comprehend the molecular basis of Dlx gene functions for limb development.
We aim to identify the upstream and downstream regulations centered on the Dlx5-Dlx6 locus, relevant for the SHFM malformation. In the past year, we focused on the p63 transcription factor, essential for epithelial stem cell functions and for the maintenance of stratified epithelia. p63 mutations in human cause the congenital disorders SHFM type IV and EEC. In cooperation with Dr. Guerrini (Univ. of Milano) we have collected evidence that p63 controls expression of Dlx5-Dlx6, a regulation needed for normal limb development.

Another line of investigation concerns possible regulations downstream of Dlx5-Dlx6. My group has collected evidence that Wnt5a and Msx2 are downregulated in the mutant limbs, and this regulation might be direct. We are pursuing this line of experiments in cooperation with Dr. Giovanni Levi (CNRS; Paris) and Dr. Benoit Robert (Pasteur Inst. Paris), which include cross-breeding of Dlx and Msx mutant mice and examine limb phenotypes and molecular alterations.
We believe that the SHFM and EEC animal models offer a unique opportunity to optimize procedures for LV-mediated gene transfer to the embryonic limb (the AER) and skin (epithelial progenitor cells), first in ex-vivo limb cultures and then in vivo. If successful, the congenital defects of SHFM and EEC could in principle be corrected in-utero.

Differentiation of Interneurons in the Basal Brain
Dlx genes are expressed in a temporal and spatial sequence in neurogenic area of the embryonic and adult forebrain (peri-ventricular region, hippocampus) and in most of the regions where GABA+ differentiation takes place, including migrating interneuron progenitors. In 2003 my group has reported that differentiation of GABA+, TH+ and CR+ interneurons is reduced in the Dlx5-null basal brain. We have then examined this defect using the neurosphere method to expand neural stem cells; the results showed that Dlx5-null NSC have a reduced neuronal differentiation potential, thus Dlx5 may control critical steps necessary for differentiation of interneuron progenitors.

We aim to clarify a molecular signature for GABA+ differentiation in vivo and in vitro, and to comprehend the role of Dlx genes in this process. Transcription profiling done in cooperation with Dr Corte (IST Genova) on Dlx5-null neurospheres has led us to recognize the secreted signaling molecule Wnt5a as a candidate for a Dlx5 target, capable of promoting interneuron differentiation. In collaboration with Drs. S. De Marchis / D. Garzotto (Univ. of Torino) and Drs. E. Cattaneo / L. Conti (Univ. of Milano) we are pursuing the functional validation of this regulation, using both primary culture of dissociated brain cells and adherent neural progenitor cells that recapitulates Dlx regulation and GABA+ interneuron differentiation, in vitro.

Axon Extension and Connectivity in the Olfactory System
During embryonic development, immature olfactory neurons extend their axons to target the anterior forebrain, amd establish the primitive connections; at the same time GnRH+ migratory neurons use these axons to reach the septum-hypothalamus area. This process is specifically impaired in the congenital disease known as Kallmann Syndrome, characterized by the inability to perceive odors and defective GnRH hormonal system. The molecules involved in guidance and connectivity of olfactory axons are not well known.

We have recently identified a Wnt-responsive cell population on the surface of the OB, involved in olfactory connectivity. Using Wnt-β-catenin reporter mice and organotypic cultures of the embryonic nasal region, we have established that Wnt-β-catenin activation in these cells requires axons to approach the OB. When the canonical Wnt signaling was exogenously blocked, the ORN axons failed to contact the FB and were often misoriented, suggesting that activation of this pathway on the surface of the OB is required for connectivity.

In the absence of Dlx5 the olfactory receptor neurons (ORN) differentiate normally, their axons elongate but fail to make contact with the olfactory bulbs of the forebrain. Thus, Dlx5 controls early steps in the guidance/connectivity of the olfactory axons. We also demonstrate that Dlx5 is expressed in migrating GnRH+ cells in the embryo. We have initiated a line of investigation on the role of Dlx5 for olfactory development, by combining transcription-profiling, analysis of patterns of conserved co-expression, computational whole-genome scanning for Dlx binding sites and organ-type cultures of the olfactory neuroepithelium.
Added value in elucidating this embryonic process resides on the peculiarity of the adult peripheral olfactory system to renew its neural component : progenitor cells give rise to new neurons, these extend axons to the OB to re-establish connections and topography. A plan is proposed to transfer the embryonic knowledge onto the adult system. On the long term, investigations on the regulatory network used for olfactory connectivity may have more general implications for the biology of nerve regeneration and repair.

Levi, G., Mantero, S., Barbieri, O., Cantatore, D., Paleari, L., Beverdam, A., Genova, F., Robert, B. and Merlo, G.R. (2006). Msx1 and Dlx5 act independently in development of craniofacial skeleton, but converge on the regulation of Bmp signaling in palate formation. Mech. Develop. 123, 3-16.

Meneghini, V., Odent, S., Platonova, N., Egeo, A., and Merlo, G.R. (2006). Novel TBX3 mutation data in families with Ulnar-Mammary syndrome indicate a genotype-phenotype relationship: mutations that do not disrupt the T-domain are associated with less severe limb defects. Eur. J. Med. Genet.  49: 151-158.

Zaghetto, A.A., Paina, S., Mantero, S., Peretto, P., Bovetti, S., Puche, A., Piccolo, S., and Merlo, G. (2006). A Wnt-beta-catenin responsive cell population that participates in the formation of olfactory axon connections in the mouse embryo. Int. J. Dev. Neurosci. 24, 584-585.

Radoja, N., Guerrini, L., LoIacono, N., Merlo, G.R., Costanzo, A., Weinberg, W.C., LaMantia, G., Calabrò, V., and Morasso, M.I. (2007). Homeobox gene Dlx3 is regulated by p63 during ectoderm development: relevance in the pathogenesis of ectodermal dysplasias. Development 134, 13-18.

Platonova, N., Scotti, M., Babich, P., Bertoli, G, Mento, E., Meneghini, V., Egeo, A., Zucchi, I., and Merlo, G.R. (2007). The TBX3 gene, mutated in Ulnar-Mammary syndrome, promotes growth of mammary epithelial cells independently of ARF and p53. Cell Tissue Res. 328, 301-316.

Vieux-Rochas, M., Coen, L., Sato, T., Kurihara, Y., Gitton, Y., Barbieri, O., Le Blay, K., Merlo, G.R., Ekker, M., Kurihara, H., Janvier, P., and Levi, G. (2007). Molecular dynamics of retinoic acid-induced craniofacial malformations: implications for the origin of the gnatostome jaw. PLoS ONE 2, e510.

Merlo, G.R., Mantero, S., Zaghetto, A.A., Peretto, P., Paina, S.. and Gozzo, M. (2007). The role of Dlx homeogenes in early development of the olfactory pathway. J. Mol. Histol. (special issue) 38(6), 612-623.

Zaghetto, A.A., Paina, S., Mantero, S., Platonova, N., Peretto, P., Bovetti, S., Puche, A.C., Piccolo, S., and Merlo, G.R. (2007). Activation of the Wnt-bcatenin pathway in a cell population on the surface of the forebrain is essential for the establishment of olfactory axon connections. J. Neurosci. 27, 9757-9768.

LoIacono, N., Mantero, S., Chiarelli, A., Garcia, E., Mills, A.A., Morasso, M.I., Costanzo, A., Levi, G., Guerrini, L. and Merlo, G.R. (2008). Regulation of Dlx5 and Dlx6 gene expression by p63 is involved in EEC and SHFM congenital limb defects. Development 135, 1377-1388.

Lopardo, T., LoIacono, N., Marinari, B., Giustizieri, M.L., Cyr, D.G., Merlo, G., Crosti, F., Costanzo, A., and Guerrini, L. (2008). Claudin-1 is a p63 target gene with a crucial role in epithelial development. PLoS One, 3(7), e2715.

Moretti F., Marinari B., LoIacono N., Botti E., Giunta A., Spallone G., Garaffo G., Vernersson-Lindhal E., Merlo G.R., Mills A.A., Ballarò C., Alemà S., Chimenti S., Guerrini L. and Costanzo A. (2010) A regulatory feed-back loop involving p63 and IRF6 links the pathogenesis of two genetically different ectodermal dysplasias. J. Clin. Invest., in press.

Vieux-Rochas M., Mantero S., Heude E., Barbieri O., Astigiano S., Couly G., Kurihara H., Levi G. and Merlo G.R. (2010) Spatio-temporal dynamics of gene expression of the Edn1-Dlx5/6 pathway during developoment of the lower jaw. Genesis, in press.

Paina S., Garzotto D., DeMarchis S., Moiana, A., Cattaneo, E., Conti L., Perera M., Corte, G., Calautti E. and Merlo G.R. (2010) Wnt5a is a transcriptional target of Dlx genes and promotes differentiation of olfactory interneuron progenitors.  J. Neurosci. submitted.