Neuron-Derived Neurotrophic Factor Is Mutated in Congenital Hypogonadotropic Hypogonadism.

Congenital hypogonadotropic hypogonadism (CHH) is a rare genetic disorder characterized by infertility and the absence of puberty. Defects in GnRH neuron migration or altered GnRH secretion and/or action lead to a severe gonadotropin-releasing hormone (GnRH) deficiency. Given the close developmental association of GnRH neurons with the olfactory primary axons, CHH is often associated with anosmia or hyposmia, in which case it is defined as Kallmann syndrome (KS). The genetics of CHH are heterogeneous, and >40 genes are involved either alone or in combination. Several CHH-related genes controlling GnRH ontogeny encode proteins containing fibronectin-3 (FN3) domains, which are important for brain and neural development. Therefore, we hypothesized that defects in other FN3-superfamily genes would underlie CHH. Next-generation sequencing was performed for 240 CHH unrelated probands and filtered for rare, protein-truncating variants (PTVs) in FN3-superfamily genes. Compared to gnomAD controls the CHH cohort was statistically enriched for PTVs in neuron-derived neurotrophic factor (NDNF) (p = 1.40 × 10-6). Three heterozygous PTVs (p.Lys62∗, p.Tyr128Thrfs∗55, and p.Trp469∗, all absent from the gnomAD database) and an additional heterozygous missense mutation (p.Thr201Ser) were found in four KS probands. Notably, NDNF is expressed along the GnRH neuron migratory route in both mouse embryos and human fetuses and enhances GnRH neuron migration. Further, knock down of the zebrafish ortholog of NDNF resulted in altered GnRH migration. Finally, mice lacking Ndnf showed delayed GnRH neuron migration and altered olfactory axonal projections to the olfactory bulb; both results are consistent with a role of NDNF in GnRH neuron development. Altogether, our results highlight NDNF as a gene involved in the GnRH neuron migration implicated in KS.

Effective high-throughput isolation of enriched platelets and circulating pro-angiogenic cells to accelerate skin-wound healing.

Delayed wound healing and chronic skin lesions represent a major health problem. Over the past years, growth factors mediated by platelet-rich plasma (PRP) and cell-based therapies were developed as effective and affordable treatment able to improve wound healing capacity. We have advanced existing concepts to develop a highly efficient high-throughput protocol with proven application for the isolation of PRP and pro-angiogenic cells (AngioPRP). This protocol outlines the effectiveness of AngioPRP in promoting the critical healing process including wound closure, re-epithelialization, granulation tissue growth, and blood vessel regeneration. We coupled this effect with normalization of mechanical properties of rescued mouse wounds, which is sustained by a correct arrangement of elastin and collagen fibers. Proteomic analysis of treated wounds demonstrated a fingerprint of AngioPRP based on the up-regulation of detoxification pathway of glutathione metabolism, correlated to a decrease in inflammatory response. Overall, these results have enabled us to provide a framework for how AngioPRP supports wound healing, opening avenues for further clinical advances.

The Emerging Roles of Long Non-Coding RNAs in Intellectual Disability and Related Neurodevelopmental Disorders.

In the human brain, long non-coding RNAs (lncRNAs) are widely expressed in an exquisitely temporally and spatially regulated manner, thus suggesting their contribution to normal brain development and their probable involvement in the molecular pathology of neurodevelopmental disorders (NDD). Bypassing the classic protein-centric conception of disease mechanisms, some studies have been conducted to identify and characterize the putative roles of non-coding sequences in the genetic pathogenesis and diagnosis of complex diseases. However, their involvement in NDD, and more specifically in intellectual disability (ID), is still poorly documented and only a few genomic alterations affecting the lncRNAs function and/or expression have been causally linked to the disease endophenotype. Considering that a significant fraction of patients still lacks a genetic or molecular explanation, we expect that a deeper investigation of the non-coding genome will unravel novel pathogenic mechanisms, opening new translational opportunities. Here, we present evidence of the possible involvement of many lncRNAs in the etiology of different forms of ID and NDD, grouping the candidate disease-genes in the most frequently affected cellular processes in which ID-risk genes were previously collected. We also illustrate new approaches for the identification and prioritization of NDD-risk lncRNAs, together with the current strategies to exploit them in diagnosis.

Neuronal Cytoskeleton in Intellectual Disability: From Systems Biology and Modeling to Therapeutic Opportunities.

Intellectual disability (ID) is a pathological condition characterized by limited intellectual functioning and adaptive behaviors. It affects 1-3% of the worldwide population, and no pharmacological therapies are currently available. More than 1000 genes have been found mutated in ID patients pointing out that, despite the common phenotype, the genetic bases are highly heterogeneous and apparently unrelated. Bibliomic analysis reveals that ID genes converge onto a few biological modules, including cytoskeleton dynamics, whose regulation depends on Rho GTPases transduction. Genetic variants exert their effects at different levels in a hierarchical arrangement, starting from the molecular level and moving toward higher levels of organization, i.e., cell compartment and functions, circuits, cognition, and behavior. Thus, cytoskeleton alterations that have an impact on cell processes such as neuronal migration, neuritogenesis, and synaptic plasticity rebound on the overall establishment of an effective network and consequently on the cognitive phenotype. Systems biology (SB) approaches are more focused on the overall interconnected network rather than on individual genes, thus encouraging the design of therapies that aim to correct common dysregulated biological processes. This review summarizes current knowledge about cytoskeleton control in neurons and its relevance for the ID pathogenesis, exploiting in silico modeling and translating the implications of those findings into biomedical research.

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.

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 ß-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.

Rho GTPases in Intellectual Disability: From Genetics to Therapeutic Opportunities.

Rho-class small GTPases are implicated in basic cellular processes at nearly all brain developmental steps, from neurogenesis and migration to axon guidance and synaptic plasticity. GTPases are key signal transducing enzymes that link extracellular cues to the neuronal responses required for the construction of neuronal networks, as well as for synaptic function and plasticity. Rho GTPases are highly regulated by a complex set of activating (GEFs) and inactivating (GAPs) partners, via protein:protein interactions (PPI). Misregulated RhoA, Rac1/Rac3 and cdc42 activity has been linked with intellectual disability (ID) and other neurodevelopmental conditions that comprise ID. All genetic evidences indicate that in these disorders the RhoA pathway is hyperactive while the Rac1 and cdc42 pathways are consistently hypoactive. Adopting cultured neurons for in vitro testing and specific animal models of ID for in vivo examination, the endophenotypes associated with these conditions are emerging and include altered neuronal networking, unbalanced excitation/inhibition and altered synaptic activity and plasticity. As we approach a clearer definition of these phenotype(s) and the role of hyper- and hypo-active GTPases in the construction of neuronal networks, there is an increasing possibility that selective inhibitors and activators might be designed via PPI, or identified by screening, that counteract the misregulation of small GTPases and result in alleviation of the cognitive condition. Here we review all knowledge in support of this possibility.

Phosphoinositide 3-Kinase-C2α Regulates Polycystin-2 Ciliary Entry and Protects against Kidney Cyst Formation.

Signaling from the primary cilium regulates kidney tubule development and cyst formation. However, the mechanism controlling targeting of ciliary components necessary for cilium morphogenesis and signaling is largely unknown. Here, we studied the function of class II phosphoinositide 3-kinase-C2α (PI3K-C2α) in renal tubule-derived inner medullary collecting duct 3 cells and show that PI3K-C2α resides at the recycling endosome compartment in proximity to the primary cilium base. In this subcellular location, PI3K-C2α controlled the activation of Rab8, a key mediator of cargo protein targeting to the primary cilium. Consistently, partial reduction of PI3K-C2α was sufficient to impair elongation of the cilium and the ciliary transport of polycystin-2, as well as to alter proliferation signals linked to polycystin activity. In agreement, heterozygous deletion of PI3K-C2α in mice induced cilium elongation defects in kidney tubules and predisposed animals to cyst development, either in genetic models of polycystin-1/2 reduction or in response to ischemia/reperfusion-induced renal damage. These results indicate that PI3K-C2α is required for the transport of ciliary components such as polycystin-2, and partial loss of this enzyme is sufficient to exacerbate the pathogenesis of cystic kidney disease.

The Dlx5 and Foxg1 transcription factors, linked via miRNA-9 and -200, are required for the development of the olfactory and GnRH system.

During neuronal development and maturation, microRNAs (miRs) play diverse functions ranging from early patterning, proliferation and commitment to differentiation, survival, homeostasis, activity and plasticity of more mature and adult neurons. The role of miRs in the differentiation of olfactory receptor neurons (ORNs) is emerging from the conditional inactivation of Dicer in immature ORN, and the depletion of all mature miRs in this system. Here, we identify specific miRs involved in olfactory development, by focusing on mice null for Dlx5, a homeogene essential for both ORN differentiation and axon guidance and connectivity. Analysis of miR expression in Dlx5(-/-) olfactory epithelium pointed to reduced levels of miR-9, miR-376a and four miRs of the -200 class in the absence of Dlx5. To functionally examine the role of these miRs, we depleted miR-9 and miR-200 class in reporter zebrafish embryos and observed delayed ORN differentiation, altered axonal trajectory/targeting, and altered genesis and position of olfactory-associated GnRH neurons, i.e. a phenotype known as Kallmann syndrome in humans. miR-9 and miR-200-class negatively control Foxg1 mRNA, a fork-head transcription factor essential for development of the olfactory epithelium and of the forebrain, known to maintain progenitors in a stem state. Increased levels of z-foxg1 mRNA resulted in delayed ORN differentiation and altered axon trajectory, in zebrafish embryos. This work describes for the first time the role of specific miR (-9 and -200) in olfactory/GnRH development, and uncovers a Dlx5-Foxg1 regulation whose alteration affects receptor neuron differentiation, axonal targeting, GnRH neuron development, the hallmarks of the Kallmann syndrome.

Wnt5a is a transcriptional target of Dlx homeogenes and promotes differentiation of interneuron progenitors in vitro and in vivo.

During brain development, neurogenesis, migration, and differentiation of neural progenitor cells are regulated by an interplay between intrinsic genetic programs and extrinsic cues. The Dlx homeogene transcription factors have been proposed to directly control the genesis and maturation of GABAergic interneurons of the olfactory bulb (OB), subpallium, and cortex. Here we provide evidence that Dlx genes promote differentiation of olfactory interneurons via the signaling molecule Wnt5a. Dlx2 and Dlx5 interact with homeodomain binding sequences within the Wnt5a locus and activate its transcription. Exogenously provided Wnt5a promotes GABAergic differentiation in dissociated OB neurons and in organ-type brain cultures. Finally, we show that the Dlx-mutant environment is unfavorable for GABA differentiation, in vivo and in vitro. We conclude that Dlx genes favor interneuron differentiation also in a non-cell-autonomous fashion, via expression of Wnt5a.

p140Cap Controls Female Fertility in Mice Acting via Glutamatergic Afference on Hypothalamic Gonadotropin-Releasing Hormone Neurons.

p140Cap, encoded by the gene SRCIN1 (SRC kinase signaling inhibitor 1), is an adaptor/scaffold protein highly expressed in the mouse brain, participating in several pre- and post-synaptic mechanisms. p140Cap knock-out (KO) female mice show severe hypofertility, delayed puberty onset, altered estrus cycle, reduced ovulation, and defective production of luteinizing hormone and estradiol during proestrus. We investigated the role of p140Cap in the development and maturation of the hypothalamic gonadotropic system. During embryonic development, migration of Gonadotropin-Releasing Hormone (GnRH) neurons from the nasal placode to the forebrain in p140Cap KO mice appeared normal, and young p140Cap KO animals showed a normal number of GnRH-immunoreactive (-ir) neurons. In contrast, adult p140Cap KO mice showed a significant loss of GnRH-ir neurons and a decreased density of GnRH-ir projections in the median eminence, accompanied by reduced levels of GnRH and LH mRNAs in the hypothalamus and pituitary gland, respectively. We examined the number of kisspeptin (KP) neurons in the rostral periventricular region of the third ventricle, the number of KP-ir fibers in the arcuate nucleus, and the number of KP-ir punctae on GnRH neurons but we found no significant changes. Consistently, the responsiveness to exogenous KP in vivo was unchanged, excluding a cell-autonomous defect on the GnRH neurons at the level of KP receptor or its signal transduction. Since glutamatergic signaling in the hypothalamus is critical for both puberty onset and modulation of GnRH secretion, we examined the density of glutamatergic synapses in p140Cap KO mice and observed a significant reduction in the density of VGLUT-ir punctae both in the preoptic area and on GnRH neurons. Our data suggest that the glutamatergic circuitry in the hypothalamus is altered in the absence of p140Cap and is required for female fertility.

Loss of ARHGAP15 affects the directional control of migrating interneurons in the embryonic cortex and increases susceptibility to epilepsy.

GTPases of the Rho family are components of signaling pathways linking extracellular signals to the control of cytoskeleton dynamics. Among these, RAC1 plays key roles during brain development, ranging from neuronal migration to neuritogenesis, synaptogenesis, and plasticity. RAC1 activity is positively and negatively controlled by guanine nucleotide exchange factors (GEFs), guanosine nucleotide dissociation inhibitors (GDIs), and GTPase-activating proteins (GAPs), but the specific role of each regulator in vivo is poorly known. ARHGAP15 is a RAC1-specific GAP expressed during development in a fraction of migrating cortical interneurons (CINs) and in the majority of adult CINs. During development, loss of ARHGAP15 causes altered directionality of the leading process of tangentially migrating CINs, along with altered morphology in vitro. Likewise, time-lapse imaging of embryonic CINs revealed a poorly coordinated directional control during radial migration, possibly due to a hyper-exploratory behavior. In the adult cortex, the observed defects lead to subtle alteration in the distribution of CALB2-, SST-, and VIP-positive interneurons. Adult Arhgap15-knock-out mice also show reduced CINs intrinsic excitability, spontaneous subclinical seizures, and increased susceptibility to the pro-epileptic drug pilocarpine. These results indicate that ARHGAP15 imposes a fine negative regulation on RAC1 that is required for morphological maturation and directional control during CIN migration, with consequences on their laminar distribution and inhibitory function.

A symphony of regulations centered on p63 to control development of ectoderm-derived structures.

The p53-related transcription factor p63 is critically important for basic cellular functions during development of the ectoderm and derived structure and tissues, including skin, limb, palate, and hair. On the one side, p63 is required to sustain the proliferation of keratinocyte progenitors, while on the other side it is required for cell stratification, commitment to differentiate, cell adhesion, and epithelial-mesenchymal signaling. Molecules that are components or regulators of the p63 pathway(s) are rapidly being identified, and it comes with no surprise that alterations in the p63 pathway lead to congenital conditions in which the skin and other ectoderm-derived structures are affected. In this paper, we summarize the current knowledge of the molecular and cellular regulations centered on p63, derived from the comprehension of p63-linked human diseases and the corresponding animal models, as well as from cellular models and high-throughput molecular approaches. We point out common themes and features, that allow to speculate on the possible role of p63 downstream events and their potential exploitation in future attempts to correct the congenital defect in preclinical studies.

Treatment with ROS detoxifying gold quantum clusters alleviates the functional decline in a mouse model of Friedreich ataxia.

Friedreich ataxia (FRDA) is caused by the reduced expression of the mitochondrial protein frataxin (FXN) due to an intronic GAA trinucleotide repeat expansion in the FXN gene. Although FRDA has no cure and few treatment options, there is research dedicated to finding an agent that can curb disease progression and address symptoms as neurobehavioral deficits, muscle endurance, and heart contractile dysfunctions. Because oxidative stress and mitochondrial dysfunctions are implicated in FRDA, we demonstrated the systemic delivery of catalysts activity of gold cluster superstructures (Au8-pXs) to improve cell response to mitochondrial reactive oxygen species and thereby alleviate FRDA-related pathology in mesenchymal stem cells from patients with FRDA. We also found that systemic injection of Au8-pXs ameliorated motor function and cardiac contractility of YG8sR mouse model that recapitulates the FRDA phenotype. These effects were associated to long-term improvement of mitochondrial functions and antioxidant cell responses. We related these events to an increased expression of frataxin, which was sustained by reduced autophagy. Overall, these results encourage further optimization of Au8-pXs in experimental clinical strategies for the treatment of FRDA.

Spatio-temporal dynamics of gene expression of the Edn1-Dlx5/6 pathway during development of the lower jaw.

The morphogenesis of the vertebrate skull results from highly dynamic integrated processes involving the exchange of signals between the ectoderm, the endoderm, and cephalic neural crest cells (CNCCs). Before migration CNCCs are not committed to form any specific skull element, molecular signals exchanged in restricted regions of tissue interaction are crucial in providing positional identity to the CNCCs mesenchyme and activate the specific morphogenetic process of different skeletal components of the head. In particular, the endothelin-1 (Edn1)-dependent activation of Dlx5 and Dlx6 in CNCCs that colonize the first pharyngeal arch (PA1) is necessary and sufficient to specify maxillo-mandibular identity. Here, to better analyze the spatio-temporal dynamics of this process, we associate quantitative gene expression analysis with detailed examination of skeletal phenotypes resulting from combined allelic reduction of Edn1, Dlx5, and Dlx6. We show that Edn1-dependent and -independent regulatory pathways act at different developmental times in distinct regions of PA1. The Edn1-->Dlx5/6-->Hand2 pathway is already active at E9.5 during early stages of CNCCs colonization. At later stages (E10.5) the scenario is more complex: we propose a model in which PA1 is subdivided into four adjacent territories in which distinct regulations are taking place. This new developmental model may provide a conceptual framework to interpret the craniofacial malformations present in several mouse mutants and in human first arch syndromes. More in general, our findings emphasize the importance of quantitative gene expression in the fine control of morphogenetic events.

Activation of the Wnt-beta catenin pathway in a cell population on the surface of the forebrain is essential for the establishment of olfactory axon connections.

A variety of signals governing early extension, guidance, and connectivity of olfactory receptor neuron (ORN) axons has been identified; however, little is known about axon-mesoderm and forebrain (FB)-mesoderm signals. Using Wnt-beta catenin reporter mice, we identify a novel Wnt-responsive resident cell population, located in a Frizzled7 expression domain at the surface of the embryonic FB, along the trajectory of incoming ORN axons. Organotypic slice cultures that recapitulate olfactory-associated Wnt-beta catenin activation show that the beta catenin response depends on a placode-derived signal(s). Likewise, in Dlx5-/- embryos, in which the primary connections fail to form, Wnt-beta catenin response on the surface of the FB is strongly reduced. The olfactory placode expresses a number of beta catenin-activating Wnt genes, and the Frizzled7 receptor transduces the "canonical" Wnt signal; using Wnt expression plasmids we show that Wnt5a and Wnt7b are sufficient to rescue beta catenin activation in the absence of incoming axons. Finally, blocking the canonical Wnt pathway with the exogenous application of the antagonists Dikkopf-1 or secreted-Frizzled-receptor protein-2 prevents ORN axon contact to the FB. These data reveal a novel function for Wnt signaling in the establishment of periphery-CNS olfactory connections and highlight a complex interplay between cells of different embryonic origin for ORN axon connectivity.

Choice of Alternative Polyadenylation Sites, Mediated by the RNA-Binding Protein Elavl3, Plays a Role in Differentiation of Inhibitory Neuronal Progenitors.

Alternative polyadenylation (APA) is a widespread mechanism involving about half of the expressed genes, resulting in varying lengths of the 3' untranslated region (3'UTR). Variations in length and sequence of the 3'UTR may underlie changes of post-transcriptional processing, localization, miRNA targeting and stability of mRNAs. During embryonic development a large array of mRNAs exhibit APA, with a prevalence of the longer 3'UTR versions in differentiating cells. Little is known about polyA+ site usage during differentiation of mammalian neural progenitors. Here we exploit a model of adherent neural stem (ANS) cells, which homogeneously and efficiently differentiate into GABAergic neurons. RNAseq data shows a global trend towards lengthening of the 3'UTRs during differentiation. Enriched expression of the longer 3'UTR variants of Pes1 and Gng2 was detected in the mouse brain in areas of cortical and subcortical neuronal differentiation, respectively, by two-probes fluorescent in situ hybridization (FISH). Among the coding genes upregulated during differentiation of ANS cells we found Elavl3, a neural-specific RNA-binding protein homologous to Drosophila Elav. In the insect, Elav regulates polyA+ site choice while interacting with paused Pol-II promoters. We tested the role of Elavl3 in ANS cells, by silencing Elavl3 and observed consistent changes in 3'UTR length and delayed neuronal differentiation. These results indicate that choice of the polyA+ site and lengthening of 3'UTRs is a possible additional mechanism of posttranscriptional RNA modification involved in neuronal differentiation.

Mutations in the Neuroblastoma Amplified Sequence gene in a family affected by Acrofrontofacionasal Dysostosis type 1.

Acrofrontofacionasal Dysostosis type 1 (AFFND1) is an extremely rare, autosomal recessive syndrome, comprising facial and skeletal abnormalities, short stature and intellectual disability. We analyzed an Indian family with two affected siblings by exome sequencing and identified a novel homozygous truncating mutation in the Neuroblastoma-Amplified Sequence (NBAS) gene in the patients' genome. Mutations in the NBAS gene have recently been associated with different phenotypes mainly involving skeletal formation, liver and cognitive development. The NBAS protein has been implicated in two key cellular processes, namely the non-sense mediated decay and the Golgi-to-Endoplasmic Reticulum retrograde traffic. Both functions were impaired in HEK293T cells overexpressing the truncated NBAS protein, as assessed by Real-Time PCR, Western blot analysis, co-immunoprecipitation, and immunofluorescence analysis. We examined the expression of NBAS protein in mouse embryos at various developmental stages by immunohistochemistry, and detected expression in developing chondrogenic and osteogenic structures of the skeleton as well as in the cortex, hippocampus and cerebellum, which is compatible with a role in bone and brain development. Functional genetics in the zebrafish model showed that depletion of endogenous z-nbas in fish embryos results in defective morphogenesis of chondrogenic cranial skeletal elements. Overall, our data point to a conserved function of NBAS in skeletal morphogenesis during development, support the hypothesis of a causative role of the mutated NBAS gene in the pathogenesis of AFFND1 and extend the spectrum of phenotypes associated with defects in this gene.

Hyperactivity of Rac1-GTPase pathway impairs neuritogenesis of cortical neurons by altering actin dynamics.

The small-GTPase Rac1 is a key molecular regulator linking extracellular signals to actin cytoskeleton dynamics. Loss-of-function mutations in RAC1 and other genes of the Rac signaling pathway have been implicated in the pathogenesis of Intellectual Disability (ID). The Rac1 activity is negatively controlled by GAP proteins, however the effect of Rac1 hyperactivity on neuronal networking in vivo has been poorly studied. ArhGAP15 is a Rac-specific negative regulator, expressed in the main subtypes of pyramidal cortical neurons. In the absence of ArhGAP15, cortical pyramidal neurons show defective neuritogenesis, delayed axonal elongation, reduced dendritic branching, both in vitro and in vivo. These phenotypes are associated with altered actin dynamics at the growth cone due to increased activity of the PAK-LIMK pathway and hyperphosphorylation of ADF/cofilin. These results can be explained by shootin1 hypo-phosphorylation and uncoupling with the adhesion system. Functionally, ArhGAP15-/- mice exhibit decreased synaptic density, altered electroencephalographic rhythms and cognitive deficits. These data suggest that both hypo- and hyperactivation of the Rac pathway due to mutations in Rac1 regulators can result in conditions of ID, and that a tight regulation of Rac1 activity is required to attain the full complexity of the cortical networks.

Msx1 and Dlx5 act independently in development of craniofacial skeleton, but converge on the regulation of Bmp signaling in palate formation.

Msx and Dlx homeoproteins control the morphogenesis and organization of craniofacial skeletal structures, specifically those derived from the pharyngeal arches. In vitro Msx and Dlx proteins have opposing transcriptional properties and form heterodimeric complexes via their homeodomain with reciprocal functional repression. In this report we examine the skeletal phenotype of Msx1; Dlx5 double knock-out (DKO) mice in relationship with their expression territories during craniofacial development. Co-expression of Dlx5 and Msx1 is only observed in embryonic tissues in which these genes have independent functions, and thus direct protein interactions are unlikely to control morphogenesis of the cranium. The DKO craniofacial phenotypes indicate a complex interplay between these genes, acting independently (mandible and middle ear), synergistically (deposition of bone tissue) or converging on the same morphogenetic process (palate growth and closure). In the latter case, the absence of Dlx5 rescues in part the Msx1-dependent defects in palate growth and elevation. At the basis of this effect, our data implicate the Bmp (Bmp7, Bmp4)/Bmp antagonist (Follistatin) signal: in the Dlx5(-/-) palate changes in the expression level of Bmp7 and Follistatin counteract the reduced Bmp4 expression. These results highlight the importance of precise spatial and temporal regulation of the Bmp/Bmp antagonist system during palate closure.

The role of Dlx homeogenes in early development of the olfactory pathway.

Development of the olfactory pathway requires interaction between cells and signals of different origin. Olfactory receptor neurons (ORN) in the olfactory placodes (OP) extend axons towards the forebrain (FB); with innervation taking place at a later time following degradation of the basement membrane. Cells from the OP migrate along ORN axons and differentiate into various elements, including ensheathing and Gonadotropin Releasing Hormone (GnRH)+ cells. The importance of the olfactory connection and migration is highlighted by the severe endocrine phenotype in Kallmann's patients who lack this migratory pathway. Little is known about the genetic control of intrinsic ORN properties. Inactivation of the distalless-related Dlx5 prevents connections between ORNs and FB. Using a grafting approach we show that misguidance and lack of connectivity is due to intrinsic defects in ORN neurites and migratory cells (MgC), and not to environmental factors. These data point to a cell-autonomous function of Dlx5 in providing ORN axons with their connectivity properties. Dlx5 also marks a population of early MgC that partly overlaps with the GnRH+ population. In the absence of Dlx5 MgCs of the Dlx5+ lineage migrate, associated with PSA-NCAM+ axons, but fail to reach the FB as a consequence of the lack of axonal connection and not an inability to migrate. These data suggests that Dlx5 is not required to initiate migration and differentiation of MgCs.