Fbrsl1 is required for heart development in Xenopus laevis and de novo variants in FBRSL1 can cause human heart defects.

De novo truncating variants in Fibrosin-like protein 1 (FBRSL1), a member of the AUTS2 gene family, cause a disability syndrome, including organ malformations such as heart defects. Here, we use Xenopus laevis to investigate whether Fbrsl1 plays a role in heart development. Xenopus laevis fbrsl1 is expressed in tissues relevant for heart development and morpholino-mediated knockdown of Fbrsl1 results in severely hypoplastic hearts. Our data suggest that Fbrsl1 is required for the development of the first heart field, which contributes to the ventricle and the atria, but not for the second heart field, which gives rise to the outflow tract. The morphant heart phenotype could be rescued using a human N-terminal FBRSL1 isoform that contains an alternative exon, but lacks the AUTS2 domain. N-terminal isoforms carrying patient variants failed to rescue. Interestingly, a long human FBRSL1 isoform, harboring the AUTS2 domain, did also not rescue the morphant heart defects. Thus, our data suggest that different FBRSL1 isoforms may have distinct functions and that only the short N-terminal isoform, appears to be critical for heart development.

The RhoGEF Trio is transported by microtubules and affects microtubule stability in migrating neural crest cells.

Directed cell migration requires a local fine-tuning of Rho GTPase activity to control protrusion formation, cell-cell contraction, and turnover of cellular adhesions. The Rho guanine nucleotide exchange factor (GEF) TRIO is ideally suited to control RhoGTPase activity because it combines two distinct catalytic domains to control Rac1 and RhoA activity in one molecule. However, at the cellular level, this molecular feature also requires a tight spatiotemporal control of TRIO activity. Here, we analyze the dynamic localization of Trio in Xenopus cranial neural crest (NC) cells, where we have recently shown that Trio is required for protrusion formation and migration. Using live cell imaging, we find that the GEF2 domain, but not the GEF1 domain of Trio, dynamically colocalizes with EB3 at microtubule plus-ends. Microtubule-mediated transport of Trio appears to be relevant for its function in NC migration, as a mutant GEF2 construct lacking the SxIP motif responsible for microtubule plus-end localization was significantly impaired in its ability to rescue the Trio loss-of-function phenotype compared to wild-type GEF2. Furthermore, by analyzing microtubule dynamics in migrating NC cells, we observed that loss of Trio function stabilized microtubules at cell-cell contact sites compared to controls, whereas they were destabilized at the leading edge of NC cells. Our data suggest that Trio is transported by microtubules to distinct subcellular locations where it has different functions in controlling microtubule stability, cell morphology, and cell-cell interaction during directed NC migration.

The H2A.Z and NuRD associated protein HMG20A controls early head and heart developmental transcription programs.

Specialized chromatin-binding proteins are required for DNA-based processes during development. We recently established PWWP2A as a direct histone variant H2A.Z interactor involved in mitosis and craniofacial development. Here, we identify the H2A.Z/PWWP2A-associated protein HMG20A as part of several chromatin-modifying complexes, including NuRD, and show that it localizes to distinct genomic regulatory regions. Hmg20a depletion causes severe head and heart developmental defects in Xenopus laevis. Our data indicate that craniofacial malformations are caused by defects in neural crest cell (NCC) migration and cartilage formation. These developmental failures are phenocopied in Hmg20a-depleted mESCs, which show inefficient differentiation into NCCs and cardiomyocytes (CM). Consequently, loss of HMG20A, which marks open promoters and enhancers, results in chromatin accessibility changes and a striking deregulation of transcription programs involved in epithelial-mesenchymal transition (EMT) and differentiation processes. Collectively, our findings implicate HMG20A as part of the H2A.Z/PWWP2A/NuRD-axis and reveal it as a key modulator of intricate developmental transcription programs that guide the differentiation of NCCs and CMs.

Comparing a Novel Malformation Syndrome Caused by Pathogenic Variants in FBRSL1 to AUTS2 Syndrome.

Truncating variants in specific exons of Fibrosin-like protein 1 (FBRSL1) were recently reported to cause a novel malformation and intellectual disability syndrome. The clinical spectrum includes microcephaly, facial dysmorphism, cleft palate, skin creases, skeletal anomalies and contractures, postnatal growth retardation, global developmental delay as well as respiratory problems, hearing impairment and heart defects. The function of FBRSL1 is largely unknown, but pathogenic variants in the FBRSL1 paralog Autism Susceptibility Candidate 2 (AUTS2) are causative for an intellectual disability syndrome with microcephaly (AUTS2 syndrome). Some patients with AUTS2 syndrome also show additional symptoms like heart defects and contractures overlapping with the phenotype presented by patients with FBRSL1 mutations. For AUTS2, a dual function, depending on different isoforms, was described and suggested for FBRSL1. Both, nuclear FBRSL1 and AUTS2 are components of the Polycomb subcomplexes PRC1.3 and PRC1.5. These complexes have essential roles in developmental processes, cellular differentiation and proliferation by regulating gene expression via histone modification. In addition, cytoplasmic AUTS2 controls neural development, neuronal migration and neurite extension by regulating the cytoskeleton. Here, we review recent data on FBRSL1 in respect to previously published data on AUTS2 to gain further insights into its molecular function, its role in development as well as its impact on human genetics.

Ptk7 Is Dynamically Localized at Neural Crest Cell-Cell Contact Sites and Functions in Contact Inhibition of Locomotion.

Neural crest (NC) cells are highly migratory cells that contribute to various vertebrate tissues, and whose migratory behaviors resemble cancer cell migration and invasion. Information exchange via dynamic NC cell-cell contact is one mechanism by which the directionality of migrating NC cells is controlled. One transmembrane protein that is most likely involved in this process is protein tyrosine kinase 7 (PTK7), an evolutionary conserved Wnt co-receptor that is expressed in cranial NC cells and several tumor cells. In Xenopus, Ptk7 is required for NC migration. In this study, we show that the Ptk7 protein is dynamically localized at cell-cell contact zones of migrating Xenopus NC cells and required for contact inhibition of locomotion (CIL). Using deletion constructs of Ptk7, we determined that the extracellular immunoglobulin domains of Ptk7 are important for its transient accumulation and that they mediate homophilic binding. Conversely, we found that ectopic expression of Ptk7 in non-NC cells was able to prevent NC cell invasion. However, deletion of the extracellular domains of Ptk7 abolished this effect. Thus, Ptk7 is sufficient at protecting non-NC tissue from NC cell invasion, suggesting a common role of PTK7 in contact inhibition, cell invasion, and tissue integrity.

Using Xenopus to analyze neurocristopathies like Kabuki syndrome.

Neurocristopathies are human congenital syndromes that arise from defects in neural crest (NC) development and are typically associated with malformations of the craniofacial skeleton. Genetic analyses have been very successful in identifying pathogenic mutations, however, model organisms are required to characterize how these mutations affect embryonic development thereby leading to complex clinical conditions. The African clawed frog Xenopus laevis provides a broad range of in vivo and in vitro tools allowing for a detailed characterization of NC development. Due to the conserved nature of craniofacial morphogenesis in vertebrates, Xenopus is an efficient and versatile system to dissect the morphological and cellular phenotypes as well as the signaling events leading to NC defects. Here, we review a set of techniques and resources how Xenopus can be used as a disease model to investigate the pathogenesis of Kabuki syndrome and neurocristopathies in a wider sense.

Caveolin 1 is required for axonal outgrowth of motor neurons and affects Xenopus neuromuscular development.

Caveolins are essential structural proteins driving the formation of caveolae, specialized invaginations of the plasma membrane. Loss of Caveolin-1 (Cav1) function in mice causes distinct neurological phenotypes leading to impaired motor control, however, the underlying developmental mechanisms are largely unknown. In this study we find that loss-of-function of Xenopus Cav1 results in a striking swimming defect characterized by paralysis of the morphants. High-resolution imaging of muscle cells revealed aberrant sarcomeric structures with disorganized actin fibers. As cav1 is expressed in motor neurons, but not in muscle cells, the muscular abnormalities are likely a consequence of neuronal defects. Indeed, targeting cav1 Morpholino oligonucleotides to neural tissue, but not muscle tissue, disrupts axonal outgrowth of motor neurons and causes swimming defects. Furthermore, inhibition of voltage-gated sodium channels mimicked the Cav1 loss-of-function phenotype. In addition, analyzing axonal morphology we detect that Cav1 loss-of-function causes excessive filopodia and lamellipodia formation. Using rescue experiments, we show that the Cav1 Y14 phosphorylation site is essential and identify a role of RhoA, Rac1, and Cdc42 signaling in this process. Taken together, these results suggest a previously unrecognized function of Cav1 in muscle development by supporting axonal outgrowth of motor neurons.

De novo mutations in FBRSL1 cause a novel recognizable malformation and intellectual disability syndrome.

We report truncating de novo variants in specific exons of FBRSL1 in three unrelated children with an overlapping syndromic phenotype with respiratory insufficiency, postnatal growth restriction, microcephaly, global developmental delay and other malformations. The function of FBRSL1 is largely unknown. Interestingly, mutations in the FBRSL1 paralogue AUTS2 lead to an intellectual disability syndrome (AUTS2 syndrome). We determined human FBRSL1 transcripts and describe protein-coding forms by Western blot analysis as well as the cellular localization by immunocytochemistry stainings. All detected mutations affect the two short N-terminal isoforms, which show a ubiquitous expression in fetal tissues. Next, we performed a Fbrsl1 knockdown in Xenopus laevis embryos to explore the role of Fbrsl1 during development and detected craniofacial abnormalities and a disturbance in neurite outgrowth. The aberrant phenotype in Xenopus laevis embryos could be rescued with a human N-terminal isoform, while the long isoform and the N-terminal isoform containing the mutation p.Gln163* isolated from a patient could not rescue the craniofacial defects caused by Fbrsl1 depletion. Based on these data, we propose that the disruption of the validated N-terminal isoforms of FBRSL1 at critical timepoints during embryogenesis leads to a hitherto undescribed complex neurodevelopmental syndrome.

The Rho guanine nucleotide exchange factor Trio is required for neural crest cell migration and interacts with Dishevelled.

Directional migration during embryogenesis and tumor progression faces the challenge that numerous external signals need to converge to precisely control cell movement. The Rho guanine exchange factor (GEF) Trio is especially well suited to relay signals, as it features distinct catalytic domains to activate Rho GTPases. Here, we show that Trio is required for Xenopus cranial neural crest (NC) cell migration and cartilage formation. Trio cell-autonomously controls protrusion formation of NC cells and Trio morphant NC cells show a blebbing phenotype. Interestingly, the Trio GEF2 domain is sufficient to rescue protrusion formation and migration of Trio morphant NC cells. We show that this domain interacts with the DEP/C-terminus of Dishevelled (DVL). DVL - but not a deletion construct lacking the DEP domain - is able to rescue protrusion formation and migration of Trio morphant NC cells. This is likely mediated by activation of Rac1, as we find that DVL rescues Rac1 activity in Trio morphant embryos. Thus, our data provide evidence for a novel signaling pathway, whereby Trio controls protrusion formation of cranial NC cells by interacting with DVL to activate Rac1.

The histone methyltransferase KMT2D, mutated in Kabuki syndrome patients, is required for neural crest cell formation and migration.

Kabuki syndrome is an autosomal dominant developmental disorder with high similarities to CHARGE syndrome. It is characterized by a typical facial gestalt in combination with short stature, intellectual disability, skeletal findings and additional features like cardiac and urogenital malformations, cleft palate, hearing loss and ophthalmological anomalies. The major cause of Kabuki syndrome are mutations in KMT2D, a gene encoding a histone H3 lysine 4 (H3K4) methyltransferase belonging to the group of chromatin modifiers. Here we provide evidence that Kabuki syndrome is a neurocrestopathy, by showing that Kmt2d loss-of-function inhibits specific steps of neural crest (NC) development. Using the Xenopus model system, we find that Kmt2d loss-of-function recapitulates major features of Kabuki syndrome including severe craniofacial malformations. A detailed marker analysis revealed defects in NC formation as well as migration. Transplantation experiments confirm that Kmt2d function is required in NC cells. Furthermore, analyzing in vivo and in vitro NC migration behavior demonstrates that Kmt2d is necessary for cell dispersion but not protrusion formation of migrating NC cells. Importantly, Kmt2d knockdown correlates with a decrease in H3K4 monomethylation and H3K27 acetylation supporting a role of Kmt2d in the transcriptional activation of target genes. Consistently, using a candidate approach, we find that Kmt2d loss-of-function inhibits Xenopus Sema3F expression, and overexpression of Sema3F can partially rescue Kmt2d loss-of-function defects. Taken together, our data reveal novel functions of Kmt2d in multiple steps of NC development and support the hypothesis that major features of Kabuki syndrome are caused by defects in NC development.

Loss of function of Kmt2d, a gene mutated in Kabuki syndrome, affects heart development in Xenopus laevis.

BACKGROUND: Kabuki syndrome is a haploinsufficient congenital multi-organ malformation syndrome, which frequently includes severe heart defects. Mutations in the histone H3K4 methyltransferase KMT2D have been identified as the main cause of Kabuki syndrome, however, the role of KMT2D in heart development remains to be characterized. RESULTS: Here we analyze the function of Kmt2d at different stages of Xenopus heart development. Xenopus Kmt2d is ubiquitously expressed at early stages of cardiogenesis, with enrichment in the anterior region including the cardiac precursor cells. Morpholino-mediated knockdown of Kmt2d led to hypoplastic hearts lacking the three-chambered structure. Analyzing different stages of cardiogenesis revealed that development of the first and second heart fields as well as cardiac differentiation were severely affected by loss of Kmt2d function. CONCLUSION: Kmt2d loss of function in Xenopus recapitulates the hypoplastic heart defects observed in Kabuki syndrome patients and shows that Kmt2d function is required for the establishment of the primary and secondary heart fields. Thus, Xenopus Kmt2d morphants can be a valuable tool to elucidate the etiology of the congenital heart defects associated with Kabuki syndrome.

Evolution of the Rho guanine nucleotide exchange factors Kalirin and Trio and their gene expression in Xenopus development.

Guanine nucleotide exchange factors (GEFs) activate Rho GTPases by accelerating their GDP/GTP exchange. Trio and its paralog Kalirin (Kalrn) are unique members of the Rho-GEFs that harbor three catalytic domains: two functional GEF domains and a serine/threonine kinase domain. The N-terminal GEF domain activates Rac1 and RhoG GTPases, while the C-terminal GEF domain acts specifically on RhoA. Trio and Kalrn have an evolutionary conserved function in morphogenetic processes including neuronal development. De novo mutations in TRIO have lately been identified in patients with intellectual disability, suggesting that this protein family plays an important role in development and disease. Phylogenetic and domain analysis revealed that a Kalrn/Trio ancestor originated in Prebilateria and duplicated in Urbilateria to yield Kalrn and Trio. Only few taxa outside the vertebrates retained both of these highly conserved proteins. To obtain first insights into their redundant or distinct functions in a vertebrate model system, we show for the first time a detailed comparative analysis of trio and kalrn expression in Xenopus laevis development. The mRNAs are maternally transcribed and expression increases starting with neurula stages. Trio and kalrn are detected in mesoderm/somites and different neuronal populations in the neural plate/tube and later also in the brain. However, only trio is expressed in migrating neural crest cells, while kalrn expression is detected in the cranial nerves, suggesting distinct functions. Thus, our expression analysis provides a good basis for further functional studies.

Sema3a plays a role in the pathogenesis of CHARGE syndrome.

CHARGE syndrome is an autosomal dominant malformation disorder caused by heterozygous loss of function mutations in the chromatin remodeler CHD7. Chd7 regulates the expression of Sema3a, which also contributes to the pathogenesis of Kallmann syndrome, a heterogeneous condition with the typical features hypogonadotropic hypogonadism and an impaired sense of smell. Both features are common in CHARGE syndrome suggesting that SEMA3A may provide a genetic link between these syndromes. Indeed, we find evidence that SEMA3A plays a role in the pathogenesis of CHARGE syndrome. First, Chd7 is enriched at the Sema3a promotor in neural crest cells and loss of function of Chd7 inhibits Sema3a expression. Second, using a Xenopus CHARGE model, we show that human SEMA3A rescues Chd7 loss of function. Third, to elucidate if SEMA3A mutations in addition to CHD7 mutations also contribute to the severity of the CHARGE phenotype, we screened 31 CHD7-positive patients and identified one patient with a heterozygous non-synonymous SEMA3A variant, c.2002A>G (p.I668V). By analyzing protein expression and processing, we did not observe any differences of the p.I668V variant compared with wild-type SEMA3A, while a pathogenic SEMA3A variant p.R66W recently described in a patient with Kallmann syndrome did affect protein secretion. Furthermore, the p.I668V variant, but not the pathogenic p.R66W variant, rescues Chd7 loss of function in Xenopus, indicating that the p.I668V variant is likely benign. Thus, SEMA3A is part of an epigenetic loop that plays a role in the pathogenesis of CHARGE syndrome, however, it seems not to act as a common direct modifier.

CHARGEd with neural crest defects.

Neural crest cells are highly migratory pluripotent cells that give rise to diverse derivatives including cartilage, bone, smooth muscle, pigment, and endocrine cells as well as neurons and glia. Abnormalities in neural crest-derived tissues contribute to the etiology of CHARGE syndrome, a complex malformation disorder that encompasses clinical symptoms like coloboma, heart defects, atresia of the choanae, retarded growth and development, genital hypoplasia, ear anomalies, and deafness. Mutations in the chromodomain helicase DNA-binding protein 7 (CHD7) gene are causative of CHARGE syndrome and loss-of-function data in different model systems have firmly established a role of CHD7 in neural crest development. Here, we will summarize our current understanding of the function of CHD7 in neural crest development and discuss possible links of CHARGE syndrome to other developmental disorders.

PTK7 Faces the Wnt in Development and Disease.

PTK7 (protein tyrosine kinase 7) is an evolutionarily conserved transmembrane receptor regulating various processes in embryonic development and tissue homeostasis. On a cellular level PTK7 affects the establishment of cell polarity, the regulation of cell movement and migration as well as cell invasion. The PTK7 receptor has been shown to interact with ligands, co-receptors, and intracellular transducers of Wnt signaling pathways, pointing to a function in the fine-tuning of the Wnt signaling network. Here we will review recent findings implicating PTK7 at the crossroads of Wnt signaling pathways in development and disease.

PTK7 localization and protein stability is affected by canonical Wnt ligands.

Protein tyrosine kinase 7 (PTK7) is an evolutionarily conserved transmembrane receptor with important roles in embryonic development and disease. Originally identified as a gene upregulated in colon cancer, it was later shown to regulate planar cell polarity (PCP) and directional cell movement. PTK7 is a Wnt co-receptor; however, its role in Wnt signaling remains controversial. Here, we find evidence that places PTK7 at the intersection of canonical and non-canonical Wnt signaling pathways. In presence of canonical Wnt ligands PTK7 is subject to caveolin-mediated endocytosis, while it is unaffected by non-canonical Wnt ligands. PTK7 endocytosis is dependent on the presence of the PTK7 co-receptor Fz7 (also known as Fzd7) and results in lysosomal degradation of PTK7. As we previously observed that PTK7 activates non-canonical PCP Wnt signaling but inhibits canonical Wnt signaling, our data suggest a mutual inhibition of canonical and PTK7 Wnt signaling. PTK7 likely suppresses canonical Wnt signaling by binding canonical Wnt ligands thereby preventing their interaction with Wnt receptors that would otherwise support canonical Wnt signaling. Conversely, if canonical Wnt proteins interact with the PTK7 receptor, they induce its internalization and degradation.

Controlled levels of canonical Wnt signaling are required for neural crest migration.

Canonical Wnt signaling plays a dominant role in the development of the neural crest (NC), a highly migratory cell population that generates a vast array of cell types. Canonical Wnt signaling is required for NC induction as well as differentiation, however its role in NC migration remains largely unknown. Analyzing nuclear localization of ß-catenin as readout for canonical Wnt activity, we detect nuclear ß-catenin in premigratory but not migratory Xenopus NC cells suggesting that canonical Wnt activity has to decrease to basal levels to enable NC migration. To define a possible function of canonical Wnt signaling in Xenopus NC migration, canonical Wnt signaling was modulated at different time points after NC induction. This was accomplished using either chemical modulators affecting ß-catenin stability or inducible glucocorticoid fusion constructs of Lef/Tcf transcription factors. In vivo analysis of NC migration by whole mount in situ hybridization demonstrates that ectopic activation of canonical Wnt signaling inhibits cranial NC migration. Further, NC transplantation experiments confirm that this effect is tissue-autonomous. In addition, live-cell imaging in combination with biophysical data analysis of explanted NC cells confirms the in vivo findings and demonstrates that modulation of canonical Wnt signaling affects the ability of NC cells to perform single cell migration. Thus, our data support the hypothesis that canonical Wnt signaling needs to be tightly controlled to enable migration of NC cells.

A PTK7/Ror2 Co-Receptor Complex Affects Xenopus Neural Crest Migration.

Neural crest cells are a highly migratory pluripotent cell population that generates a wide array of different cell types and failure in their migration can result in severe birth defects and malformation syndromes. Neural crest migration is controlled by various means including chemotaxis, repellent guidance cues and cell-cell interaction. Non-canonical Wnt PCP (planar cell polarity) signaling has previously been shown to control cell-contact mediated neural crest cell guidance. PTK7 (protein tyrosine kinase 7) is a transmembrane pseudokinase and a known regulator of Wnt/PCP signaling, which is expressed in Xenopus neural crest cells and required for their migration. PTK7 functions as a Wnt co-receptor; however, it remains unclear by which means PTK7 affects neural crest migration. Expressing fluorescently labeled proteins in Xenopus neural crest cells we find that PTK7 co-localizes with the Ror2 Wnt-receptor. Further, co-immunoprecipitation experiments demonstrate that PTK7 interacts with Ror2. The PTK7/Ror2 interaction is likely relevant for neural crest migration, because Ror2 expression can rescue the PTK7 loss of function migration defect. Live cell imaging of explanted neural crest cells shows that PTK7 loss of function affects the formation of cell protrusions as well as cell motility. Co-expression of Ror2 can rescue these defects. In vivo analysis demonstrates that a kinase dead Ror2 mutant cannot rescue PTK7 loss of function. Thus, our data suggest that Ror2 can substitute for PTK7 and that the signaling function of its kinase domain is required for this effect.

The PDZ domain protein Mcc is a novel effector of non-canonical Wnt signaling during convergence and extension in zebrafish.

During vertebrate gastrulation, a complex set of mass cellular rearrangements shapes the embryonic body plan and appropriately positions the organ primordia. In zebrafish and Xenopus, convergence and extension (CE) movements simultaneously narrow the body axis mediolaterally and elongate it from head to tail. This process is governed by polarized cell behaviors that are coordinated by components of the non-canonical, ß-catenin-independent Wnt signaling pathway, including Wnt5b and the transmembrane planar cell polarity (PCP) protein Vangl2. However, the intracellular events downstream of Wnt/PCP signals are not fully understood. Here, we show that zebrafish mutated in colorectal cancer (mcc), which encodes an evolutionarily conserved PDZ domain-containing putative tumor suppressor, is required for Wnt5b/Vangl2 signaling during gastrulation. Knockdown of mcc results in CE phenotypes similar to loss of vangl2 and wnt5b, whereas overexpression of mcc robustly rescues the depletion of wnt5b, vangl2 and the Wnt5b tyrosine kinase receptor ror2. Biochemical experiments establish a direct physical interaction between Mcc and the Vangl2 cytoplasmic tail. Lastly, CE defects in mcc morphants are suppressed by downstream activation of RhoA and JNK. Taken together, our results identify Mcc as a novel intracellular effector of non-canonical Wnt5b/Vangl2/Ror2 signaling during vertebrate gastrulation.

CHD7, the gene mutated in CHARGE syndrome, regulates genes involved in neural crest cell guidance.

Heterozygous loss of function mutations in CHD7 (chromodomain helicase DNA-binding protein 7) lead to CHARGE syndrome, a complex developmental disorder affecting craniofacial structures, cranial nerves and several organ systems. Recently, it was demonstrated that CHD7 is essential for the formation of multipotent migratory neural crest cells, which migrate from the neural tube to many regions of the embryo, where they differentiate into various tissues including craniofacial and heart structures. So far, only few CHD7 target genes involved in neural crest cell development have been identified and the role of CHD7 in neural crest cell guidance and the regulation of mesenchymal-epithelial transition are unknown. Therefore, we undertook a genome-wide microarray expression analysis on wild-type and CHD7 deficient (Chd7 (Whi/+) and Chd7 (Whi/Whi)) mouse embryos at day 9.5, a time point of neural crest cell migration. We identified 98 differentially expressed genes between wild-type and Chd7 (Whi/Whi) embryos. Interestingly, many misregulated genes are involved in neural crest cell and axon guidance such as semaphorins and ephrin receptors. By performing knockdown experiments for Chd7 in Xenopus laevis embryos, we found abnormalities in the expression pattern of Sema3a, a protein involved in the pathogenesis of Kallmann syndrome, in vivo. In addition, we detected non-synonymous SEMA3A variations in 3 out of 45 CHD7-negative CHARGE patients. In summary, we discovered for the first time that Chd7 regulates genes involved in neural crest cell guidance, demonstrating a new aspect in the pathogenesis of CHARGE syndrome. Furthermore, we showed for Sema3a a conserved regulatory mechanism across different species, highlighting its significance during development. Although we postulated that the non-synonymous SEMA3A variants which we found in CHD7-negative CHARGE patients alone are not sufficient to produce the phenotype, we suggest an important modifier role for SEMA3A in the pathogenesis of this multiple malformation syndrome.