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1.
Genesis ; 59(1-2): e23404, 2021 02.
Article in English | MEDLINE | ID: mdl-33351273

ABSTRACT

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.


Subject(s)
Abnormalities, Multiple/genetics , Disease Models, Animal , Face/abnormalities , Hematologic Diseases/genetics , Vestibular Diseases/genetics , Xenopus laevis/genetics , Abnormalities, Multiple/pathology , Animals , Face/pathology , Hematologic Diseases/pathology , Histone Demethylases/genetics , Histone-Lysine N-Methyltransferase/genetics , Neural Crest/metabolism , Neural Crest/pathology , Vestibular Diseases/pathology , Xenopus Proteins/genetics , Xenopus laevis/physiology
2.
Hum Mol Genet ; 29(2): 305-319, 2020 01 15.
Article in English | MEDLINE | ID: mdl-31813957

ABSTRACT

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.


Subject(s)
Abnormalities, Multiple/enzymology , Face/abnormalities , Hematologic Diseases/enzymology , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Neural Crest/metabolism , Vestibular Diseases/enzymology , Xenopus Proteins/genetics , Xenopus Proteins/metabolism , Abnormalities, Multiple/genetics , Abnormalities, Multiple/metabolism , Abnormalities, Multiple/pathology , Acetylation , Animals , Cell Movement/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Face/pathology , Hematologic Diseases/genetics , Hematologic Diseases/metabolism , Hematologic Diseases/pathology , Histones/metabolism , Loss of Function Mutation , Methylation , Mutation , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Neural Crest/enzymology , Neural Crest/pathology , Neural Plate/growth & development , Neural Plate/metabolism , Neural Plate/pathology , Semaphorins/genetics , Semaphorins/metabolism , Vestibular Diseases/genetics , Vestibular Diseases/metabolism , Vestibular Diseases/pathology , Xenopus/embryology , Xenopus/genetics , Xenopus/metabolism , Xenopus Proteins/physiology
3.
Dev Dyn ; 248(6): 465-476, 2019 06.
Article in English | MEDLINE | ID: mdl-30980591

ABSTRACT

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.


Subject(s)
Abnormalities, Multiple/genetics , Face/abnormalities , Heart/growth & development , Hematologic Diseases/genetics , Histone-Lysine N-Methyltransferase/physiology , Vestibular Diseases/genetics , Xenopus Proteins/physiology , Xenopus laevis/growth & development , Animals , Heart Defects, Congenital/genetics , Histone-Lysine N-Methyltransferase/genetics , Loss of Function Mutation , Xenopus Proteins/genetics
4.
Hum Mol Genet ; 27(8): 1343-1352, 2018 04 15.
Article in English | MEDLINE | ID: mdl-29432577

ABSTRACT

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.


Subject(s)
CHARGE Syndrome/genetics , DNA Helicases/genetics , DNA-Binding Proteins/genetics , Epigenesis, Genetic , Neural Crest/metabolism , Semaphorin-3A/genetics , Animals , CHARGE Syndrome/metabolism , CHARGE Syndrome/pathology , DNA Helicases/metabolism , DNA-Binding Proteins/metabolism , Disease Models, Animal , Embryo, Nonmammalian , Genetic Complementation Test , HEK293 Cells , Homeobox Protein Nkx-2.5/genetics , Homeobox Protein Nkx-2.5/metabolism , Humans , Kallmann Syndrome/genetics , Kallmann Syndrome/metabolism , Kallmann Syndrome/pathology , Mutation , Neural Crest/pathology , Promoter Regions, Genetic , Semaphorin-3A/metabolism , Severity of Illness Index , Xenopus laevis
5.
Development ; 141(18): 3505-16, 2014 Sep.
Article in English | MEDLINE | ID: mdl-25183869

ABSTRACT

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.


Subject(s)
Gastrulation/physiology , Genes, MCC/genetics , Morphogenesis/physiology , Wnt Signaling Pathway/physiology , Zebrafish/embryology , Animals , Blotting, Western , Cell Polarity/physiology , Immunoprecipitation , In Situ Hybridization , Luciferases , Membrane Proteins/metabolism , Microscopy, Confocal , PDZ Domains/genetics , Polymerase Chain Reaction , Receptor Tyrosine Kinase-like Orphan Receptors/metabolism , Wnt Proteins/metabolism , Wnt-5a Protein , Zebrafish Proteins/metabolism
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