CDK19-related disorder results from both loss-of-function and gain-of-function de novo missense variants

Purpose To expand the recent description of a new neurodevelopmental syndrome related to alterations in CDK19. Methods Individuals were identified through international collaboration. Functional studies included autophosphorylation assays for CDK19 Gly28Arg and Tyr32His variants and in vivo zebrafish assays of the CDK19(G28R) and CDK19(Y32H). Results We describe 11 unrelated individuals (age range: 9 months to 14 years) with de novo missense variants mapped to the kinase domain of CDK19, including two recurrent changes at residues Tyr32 and Gly28. In vitro autophosphorylation and substrate phosphorylation assays revealed that kinase activity of protein was lower for p.Gly28Arg and higher for p.Tyr32His substitutions compared with that of the wild-type protein. Injection of CDK19 messenger RNA (mRNA) with either the Tyr32His or the Gly28Arg variants using in vivo zebrafish model significantly increased fraction of embryos with morphological abnormalities. Overall, the phenotype of the now 14 individuals with CDK19-related disorder includes universal developmental delay and facial dysmorphism, hypotonia (79%), seizures (64%), ophthalmologic anomalies (64%), and autism/autistic traits (56%). Conclusion CDK19 de novo missense variants are responsible for a novel neurodevelopmental disorder. Both kinase assay and zebrafish experiments showed that the pathogenetic mechanism may be more diverse than previously thought.Peer reviewe

Mesp2 and Tbx6 cooperatively establish periodic patterns, coupled with the clock machinery during mouse somitogenesis

　　During mouse embryogenesis, many morphogenetic events occur sequentially according to the scheduled time, indicating that these sequential events are linked with the precise temporal regulation. Such regulations must exist throughout embryogenesis to coordinate many developmental processes, although the molecular nature coordinating such temporal regulation is largely unknown. 　　The vertebrate body is subdivided into repeating segments along the anterior-posterior (AP) axis. This segmental or metameric pattern is established early in embryogenesis by the process of somitogenesis. Somites are blocks of paraxial mesoderm cells that give rise to the axial skeleton and their associated muscles and tendons, which retain a metameric pattern. During development, somitogenesis is tightly coupled with axis elongation. Precursors of the somites, called presomitic mesoderm (PSM), arise from the posterior end of embryo, called tail bud. Somites are aligned along the neural tube, and budding off from the anterior-most end of the unsegmented presomitic mesoderm at the regular time. Therefore, somitogenesis is an event that occurs according to the scheduled time, and it is believed that somitogenesis is under the precise control of temporal information. 　　The timing of somitogenesis is regulated by the so-called `segmentation clock\u27, which is associated with a periodic activation of Notch signal pathway in PSM cells. Notch signal .activates the target genes, Hes7 and L-fng. The transcription factor Hes7 (hairy and enhancer of split 7) in turn represses own transcription as well as that of L-fng, making negative feedback loops. L-fng encodes a glycosyltransferase that acts as a negative regulator of Notch activity, which generates the oscillation of Notch signal activity within the PSM. However, the oscillation itself does not make a segmental boundary, as exemplified by a pendulum clock in which the correct time is not provided by the rhythm of pendulum. This temporal information has therefore to be accurately translated into a spatial pattern during somitogenesis. 　　The basic helix-loop-helix (bHLH) protein Mesp2 is a crucial factor in this process. Mesp2 expression is periodically observed only in the anterior PSM, and the anterior border of the Mesp2 expression domain determines the next somite segmental border. To understand dynamic expression of Mesp2, the enhancer sequence, which is required for the expression in the PSM, has been mapped within 185bp upstream region in the 5\u27 flanking region of Mesp2 gene, and it has been shown that a T-box transcriptional factor, Tbx6, directly binds to the enhancer elements, and is essential for the activation of Mesp2. Furthermore, it is shown that Notch signaling synergistically works with Tbx6 and enhances Mesp2 activation when these factors coexist. However, since the enhancer analysis was mainly conducted using the cultured cell system, mechanisms involved in the spatial restriction and periodic regulation of Mesp2 expression remain elusive. 　　In this study, I have employed high resolution fluorescent in situ hybridization in conjunction with immunohistochemical methods to analyze sections derived from single specimens. These methods have enabled me to determine the spatio-temporal relationship among several factors involved in mouse somitogenesis. Initially I show that the timing of Mesp2 expression is determined by the periodic waves of Notch activity, indicating the temporal link between Notch signal oscillation and Mesp2 transcription cycle. Next, I find that Tbx6 defines the anterior limit of Mesp2 expression domain by serving as an important transcription activator. Intriguingly, Mesp2 mRNA initially shares an identical anterior border, but that once translated, the Mesp2 protein is found to suppress Tbx6 expression post-translationally. This was strongly supported by the fact that Tbx6 protein expression was expanded to the anterior somitic region in the Mesp2-null embryo without altering expression pattern of the transcript. The negative regulation of the Tbx6 by Mesp2 is critically important to set up the next anterior border of Mesp2 expression domain. These results indicate that interactions of three factors, Mesp2, Tbx6 and Notch activity are critically important to translate temporal information to the spatial patterning. I also find that onset of Mesp2 transcription is intimately linked with the initiation of Notch signal oscillation, indicating that the relationship of three factors appears to be established in the early stage embryo via initial Notch oscillation. I further show that the lack of FGF signaling results in the posterior shift of Mesp2 expression domain, indicating that FGF signaling provides a spatial cue to position the posterior border of Mesp2 expresslon. 　　Furthermore, to reveal the mechanism of post-translational Tbx6 suppression downstream of Mesp2, I tried to determine the domain of Tbx6 protein that was required for the suppression process. I generated transgenic mice harboring several types of Tbx6 protein that had truncation in several domains, under the control of endogenous promoter and enhancers of Tbx6 using a BAC-base transgenic mouse technology. These results indicate that the T-box domain containing a DNA-binding motif, is essential and sufficient for the suppression of Tbx6 expression. In good agreement with these results, I find that Mesp2 also suppresses the expression of Brachyury, the other T-box factor protein, by the post-translational mechanism. 　　Taken together, I conclude that Mesp2 is the final output signal by which the temporal information from the segmentation clock is translated to the segmental patterning, and reciprocal regulation between Mesp2 and Tbx6 creates the periodic pattern during somitogenesis. <br /

Recapitulating early development of mouse musculoskeletal precursors of the paraxial mesoderm in vitro

The work described in this article is partially covered by patent application PCT/EP2012/066793 (publication number WO2013030243 A1). O.P., J.C. and M.K. are co-founders and shareholders of Anagenesis Biotechnologies, a start-up company specializing in the production of muscle cells in vitro for cell therapy and drug screeninInternational audienceBody skeletal muscles derive from the paraxial mesoderm, which forms in the posterior region of the embryo. Using microarrays, we characterize novel mouse presomitic mesoderm (PSM) markers and show that, unlike the abrupt transcriptome reorganization of the PSM, neural tube differentiation is accompanied by progressive transcriptome changes. The early paraxial mesoderm differentiation stages can be efficiently recapitulated in vitro using mouse and human pluripotent stem cells. While Wnt activation alone can induce posterior PSM markers, acquisition of a committed PSM fate and efficient differentiation into anterior PSM Pax3+ identity further requires BMP inhibition to prevent progenitors from drifting to a lateral plate mesoderm fate. When transplanted into injured adult muscle, these precursors generated large numbers of immature muscle fibers. Furthermore, exposing these mouse PSM-like cells to a brief FGF inhibition step followed by culture in horse serum-containing medium allows efficient recapitulation of the myogenic program to generate myotubes and associated Pax7+ cells. This protocol results in improved in vitro differentiation and maturation of mouse muscle fibers over serum-free protocols and enables the study of myogenic cell fusion and satellite cell differentiation

Differentiation of pluripotent stem cells to muscle fiber to model Duchenne muscular dystrophy

International audienceKey cell types including skeletal muscle have proven difficult to differentiate invitro from pluripotent cells. During embryonic development, skeletal musclesarise from somites, which derive from the presomitic mesoderm (PSM). Based onour understanding of PSM development, we established serum-free conditionsallowing efficient differentiation of monolayer cultures of mouse embryonic stem(ES) cells into PSM-like cells without introduction of exogenous genetic materialor cell sorting. We show that primary and secondary skeletal myogenesis can berecapitulated in vitro from these PSM-like cells. Our strategy allowed for theproduction of striated contractile fibers from mouse and human pluripotent cellsin vitro with an efficiency comparing with current cardiomyocytes differentiationprotocols. We also differentiated ES cells into Pax7-positive cells with satellitecell characteristics, including the ability to generate dystrophin-positive fiberswhen grafted into muscles from dystrophin-deficient mdx mice. We show thatdifferentiated ES cells derived from mdx mice exhibit a striking branchedphenotype resembling that described in vivo, thus providing an attractive modelto study the origin of the pathological defects associated with DuchenneMuscular Dystrophy

CDK19-related disorder results from both loss-of-function and gain-of-function de novo missense variants

Purpose To expand the recent description of a new neurodevelopmental syndrome related to alterations in CDK19. Methods Individuals were identified through international collaboration. Functional studies included autophosphorylation assays for CDK19 Gly28Arg and Tyr32His variants and in vivo zebrafish assays of the CDK19(G28R) and CDK19(Y32H). Results We describe 11 unrelated individuals (age range: 9 months to 14 years) with de novo missense variants mapped to the kinase domain of CDK19, including two recurrent changes at residues Tyr32 and Gly28. In vitro autophosphorylation and substrate phosphorylation assays revealed that kinase activity of protein was lower for p.Gly28Arg and higher for p.Tyr32His substitutions compared with that of the wild-type protein. Injection of CDK19 messenger RNA (mRNA) with either the Tyr32His or the Gly28Arg variants using in vivo zebrafish model significantly increased fraction of embryos with morphological abnormalities. Overall, the phenotype of the now 14 individuals with CDK19-related disorder includes universal developmental delay and facial dysmorphism, hypotonia (79%), seizures (64%), ophthalmologic anomalies (64%), and autism/autistic traits (56%). Conclusion CDK19 de novo missense variants are responsible for a novel neurodevelopmental disorder. Both kinase assay and zebrafish experiments showed that the pathogenetic mechanism may be more diverse than previously thought.Peer reviewe