A novel inhibitor rescues cerebellar defects in a zebrafish model of Down syndrome-associated kinase Dyrk1A overexpression.

The highly conserved dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A) plays crucial roles during central nervous system development and homeostasis. Furthermore, its hyperactivity is considered responsible for some neurological defects in individuals with Down syndrome. We set out to establish a zebrafish model expressing human Dyrk1A that could be further used to characterize the interaction between Dyrk1A and neurological phenotypes. First, we revealed the prominent expression of dyrk1a homologs in cerebellar neurons in the zebrafish larval and adult brains. Overexpression of human dyrk1a in postmitotic cerebellar Purkinje neurons resulted in a structural misorganization of the Purkinje cells in cerebellar hemispheres and a compaction of this cell population. This impaired Purkinje cell organization was progressive, leading to an age-dependent dispersal of Purkinje neurons throughout the cerebellar molecular layer with larval swim deficits resulting in miscoordination of swimming and reduced exploratory behavior in aged adults. We also found that the structural misorganization of the larval Purkinje cell layer could be rescued by pharmacological treatment with Dyrk1A inhibitors. We further reveal the in vivo efficiency of a novel selective Dyrk1A inhibitor, KuFal194. These findings demonstrate that the zebrafish is a well-suited vertebrate organism to genetically model severe neurological diseases with single cell type specificity. Such models can be used to relate molecular malfunction to cellular deficits, impaired tissue formation, and organismal behavior and can also be used for pharmacological compound testing and validation.

Copy number variation of two separate regulatory regions upstream of SOX9 causes isolated 46,XY or 46,XX disorder of sex development.

BACKGROUND: SOX9 mutations cause the skeletal malformation syndrome campomelic dysplasia in combination with XY sex reversal. Studies in mice indicate that SOX9 acts as a testis-inducing transcription factor downstream of SRY, triggering Sertoli cell and testis differentiation. An SRY-dependent testis-specific enhancer for Sox9 has been identified only in mice. A previous study has implicated copy number variations (CNVs) of a 78 kb region 517-595 kb upstream of SOX9 in the aetiology of both 46,XY and 46,XX disorders of sex development (DSD). We wanted to better define this region for both disorders. RESULTS: By CNV analysis, we identified SOX9 upstream duplications in three cases of SRY-negative 46,XX DSD, which together with previously reported duplications define a 68 kb region, 516-584 kb upstream of SOX9, designated XXSR (XX sex reversal region). More importantly, we identified heterozygous deletions in four families with SRY-positive 46,XY DSD without skeletal phenotype, which define a 32.5 kb interval 607.1-639.6 kb upstream of SOX9, designated XY sex reversal region (XYSR). To localise the suspected testis-specific enhancer, XYSR subfragments were tested in cell transfection and transgenic experiments. While transgenic experiments remained inconclusive, a 1.9 kb SRY-responsive subfragment drove expression specifically in Sertoli-like cells. CONCLUSIONS: Our results indicate that isolated 46,XY and 46,XX DSD can be assigned to two separate regulatory regions, XYSR and XXSR, far upstream of SOX9. The 1.9 kb SRY-responsive subfragment from the XYSR might constitute the core of the Sertoli-cell enhancer of human SOX9, representing the so far missing link in the genetic cascade of male sex determination.

Zebrafish jam-b2 Gal4-enhancer trap line recapitulates endogenous jam-b2 expression in extraocular muscles.

BACKGROUND: Members of the junctional adhesion molecule (JAM) family function as cell adhesion molecules and cell surface receptors. The zebrafish genome contains six different jam genes, and jam-b and jam-c were shown to be essential for myoblast fusion during skeletal muscle development. However, little is known about jam-b2 expression and function. RESULTS: We isolated the cDNA of zebrafish jam-b2. jam-b2 is expressed specifically in extraocular muscles (EOMs), jaw muscles, and pectoral fins in zebrafish larvae, but not in trunk muscles. The identified jam-b2 expression pattern is supported by the analysis of a zebrafish Gal4-enhancer trap line, in which the coding sequence of the transcriptional activator KalTA4 together with a Gal4-dependent UAS-mCherry expression cassette was inserted into the jam-b2 locus. Intercrosses with an UAS:EGFP strain proves the possibility for targeting transgene expression to EOMs, jaw muscles and fins. Finally, we characterized the concerted contraction pattern of EOMs in larvae performing an optokinetic response. CONCLUSIONS: The expression pattern of jam-b2 suggests that it may contribute different properties to EOMs, jaw muscles, and pectoral fins. The jam-b2:KalTA4-UAS-mCherry transgenic strain serves a dual role as both a reporter for these muscles and as a valuable genetic tool for targeting transgene expression to EOMs.

A homeo-paired domain-binding motif directs Myf5 expression in progenitor cells of limb muscle.

Recruitment of multipotent mesodermal cells to the myogenic lineage is mediated by the transcription factor Myf5, the first of the myogenic regulatory factors to be expressed in most sites of myogenesis in the mouse embryo. Among numerous elements controlling the spatiotemporal pattern of Myf5 expression, the -58/-56 kb distal Myf5 enhancer directs expression in myogenic progenitor cells in limbs and in somites. Here, we show by site-directed mutagenesis within this enhancer that a predicted homeobox adjacent to a putative paired domain-binding site is required for the activity in muscle precursor cells in limbs and strongly contributes to expression in somites. By contrast, predicted binding sites for Tcf/Lef, Mef3 and Smad transcription factors play no apparent role for the expression in limbs but might participate in the control in somites. A 30mer oligonucleotide sequence containing and surrounding the homeo and paired domain-binding motifs directs faithful expression in myogenic cells in limbs and also enhances myotomal expression in somites. Pax3 and Meox2 transcription factors can bind to these consensus sites in vitro and therefore constitute potential regulators. However, genetic evidence in the Meox2-deficient mouse mutant argues against a role for Meox2 in the regulation of Myf5 expression. The data presented here demonstrate that a composite homeo and paired domain-binding motif within the -58/-56 enhancer is required and sufficient for activation of the Myf5 gene in muscle progenitor cells in the limb. Although Pax3 constitutes a potential cognate transcription factor for the enhancer, it fails to transactivate the site in transfection experiments.

Myf5 expression in somites and limb buds of mouse embryos is controlled by two distinct distal enhancer activities.

The initiation of skeletal muscle development in the mouse embryo is strictly associated with the expression of the muscle-specific transcription factor Myf5, the first of four myogenic regulatory factors (MRFs) to be expressed in muscle progenitors, and ablation of the Myf5 gene prevents myogenesis. The complex spatiotemporal expression pattern of Myf5 depends on many discrete regulatory elements that are dispersed over long distances throughout the gene locus. These multiple control modules act differently in the various muscle precursor populations, presumably in response to diverse signals that control myogenesis. A potent enhancer region regulating Myf5 expression in limb muscles and somites has been identified previously at -58/-48 kb upstream of the transcriptional start site (Hadchouel et al., 2000). Here, we focus on the physical and functional dissection of this control region. We demonstrate that a conserved sequence of 270 bp located around -57 kb is required and sufficient to drive Myf5 expression in limbs and to maintain it in somites. A second enhancer nearby is responsible for Myf5 transcription in occipital/cranial somites. This enhancer activity also directs expression accurately to the myotome, preventing ectopic expression in the dermomyotome during the second phase of Myf5 gene activation in somites. Our data suggest that the enhancer identified here collaborates with other somitic enhancers to ensure correct myotomal Myf5 expression. Moreover, it constitutes an important element that mediates somitic expression after the initial and transient Myf5 activation through a previously described sonic hedgehog-dependent early epaxial enhancer.

Dynamic expression of chicken cMeso2 in segmental plate and somites.

Abstract Somitogenesis in vertebrates involves prepatterning of paraxial mesoderm into somitomeres, establishing of anteroposterior polarity within somite primordia, and boundary formation between individual somites. cMeso2 is a newly identified chicken gene encoding a bHLH transcription factor, which is expressed in a transient stripe pattern in anterior presomitic mesoderm before segmentation of somites. The expression pattern overlaps with that of cMeso1 and correlates in time with the formation cycle of somites, suggesting that it may have a role in this process. Unlike its homologues in other organisms cMeso2 transcripts in chicken locate to the posterior aspects of somitomeres and constitute a marker for the caudal half of somites. Initiation of cMeso2 expression in presomitic mesoderm as well as its maintenance appears to be independent from influences by surrounding tissues, suggesting that it is part of the intrinsic program underlying segmentation. Although cMeso1 contains a C-terminal activator domain that can be transferred onto an independent DNA-binding domain, no evidence for such a transactivator domain can be found in cMeso2. In contrast, cMeso2 exerts transcriptional inhibition when coexpressed with the cMeso1 transactivator and seems to contain a repressor domain. Thus, cMeso1 and cMeso2 may function in an antagonistic manner during somitogenesis.