Modeling Bainbridge-Ropers Syndrome in Xenopus laevis Embryos.

The Additional sex combs-like (ASXL1-3) genes are linked to human neurodevelopmental disorders. The de novo truncating variants in ASXL1-3 proteins serve as the genetic basis for severe neurodevelopmental diseases such as Bohring-Opitz, Shashi-Pena, and Bainbridge-Ropers syndromes, respectively. The phenotypes of these syndromes are similar but not identical, and include dramatic craniofacial defects, microcephaly, developmental delay, and severe intellectual disability, with a loss of speech and language. Bainbridge-Ropers syndrome resulting from ASXL3 gene mutations also includes features of autism spectrum disorder. Human genomic studies also identified missense ASXL3 variants associated with autism spectrum disorder, but lacking more severe Bainbridge-Ropers syndromic features. While these findings strongly implicate ASXL3 in mammalian brain development, its functions are not clearly understood. ASXL3 protein is a component of the polycomb deubiquitinase complex that removes mono-ubiquitin from Histone H2A. Dynamic chromatin modifications play important roles in the specification of cell fates during early neural patterning and development. In this study, we utilize the frog, Xenopus laevis as a simpler and more accessible vertebrate neurodevelopmental model system to understand the embryological cause of Bainbridge-Ropers syndrome. We have found that ASXL3 protein knockdown during early embryo development highly perturbs neural cell fate specification, potentially resembling the Bainbridge-Ropers syndrome phenotype in humans. Thus, the frog embryo is a powerful tool for understanding the etiology of Bainbridge-Ropers syndrome in humans.

XenMine: A genomic interaction tool for the Xenopus community.

The Xenopus community has embraced recent advances in sequencing technology, resulting in the accumulation of numerous RNA-Seq and ChIP-Seq datasets. However, easily accessing and comparing datasets generated by multiple laboratories is challenging. Thus, we have created a central space to view, search and analyze data, providing essential information on gene expression changes and regulatory elements present in the genome. XenMine (www.xenmine.org) is a user-friendly website containing published genomic datasets from both Xenopus tropicalis and Xenopus laevis. We have established an analysis pipeline where all published datasets are uniformly processed with the latest genome releases. Information from these datasets can be extracted and compared using an array of pre-built or custom templates. With these search tools, users can easily extract sequences for all putative regulatory domains surrounding a gene of interest, identify the expression values of a gene of interest over developmental time, and analyze lists of genes for gene ontology terms and publications. Additionally, XenMine hosts an in-house genome browser that allows users to visualize all available ChIP-Seq data, extract specifically marked sequences, and aid in identifying important regulatory elements within the genome. Altogether, XenMine is an excellent tool for visualizing, accessing and querying analyzed datasets rapidly and efficiently.

FoxH1 mediates a Grg4 and Smad2 dependent transcriptional switch in Nodal signaling during Xenopus mesoderm development.

In the vertebrate blastula and gastrula the Nodal pathway is essential for formation of the primary germ layers and the organizer. Nodal autoregulatory feedback potentiates signaling activity, but mechanisms limiting embryonic Nodal ligand transcription are poorly understood. Here we describe a transcriptional switch mechanism mediated by FoxH1, the principle effector of Nodal autoregulation. FoxH1 contains a conserved engrailed homology (EH1) motif that mediates direct binding of groucho-related gene 4 (Grg4), a Groucho family corepressor. Nodal-dependent gene expression is suppressed by FoxH1, but enhanced by a FoxH1 EH1 mutant, indicating that the EH1 motif is necessary for repression. Grg4 blocks Nodal-induced mesodermal gene expression and Nodal autoregulation, suggesting that Grg4 limits Nodal pathway activity. Conversely, blocking Grg4 function in the ectoderm results in ectopic expression of Nodal target genes. FoxH1 and Grg4 occupy the Xnr1 enhancer, and Grg4 occupancy is dependent on the FoxH1 EH1 motif. Grg4 occupancy at the Xnr1 enhancer significantly decreases with Nodal activation or Smad2 overexpression, while FoxH1 occupancy is unaffected. These results suggest that Nodal-activated Smad2 physically displaces Grg4 from FoxH1, an essential feature of the transcriptional switch mechanism. In support of this model, when FoxH1 is unable to bind Smad2, Grg4 occupancy is maintained at the Xnr1 enhancer, even in the presence of Nodal signaling. Our findings reveal that FoxH1 mediates both activation and repression of Nodal gene expression. We propose that this transcriptional switch is essential to delimit Nodal pathway activity in vertebrate germ layer formation.

Transcriptional integration of Wnt and Nodal pathways in establishment of the Spemann organizer.

Signaling inputs from multiple pathways are essential for the establishment of distinct cell and tissue types in the embryo. Therefore, multiple signals must be integrated to activate gene expression and confer cell fate, but little is known about how this occurs at the level of target gene promoters. During early embryogenesis, Wnt and Nodal signals are required for formation of the Spemann organizer, which is essential for germ layer patterning and axis formation. Signaling by both Wnt and Nodal pathways is required for the expression of multiple organizer genes, suggesting that integration of these signals is required for organizer formation. Here, we demonstrate transcriptional cooperation between the Wnt and Nodal pathways in the activation of the organizer genes Goosecoid (Gsc), Cerberus (Cer), and Chordin (Chd). Combined Wnt and Nodal signaling synergistically activates transcription of these organizer genes. Effectors of both pathways occupy the Gsc, Cer and Chd promoters and effector occupancy is enhanced with active Wnt and Nodal signaling. This suggests that, at organizer gene promoters, a stable transcriptional complex containing effectors of both pathways forms in response to combined Wnt and Nodal signaling. Consistent with this idea, the histone acetyltransferase p300 is recruited to organizer promoters in a Wnt and Nodal effector-dependent manner. Taken together, these results offer a mechanism for spatial and temporal restriction of organizer gene transcription by the integration of two major signaling pathways, thus establishing the Spemann organizer domain.

Siamois and Twin are redundant and essential in formation of the Spemann organizer.

The Spemann organizer is an essential signaling center in Xenopus germ layer patterning and axis formation. Organizer formation occurs in dorsal blastomeres receiving both maternal Wnt and zygotic Nodal signals. In response to stabilized ßcatenin, dorsal blastomeres express the closely related transcriptional activators, Siamois (Sia) and Twin (Twn), members of the paired homeobox family. Sia and Twn induce organizer formation and expression of organizer-specific genes, including Goosecoid (Gsc). In spite of the similarity of Sia and Twn sequence and expression pattern, it is unclear whether these factors function equivalently in promoter binding and subsequent transcriptional activation, or if Sia and Twn are required for all aspects of organizer function. Here we report that Sia and Twn activate Gsc transcription by directly binding to a conserved P3 site within the Wnt-responsive proximal element of the Gsc promoter. Sia and Twn form homodimers and heterodimers by direct homeodomain interaction and dimer forms are indistinguishable in both DNA-binding and activation functions. Sequential chromatin immunoprecipitation reveals that the endogenous Gsc promoter can be occupied by either Sia or Twn homodimers or Sia-Twn heterodimers. Knockdown of Sia and Twn together, but not individually, results in a failure of organizer gene expression and a disruption of axis formation, consistent with a redundant role for Sia and Twn in organizer formation. Furthermore, simultaneous knockdown of Sia and Twn blocks axis induction in response to ectopic Wnt signaling, demonstrating an essential role for Sia and Twn in mediating the transcriptional response to the maternal Wnt pathway. The results demonstrate the functional redundancy of Sia and Twn and their essential role in direct transcriptional responses necessary for Spemann organizer formation.

Chromatin immunoprecipitation in early Xenopus laevis embryos.

Chromatin immunoprecipitation (ChIP) is a powerful method for analyzing the interaction of regulatory proteins with genomic loci, but has been difficult to apply to studies on early embryos due to the limiting amount of genomic material in these samples. Here, we present a comprehensive technique for performing ChIP on blastula and gastrula stage Xenopus embryos. We also describe methods for optimizing crosslinking and chromatin shearing, verifying antibody specificity, maximizing PCR sensitivity, and quantifying PCR results, allowing for the use of as few as 50 early blastula stage embryos (approximately 5x10(4) cells) per experimental condition. Finally, we demonstrate the predicted binding of endogenous beta-catenin to the nodal-related 6 promoter, binding of tagged Fast-1/FoxH1 to the goosecoid promoter, and binding of tagged Tcf3 to the siamois and nodal-related 6 promoters as examples of the potential application of ChIP to embryological investigations. Developmental Dynamics 238:1422-1432, 2009. (c) 2009 Wiley-Liss, Inc.