Darwin S. Dichmann

The Genome of the Western Clawed Frog Xenopus tropicalis

Frog Genome The African clawed frog Xenopus tropicalis is the first amphibian to have its genome sequenced. Hellsten et al. (p. 633, see the cover) present an analysis of a draft assembly of the genome. The genome of the frog, which is an important model system for developmental biology, encodes over 20,000 protein-coding genes, of which more than 1700 genes have identified human disease associations. Detailed comparison of the content of protein-coding genes with other tetrapods—human and chicken—reveals extensive shared synteny, occasionally spanning entire chromosomes. Assembly, annotation, and analysis of the frog genome compares gene content and synteny with the human and chicken genomes. The western clawed frog Xenopus tropicalis is an important model for vertebrate development that combines experimental advantages of the African clawed frog Xenopus laevis with more tractable genetics. Here we present a draft genome sequence assembly of X. tropicalis. This genome encodes more than 20,000 protein-coding genes, including orthologs of at least 1700 human disease genes. Over 1 million expressed sequence tags validated the annotation. More than one-third of the genome consists of transposable elements, with unusually prevalent DNA transposons. Like that of other tetrapods, the genome of X. tropicalis contains gene deserts enriched for conserved noncoding elements. The genome exhibits substantial shared synteny with human and chicken over major parts of large chromosomes, broken by lineage-specific chromosome fusions and fissions, mainly in the mammalian lineage.

Genome evolution in the allotetraploid frog Xenopus laevis

To explore the origins and consequences of tetraploidy in the African clawed frog, we sequenced the Xenopus laevis genome and compared it to the related diploid X. tropicalis genome. We characterize the allotetraploid origin of X. laevis by partitioning its genome into two homoeologous subgenomes, marked by distinct families of ‘fossil’ transposable elements. On the basis of the activity of these elements and the age of hundreds of unitary pseudogenes, we estimate that the two diploid progenitor species diverged around 34 million years ago (Ma) and combined to form an allotetraploid around 17–18 Ma. More than 56% of all genes were retained in two homoeologous copies. Protein function, gene expression, and the amount of conserved flanking sequence all correlate with retention rates. The subgenomes have evolved asymmetrically, with one chromosome set more often preserving the ancestral state and the other experiencing more gene loss, deletion, rearrangement, and reduced gene expression.

Expression patterns of Wnts, Frizzleds, sFRPs, and misexpression in transgenic mice suggesting a role for Wnts in pancreas and foregut pattern formation.

It is well established that gut and pancreas development depend on epithelial‐mesenchymal interactions. We show here that several Wnt, Frizzled, and secreted frizzled‐related protein (sFRP) encoding mRNAs are present during mouse pancreatic morphogenesis. Wnt5a and 7b mRNA is broadly expressed in foregut mesenchyme starting around embryonic day 10 in mice. Other members expressed are Wnt2b, Wnt5b, and Wnt11. In addition, genes for the Wnt receptors, Frizzled2, 3, 4, 5, 6, 7, 8, and 9 are expressed. To understand potential Wnt functions in pancreas and foregut development in vivo, we analyzed transgenic F0 mouse fetuses expressing Wnt1 and 5a cDNAs under control of the PDX‐1 gene promoter. In PDX‐Wnt1 fetuses, the foregut region normally comprising the proximal duodenum instead resembles a posterior extension of the stomach, often associated with complete pancreatic and splenic agenesis. Furthermore, the boundary between expression domains of gastric and duodenal markers is shifted in a posterior direction. In PDX‐Wnt5a fetuses, several structures derived from the proximal foregut are reduced in size, including the pancreas, spleen, and stomach, without any apparent shift in the stomach to duodenum transition. In these fetuses, overall pancreatic morphology is changed and the pancreatic epithelium is dense and compact, consistent with Wnt5A effects on cell movements and/or attachment. Taken together, these results suggest that Wnt genes participate in epithelial‐mesenchymal signaling and may specify region identity in the anterior foregut.

Expression and misexpression of members of the FGF and TGFβ families of growth factors in the developing mouse pancreas

We have performed a high‐capacity, semiquantitative, reverse transcriptase‐polymerase chain reaction screen for expression of fibroblast growth factor (FGF) and transforming growth factor β (TGFβ) family genes as well as their cognate receptors. By using cDNA prepared from embryonic day 12 to postnatal day 0 embryonic mouse pancreas, we have identified several factors potentially involved in the development of the endocrine pancreas. We find high‐level early expression of TGFβ‐1 and ‐2, and constitutive expression of TGFβ‐3 and their receptors. Of the Inhibin/Activin members, we found exclusively Inhibin‐α and Activin‐βB to be expressed, and the BMP family was represented by BMP4, BMP5, and BMP7. The predominant forms of the BMP and Activin type II receptors were ActR‐IIB and BMPR‐II and of the type I receptors, BMPR‐1A and ‐1B were the highest expressed. FGF1, FGF7, FGF9, FGF10, FGF11, and FGF18 were also expressed in the pancreas at varying time points and levels, as well as FGF receptor forms FGFR1b, FGFR1c, FGFR2b, FGFR2c, FGFR3b, and FGFR4. To gain insight into the biological function, we misexpressed members of these families in the pancreas by using the early pancreas promoter Pdx1. Misexpression of FGF4 results in disruption of the pancreas morphology with epithelial structures interspersed in stroma tissue. The endocrine compartment was reduced to scattered single cells, and the exocrine consisted of unbranched ductal epithelia with acinar structures budding off. In contrast, misexpression of BMP‐6 resulted in complete agenesis of the pancreas and reduced the size of the stomach and spleen dramatically and caused fusion of the liver and duodenum. Developmental Dynamics 226:663–674, 2003.

Analysis of pancreatic endocrine development in GDF11-deficient mice.

Here, we examine the role of GDF11 in pancreatic development. Using in situ hybridization and reverse transcriptase‐polymerase chain reaction analyses, we show that Gdf11 transcripts are expressed in embryonic pancreas epithelium before the secondary transition but decrease rapidly afterward. To determine the function of GDF11 during pancreas development, we analyzed Gdf11−/− mouse embryos. In such embryos, pancreas size is twofold reduced at embryonic day (E) 18 compared with wild‐type littermates. Quantification of the different tissue compartments shows a specific hypoplasia of the exocrine compartment, while the endocrine and ductal compartments are unaffected. Notably, NGN3+ endocrine precursor cells are increased fourfold at E18, although the amount of endocrine cells in the pancreas of these animals is unchanged compared with wild‐type littermates. Similarly, the maturation of endocrine cells as well as the ratio between α‐ and β‐cells appears normal. Developmental Dynamics 235:3016–3025, 2006.

fus/TLS orchestrates splicing of developmental regulators during gastrulation

Here we investigated the function of the atypical RNA-binding protein fus/TLS (fused in sarcoma/translocated in sarcoma) during early frog development. We found that fus is necessary for proper mRNA splicing of a set of developmental regulatory genes during early frog development and gastrulation. Upon fus knockdown, embryos fail to gastrulate and show mesodermal differentiation defects that we connect to intron retention in fgf8 (fibroblast growth factor 8) and fgfr2 (fgf receptor 2) transcripts. During gastrulation, the animal and marginal regions dissociate, and we show that this is caused, at least in part, by intron retention in cdh1 transcripts. We confirm the specificity of splicing defects at a genomic level using analysis of RNA sequencing (RNA-seq) and show that 3%-5% of all transcripts display intron retention throughout the pre-mRNA. By analyzing gene ontology slim annotations, we show that the affected genes are enriched for developmental regulators and therefore represent a biologically coherent set of targets for fus regulation in embryogenesis. This shows that fus is central to embryogenesis and may provide information on its function in neurodegenerative disease.

Expression cloning in Xenopus identifies RNA-binding proteins as regulators of embryogenesis and Rbmx as necessary for neural and muscle development.

We have performed an expression cloning screen in Xenopus laevis with the aim of isolating novel gene activities from the neural plate. Of 8,064 clones screened, we isolated 61 clones that affected either neural plate patterning or tadpole morphology. Of these, 20 clones encoded RNA binding proteins, and the majority of these are heterogeneous nuclear ribonucleoproteins (hnRNPs) or SR‐proteins, which are associated with alternative splicing. All of these genes are expressed in the nervous system, and in several cases specific to neural tissue. Injecting mRNA encoding these proteins results in neural plate mispatterning and abnormal muscle segmentation. To initiate characterization of these proteins, we selected Rbmx as a candidate for deeper analysis. Using morpholino mediated knockdown, we show that Rbmx is necessary for normal anterior neural plate patterning, neurogenesis, neural crest development, and muscle segmentation. Developmental Dynamics 237:1755–1766, 2008.

Nkx6 genes pattern the frog neural plate and Nkx6.1 is necessary for motoneuron axon projection.

Neuronal subtypes originate from an undifferentiated neural epithelium that is progressively divided into progenitor domains by homeodomain transcription factors such as members of the Nkx family. Here we report the functional analysis of Nkx6.1 and Nkx6.2 in Xenopus. While Nkx6.2 is expressed early in a large region of the medial neural plate, Nkx6.1 is restricted to a region overlapping with the region of motor neuron formation. By mRNA injection we show that both can inhibit primary neurogenesis as well as expression of intermediate neural plate markers. However, they do not form auto-regulatory loops and fail to induce ectopic motor neurons as they do in the chick. Using morpholino-mediated knockdown in Xenopus laevis and Xenopus tropicalis we show that Nkx6.1 knockdown results in paralyzed tadpoles. Using DiI labeling and immunohistochemistry we show that the underlying mechanism is a failure of spinal motor neurons to extend axons to their targets. Analysis of neural pattern reveals that ventral Lhx3+ and Pax2+ interneurons are dependent on Nkx6.1 function, but overall neural patterning is not. This study illustrates that while important aspects of Nkx6 gene function are conserved in vertebrate neural patterning, others are not.

Unspecific Labeling of Pancreatic Islets by Antisera Against Fibroblast Growth Factors and Their Receptors

Six distinct fibroblast growth factors (FGF5) have been detected in pancreatic islets by immunohistochemistry (IHC) using commercially available antisera. We show here that these antisera are useful for Western blotting but that only two are suited for IHC. By Western blotting, these antisera detect recombinant FGFs. Detection can be eliminated by preabsorption with immunizing peptide but not with irrelevant peptide. By IHC we find specific labeling of islets with anti-FGF1 and anti-FGF2 antisera. Labeling can be abolished by preabsorption with the immunizing peptides. In contrast, prominent staining of islets by anti-FGF4, −FGF5, −FGF7, and −FGF10 antisera is unspecific because the staining cannot be competed by preabsorption with the immunizing peptides.

The Alternative Splicing Regulator Tra2b Is Required for Somitogenesis and Regulates Splicing of an Inhibitory Wnt11b Isoform

Alternative splicing is pervasive in vertebrates, yet little is known about most isoforms or their regulation. transformer-2b (tra2b) encodes a splicing regulator whose endogenous function is poorly understood. Tra2b knockdown in Xenopus results in embryos with multiple defects, including defective somitogenesis. Using RNA sequencing, we identify 142 splice changes (mostly intron retention and exon skipping), 89% of which are not in current annotations. A previously undescribed isoform of wnt11b retains the last intron, resulting in a truncated ligand (Wnt11b-short). We show that this isoform acts as a dominant-negative ligand in cardiac gene induction and pronephric tubule formation. To determine the contribution of Wnt11b-short to the tra2b phenotype, we induce retention of intron 4 in wnt11b, which recapitulates the failure to form somites but not other tra2b morphant defects. This alternative splicing of a Wnt ligand adds intricacy to a complex signaling pathway and highlights intron retention as a regulatory mechanism.