Xenopus Rnd1 and Rnd3 GTP‐binding proteins are expressed under the control of segmentation clock and required for somite formation

The process of segmentation in vertebrates is described by a clock and wavefront model consisting of a Notch signal and an fibroblast growth factor‐8 (FGF8) gradient, respectively. To further investigate the segmentation process, we screened gene expression profiles for downstream targets of the segmentation clock. The Rnd1 and Rnd3 GTP‐binding proteins comprise a subgroup of the Rho GTPase family that show a specific expression pattern similar to the Notch signal component ESR5, suggesting an association between Rnd1/3 and the segmentation clock. Rnd1/3 expression patterns are disrupted by overexpression of dominant‐negative or active forms of Notch signaling genes, and responds to the FGF inhibitor SU5402 by a posterior shift analogous to other segmentation‐related genes, suggesting that Rnd1/3 expressions are regulated by the segmentation clock machinery. We also show that antisense morpholino oligonucleotides to Rnd1/3 inhibit somite segmentation and differentiation in Xenopus embryos. These results suggest that Rnd1/3 are required for Xenopus somitogenesis. Developmental Dynamics 238:2867–2876, 2009. © 2009 Wiley‐Liss, Inc.     

The functions of maternal Dishevelled 2 and 3 in the Early Xenopus embryo

Of the three Dishevelled (Dvl) genes, only Dvl2 and Dvl3 are maternally encoded in the frog, Xenopus laevis. We show here by loss of function analysis that single depletion of either Dvl2 or Dvl3 from the oocyte causes the same embryonic phenotype. We find that the effects of loss of function of Dvl2 and 3 together are additive, and that the proteins physically interact, suggesting that both are required in the same complex. We show that maternal Dvl2 and 3 are required for convergence extension movements downstream of the dorsally localized signaling pathway activated by Xnr3, but not downstream of the pathway activated by activin. Also, depletion of maternal Dvl2 and 3 mRNAs causes the up‐regulation of a subset of zygotic ectodermal genes, including Foxi1e, with surprisingly no significant effect on the canonical Wnt direct target genes Siamois and Xnr3. We suggest that the likely reason for continued expression of the Wnt target genes in Dvl2/3‐depleted embryos is that maternal Dvl mRNA depletion is insufficient to deplete stored punctae of Dvl protein in the oocyte cortex, which may transduce dorsal signaling after fertilization. Developmental Dynamics 240:1727–1736, 2011. © 2011 Wiley‐Liss, Inc.     

Developmental expression of sideroflexin family genes in Xenopus embryos

Emerging evidence indicates that mitochondrial carriers are not only crucial for metabolism, but also important for embryonic development. Sideroflexin is a novel family of mitochondrial tricarboxylate carrier proteins, of which the in vivo function is largely unknown. Here, we report on the expression patterns of five sideroflexin genes in Xenopus embryos. Whole‐mount in situ hybridization analysis reveals that while sideroflexin2 is expressed in the pancreas, sideroflexin1 and 3 display a complex expression in the central nervous system, somites, pronephros, liver, and pancreas. In contrast, only a weak expression of sideroflexin4 and 5 was detected in embryonic brain. Taken together, the five sideroflexin genes show both overlapping and nonoverlapping expression during Xenopus embryogenesis. As the primary structures of the five sideroflexin proteins are also quite similar, their functional redundancy should be taken into consideration for gene targeting studies. Developmental Dynamics 239:2742–2747, 2010. © 2010 Wiley‐Liss, Inc.     

Cis‐regulatory mechanisms of gene expression in an olfactory neuron type in Caenorhabditis elegans

The generation of cellular diversity is dependent on the precise spatiotemporal regulation of gene expression by both cis‐ and trans‐acting mechanisms. The developmental principles regulating expression of specific gene subsets in individual cell types are not fully understood. Here we define the cis‐regulatory mechanisms driving expression of cell‐selective and broadly expressed genes in vivo in the AWB olfactory neuron subtype in C. elegans. We identify an element that is necessary to drive expression of neuron‐selective chemoreceptor genes in the AWB neurons, and show that this element functions in a context‐dependent manner. We find that the expression of broadly expressed sensory neuronal genes in the AWB neurons is regulated by diverse cis‐ and trans‐regulatory mechanisms that act partly in parallel to the pathways governing expression of AWB‐selective genes. We further demonstrate that cis‐acting mechanisms driving gene expression in the AWB neurons appear to have diverged in related nematode species. Our results provide insights into the cis‐regulatory logic driving cell‐specific gene expression, and suggest that variations in this logic contribute to the generation of functional diversity. Developmental Dynamics 238:3080–3092, 2009. © 2009 Wiley‐Liss, Inc.     

Manic fringe is not required for embryonic development,and fringe family members do not exhibit redundant functions in the axial skeleton,limb, or hindbrain

Tight regulation of Notch pathway signaling is important in many aspects of embryonic development. Notch signaling can be modulated by expression of fringe genes, encoding glycosyltransferases that modify EGF repeats in the Notch receptor. Although Lunatic fringe (Lfng) has been shown to play important roles in vertebrate segmentation, comparatively little is known regarding the developmental functions of the other vertebrate fringe genes, Radical fringe (Rfng) and Manic fringe (Mfng). Here we report that Mfng expression is not required for embryonic development. Further, we find that despite significant overlap in expression patterns, we detect no obvious synergistic defects in mice in the absence of two, or all three, fringe genes during development of the axial skeleton, limbs, hindbrain, and cranial nerves. Developmental Dynamics 238:1803–1812, 2009. © 2009 Wiley‐Liss, Inc.     

Sebox regulates mesoderm formation in early amphibian embryos

Background: Mix/Bix genes are important regulators of mesendoderm formation during vertebrate embryogenesis. Sebox, an additional member of this gene family, has been implicated in endoderm formation during early embryogenesis in zebrafish. However, it remains unclear whether Sebox plays a unique role in early Xenopus embryos. Results: In this study, we provide evidence that Sebox is uniquely required for the formation of mesoderm during early Xenopus embryogenesis. Sebox is dynamically expressed in the involuted mesoderm during gastrulation. It is activated by Nodal/Activin signaling and modulated by zygotic Wnt/β‐catenin signaling. Overexpression of Sebox perturbs movements during convergent extension and inhibits the expression of mesodermal, but not endodermal, genes induced by Nodal/Activin signaling. Depletion of Sebox using a specific morpholino increases the expression of noncanonical wnt5a, wnt5b, and wnt11b. Depletion of Sebox also up‐regulates the expression of pcdh8.2, a paraxial mesoderm‐specific protocadherin, in a Wnt11B‐dependent manner. Sebox morphants display reduced development of the head and notochord. Conclusions: Our findings illustrate that Sebox, a unique member of the Mix/Bix gene family, functions downstream of Nodal/Activin signaling and is required for the proper expression of noncanonical Wnt ligands and the normal development of mesoderm in Xenopus. Developmental Dynamics 244:1415–1426, 2015. © 2015 Wiley Periodicals, Inc.     

Gene expression analysis identifies over‐expression of CXCL1, SPARC,SPP1, and SULF1 in gastric cancer

To elucidate gene expression signatures associated with gastric carcinogenesis, we performed a genome‐wide expression analysis of 46 Finnish and 20 Japanese gastric tissues. Comparative analysis between Finnish and Japanese datasets identified 58 common genes that were differentially expressed between cancerous and non‐neoplastic gastric tissues. Twenty‐six of these genes were up‐regulated in cancer and 32 down‐regulated. Of these genes, 64% were also differentially expressed in another unrelated publicly available dataset. The expression levels of four of the up‐regulated genes, CXCL1, SPARC, SPP1 and SULF, were further analyzed in 82 gastric tissues using quantitative real‐time RT‐PCR. This analysis validated the results from the microarray analysis as the expression of these four genes was significantly higher in the cancerous tissue compared with the normal tissue (fold change 3.4–8.9). Over‐expression of CXCL1 also positively correlated with improved survival. To conclude, irrespective of the microarray platform or patient population, a common gastric cancer gene expression signature of 58 genes, including CXCL1, SPARC, SPP1, and SULF, was identified. These genes represent potential biomarkers for gastric cancer. © 2009 Wiley‐Liss, Inc.     

Sequence and expression of the zebrafish alpha‐actinin gene family reveals conservation and diversification among vertebrates

alpha‐actinins are actin microfilament crosslinking proteins. Vertebrate actinins fall into two classes: the broadly‐expressed actinins 1 and 4 (actn1 and actn4) and muscle‐specific actinins, actn2 and actn3. Members of this family have numerous roles, including regulation of cell adhesion, cell differentiation, directed cell motility, intracellular signaling, and stabilization of f‐actin at the sarcomeric Z‐line in muscle. Here we identify five zebrafish actinin genes including two paralogs of ACTN3. We describe the temporal and spatial expression patterns of these genes through embryonic development. All zebrafish actinin genes have unique expression profiles, indicating specialization of each gene. In particular, the muscle actinins display preferential expression in different domains of axial, pharyngeal, and cranial musculature. There is no identified avian actn3 and approximately 16% of humans are null for ACTN3. Duplication of actn3 in the zebrafish indicates that variation in actn3 expression may promote physiological diversity in muscle function among vertebrates. Developmental Dynamics 238:2936–2947, 2009. © 2009 Wiley‐Liss, Inc.     

Duplicated major histocompatibility complex class II genes in the tongue sole (Cynoglossus semilaevis)

The major histocompatibility complex (MHC) molecule plays an important role in the vertebrate immune system. However, we have a limited understanding of the MHC genomic structure in teleosts. Using gene cloning and family analysis, we isolate the MHC class II genes in the tongue sole (Cynoglossus semilaevis) and find that both class II A and class II B genes are duplicated (named Cyse‐DAA and Cyse‐DBA, Cyse‐DAB and Cyse‐DBB, respectively). The class II A genes consist of four exons with a highly conserved genomic structure, but each gene has unique and defining exon 2 and intron 2 sequences. The class II B genes have a conserved six‐exon genomic structure, with intron 3 splitting the β2 encoding region into two exons. Each class II B gene has unique variations in exon 2 and intron 1 sequences. The two class II A genes have similar expression patterns among tissues, with high levels in spleen and gill. Both class II B genes have similar patterns, with high expression in spleen, gill and intestine. The alleles of MHC class II have wide distribution and reliable inheritance in the families analysed. This indicates that the duplicated MHC genes are all classical class II genes. The class II gene duplication with divergent exon and intron sequences, but similar expression patterns in tongue sole provides new insights into MHC evolution.     

The Prx1 limb enhancers: Targeted gene expression in developing zebrafish pectoral fins

Limbs represent an excellent model to study the induction, growth, and patterning of several organs. A breakthrough to study gene function in various tissues has been the characterization of regulatory elements that allow tissue‐specific interference of gene function. The mouse Prx1 promoter has been used to generate limb‐specific mutants and overexpress genes in tetrapod limbs. Although zebrafish possess advantages that favor their use to study limb morphogenesis, there is no driver described suitable for specifically interfering with gene function in developing fins. We report the generation of zebrafish lines that express enhanced green fluorescent protein (EGFP) driven by the mouse Prx1 enhancer in developing pectoral fins. We also describe the expression pattern of the zebrafish prrx1 genes and identify three c onserved n on‐coding e lements (CNEs) that we use to generate fin‐specific EGFP reporter lines. Finally, we show that the mouse and zebrafish regulatory elements may be used to modify gene function in pectoral fins. Developmental Dynamics 240:1977–1988, 2011. © 2011 Wiley‐Liss, Inc.     

Characterization of three synuclein genes in Xenopus laevis

The synuclein family consists of three small intracellular proteins mainly expressed in neural tissues, and has been associated with human neurodegenerative diseases. We have examined the spatial and temporal expression patterns of three synuclein genes during embryogenesis of Xenopus laevis. The Xenopus synucleins were firstly expressed in the developing nervous system at the tail bud stages. At tadpole stages, Xenopus snca was expressed in the brain, branchial arch and somite, and sncbb signals were detected in entire brain and spinal cord. However, sncg was only expressed in the peripheral nervous system including trigeminal nerve and dorsal root ganglion. RT‐PCR indicated that expression of synucleins was up‐regulated at the end of neurulation, and then maintained at later examined stages. Our study provides the spatiotemporal expression patterns of the synuclein family genes in Xenopus embryos, and forms a basis for further functional analysis of synucleins. Developmental Dynamics 240:2028–2033, 2011. © 2011 Wiley‐Liss, Inc.