Scube2 mediates Hedgehog signalling in the zebrafish embryo

The Hedgehog family of secreted morphogens specifies the fate of a large number of different cell types within invertebrate and vertebrate embryos, including the muscle cell precursors of the embryonic myotome of zebrafish. Formation of Hedgehog-sensitive muscle fates is disrupted within homozygous zebrafish mutants of the "you"-type class, the majority of which disrupt components of the Hedgehog (HH) signal transduction pathway. We have undertaken a phenotypic and molecular characterisation of one of these mutants, you, which we show results from mutations within the zebrafish orthologue of the mammalian gene scube2. This gene encodes a member of the Scube family of proteins, which is characterised by several protein motifs including EGF and CUB domains. Epistatic and molecular analyses position Scube2 function upstream of Smoothened (Smoh), the signalling component of the HH receptor complex, suggesting that Scube2 may act during HH signal transduction prior to, or during, receipt of the HH signal at the plasma membrane. In support of this model we show that scube2 has homology to cubilin, which encodes an endocytic receptor involved in protein trafficking suggesting a possible mode of function for Scube2 during HH signal transduction.     

SCUBE3 (Signal Peptide-CUB-EGF Domain-containing Protein 3) Modulates Fibroblast Growth Factor Signaling during Fast Muscle Development

SCUBE3 (signal peptide CUB-EGF-like domain-containing protein 3) belongs to a newly identified secreted and cell membrane-associated SCUBE family, which is evolutionarily conserved in vertebrates. Scube3 is predominantly expressed in a variety of developing tissues in mice such as somites, neural tubes, and limb buds. However, its function during development remains unclear. In this study, we first showed that knockdown of SCUBE3 in C2C12 myoblasts inhibited FGF receptor 4 expression and FGF signaling, thus resulting in reduced myogenic differentiation. Furthermore, knockdown of zebrafish scube3 by antisense morpholino oligonucleotides specifically suppressed the expression of the myogenic marker myod1 within the lateral fast muscle precursors, whereas its expression in the adaxial slow muscle precursors was largely unaffected. Consistent with these findings, immunofluorescent staining of fast but not slow muscle myosin was markedly decreased in scube3 morphants. Further genetic studies identified fgf8 as a key regulator in scube3-mediated fast muscle differentiation in zebrafish. Biochemical and molecular analysis showed that SCUBE3 acts as a FGF co-receptor to augment FGF8 signaling. Scube3 may be a critical upstream regulator of fast fiber myogenesis by modulating fgf8 signaling during zebrafish embryogenesis.     

Zebrafish scube1 (Signal Peptide-CUB (Complement Protein C1r/C1s,Uegf, and Bmp1)-EGF (Epidermal Growth Factor) Domain-containing Protein 1) Is Involved in Primitive Hematopoiesis

scube1 (signal peptide-CUB (complement protein C1r/C1s, Uegf, and Bmp1)-EGF domain-containing protein 1), the founding member of a novel secreted and cell surface SCUBE protein family, is expressed predominantly in various developing tissues in mice. However, its function in primitive hematopoiesis remains unknown. In this study, we identified and characterized zebrafish scube1 and analyzed its function by injecting antisense morpholino-oligonucleotide into embryos. Whole-mount in situ hybridization revealed that zebrafish scube1 mRNA is maternally expressed and widely distributed during early embryonic development. Knockdown of scube1 by morpholino-oligonucleotide down-regulated the expression of marker genes associated with early primitive hematopoietic precursors (scl) and erythroid (gata1 and hbbe1), as well as early (pu.1) and late (mpo and l-plastin) myelomonocytic lineages. However, the expression of an early endothelial marker fli1a and vascular morphogenesis appeared normal in scube1 morphants. Overexpression of bone morphogenetic protein (bmp) rescued the expression of scl in the posterior lateral mesoderm during early primitive hematopoiesis in scube1 morphants. Biochemical and molecular analysis revealed that Scube1 could be a BMP co-receptor to augment BMP signaling. Our results suggest that scube1 is critical for and functions at the top of the regulatory hierarchy of primitive hematopoiesis by modulating BMP activity during zebrafish embryogenesis.     

The zebrafish-secreted matrix protein you/scube2 is implicated in long-range regulation of hedgehog signaling

The Hedgehog (Hh) signal plays a pivotal role in induction of ventral neuronal and muscle cell types around the midline during vertebrate development [1]. We report that the gene disrupted in zebrafish you mutants, in which Hh signaling is impaired, encodes the secreted matrix protein Scube2. Consistently, epistasis analyses suggested that Scube2 functions upstream of Hh ligands or through a parallel pathway. In addition, overexpression analyses suggested that Scube2 is an essential, but a permissive, mediator of Hh signaling in zebrafish embryos. Surprisingly, the you gene is expressed in the dorsal neural tube, raising the possibility that Scube2 could indirectly act via a long-range regulator of Hh signaling. The dorsal Bmps have a long-range and opposing influence on Hh signaling [2-5]. We show that neural plate patterning is affected in you mutants in a way that is consistent with the aberrant long-range action of a Bmp-dependent signal. We further show that Bmp activity can be attenuated by the coexpression of Scube2. Our data support the idea that Scube2 can modulate the long-range action of Bmp-dependent signaling in the neural tube and somites.     

Wnt to notch relay signaling induces definitive hematopoiesis

The molecular mechanisms specifying hematopoietic stem cells (HSCs) in the vertebrate embryo remain poorly understood. Recently in Nature, Traver and colleagues demonstrate that timed wnt to Notch relay signaling across multiple cell types serves as an early upstream mechanism of HSC induction in zebrafish (Clements et?al., 2011).     

Zebrafish inositol polyphosphate kinases: New effectors of cilia and developmental signaling

We described here our recent findings that Ipk1 catalyzed production of IP₆ regulates LR-axis specification (Sarmah et al., 2005) and that IP₆ is an essential effector of ciliary beating and length maintenance in zebrafish (Sarmah et al., 2007). We have also uncovered a novel role for the IP-kinase IP₆k2 in craniofacial development, neural crest cell migration, and hedgehog signal transduction (B.S. and S.R.W., unpublished). Together, these findings place IP production as a key mediator for cellular signaling mechanisms that regulate vital cellular and developmental processes. How these and other IPs are integrated with cell–cell signaling networks during complex processes, such as, tissue morphogenesis and maintenance of cell fate and function? We propose that with its enormous resource and unique set of structural, functional, and sensory attributes, cilium provides a platform for executing IP-based signaling functions. Given the evolutionary conservation of the IP repertoire and pathways, the developmental and molecular events uncovered in our studies in the zebrafish system could be applicable in other vertebrates including humans. This unbiased approach of systematic identification of IP functions in cilia and development will aid in understanding of multiple disease pathologies including ciliopathies and dysmorphic syndromes.     

All roads lead to FoxO

Stress-activated kinases control metabolism by antagonizing the early steps of insulin signal transduction. Two papers now demonstrate that Jnk, the prototypical stress-activated kinase, controls life span in Drosophila and C. elegans by promoting phosphorylation of the forkhead protein FoxO (Oh et al., 2005; Wang et al., 2005). The findings provide yet another mechanism by which metabolic and stress responses are integrated via phosphorylation of FoxO proteins.     

Subcellular localization of Hairless protein shows a major focus of activity within the nucleus

Hairless, a major antagonist of the Notch signaling-pathway in Drosophila (Bang and Posakony, 1992; Maier et al., 1992), associates with Suppressor of Hairless [Su(H)], thereby inhibiting trans-activation of Notch target genes (Brou et al., 1994). These molecular interactions could occur either at the step of signal transduction in the cytoplasm or during implementation of the signal within the nucleus. We examined the subcellular distribution of Hairless, showing that it is a low abundant, ubiquitous protein that is cytosolic as well as nuclear. High levels of Hairless cause nuclear retention of Su(H), loss of Hairless reduces the amount of Su(H) in the nucleus.     

Differential effects of mutations in NS4B on West Nile virus replication and inhibition of interferon signaling

West Nile virus (WNV) is a human pathogen that can cause symptomatic infections associated with meningitis and encephalitis. Previously, we demonstrated that replication of WNV inhibits the interferon (IFN) signal transduction pathway by preventing the accumulation of phosphorylated Janus kinase 1 (JAK1) and tyrosine kinase 2 (Tyk2) (J. T. Guo et al., J. Virol. 79:1343-1350, 2005). Through a genetic analysis, we have now identified a determinant on the nonstructural protein 4B (NS4B) that controls IFN resistance in HeLa cells expressing subgenomic WNV replicons lacking the structural genes. However, in the context of infectious genomes, the same determinant did not influence IFN signaling. Thus, our results indicate that NS4B may be sufficient to inhibit the IFN response in replicon cells and suggest a role for structural genes, or as yet unknown interactions, in the inhibition of the IFN signaling pathway during WNV infections.     

A novel role for Greatwall kinase in recovery from DNA damage

Activation of the DNA damage response (DDR) is critical for genomic integrity and tumor suppression. The occurrence of DNA damage quickly evokes the DDR through ATM/ATR-dependent signal transduction, which promotes DNA repair and activates the checkpoint to halt cell cycle progression (Halazonetis et al., 2008; Motoyama and Naka, 2004; Zhou and Elledge, 2000). The "turn off" process of the DDR upon satisfaction of DNA repair, also known as "checkpoint recovery", involves deactivation of DDR elements, but the mechanism is poorly understood. Greatwall kinase (Gwl) has been identified as a key element in the G2/M transition (Archambault et al., 2007; Jackson, 2006; Zhao et al., 2008; Yu et al., 2004; Yu et al., 2006; Zhao et al., 2006) and helps maintain M phase through inhibition of PP2A/B55δ (Burgess et al., 2010; Castilho et al., 2009; Goldberg, 2010; Lorca et al., 2010; Vigneron et al., 2009), the principal phosphatase for Cdk-phosphorylated substrates. Here we show that Gwl also promotes recovery from DNA damage and is itself directly inhibited by the DNA damage response (DDR). In Xenopus egg extracts, immunodepletion of Gwl increased the DDR to damaged DNA, whereas addition of wild type, but not kinase dead Gwl, inhibited the DDR. The removal of damaged DNA from egg extracts leads to recovery from checkpoint arrest and entry into mitosis, a process impaired by Gwl depletion and enhanced by Gwl over-expression. Moreover, activation of Cdk1 after the removal of damaged DNA is regulated by Gwl. Collectively, these results defines Gwl as a new regulator of the DDR, which plays an important role in recovery from DNA     

植物抗旱相关基因研究进展

遭遇极端温度、干旱、高盐等胁迫时,植物需要调控多种基因,通过多种途径来抵御非生物胁迫的伤害。综述了植物在干旱胁迫发生时,信号传导和转录因子相关调控基因以及在水分运输、抗脱水、渗透调节以调节气孔开关等功能相关基因克隆的研究进展,并提出了今后开展植物抗逆研究的建议。