C. elegans ksr-1 and ksr-2 have both unique and redundant functions and are required for MPK-1 ERK phosphorylation

Kinase Suppressor of Ras (KSR) is a conserved protein that positively regulates Ras signaling and may function as a scaffold for Raf, MEK, and ERK. However, the precise role of KSR is not well understood, and some observations have suggested that KSR might act in a parallel pathway. In C. elegans, ksr-1 is only required for a specific Ras-mediated process (sex myoblast migration) and is a nonessential positive regulator of other Ras-mediated developmental events. We report the existence of a second C. elegans ksr gene, ksr-2, which is required for Ras-mediated signaling during germline meiotic progression and functions redundantly with ksr-1 during development of the excretory system, hermaphrodite vulva, and male spicules. Thus, while the ksr-1 and ksr-2 genes are individually required only for specific Ras-dependent processes, together these two genes appear necessary for most aspects of Ras-mediated signaling in C. elegans. The finding that ksr-2; ksr-1 double mutants have strong ras-like phenotypes and severely reduced or absent levels of diphosphorylated MPK-1 ERK strongly supports models where KSR acts to promote the activation or maintenance of the Raf/MEK/ERK kinase cascade.     

Regulation of isthmic Fgf8 signal by sprouty2

Fgf8 functions as an organizer at the mes/metencephalic boundary (isthmus). We showed that a strong Fgf8 signal activates the Ras-ERK signaling pathway to organize cerebellar differentiation. Sprouty2 is expressed in an overlapping manner to Fgf8, and is induced by Fgf8. Its function, however, is indicated to antagonize Ras-ERK signaling. Here, we show the regulation of Fgf8 signaling in relation to Sprouty2. sprouty2 expression was induced very rapidly by Fgf8b, but interfered with ERK activation. sprouty2 misexpression resulted in a fate change of the presumptive metencephalon to the mesencephalon. Misexpression of a dominant negative form of Sprouty2 augmented ERK activation, and resulted in anterior shift of the posterior border of the tectum. The results indicate that Fgf8 activates the Ras-ERK signaling pathway to differentiate the cerebellum, and that the hyper- or hypo-signaling of this pathway affects the fate of the brain vesicles. Sprouty2 may regulate the Fgf8-Ras-ERK signaling pathway for the proper regionalization of the metencephalon and mesencephalon.     

The CNS midline cells control the spitz class and Egfr signaling genes to establish the proper cell fate of the Drosophila ventral neuroectoderm.

The spitz class genes, pointed (pnt), rhomboid frho), single-minded (sim), spitz (spi)and Star (S), as well as the Drosophila epidermal growth factor receptor (Egfr) signaling genes, argos (aos), Egfr, orthodenticle (otd) and vein (vn), are required for the proper establishment of ventral neuroectodermal cell fate. The roles of the CNS midline cells, spitz class and Egfr signaling genes in cell fate determination of the ventral neuroectoderm were determined by analyzing the spatial and temporal expression patterns of each individual gene in spitz class and Egfr signaling mutants. This analysis showed that the expression of all the spitz class and Egfrsignaling genes is affected by the sim gene, which indicates that sim acts upstream of all the spitz class and Egfr signaling genes. It was shown that overexpression of sim in midline cells fails to induce the ectodermal fate in the spi and Egfr mutants. On the other hand, overexpression of spi and Draf causes ectopic expression of the neuroectodermal markers in the sim mutant. Ectopic expression of sim in the en-positive cells induces the expression of downstream genes such as otd, pnt, rho, and vn, which clearly demonstrates that the sim gene activates the EGFR signaling pathway and that CNS midline cells, specified by sim, provide sufficient positional information for the establishment of ventral neuroectodermal fate. These results reveal that the CNS midline cells are one of the key regulators for the proper patterning of the ventral neuroectoderm by controlling EGFR activity through the regulation of the expression of spitz class genes and Egfr signaling genes.     

Spred2 is involved in imatinib-induced cytotoxicity in chronic myeloid leukemia cells

Spreds, a recently established class of negative regulators of the Ras-ERK (extracellular signal-regulated kinase) pathway, are involved in hematogenesises, allergic disorders and tumourigenesis. However, their role in hematologic neoplasms is largely unknown. Possible effects of Spreds on other signal pathways closely related to Ras-ERK have been poorly investigated. In this study, we investigated the in vitro effects of Spred2 on chronic myeloid leukemia (CML) cells. In addition to inhibiting the well-established Ras-ERK cascade, adenovirus-mediated Spred2 over-expression inhibits constitutive and stem cell factor (SCF)-stimulated sphingosine kinase-1 (SPHK1) and Mcl-1 expression, as well as inhibiting proliferation and inducing apoptosis in CML cells. In K562 cells and primary CML cells, imatinib induces endogenous Spred2 expression. Spred2 silencing by stable RNA interference partly protects K562 cells against imatinib-induced apoptosis. Together, these data implicate Spred2 in imatinib-induced cytotoxicity in CML cells, possibly by inhibiting the Ras-ERK cascade and the pro-survival signaling molecules SPHK1 and Mcl-1. These findings reveal potential targets for selective therapy of CML.     

Comprehensive Expression of Wnt Signaling Pathway Genes during Development and Maturation of the Mouse Cochlea

Background

In the inner ear Wnt signaling is necessary for proliferation, cell fate determination, growth of the cochlear duct, polarized orientation of stereociliary bundles, differentiation of the periotic mesenchyme, and homeostasis of the stria vascularis. In neonatal tissue Wnt signaling can drive proliferation of cells in the sensory region, suggesting that Wnt signaling could be used to regenerate the sensory epithelium in the damaged adult inner ear. Manipulation of Wnt signaling for regeneration will require an understanding of the dynamics of Wnt pathway gene expression in the ear. We present a comprehensive screen for 84 Wnt signaling related genes across four developmental and postnatal time points.

Results

We identified 72 Wnt related genes expressed in the inner ear on embryonic day (E) 12.5, postnatal day (P) 0, P6 and P30. These genes included secreted Wnts, Wnt antagonists, intracellular components of canonical signaling and components of non-canonical signaling/planar cell polarity.

Conclusion

A large number of Wnt signaling molecules were dynamically expressed during cochlear development and in the early postnatal period, suggesting complex regulation of Wnt transduction. The data revealed several potential key regulators for further study.     

Repression of germline RNAi pathways in somatic cells by retinoblastoma pathway chromatin complexes

The retinoblastoma (Rb) tumor suppressor acts with a number of chromatin cofactors in a wide range of species to suppress cell proliferation. The Caenorhabditis elegans retinoblastoma gene and many of these cofactors, called synMuv B genes, were identified in genetic screens for cell lineage defects caused by growth factor misexpression. Mutations in many synMuv B genes, including lin-35/Rb, also cause somatic misexpression of the germline RNA processing P granules and enhanced RNAi. We show here that multiple small RNA components, including a set of germline-specific Argonaute genes, are misexpressed in the soma of many synMuv B mutant animals, revealing one node for enhanced RNAi. Distinct classes of synMuv B mutants differ in the subcellular architecture of their misexpressed P granules, their profile of misexpressed small RNA and P granule genes, as well as their enhancement of RNAi and the related silencing of transgenes. These differences define three classes of synMuv B genes, representing three chromatin complexes: a LIN-35/Rb-containing DRM core complex, a SUMO-recruited Mec complex, and a synMuv B heterochromatin complex, suggesting that intersecting chromatin pathways regulate the repression of small RNA and P granule genes in the soma and the potency of RNAi. Consistent with this, the DRM complex and the synMuv B heterochromatin complex were genetically additive and displayed distinct antagonistic interactions with the MES-4 histone methyltransferase and the MRG-1 chromodomain protein, two germline chromatin regulators required for the synMuv phenotype and the somatic misexpression of P granule components. Thus intersecting synMuv B chromatin pathways conspire with synMuv B suppressor chromatin factors to regulate the expression of small RNA pathway genes, which enables heightened RNAi response. Regulation of small RNA pathway genes by human retinoblastoma may also underlie its role as a tumor suppressor gene.     

Tissue-specific direct targets of Caenorhabditis elegans Rb/E2F dictate distinct somatic and germline programs

Background

The tumor suppressor Rb/E2F regulates gene expression to control differentiation in multiple tissues during development, although how it directs tissue-specific gene regulation in vivo is poorly understood.

Results

We determined the genome-wide binding profiles for Caenorhabditis elegans Rb/E2F-like components in the germline, in the intestine and broadly throughout the soma, and uncovered highly tissue-specific binding patterns and target genes. Chromatin association by LIN-35, the C. elegans ortholog of Rb, is impaired in the germline but robust in the soma, a characteristic that might govern differential effects on gene expression in the two cell types. In the intestine, LIN-35 and the heterochromatin protein HPL-2, the ortholog of Hp1, coordinately bind at many sites lacking E2F. Finally, selected direct target genes contribute to the soma-to-germline transformation of lin-35 mutants, including mes-4, a soma-specific target that promotes H3K36 methylation, and csr-1, a germline-specific target that functions in a 22G small RNA pathway.

Conclusions

In sum, identification of tissue-specific binding profiles and effector target genes reveals important insights into the mechanisms by which Rb/E2F controls distinct cell fates in vivo.     

Regulation of growth and cell fate during tissue regeneration by the two SWI/SNF chromatin-remodeling complexes of Drosophila

To regenerate, damaged tissue must heal the wound, regrow to the proper size, replace the correct cell types, and return to the normal gene-expression program. However, the mechanisms that temporally and spatially control the activation or repression of important genes during regeneration are not fully understood. To determine the role that chromatin modifiers play in regulating gene expression after tissue damage, we induced ablation in Drosophila melanogaster imaginal wing discs, and screened for chromatin regulators that are required for epithelial tissue regeneration. Here, we show that many of these genes are indeed important for promoting or constraining regeneration. Specifically, the two SWI/SNF chromatin-remodeling complexes play distinct roles in regulating different aspects of regeneration. The PBAP complex regulates regenerative growth and developmental timing, and is required for the expression of JNK signaling targets and the growth promoter Myc. By contrast, the BAP complex ensures correct patterning and cell fate by stabilizing the expression of the posterior gene engrailed. Thus, both SWI/SNF complexes are essential for proper gene expression during tissue regeneration, but they play distinct roles in regulating growth and cell fate.     

Essential function of Wnt-4 for tubulogenesis in the Xenopus pronephric kidney

In the vertebrate embryo, development of the excretory system is characterized by the successive formation of three distinct kidneys: the pronephros, mesonephros, and metanephros. While tubulogenesis in the metanephric kidney is critically dependent on the signaling molecule Wnt-4, it is unknown whether Wnt signaling is equally required for the formation of renal epithelia in the other embryonic kidney forms. We therefore investigated the expression of Wnt genes during the pronephric kidney development in Xenopus. Wnt4 was found to be associated with developing pronephric tubules, but was absent from the pronephric duct. Onset of pronephric Wnt-4 expression coincided with mesenchyme-to-epithelium transformation. To investigate Wnt-4 gene function, we performed gain- and loss-of-function experiments. Misexpression of Wnt4 in the intermediate and lateral mesoderm caused abnormal morphogenesis of the pronephric tubules, but was not sufficient to initiate ectopic tubule formation. We used a morpholino antisense oligonucleotide-based gene knockdown strategy to disrupt Wnt-4 gene function. Xenopus embryos injected with antisense Wnt-4 morpholinos developed normally, but marker gene and morphological analysis revealed a complete absence of pronephric tubules. Pronephric duct development was largely unaffected, indicating that ductogenesis may occur normally in the absence of pronephric tubules. Our results show that, as in the metanephric kidney, Wnt-4 is critically required for tubulogenesis in the pronephric kidney, indicating that a common, evolutionary conserved gene regulatory network may control tubulogenesis in different vertebrate excretory organs.     

kin-19/casein kinase Iα has dual functions in regulating asymmetric division and terminal differentiation in C. elegans epidermal stem cells

Casein Kinase I (CKI) is a conserved component of the Wnt signaling pathway that regulates cell fate determination in metazoans. We show that post-embryonic asymmetric division and fate specification of C. elegans epidermal stem cells are controlled by a non-canonical Wnt/β-catenin signaling pathway, involving the β-catenins WRM-1 and SYS-1, and that C. elegans kin-19/CKIα functions in this pathway. Furthermore, we find that kin-19 is the only member of the Wnt asymmetry pathway that functions with, or in parallel to, the heterochronic temporal patterning pathway to control withdrawal from self-renewal and subsequent terminal differentiation of epidermal stem cells. We show that, except in the case of kin-19, the Wnt asymmetry pathway and the heterochronic pathway function separately and in parallel to control different aspects of epidermal stem cell fate specification. However, given the function of kin-19/CKIα in both pathways, and that CKI, Wnt signaling pathway and heterochronic pathway genes are widely conserved in animals, our findings suggest that CKIα may function as a regulatory hub through which asymmetric division and terminal differentiation are coordinated in adult stem cells of vertebrates.Key words: C. elegans, kin-19, casein kinase Ialpha (CKIα), Wnt, stem cell, asymmetric cell division, heterochronic, temporal identity, terminal differentiation, self-renewal     

BTG Interacts with Retinoblastoma to Control Cell Fate in Dictyostelium

Background

In the genesis of many tissues, a phase of cell proliferation is followed by cell cycle exit and terminal differentiation. The latter two processes overlap: genes involved in the cessation of growth may also be important in triggering differentiation. Though conceptually distinct, they are often causally related and functional interactions between the cell cycle machinery and cell fate control networks are fundamental to coordinate growth and differentiation. A switch from proliferation to differentiation may also be important in the life cycle of single-celled organisms, and genes which arose as regulators of microbial differentiation may be conserved in higher organisms. Studies in microorganisms may thus contribute to understanding the molecular links between cell cycle machinery and the determination of cell fate choice networks.

Methodology/Principal Findings

Here we show that in the amoebozoan D. discoideum, an ortholog of the metazoan antiproliferative gene btg controls cell fate, and that this function is dependent on the presence of a second tumor suppressor ortholog, the retinoblastoma-like gene product. Specifically, we find that btg-overexpressing cells preferentially adopt a stalk cell (and, more particularly, an Anterior-Like Cell) fate. No btg-dependent preference for ALC fate is observed in cells in which the retinoblastoma-like gene has been genetically inactivated. Dictyostelium btg is the only example of non-metazoan member of the BTG family characterized so far, suggesting that a genetic interaction between btg and Rb predated the divergence between dictyostelids and metazoa.

Conclusions/Significance

While the requirement for retinoblastoma function for BTG antiproliferative activity in metazoans is known, an interaction of these genes in the control of cell fate has not been previously documented. Involvement of a single pathway in the control of mutually exclusive processes may have relevant implication in the evolution of multicellularity.     

A mutation in the pale aleurone color1 gene identifies a novel regulator of the maize anthocyanin pathway.

By screening for new seed color mutations, we have identified a new gene, pale aleurone color1 (pac1), which when mutated causes a reduction in anthocyanin pigmentation. The pac1 gene is not allelic to any known anthocyanin biosynthetic or regulatory gene. The pac1-ref allele is recessive, nonlethal, and only reduces pigment in kernels, not in vegetative tissues. Genetic and molecular evidence shows that the pac1-ref allele reduces pigmentation by reducing RNA levels of the biosynthetic genes in the pathway. The mutant does not reduce the RNA levels of either of the two regulatory genes, b and c1. Introduction of an anthocyanin structural gene promoter (a1) driving a reporter gene into maize aleurones shows that pac1-ref kernels have reduced expression resulting from the action of the a1 promoter. Introduction of the reporter gene with constructs that express the regulatory genes b and c1 or the phlobaphene pathway regulator p shows that this reduction in a1-driven expression occurs in both the presence and absence of these regulators. Our results imply that pac1 is required for either b/c1 or p activation of anthocyanin biosynthetic gene expression and that pac1 acts independently of these regulatory genes.