Gata3 is required for early morphogenesis and Fgf10 expression during otic development

Inner ear develops from an induced surface ectoderm placode that invaginates and closes to form the otic vesicle, which then undergoes a complex morphogenetic process to form the membranous labyrinth. Inner ear morphogenesis is severely affected in Gata3 deficient mouse embryos, but the onset and basis of the phenotype has not been known. We show here that Gata3 deficiency leads to severe and unique abnormalities during otic placode invagination. The invagination problems are accompanied often by the formation of a morphological boundary between the dorsal and ventral otic cup and by the precocious appearance of dorsal endolymphatic characteristics. In addition, the endolymphatic domain often detaches from the rest of the otic epithelium during epithelial closure. The expression of several cell adhesion mediating genes is altered in Gata3 deficient ears suggesting that Gata3 controls adhesion and morphogenetic movements in early otic epithelium. Inactivation of Gata3 leads also to a loss of Fgf10 expression in otic epithelium and auditory ganglion demonstrating that Gata3 is an important regulator of Fgf-signalling during otic development.     

The Jnk1 and Jnk2 protein kinases are required for regional specific apoptosis during early brain development

The c-Jun NH2-terminal kinase (Jnk) family is implicated in apoptosis, but its function in brain development is unclear. Here, we address this issue using mutant mice lacking different members of the family (Jnk1, Jnk2, and Jnk3). Mice deficient in Jnk1, Jnk2, Jnk3, and Jnk1/Jnk3 or Jnk2/Jnk3 double mutants all survived normally. Compound mutants lacking Jnk1 and Jnk2 genes were embryonic lethal and had severe dysregulation of apoptosis in brain. Specifically, there was a reduction of cell death in the lateral edges of hindbrain prior to neural tube closure. In contrast, increased apoptosis and caspase activation were found in the mutant forebrain, leading to precocious degeneration. These results suggest that Jnk1 and Jnk2 regulate region-specific apoptosis during early brain development.     

SPATULA and ALCATRAZ, are partially redundant, functionally diverging bHLH genes required for Arabidopsis gynoecium and fruit development

The Arabidopsis gynoecium is a complex organ that facilitates fertilization, later developing into a dehiscent silique that protects seeds until their dispersal. Identifying genes important for development is often hampered by functional redundancy. We report unequal redundancy between two closely related genes, SPATULA (SPT) and ALCATRAZ (ALC), revealing previously unknown developmental roles for each. SPT is known to support septum, style and stigma development in the flower, whereas ALC is involved in dehiscence zone development in the fruit. ALC diverged from a SPT-like ancestor following gene duplication coinciding with the At-β polyploidy event. Here we show that ALC is also involved in early gynoecium development, and SPT in later valve margin generation in the silique. Evidence includes the increased severity of early gynoecium disruption, and of later valve margin defects, in spt-alc double mutants. In addition, a repressive version of SPT (35S:SPT-SRDX) disrupts both structures. Consistent with redundancy, ALC and SPT expression patterns overlap in these tissues, and the ALC promoter carries two atypical E-box elements identical to one in SPT required for valve margin expression. Further, SPT can heterodimerize with ALC, and 35S:SPT can fully complement dehiscence defects in alc mutants, although 35S:ALC can only partly complement spt gynoecium disruptions, perhaps associated with its sequence simplification. Interactions with FRUITFULL and SHATTERPROOF genes differ somewhat between SPT and ALC, reflecting their different specializations. These two genes are apparently undergoing subfunctionalization, with SPT essential for earlier carpel margin tissues, and ALC specializing in later dehiscence zone development.     

Polyamines are required for phospholipase C-gamma1 expression promoting intestinal epithelial restitution after wounding

Intestinal mucosal restitution occurs by epithelial cell migration, rather than by proliferation, to reseal superficial wounds after injury. Polyamines are essential for the stimulation of intestinal epithelial cell (IEC) migration during restitution in association with their ability to regulate Ca2+ homeostasis, but the exact mechanism by which polyamines induce cytosolic free Ca2+ concentration ([Ca2+]cyt) remains unclear. Phospholipase C (PLC)-gamma1 catalyzes the formation of inositol (1,4,5)-trisphosphate (IP3), which is implicated in the regulation of [Ca2+]cyt by modulating Ca2+ store mobilization and Ca2+ influx. The present study tested the hypothesis that polyamines are involved in PLC-gamma1 activity, regulating [Ca2+]cyt and cell migration after wounding. Depletion of cellular polyamines by alpha-difluoromethylornithine inhibited PLC-gamma1 expression in differentiated IECs (stable Cdx2-transfected IEC-6 cells), as indicated by substantial decreases in levels of PLC-gamma1 mRNA and protein and its enzyme product IP3. Polyamine-deficient cells also displayed decreased [Ca2+]cyt and inhibited cell migration. Decreased levels of PLC-gamma1 by treatment with U-73122 or transfection with short interfering RNA specifically targeting PLC-gamma1 also decreased IP3, reduced resting [Ca2+]cyt and Ca2+ influx after store depletion, and suppressed cell migration in control cells. In contrast, stimulation of PLC-gamma1 by 2,4,6-trimethyl-N-(meta-3-trifluoromethylphenyl)-benzenesulfonamide induced IP3, increased [Ca2+]cyt, and promoted cell migration in polyamine-deficient cells. These results indicate that polyamines are absolutely required for PLC-gamma1 expression in IECs and that polyamine-mediated PLC-gamma1 signaling stimulates cell migration during restitution as a result of increased [Ca2+]cyt.     

Laminin alpha5 chain is required for intestinal smooth muscle development

Laminins (comprised of alpha, beta, and gamma chains) are heterotrimeric glycoproteins integral to all basement membranes. The function of the laminin alpha5 chain in the developing intestine was defined by analysing laminin alpha5(-/-) mutants and by grafting experiments. We show that laminin alpha5 plays a major role in smooth muscle organisation and differentiation, as excessive folding of intestinal loops and delay in the expression of specific markers are observed in laminin alpha5(-/-) mice. In the subepithelial basement membrane, loss of alpha5 expression was paralleled by ectopic or accelerated deposition of laminin alpha2 and alpha4 chains; this may explain why no obvious defects were observed in the villous form and enterocytic differentiation. This compensation process is attributable to mesenchyme-derived molecules as assessed by chick/mouse alpha5(-/-) grafted associations. Lack of the laminin alpha5 chain was accompanied by a decrease in epithelial alpha3beta1 integrin receptor expression adjacent to the epithelial basement membrane and of Lutheran blood group glycoprotein in the smooth muscle cells, indicating that these receptors are likely mediating interactions with laminin alpha5-containing molecules. Taken together, the data indicate that the laminin alpha5 chain is essential for normal development of the intestinal smooth muscle and point to possible mesenchyme-derived compensation to promote normal intestinal morphogenesis when laminin alpha5 is absent.     

Rhizobium leguminosarum genes required for expression and transfer of host specific nodulation

The contributions of various nod genes from Rhizobium leguminosarum biovar viceae to host-specific nodulation have been assessed by transferring specific genes and groups of genes to R. leguminosarum bv. trifolii and testing the levels of nodulation on Pisum sativum (peas) and Vicia hirsuta. Many of the nod genes are important in determination of host-specificity; the nodE gene plays a key (but not essential) role and the efficiency of transfer of host specific nodulation increased with additional genes such that nodFE < nodFEL < nodFELMN. In addition the nodD gene was shown to play an important role in host-specific nodulation of peas and Vicia whilst other genes in the nodABCIJ gene region also appeared to be important. In a reciprocal series of experiments involving nod genes cloned from R. leguminosarum bv. trifolii it was found that the nodD gene enabled bv. viciae to nodulate Trifolium pratense (red clover) but the nodFEL gene region did not. The bv. trifolii nodD or nodFEL genes did significantly increase nodulation of Trifolium subterraneum (sub-clover) by R. leguminosarum bv. viciae. It is concluded that host specificity determinants are encoded by several different nod genes.     

Six1 and Six4 homeoproteins are required for Pax3 and Mrf expression during myogenesis in the mouse embryo

In mammals, Six5, Six4 and Six1 genes are co-expressed during mouse myogenesis. Six4 and Six5 single knockout (KO) mice have no developmental defects, while Six1 KO mice die at birth and show multiple organ developmental defects. We have generated Six1Six4 double KO mice and show an aggravation of the phenotype previously reported for the single Six1 KO. Six1Six4 double KO mice are characterized by severe craniofacial and rib defects, and general muscle hypoplasia. At the limb bud level, Six1 and Six4 homeogenes control early steps of myogenic cell delamination and migration from the somite through the control of Pax3 gene expression. Impaired in their migratory pathway, cells of the somitic ventrolateral dermomyotome are rerouted, lose their identity and die by apoptosis. At the interlimb level, epaxial Met expression is abolished, while it is preserved in Pax3-deficient embryos. Within the myotome, absence of Six1 and Six4 impairs the expression of the myogenic regulatory factors myogenin and Myod1, and Mrf4 expression becomes undetectable. Myf5 expression is correctly initiated but becomes restricted to the caudal region of each somite. Early syndetomal expression of scleraxis is reduced in the Six1Six4 embryo, while the myotomal expression of Fgfr4 and Fgf8 but not Fgf4 and Fgf6 is maintained. These results highlight the different roles played by Six proteins during skeletal myogenesis.     

RSC1 and RSC2 are required for expression of mid-late sporulation-specific genes in Saccharomyces cerevisiae

Rsc1 and Rsc2 are alternative bromodomain-containing subunits of the ATP-dependent RSC chromatin remodeling complex in Saccharomyces cerevisiae. Smk1 is a sporulation-specific mitogen-activated protein kinase homolog that is required for the postmeiotic events of spore formation. In this study we show that RSC1 and RSC2 are haploinsufficient for spore formation in a smk1 hypomorph. Moreover, diploids lacking Rsc1 or Rsc2 show a subset of smk1-like phenotypes. High-copy-number RSC1 plasmids do not suppress rsc2-Delta/rsc2-Delta sporulation defects, and high-copy-number RSC2 plasmids do not suppress rsc1-Delta/rsc1-Delta sporulation defects. Mid-late sporulation-specific genes, which are normally expressed while key steps in spore assembly occur and which include genes that are required for spore wall formation, are not expressed in cells lacking Rsc1 or Rsc2. We speculate that the combined action of Rsc1 and Rsc2 at mid-late promoters is specifically required for the proper expression of this uniquely timed set of genes. Our data suggest that Smk1 and Rsc1/2 define parallel pathways that converge to provide signaling information and the expression of gene products, respectively, that are required for spore morphogenesis.     

Both subtelomeric regions are required and sufficient for specific DNA fragmentation during macronuclear development in Stylonychia lemnae

Background  

Programmed DNA-reorganization and DNA-elimination events take place frequently during cellular differentiation. An extreme form of such processes, involving DNA reorganization, DNA elimination and DNA fragmentation, is found during macronuclear differentiation in hypotrichous ciliates. Ciliated protozoa can therefore serve as a model system to analyze the molecular basis of these processes during cellular differentiation in eukaryotic cells.     

Five SWI genes are required for expression of the HO gene in yeast

High-frequency mating type interconversion in yeast requires the HO gene, which encodes a site-specific endonuclease that initiates the switching process. We have isolated and analyzed switching-defective mutants. These mutants define five complementation and linkage groups, SWI 1 to SWI 5. We have shown by two assays, Northern hybridization and beta-galactosidase activity in strains containing an HO-lacZ fusion, that mutants defective any SWI gene fail to express the HO gene. In addition, all of the swi mutants exhibit other phenotypes, the most notable being the inviability of double mutants defective in SWI 4 and in either SWI 1, SWI 2 or SWI 3. These results indicate that the SWI genes function in some way as positive regulators of HO expression and have additional cellular roles.