Wnt1 and wnt10b function redundantly at the zebrafish midbrain-hindbrain boundary

Wnt signals have been shown to be involved in multiple steps of vertebrate neural patterning, yet the relative contributions of individual Wnts to the process of brain regionalization is poorly understood. Wnt1 has been shown in the mouse to be required for the formation of the midbrain and the anterior hindbrain, but this function of wnt1 has not been explored in other model systems. Further, wnt1 is part of a Wnt cluster conserved in all vertebrates comprising wnt1 and wnt10b, yet the function of wnt10b during embryogenesis has not been explored. Here, we report that in zebrafish wnt10b is expressed in a pattern overlapping extensively with that of wnt1. We have generated a deficiency allele for these closely linked loci and performed morpholino antisense oligo knockdown to show that wnt1 and wnt10b provide partially redundant functions in the formation of the midbrain-hindbrain boundary (MHB). When both loci are deleted, the expression of pax2.1, en2, and her5 is lost in the ventral portion of the MHB beginning at the 8-somite stage. However, wnt1 and wnt10b are not required for the maintenance of fgf8, en3, wnt8b, or wnt3a expression. Embryos homozygous for the wnt1-wnt10b deficiency display a mild MHB phenotype, but are sensitized to reductions in either Pax2.1 or Fgf8; that is, in combination with mutant alleles of either of these loci, the morphological MHB is lost. Thus, wnt1 and wnt10b are required to maintain threshold levels of Pax2.1 and Fgf8 at the MHB.     

ced-10 Rac and mig-2 function redundantly and act with unc-73 trio to control the orientation of vulval cell divisions and migrations in Caenorhabditis elegans.

Vulval development in the nematode Caenorhabditis elegans can be divided into a fate specification phase controlled in part by let-60 Ras, and a fate execution phase involving stereotypical patterns of cell division and migration controlled in part by lin-17 Frizzled. Since the small GTPase Rac has been implicated as a downstream target of both Ras and Frizzled and influences cytoskeletal dynamics, we investigated the role of Rac signaling during each phase of vulval development. We show that the Rac gene ced-10 and the Rac-related gene mig-2 are redundantly required for the proper orientation of certain vulval cell divisions, suggesting a role in spindle positioning. ced-10 Rac and mig-2 are also redundantly required for vulval cell migrations and play a minor role in vulval fate specification. Constitutively active and dominant-negative mutant forms of mig-2 cause vulval defects that are very similar to those seen in ced-10;mig-2 double loss-of-function mutants, indicating that they interfere with the functions of both ced-10 Rac and mig-2. Mutations in unc-73 (a Trio-like guanine nucleotide exchange factor) cause similar vulval defects, suggesting that UNC-73 is an exchange factor for both CED-10 and MIG-2. We discuss the similarities and differences between the cellular defects seen in Rac mutants and let-60 Ras or lin-17 Frizzled mutants.     

SAX-7/L1CAM and HMR-1/cadherin function redundantly in blastomere compaction and non-muscle myosin accumulation during Caenorhabditis elegans gastrulation

Gastrulation is the first major morphogenetic movement in development and requires dynamic regulation of cell adhesion and the cytoskeleton. Caenorhabditis elegans gastrulation begins with the migration of the two endodermal precursors, Ea and Ep, from the surface of the embryo into the interior. Ea/Ep migration provides a relatively simple system to examine the intersection of cell adhesion, cell signaling, and cell movement. Ea/Ep ingression depends on correct cell fate specification and polarization, apical myosin accumulation, and Wnt activated actomyosin contraction that drives apical constriction and ingression (Lee et al., 2006; Nance et al., 2005). Here, we show that Ea/Ep ingression also requires the function of either HMR-1/cadherin or SAX-7/L1CAM. Both cadherin complex components and L1CAM are localized at all sites of cell-cell contact during gastrulation. Either system is sufficient for Ea/Ep ingression, but loss of both together leads to a failure of apical constriction and ingression. Similar results are seen with isolated blastomeres. Ea/Ep are properly specified and appear to display correct apical-basal polarity in sax-7(eq1);hmr-1(RNAi) embryos. Significantly, in sax-7(eq1);hmr-1(RNAi) embryos, Ea and Ep fail to accumulate myosin (NMY-2∷GFP) at their apical surfaces, but in either sax-7(eq1) or hmr-1(RNAi) embryos, apical myosin accumulation is comparable to wild type. Thus, the cadherin and L1CAM adhesion systems are redundantly required for localized myosin accumulation and hence for actomyosin contractility during gastrulation. We also show that sax-7 and hmr-1 function are redundantly required for Wnt-dependent spindle polarization during division of the ABar blastomere, indicating that these cell surface proteins redundantly regulate multiple developmental events in early embryos.     

The ventralized ogon mutant phenotype is caused by a mutation in the zebrafish homologue of Sizzled,a secreted Frizzled-related protein

The BMP signaling pathway plays a key role during dorsoventral pattern formation of vertebrate embryos. In zebrafish, all cloned mutants affecting this process are deficient in members of the BMP pathway. In a search for factors differentially expressed in swirl/bmp2b mutants compared with wild type, we isolated zebrafish Sizzled, a member of the secreted Frizzled-related protein family and putative Wnt inhibitor. The knockdown of sizzled using antisense morpholino phenocopied the ventralized mutant ogon (formerly also known as mercedes and short tail). By sequencing and rescue experiments, we demonstrate that ogon encodes sizzled. Overexpression of sizzled, resulting in strongly dorsalized phenotypes, and the expression domains of sizzled in wild type embryos, localized in the ventral side during gastrulation and restricted to the posterior end during segmentation stages, correlate with its role in dorsoventral patterning. The expanded expression domain of sizzled in ogon and chordino together with its downregulation in swirl suggests a BMP2b-dependent negative autoregulation of sizzled. Indicating a novel role for a secreted Frizzled-related protein, we show that, in addition to the BMP pathway, a component of the Wnt signaling pathway is required for dorsoventral pattern formation in zebrafish.     

C. elegans HIM-8 functions outside of meiosis to antagonize EGL-13 Sox protein function

egl-13 encodes a Sox domain protein that is required for proper uterine seam cell development in Caenorhabditis elegans. We demonstrate that mutations of the C2H2 zinc fingers encoded by the him-8 (high incidence of males) gene partially suppress the egg-laying and connection-of-gonad morphology defects caused by incompletely penetrant alleles of egl-13. him-8 alleles have previously characterized recessive effects on recombination and segregation of the X chromosome during meiosis due to failure of X chromosome homolog pairing and subsequent synapsis. However, we show that him-8 alleles are semi-dominant suppressors of egl-13, and the semi-dominant effect is due to haplo-insufficiency of the him-8 locus. Thus, we conclude that the wild-type him-8 gene product acts antagonistically to EGL-13. Null alleles of egl-13 cannot be suppressed, suggesting that this antagonistic interaction most likely occurs either upstream of or in parallel with EGL-13. Moreover, we conclude that suppression of egl-13 is due to a meiosis-independent function of him-8 because suppression is observed in mutants that have severely reduced meiotic germ cell populations and suppression does not depend on the function of him-8 in the maternal germ line. We also show that the chromosomal context of egl-13 seems important in the him-8 suppression mechanism. Interactions between these genes can give insight into function of Sox family members, which are important in many aspects of metazoan development, and into functions of him-8 outside of meiosis.     

crm-1 facilitates BMP signaling to control body size in Caenorhabditis elegans

We have identified in Caenorhabditis elegans a homologue of the vertebrate Crim1, crm-1, which encodes a putative transmembrane protein with multiple cysteine-rich (CR) domains known to have bone morphogenetic proteins (BMPs) binding activity. Using the body morphology of C. elegans as an indicator, we showed that attenuation of crm-1 activity leads to a small body phenotype reminiscent of that of BMP pathway mutants. We showed that the crm-1 loss-of-function phenotype can be rescued by constitutive supply of sma-4 activity. crm-1 can enhance BMP signaling and this activity is dependent on the presence of the DBL-1 ligand and its receptors. crm-1 is expressed in neurons at the ventral nerve cord, where the DBL-1 ligand is produced. However, ectopic expression experiments reveal that crm-1 gene products act outside the DBL-1 producing cells and function non-autonomously to facilitate dbl/sma pathway signaling to control body size.     

Modulation of BMP activity in dorsal-ventral pattern formation by the chordin and ogon antagonists

We analyzed the interactions between mutations in antagonistic BMP pathway signaling components to examine the roles that the antagonists play in regulating BMP signaling activity. The dorsalized mutants swirl/bmp2b, snailhouse/bmp7, lost-a-fin/alk8, and mini fin/tolloid were each analyzed in double mutant combinations with the ventralized mutants chordino/chordin and ogon, whose molecular nature is not known. Similar to the BMP antagonist chordino, we found that the BMP ligand mutants swirl/bmp2b and snailhouse/bmp7 are also epistatic to the putative BMP pathway antagonist, ogon, excluding a class of intracellular antagonists as candidates for ogon. In ogon;mini fin double mutants, we observed a mutual suppression of the ogon and mini fin mutant phenotypes, frequently to a wild type phenotype. Thus, the Tolloid/Mini fin metalloprotease that normally cleaves and inhibits Chordin activity is dispensable, when Ogon antagonism is reduced. These results suggest that Ogon encodes a Tolloid and Chordin-independent antagonistic function. By analyzing genes whose expression is very sensitive to BMP signaling levels, we found that the absence of Ogon or Chordin antagonism did not increase the BMP activity remaining in swirl/bmp2b or hypomorphic snailhouse/bmp7 mutants. These results, together with other studies, suggest that additional molecules or mechanisms are essential in generating the presumptive gastrula BMP activity gradient that patterns the dorsal-ventral axis. Lastly we observed a striking increased penetrance of the swirl/bmp2b dominant dorsalized phenotype, when Chordin function is also absent. Loss of the BMP antagonist Chordin is expected to increase BMP signaling levels in a swirl heterozygote, but instead we observed an apparent decrease in BMP signaling levels and a loss of ventral tail tissue. As has been proposed for the fly orthologue of chordin, short gastrulation, our paradoxical results can be explained by a model whereby Chordin both antagonizes and promotes BMP activity.     

Twisted gastrulation is required for forebrain specification and cooperates with Chordin to inhibit BMP signaling during X. tropicalis gastrulation

In the developing vertebrate embryo, proper dorsal-ventral patterning relies on BMP antagonists secreted by the organizer during gastrulation. The BMP antagonist chordin has a complex interaction with BMPs that is governed in part by its interaction with the secreted protein twisted gastrulation (tsg). In different contexts, tsg has activity as either a BMP agonist or as a BMP antagonist. Using morpholino oligonucleotides in Xenopus tropicalis, we show that reducing tsg gene product results in a ventralized embryo, and that tsg morphants specifically lack a forebrain. We provide new evidence that tsg acts as a BMP antagonist during X. tropicalis gastrulation since the tsg depletion phenotype can be rescued in two ways: by chordin overexpression and by BMP depletion. We conclude that tsg acts as a BMP antagonist in the context of the frog gastrula, and that it acts cooperatively with chordin to establish dorsal structures and particularly forebrain tissue during development.     

Additional sex combs-like 1 belongs to the enhancer of trithorax and polycomb group and genetically interacts with Cbx2 in mice

The Additional sex combs (Asx) gene of Drosophila behaves genetically as an enhancer of trithorax and polycomb (ETP) in displaying bidirectional homeotic phenotypes, suggesting that is required for maintenance of both activation and silencing of Hox genes. There are three murine homologs of Asx called Additional sex combs-like1, 2, and 3. Asxl1 is required for normal adult hematopoiesis; however, its embryonic function is unknown. We used a targeted mouse mutant line Asxl1^tm1Bc to determine if Asxl1 is required to silence and activate Hox genes in mice during axial patterning. The mutant embryos exhibit simultaneous anterior and posterior transformations of the axial skeleton, consistent with a role for Asxl1 in activation and silencing of Hox genes. Transformations of the axial skeleton are enhanced in compound mutant embryos for the polycomb group gene M33/Cbx2. Hoxa4, Hoxa7, and Hoxc8 are derepressed in Asxl1^tm1Bc mutants in the antero-posterior axis, but Hoxc8 expression is reduced in the brain of mutants, consistent with Asxl1 being required both for activation and repression of Hox genes. We discuss the genetic and molecular definition of ETPs, and suggest that the function of Asxl1 depends on its cellular context.     

Structural and functional evidences for a type 1 TGF-beta sensu stricto receptor in the lophotrochozoan Crassostrea gigas suggest conserved molecular mechanisms controlling mesodermal patterning across bilateria

The transforming growth factor beta (TGFbeta) superfamily includes bone morphogenetic proteins, activins and TGF-betasensu stricto (s.s.). These ligands have been shown to play a key role in numerous biological processes including early embryonic development and immune regulation. They transduce their signal through a hetromeric complex of type I and type II receptors. Such receptors have been identified in ecdysozoans but none have been found as yet in the other major protostomal clade, the lophotrochozoans. Here, we report the identification of the first lophotrochozoan TGFbetas.s. type I receptor (Cg-TGFbetaRI) from the mollusk Crassostrea gigas. The phylogenetic and structural analyses as well as the expression pattern during early development suggest Cg-TGFbetaRI to belong to the TGFbetas.s./activin type I receptor clade and functional studies corroborate these deductions. The use of the zebrafish embryo as a reporter organism reveals that either Cg-TGFbetaRI or its dominant negative acting truncated form, when overexpressed during gastrulation, resulted in a range of phenotypes displaying severe disturbance of anterioposterior patterning due to a strong modulation of ventrolateral mesoderm patterning. Finally, a Cg-TGFbetaRI cytokine activity during immune regulation in C. gigas has been investigated by real-time PCR in haemocytes and mantle edge during an in vivo bacterial LPS challenge. One piece of evidence from this study suggests that the molecular mechanisms controlling mesodermal patterning and some immune regulations across all bilateria could be conserved through a functional TGF-beta s.s. pathway in lophotrochozoans.