Non-canonical ubiquitylation of the proneural protein Ngn2 occurs in both Xenopus embryos and mammalian cells

Poly-ubiquitin chains targeting proteins for 26S proteasomal degradation are classically anchored on internal lysines of substrates via iso-peptide linkages. However recently, linkage of ubiquitin moieties to non-canonical nucleophilic residues, such as cysteines, serines and threonines, has been demonstrated in a small number of cases.Non-canonical ubiquitylation of the proneural protein Ngn2 has previously been seen in Xenopus egg extract, but it was not clear whether such highly unusual modes of ubiquitylation were restricted to the environment of egg cytoplasm. Here we show that Ngn2 is, indeed, ubiquitylated on non-canonical sites in extracts from neurula stage Xenopus embryos, when Ngn2 is usually active. Moreover, in the P19 mammalian embryonal carcinoma cell line capable of differentiating into neurons, xNgn2 is ubiquitylated on both canonical and non-canonical sites. We see that mutation of cysteines alone results stabilisation of the protein in P19 cells, indicating that non-canonical ubiquitylation on these residues normally contributes to the fast turnover of xNgn2 in mammalian cells.     

Near complete loss of retinal ganglion cells in the math5/brn3b double knockout elicits severe reductions of other cell types during retinal development

Retinal ganglion cells (RGCs) are the first cell type to differentiate during retinal histogenesis. It has been postulated that specified RGCs subsequently influence the number and fate of the remaining progenitors to produce the rest of the retinal cell types. However, several genetic knockout models have argued against this developmental role for RGCs. Although it is known that RGCs secrete cellular factors implicated in cell proliferation, survival, and differentiation, until now, limited publications have shown that reductions in the RGC number cause significant changes in these processes. In this study, we observed that Math5 and Brn3b double null mice exhibited over a 99% reduction in the number of RGCs during development. This severe reduction of RGCs is accompanied by a drastic loss in the number of all other retinal cell types that was never seen before. Unlike Brn3b null or Math5 null animals, mice null for both alleles lack an optic nerve and have severe retinal dysfunction. Results of this study support the hypothesis that RGCs play a pivotal role in the late phase of mammalian retina development.     

ELONGATA3 is required for shoot meristem cell cycle progression in Arabidopsis thaliana seedlings

A key feature of the development of a higher plant is the continuous formation of new organs from the meristems. Originally patterned during embryogenesis, the meristems must activate cell division de novo at the time of germination, in order to initiate post-embryonic development. In a mutagenesis screen aimed at finding new players in early seedling cell division control, we identified ELONGATA3 (ELO3) as a key regulator of meristem cell cycle activation in Arabidopsis. Our results show that plants carrying a hypomorphic allele of ELO3 fail to activate cell division in the meristems following germination, which leads to seedling growth arrest and lethality. Further analyses suggest that this is due to a failure in DNA replication, followed by cell cycle arrest, in the meristematic tissue. Interestingly, the meristem cell cycle arrest in elo3 mutants, but not the later leaf developmental defects that have been linked to the loss of ELO3 activities, can be relieved by the addition of metabolic sugars in the growth medium. This finding points to a new role by which carbohydrate availability promotes meristem growth. Furthermore, growth arrested elo3 mutants suffer a partial loss of shoot meristem identity, which provides further evidence that cell cycle activities can influence the control of tissue identity.     

Transcriptional cascade from Math1 to Mbh1 and Mbh2 is required for cerebellar granule cell differentiation

Cerebellar granule cells (CGCs) are the most abundant neuronal type in the mammalian brain, and their differentiation is regulated by the basic helix-loop-helix gene, Math1. However, little is known about downstream genes of Math1 and their functions in the cerebellum. To investigate them, we have here established an electroporation-based in vivo gene transfer method in the developing mouse cerebellum. Misexpression of Math1 ectopically induced expression of Bar-class homeobox genes, Mbh1 and Mbh2, which are expressed by CGCs. Conversely, their expression was repressed in CGCs by knockdown of Math1. These findings, taken together with chromatin immunoprecipitation assays, suggest that Math1 directly regulates the Mbh genes in CGCs. Furthermore, a dominant-negative form of the Mbh proteins disrupted proper formation of the external granule layer and differentiation of CGCs, whereas misexpression of the Mbh genes ectopically induced expression of a CGC marker in nonneuronal cells, indicating that the Mbh proteins are required for the differentiation of CGCs.     

A Xenopus tribbles orthologue is required for the progression of mitosis and for development of the nervous system

The product of the Drosophila gene tribbles inhibits cell division in the ventral furrow of the embryo and thereby allows the normal prosecution of gastrulation. Cell division is also absent in involuting dorsal mesoderm during gastrulation in Xenopus, and to ask whether the two species employ similar mechanisms to coordinate morphogenesis and the cell cycle, we isolated a putative Xenopus homologue of tribbles which we call Xtrb2. Extensive cDNA cloning identified long and short forms of Xtrb2, termed Xtrb2-L and Xtrb2-S, respectively. Xtrb2 is expressed maternally and in mesoderm and ectoderm at blastula and gastrula stages. Later, it is expressed in dorsal neural tube, eyes, and cephalic neural crest. Time-lapse imaging of GFP-tagged Xtrb2-L suggests that during cell division, it is associated with mitotic spindles. Knockdown of Xtrb2 by antisense morpholino oligonucleotides (MOs) disrupted synchronous cell divisions during blastula stages, apparently as a result of delayed progression through mitosis and cytokinesis. At later stages, tissues expressing the highest levels of Xtrb2 were most markedly affected by morpholino knockdown, with perturbation of neural crest and eye development.     

The anaphase-promoting complex is required in both dividing and quiescent cells during zebrafish development

The anaphase-promoting complex/cyclosome (APC/C) regulates multiple stages of the cell cycle, most prominently mitosis. We describe zebrafish with mutations in two APC/C subunits, Cdc16 and Cdc26, whose phenotypes reveal a multifaceted set of defects resulting from the gradual depletion of the APC/C. First, loss of the APC/C in dividing cells results in mitotic arrest, followed by apoptosis. This defect becomes detectable in different organs at different larval ages, because the subunits of the APC/C are maternally deposited, are unusually stable, and are depleted at uneven rates in different tissues. Second, loss of the APC/C in quiescent or differentiated cells results in improper re-entry into the cell cycle, again in an apparently tissue-specific manner. This study is the first demonstration of both functions of the APC/C in a vertebrate organism and also provides an illustration of the surprisingly complex effects that essential, maternally supplied factors can have on the growing animal over a period of 10 days or longer.     

The C. elegans Frizzled CFZ-2 is required for cell migration and interacts with multiple Wnt signaling pathways

Members of the Frizzled family of integral membrane proteins are implicated in many developmental events, including specifying cell fate, orienting cell and planar polarity, and directing cell migration. Frizzleds function as cell surface receptors for secreted Wnt proteins. We report here the isolation of a mutation in cfz-2, a Caenorhabditis elegans Frizzled gene. Mutation of cfz-2 causes defective cell migration, disorganization of head neurons, and can cause ectopic axon outgrowth. Analysis of mosaic animals shows that CFZ-2 functions cell nonautonomously, but does not rule out an autonomous role. CFZ-2 is expressed primarily in the anterior of embryos and in several cells in the head of adults. Our analysis of interactions between CFZ-2 and other Wnt pathways reveals that three Wnts, CWN-1, CWN-2 and EGL-20, and a Frizzled, MOM-5, function redundantly with one another and with CFZ-2 for specific cell migrations. In contrast, CWN-1, CWN-2, EGL-20, CFZ-2, and MOM-5 antagonize one another for other migrations. Therefore, CFZ-2 functions by collaborating with and/or antagonizing other Wnt signaling pathways to regulate specific cell migrations.     

The minibrain kinase homolog, mbk-2, is required for spindle positioning and asymmetric cell division in early C. elegans embryos

In the newly fertilized Caenorhabditis elegans zygote, cytoplasmic determinants become localized asymmetrically along the anterior-posterior (A-P) axis of the embryo. The mitotic apparatus then orients so as to cleave the embryo into anterior and posterior blastomeres that differ in both size and developmental potential. Here we describe a role for MBK-2, a member of the Dyrk family of protein kinases, in asymmetric cell division in C. elegans. In mbk-2 mutants, the initial mitotic spindle is misplaced and cytoplasmic factors, including the germline-specific protein PIE-1, are mislocalized. Our findings support a model in which MBK-2 down-regulates the katanin-related protein MEI-1 to control spindle positioning and acts through distinct, as yet unknown factors, to control the localization of cytoplasmic determinants. These findings in conjunction with work from Schizosaccharomyces pombe indicate a possible conserved role for Dyrk family kinases in the regulation of spindle placement during cell division.     

Retinoic acid signalling is required for specification of pronephric cell fate

The mechanisms by which a subset of mesodermal cells are committed to a nephrogenic fate are largely unknown. In this study, we have investigated the role of retinoic acid (RA) signalling in this process using Xenopus laevis as a model system and Raldh2 knockout mice. Pronephros formation in Xenopus embryo is severely impaired when RA signalling is inhibited either through expression of a dominant-negative RA receptor, or by expressing the RA-catabolizing enzyme XCyp26 or through treatment with chemical inhibitors. Conversely, ectopic RA signalling expands the size of the pronephros. Using a transplantation assay that inhibits RA signalling specifically in pronephric precursors, we demonstrate that this signalling is required within this cell population. Timed antagonist treatments show that RA signalling is required during gastrulation for expression of Xlim-1 and XPax-8 in pronephric precursors. Moreover, experiments conducted with a protein synthesis inhibitor indicate that RA may directly regulate Xlim-1. Raldh2 knockout mouse embryos fail to initiate the expression of early kidney-specific genes, suggesting that implication of RA signalling in the early steps of kidney formation is evolutionary conserved in vertebrates.     

bHLH genes cath5 and cNSCL1 promote bFGF-stimulated RPE cells to transdifferentiate toward retinal ganglion cells

The molecular mechanism of retinal ganglion cell (RGC) genesis and development is not well understood. Published data suggest that the process may involve two bHLH genes, ath5 and NSCL1. Gain-of-function studies show that ath5 increases RGC production in the developing retina. We examined whether two chick genes, cath5 and cNSCL1, can guide retinal pigment epithelial (RPE) cells to transdifferentiate toward RGCs. Ectopic expression of cath5 and cNSCL1 in cultured chick RPE cells was achieved through retroviral transduction. cath5 alone was unable to induce de novo expression of early RGC markers, such as RA4 antigen, neurofilament (160 kDa), and a neurofilament-associated antigen. However, cath5 induced the expression of these proteins when the RPE cells were cultured with medium supplemented with bFGF. Since bFGF alone can induce only RA4 antigen, the expression of the additional RGC markers reflects a synergism between cath5 and bFGF in promoting RPE transdifferentiation toward RGCs. Morphologically, the RA4(+) cells in bFGF + cath5 cultures appeared more neuron-like than those generated by bFGF alone. cNSCL1 also promoted bFGF-stimulated RPE cells to transdifferentiate toward RGCs that expressed RA4 antigen, N-CAM, Islet-1, neurofilament, and neurofilament-associated antigen. We found that cath5 induced cNSCL1 expression, but not vice versa. Our data suggest that cath5 or cNSCL1 alone was insufficient to induce RPE transdifferentiation into RGCs, but could further neural differentiation initiated by bFGF. We propose that intrinsic factors act synergistically with extrinsic factors during RGC genesis and development.     

gcm2 promotes glial cell differentiation and is required with glial cells missing for macrophage development in Drosophila

glial cells missing (gcm) is the primary regulator of glial cell fate in Drosophila. In addition, gcm has a role in the differentiation of the plasmatocyte/macrophage lineage of hemocytes. Since mutation of gcm causes only a decrease in plasmatocyte numbers without changing their ability to convert into macrophages, gcm cannot be the sole determinant of plasmatocyte/macrophage differentiation. We have characterized a gcm homolog, gcm2. gcm2 is expressed at low levels in glial cells and hemocyte precursors. We show that gcm2 has redundant functions with gcm and has a minor role promoting glial cell differentiation. More significant, like gcm, mutation of gcm2 leads to reduced plasmatocyte numbers. A deletion removing both genes has allowed us to clarify the role of these redundant genes in plasmatocyte development. Animals deficient for both gcm and gcm2 fail to express the macrophage receptor Croquemort. Plasmatocytes are reduced in number, but still express the early marker Peroxidasin. These Peroxidasin-expressing hemocytes fail to migrate to their normal locations and do not complete their conversion into macrophages. Our results suggest that both gcm and gcm2 are required together for the proliferation of plasmatocyte precursors, the expression of Croquemort protein, and the ability of plasmatocytes to convert into macrophages.