Regional neural tube closure defined by the Grainy head-like transcription factors

Primary neurulation in mammals has been defined by distinct anatomical closure sites, at the hindbrain/cervical spine (closure 1), forebrain/midbrain boundary (closure 2), and rostral end of the forebrain (closure 3). Zones of neurulation have also been characterized by morphologic differences in neural fold elevation, with non-neural ectoderm-induced formation of paired dorso-lateral hinge points (DLHP) essential for neural tube closure in the cranial and lower spinal cord regions, and notochord-induced bending at the median hinge point (MHP) sufficient for closure in the upper spinal region. Here we identify a unifying molecular basis for these observations based on the function of the non-neural ectoderm-specific Grainy head-like genes in mice. Using a gene-targeting approach we show that deletion of Grhl2 results in failed closure 3, with mutants exhibiting a split-face malformation and exencephaly, associated with failure of neuro-epithelial folding at the DLHP. Loss of Grhl3 alone defines a distinct lower spinal closure defect, also with defective DLHP formation. The two genes contribute equally to closure 2, where only Grhl gene dosage is limiting. Combined deletion of Grhl2 and Grhl3 induces severe rostral and caudal neural tube defects, but DLHP-independent closure 1 proceeds normally in the upper spinal region. These findings provide a molecular basis for non-neural ectoderm mediated formation of the DLHP that is critical for complete neuraxis closure.     

Grainyhead-like 2 regulates neural tube closure and adhesion molecule expression during neural fold fusion

Defects in closure of embryonic tissues such as the neural tube, body wall, face and eye lead to severe birth defects. Cell adhesion is hypothesized to contribute to closure of the neural tube and body wall; however, potential molecular regulators of this process have not been identified. Here we identify an ENU-induced mutation in mice that reveals a molecular pathway of embryonic closure. Line2F homozygous mutant embryos fail to close the neural tube, body wall, face, and optic fissure, and they also display defects in lung and heart development. Using a new technology of genomic sequence capture and high-throughput sequencing of a 2.5 Mb region of the mouse genome, we discovered a mutation in the grainyhead-like 2 gene (Grhl2). Microarray analysis revealed Grhl2 affects the expression of a battery of genes involved in cell adhesion and E-cadherin protein is drastically reduced in tissues that require Grhl2 function. The tissue closure defects in Grhl2 mutants are similar to that of AP-2α null mutants and AP-2α has been shown to bind to the promoter of E-cadherin. Therefore, we tested for a possible interaction between these genes. However, we find that Grhl2 and AP-2α do not regulate each other's expression, E-cadherin expression is normal in AP-2α mutants during neural tube closure, and Grhl2;AP-2α trans-heterozygous embryos are morphologically normal. Taken together, our studies point to a complex regulation of neural tube fusion and highlight the importance of comparisons between these two models to understand more fully the molecular pathways of embryonic tissue closure.     

d4 family genes: Genomic organization and expression

A family of closely related genes, named the d4 family, has been previously identified in mammals. It comprises three genes encoding structurally related proteins. The hallmark of the family is D4 domain—a double-paired finger motif that consists of two tandemly arranged PHD finger domains. These genes are expressed in various tissues and at various developmental stages. Two of those, neuro-d4 and cer-d4, are strictly neurospecific and their expression is developmentally regulated. Another gene, ubi-d4/Requiem is ubiquitously expressed in all embryonic and adult tissues at the same levels. d4 family genes are evolutionary conserved. Human, mouse, rat, and chicken d4 genes have been cloned. The only d4-like gene was found in the genome of nematode C. elegans and hydra H. magnipapillata. The sole member of d4 family was identified also in the genome of D. melanogaster. However, d4 genes are not believed to be present in the genomes of prokaryotes and yeast. This review describes genomic organization and expression of d4 family genes in vertebrates.     

Grhl2 is required in nonneural tissues for neural progenitor survival and forebrain development

Grainyhead‐like genes are part of a highly conserved gene family that play a number of roles in ectoderm development and maintenance in mammals. Here we identify a novel allele of Grhl2, cleft‐face 3 (clft3), in a mouse line recovered from an ENU mutagenesis screen for organogenesis defects. Homozygous clft3 mutants have a number of phenotypes in common with other alleles of Grhl2. We note a significant effect of genetic background on the clft3 phenotype. One of these is a reduction in size of the telencephalon where we find abnormal patterns of neural progenitor mitosis and apoptosis in mutant brains. Interestingly, Grhl2 is not expressed in the developing forebrain, suggesting this is a survival factor for neural progenitors exerting a paracrine effect on the neural tissue from the overlying ectoderm where Grhl2 is highly expressed. genesis 53:573–582, 2015. © 2015 Wiley Periodicals, Inc.     

The Caenorhabditis elegans Ste20 kinase, GCK-3, is essential for postembryonic developmental timing and regulates meiotic chromosome segregation

Ste20 kinases constitute a large family of serine/threonine kinases with a plethora of biological functions. Members of the GCK-VI subfamily have been identified as important regulators of osmohomeostasis across species functioning upstream of ion channels. Although the expression of the two highly similar mammalian GCK-VI kinases is eminent in a wide variety of tissues, which includes also the testis, their potential roles in development remain elusive. Caenorhabditis elegans contains a single ancestral ortholog termed GCK-3. Here, we report a comprehensive analysis of gck-3 function and demonstrate its requirement for several developmental processes independent of ion homeostasis, i.e., larval progression, vulva, and germ line formation. Consistent with a wide range of gck-3 function we find that endogenous GCK-3 is expressed ubiquitously. The serine/threonine kinase activity of GCK-3, but not its presumed C-terminal substrate interaction domain, is essential for gck-3 gene function. Although expressed in female germ cells, we find GCK-3 progressively accumulating during spermatogenesis where it promotes the first meiotic cell division and facilitates faithful chromosome segregation. In particular, we find that different levels of gck-3 activity appear to be important for various aspects of germ line development. Taken together, our findings suggest that members of the GCK-VI kinase subfamily may act as key regulators of many developmental processes and that this newly described role in meiotic progression might be conserved and an important part of sexual reproduction.     

Predictive screening for regulators of conserved functional gene modules (gene batteries) in mammals

Background

Hematopoiesis is a complex developmental process controlled by a large number of factors that regulate stem cell renewal, lineage commitment and differentiation. Secreted proteins, including the hematopoietic growth factors, play critical roles in these processes and have important biological and clinical significance. We have employed representational difference analysis to identify genes that are differentially expressed during experimentally induced myeloid differentiation in the murine EML hematopoietic stem cell line.

Results

One identified clone encoded a previously unidentified protein of 541 amino acids that contains an amino terminal signal sequence but no other characterized domains. This protein is a member of family of related proteins that has been named family with sequence similarity 20 (FAM20) with three members (FAM20A, FAM20B and FAM20C) in mammals. Evolutionary comparisons revealed the existence of a single FAM20 gene in the simple vertebrate Ciona intestinalis and the invertebrate worm Caenorhabditis elegans and two genes in two insect species, Drosophila melanogaster and Anopheles gambiae. Six FAM20 family members were identified in the genome of the pufferfish, Fugu rubripes and five members in the zebrafish, Danio rerio. The mouse Fam20a protein was ectopically expressed in a mammalian cell line and found to be a bona fide secreted protein and efficient secretion was dependent on the integrity of the signal sequence. Expression analysis revealed that the Fam20a gene was indeed differentially expressed during hematopoietic differentiation and that the other two family members (Fam20b and Fam20c) were also expressed during hematcpoiesis but that their mRNA levels did not vary significantly. Likewise FAM20A was expressed in more limited set of human tissues than the other two family members.

Conclusions

The FAM20 family represents a new family of secreted proteins with potential functions in regulating differentiation and function of hematopoietic and other tissues. The Fam20a mRNA was only expressed during early stages of hematopoietic development and may play a role in lineage commitment or proliferation. The expansion in gene number in different species suggests that the family has evolved as a result of several gene duplication events that have occurred in both vertebrates and invertebrates.