Fuz regulates craniofacial development through tissue specific responses to signaling factors

The planar cell polarity effector gene Fuz regulates ciliogenesis and Fuz loss of function studies reveal an array of embryonic phenotypes. However, cilia defects can affect many signaling pathways and, in humans, cilia defects underlie several craniofacial anomalies. To address this, we analyzed the craniofacial phenotype and signaling responses of the Fuz(-/-) mice. We demonstrate a unique role for Fuz in regulating both Hedgehog (Hh) and Wnt/β-catenin signaling during craniofacial development. Fuz expression first appears in the dorsal tissues and later in ventral tissues and craniofacial regions during embryonic development coincident with cilia development. The Fuz(-/-) mice exhibit severe craniofacial deformities including anophthalmia, agenesis of the tongue and incisors, a hypoplastic mandible, cleft palate, ossification/skeletal defects and hyperplastic malformed Meckel's cartilage. Hh signaling is down-regulated in the Fuz null mice, while canonical Wnt signaling is up-regulated revealing the antagonistic relationship of these two pathways. Meckel's cartilage is expanded in the Fuz(-/-) mice due to increased cell proliferation associated with the up-regulation of Wnt canonical target genes and decreased non-canonical pathway genes. Interestingly, cilia development was decreased in the mandible mesenchyme of Fuz null mice, suggesting that cilia may antagonize Wnt signaling in this tissue. Furthermore, expression of Fuz decreased expression of Wnt pathway genes as well as a Wnt-dependent reporter. Finally, chromatin IP experiments demonstrate that β-catenin/TCF-binding directly regulates Fuz expression. These data demonstrate a new model for coordination of Hh and Wnt signaling and reveal a Fuz-dependent negative feedback loop controlling Wnt/β-catenin signaling.     

Mutations in Planar Cell Polarity Gene SCRIB Are Associated with Spina Bifida

Neural tube defects (NTDs) (OMIM #182940) including anencephaly, spina bifida and craniorachischisis, are severe congenital malformations that affect 0.5–1 in 1,000 live births in the United States, with varying prevalence around the world. Mutations in planar cell polarity (PCP) genes are believed to cause a variety of NTDs in both mice and humans. SCRIB is a PCP-associated gene. Mice that are homozygous for the Scrib p.I285K and circletail (Crc) mutations, present with the most severe form of NTDs, namely craniorachischisis. A recent study reported that mutations in SCRIB were associated with craniorachischisis in humans, but whether SCRIB mutations contribute to increased spina bifida risk is still unknown. We sequenced the SCRIB gene in 192 infants with spina bifida and 190 healthy controls. Among the spina bifida patients, we identified five novel missense mutations that were predicted-to-be-deleterious by the PolyPhen software. Of these five mutations, three of them (p.P1043L, p.P1332L, p.L1520R) significantly affected the subcellular localization of SCRIB. In addition, we demonstrated that the craniorachischisis mouse line-90 mutation I285K, also affected SCRIB subcellular localization. In contrast, only one novel missense mutation (p.A1257T) was detected in control samples, and it was predicted to be benign. This study demonstrated that rare deleterious mutations of SCRIB may contribute to the multifactorial risk for human spina bifida.     

The unaccounted yet abundant nitrous oxide-reducing microbial community: a potential nitrous oxide sink

Nitrous oxide (N₂O) is a major radiative forcing and stratospheric ozone-depleting gas emitted from terrestrial and aquatic ecosystems. It can be transformed to nitrogen gas (N₂) by bacteria and archaea harboring the N₂O reductase (N₂OR), which is the only known N₂O sink in the biosphere. Despite its crucial role in mitigating N₂O emissions, knowledge of the N₂OR in the environment remains limited. Here, we report a comprehensive phylogenetic analysis of the nosZ gene coding the N₂OR in genomes retrieved from public databases. The resulting phylogeny revealed two distinct clades of nosZ, with one unaccounted for in studies investigating N₂O-reducing communities. Examination of N₂OR structural elements not considered in the phylogeny revealed that the two clades differ in their signal peptides, indicating differences in the translocation pathway of the N₂OR across the membrane. Sequencing of environmental clones of the previously undetected nosZ lineage in various environments showed that it is widespread and diverse. Using quantitative PCR, we demonstrate that this clade was most often at least as abundant as the other, thereby more than doubling the known extent of the overall N₂O-reducing community in the environment. Furthermore, we observed that the relative abundance of nosZ from either clade varied among habitat types and environmental conditions. Our results indicate a physiological dichotomy in the diversity of N₂O-reducing microorganisms, which might be of importance for understanding the relationship between the diversity of N₂O-reducing microorganisms and N₂O reduction in different ecosystems.     

Analysis of archived residual newborn screening blood spots after whole genome amplification

Background

Deidentified newborn screening bloodspot samples (NBS) represent a valuable potential resource for genomic research if impediments to whole exome sequencing of NBS deoxyribonucleic acid (DNA), including the small amount of genomic DNA in NBS material, can be overcome. For instance, genomic analysis of NBS could be used to define allele frequencies of disease-associated variants in local populations, or to conduct prospective or retrospective studies relating genomic variation to disease emergence in pediatric populations over time. In this study, we compared the recovery of variant calls from exome sequences of amplified NBS genomic DNA to variant calls from exome sequencing of non-amplified NBS DNA from the same individuals.

Results

Using a standard alignment-based Genome Analysis Toolkit (GATK), we find 62,000–76,000 additional variants in amplified samples. After application of a unique kmer enumeration and variant detection method (RUFUS), only 38,000–47,000 additional variants are observed in amplified gDNA. This result suggests that roughly half of the amplification-introduced variants identified using GATK may be the result of mapping errors and read misalignment.

Conclusions

Our results show that it is possible to obtain informative, high-quality data from exome analysis of whole genome amplified NBS with the important caveat that different data generation and analysis methods can affect variant detection accuracy, and the concordance of variant calls in whole-genome amplified and non-amplified exomes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1747-2) contains supplementary material, which is available to authorized users.     

Degradation of extracellular matrix by mouse trophoblast outgrowths: a model for implantation

During implantation the embryo attaches to the endometrial surface and trophoblast traverses the uterine epithelium, anchoring in the uterine connective tissue. To determine whether trophoblast can facilitate invasion of the uterus by degrading components of normal uterine extracellular matrix, mouse blastocysts were cultured on a radio-labeled extracellular matrix that contained glycoproteins, elastin, and collagen. The embryos attached to the matrix, and trophoblast spread over the surface. Starting on day 5 of culture there was a release of labeled peptides into the medium. The radioactive peptides released from the matrix by the embryos had molecular weights ranging from more than 25,000 to more than 200. By day 7 there were areas where individual trophoblast cells had separated from one another, revealing the underlying substratum that was cleared of matrix. When trophoblast cells were lysed with NH(4)OH on day 8, it was apparent that the area underneath the trophoblast outgrowth had been cleared of matrix. Scanning electron microscopy and time-lapse cinemicrography confirmed that the digestion of matrix was highly localized, taking place only underneath the trophoblast, with no evidence of digestion of the matrix beyond the periphery of the trophoblast outgrowth. The sharp boundaries of degredation observed may be due to localized proteinase secretion by trophoblast, to membrane proteinases on the surface of trophoblast, or to endocytosis. Digestion of the matrix was not dependent on plasminogen, thus ruling out a role for plasminogen activator. Digestion was not inhibited by a variety of hormones and inhibitors, including progesterone, 17β-estradiol, leupeptin, EDTA, colchicine, NH(4)Cl, or ε-aminocaproic acid. This system of culturing embryos on extracellular matrix may be useful in determining the processes that regulate trophoblast migration and invasion into the maternal tissues during implantation.0     

Genetic Differences in the Duration of the Lymphocyte Heat Shock Response in Mice

Lymphocytes from adult mice bearing a known difference in genetic susceptibility to teratogen-induced exencephaly (SWV/SD, and DBA/2J) were evaluated for changes in protein synthesis following an in vivo heat treatment. Particular attention was paid to changes indicative of the heat shock response, a highly conserved response to environmental insult consisting of induction of a few, highly conserved proteins with simultaneous decreases in normal protein synthesis. The duration of heat shock protein induction in lymphocytes was found to be increased by 1 hr in the teratogen-sensitive SWV/SD strain as compared to the resistant DBA/2J strain. Densitometric analysis revealed a significant decrease in the relative synthesis of at least two non-heat shock proteins (36 kD and 45 kD) in the SWV/SD lymphocytes as compared to DBA/2J cells. The increased sensitivity of protein synthesis to hyperthermia in the SWV/SD lymphocytes were lost in the F1 progeny of reciprocal crosses between SWV/SD and DBA/2J mouse strains. Sensitivity to hyperthermia-induced exencephaly is recessive to resistance in these crosses. The relationship between altered protein synthesis and teratogen susceptibility is discussed.     

State of the art in developmental toxicity screening methods and a way forward: a meeting report addressing embryonic stem cells, whole embryo culture, and zebrafish

A meeting was convened so that users of three models for in vitro developmental toxicity (embryonic stem cells, whole embryo culture, and zebrafish) could share their experiences with each model, and explore the areas for improvement. We present a summary of this meeting and the recommendations of the group.     

PP2A dephosphorylates Elf-1 and determines the expression of CD3zeta and FcRgamma in human systemic lupus erythematosus T cells

T cells from patients with systemic lupus erythematosus are characterized by decreased expression of CD3zeta-chain and increased expression of FcRgamma-chain, which becomes part of the CD3 complex and contributes to aberrant signaling. Elf-1 enhances the expression of CD3zeta, whereas it suppresses the expression of FcRgamma gene and lupus T cells have decreased amounts of DNA-binding 98 kDa form of Elf-1. We show that the aberrantly increased PP2A in lupus T cells dephosphorylates Elf-1 at Thr-231. Dephosphorylation results in limited expression and binding of the 98 kDa Elf-1 form to the CD3zeta and FcRgamma promoters. Suppression of the expression of the PP2A leads to increased expression of CD3zeta and decreased expression of FcRgamma genes and correction of the early signaling response. Therefore, PP2A serves as a central determinant of abnormal T cell function in human lupus and may represent an appropriate treatment target.