RRP7A links primary microcephaly to dysfunction of ribosome biogenesis, resorption of primary cilia, and neurogenesis.

Primary microcephaly (MCPH) is characterized by reduced brain size and intellectual disability. The exact pathophysiological mechanism underlying MCPH remains to be elucidated, but dysfunction of neuronal progenitors in the developing neocortex plays a major role. We identified a homozygous missense mutation (p.W155C) in Ribosomal RNA Processing 7 Homolog A, RRP7A, segregating with MCPH in a consanguineous family with 10 affected individuals. RRP7A is highly expressed in neural stem cells in developing human forebrain, and targeted mutation of Rrp7a leads to defects in neurogenesis and proliferation in a mouse stem cell model. RRP7A localizes to centrosomes, cilia and nucleoli, and patient-derived fibroblasts display defects in ribosomal RNA processing, primary cilia resorption, and cell cycle progression. Analysis of zebrafish embryos supported that the patient mutation in RRP7A causes reduced brain size, impaired neurogenesis and cell proliferation, and defective ribosomal RNA processing. These findings provide novel insight into human brain development and MCPH.

Intragastric balloon outcomes in super-obesity: a 16-year city center hospital series.

BACKGROUND: Intragastric balloons represent an endoscopic therapy aimed at achieving weight loss by mechanical induction of satiety. Their exact role within the bariatric armamentarium remains uncertain. OBJECTIVE: Our study aimed to evaluate the use of intragastric balloon therapy alone and before definitive bariatric surgery over a 16-year period. SETTING: A large city academic bariatric center for super-obese patients. METHODS: Between January 2000 and February 2016, 207 patients underwent ORBERA intragastric balloon placement at esophagogastroduodenoscopy. Four surgeons performed the procedures, and data were entered prospectively into a dedicated bariatric database. Patients' weight loss data were measured through body mass index (BMI) and excess weight loss and recorded at each clinic review for up to 5 years (60 mo). Treatment arms included intragastric balloon alone with lifestyle therapy or intragastric balloon and definitive bariatric surgery: gastric banding, sleeve gastrectomy, or Roux-en-Y gastric bypass. An additional treatment arm of analysis included the overall results from intragastric balloon followed by stapled procedure. RESULTS: One hundred twenty-nine female and 78 male patients had a mean age of 44.5 (±11.3) years and a mean BMI of 57.3 (±9.7) kg/m2. Fifty-eight percent of patients suffered from type 2 diabetes. Time from initial or first balloon insertion to definitive surgical therapy ranged between 9 and 13 months. Seventy-six patients had intragastric balloon alone, and 131 had intragastric balloon followed by definitive procedure. At 60 months postoperatively the intragastric balloon alone with lifestyle changes demonstrated an excess weight loss of 9.04% and BMI drop of 3.8; intragastric balloon with gastric banding demonstrated an excess weight loss of 32.9% and BMI drop of 8.9. Intragastric balloon and definitive stapled procedure demonstrated a BMI drop of 17.6 and an excess weight loss of 52.8%. Overall, there were 3 deaths (1.4%), 2 within 10 days due to acute gastric perforation secondary to vomiting and 1 cardiac arrest at 4 weeks postoperatively. CONCLUSION: Intragastric balloons can offer effective weight loss in selected super-obese patients within a dedicated bariatric center offering multidisciplinary support. Balloon insertion alone offers only short-term weight loss; however, when combined with definitive bariatric surgical approaches, durable weight loss outcomes can be achieved. A strategy of early and continual vigilance for side effects and a low threshold for removal should be implemented. Surgeon and unit experience with intragastric balloons can contribute to "kick starting" successful weight loss as a bridge to definitive therapy in an established bariatric surgical pathway.

CEP128 Localizes to the Subdistal Appendages of the Mother Centriole and Regulates TGF-ß/BMP Signaling at the Primary Cilium.

The centrosome is the main microtubule-organizing center in animal cells and comprises a mother and daughter centriole surrounded by pericentriolar material. During formation of primary cilia, the mother centriole transforms into a basal body that templates the ciliary axoneme. Ciliogenesis depends on mother centriole-specific distal appendages, whereas the role of subdistal appendages in ciliary function is unclear. Here, we identify CEP128 as a centriole subdistal appendage protein required for regulating ciliary signaling. Loss of CEP128 did not grossly affect centrosomal or ciliary structure but caused impaired transforming growth factor-ß/bone morphogenetic protein (TGF-ß/BMP) signaling in zebrafish and at the primary cilium in cultured mammalian cells. This phenotype is likely the result of defective vesicle trafficking at the cilium as ciliary localization of RAB11 was impaired upon loss of CEP128, and quantitative phosphoproteomics revealed that CEP128 loss affects TGF-ß1-induced phosphorylation of multiple proteins that regulate cilium-associated vesicle trafficking.

Forward Genetic Screens in Zebrafish Identify Pre-mRNA-Processing Pathways Regulating Early T Cell Development.

Lymphocytes represent basic components of vertebrate adaptive immune systems, suggesting the utility of non-mammalian models to define the molecular basis of their development and differentiation. Our forward genetic screens in zebrafish for recessive mutations affecting early T cell development revealed several major genetic pathways. The identification of lineage-specific transcription factors and specific components of cytokine signaling and DNA replication and/or repair pathways known from studies of immunocompromised mammals provided an evolutionary cross-validation of the screen design. Unexpectedly, however, genes encoding proteins required for pre-mRNA processing were enriched in the collection of mutants identified here. In both zebrafish and mice, deficiency of the splice regulator TNPO3 impairs intrathymic T cell differentiation, illustrating the evolutionarily conserved and cell-type-specific functions of certain pre-mRNA-processing factors for T cell development.

Cohesin and CTCF differentially regulate spatiotemporal runx1 expression during zebrafish development.

Runx1 is a transcription factor essential for definitive hematopoiesis. In all vertebrates, the Runx1 gene is transcribed from two promoters: a proximal promoter (P2), and a distal promoter (P1). We previously found that runx1 expression in a specific hematopoietic cell population in zebrafish embryos depends on cohesin. Here we show that zebrafish runx1 is directly bound by cohesin and CCCTC binding factor (CTCF) at the P1 and P2 promoters, and within the intron between P1 and P2. Cohesin initiates expression of runx1 in the posterior lateral mesoderm and influences promoter use, while CTCF represses its expression in the newly emerging cells of the tail bud. The intronic binding sites for cohesin and CTCF coincide with histone modifications that confer enhancer-like properties, and two of the cohesin/CTCF sites behaved as insulators in an in vivo assay. The identified cohesin and CTCF binding sites are likely to be cis-regulatory elements (CREs) for runx1 since they also recruit RNA polymerase II (RNAPII). CTCF depletion excluded RNAPII from two intronic CREs but not the promoters of runx1. We propose that cohesin and CTCF have distinct functions in the regulation of runx1 during zebrafish embryogenesis, and that these regulatory functions are likely to involve runx1 intronic CREs. Cohesin (but not CTCF) depletion enhanced RUNX1 expression in a human leukemia cell line, suggesting conservation of RUNX1 regulation through evolution.

Diverse developmental disorders from the one ring: distinct molecular pathways underlie the cohesinopathies.

The multi-subunit protein complex, cohesin, is responsible for sister chromatid cohesion during cell division. The interaction of cohesin with DNA is controlled by a number of additional regulatory proteins. Mutations in cohesin, or its regulators, cause a spectrum of human developmental syndromes known as the "cohesinopathies." Cohesinopathy disorders include Cornelia de Lange Syndrome and Roberts Syndrome. The discovery of novel roles for chromatid cohesion proteins in regulating gene expression led to the idea that cohesinopathies are caused by dysregulation of multiple genes downstream of mutations in cohesion proteins. Consistent with this idea, Drosophila, mouse, and zebrafish cohesinopathy models all show altered expression of developmental genes. However, there appears to be incomplete overlap among dysregulated genes downstream of mutations in different components of the cohesion apparatus. This is surprising because mutations in all cohesion proteins would be predicted to affect cohesin's roles in cell division and gene expression in similar ways. Here we review the differences and similarities between genetic pathways downstream of components of the cohesion apparatus, and discuss how such differences might arise, and contribute to the spectrum of cohesinopathy disorders. We propose that mutations in different elements of the cohesion apparatus have distinct developmental outcomes that can be explained by sometimes subtly different molecular effects.

RAD21 mutations cause a human cohesinopathy.

The evolutionarily conserved cohesin complex was originally described for its role in regulating sister-chromatid cohesion during mitosis and meiosis. Cohesin and its regulatory proteins have been implicated in several human developmental disorders, including Cornelia de Lange (CdLS) and Roberts syndromes. Here we show that human mutations in the integral cohesin structural protein RAD21 result in a congenital phenotype consistent with a "cohesinopathy." Children with RAD21 mutations display growth retardation, minor skeletal anomalies, and facial features that overlap findings in individuals with CdLS. Notably, unlike children with mutations in NIPBL, SMC1A, or SMC3, these individuals have much milder cognitive impairment than those with classical CdLS. Mechanistically, these mutations act at the RAD21 interface with the other cohesin proteins STAG2 and SMC1A, impair cellular DNA damage response, and disrupt transcription in a zebrafish model. Our data suggest that, compared to loss-of-function mutations, dominant missense mutations result in more severe functional defects and cause worse structural and cognitive clinical findings. These results underscore the essential role of RAD21 in eukaryotes and emphasize the need for further understanding of the role of cohesin in human development.

A zebrafish model of Roberts syndrome reveals that Esco2 depletion interferes with development by disrupting the cell cycle.

The human developmental diseases Cornelia de Lange Syndrome (CdLS) and Roberts Syndrome (RBS) are both caused by mutations in proteins responsible for sister chromatid cohesion. Cohesion is mediated by a multi-subunit complex called cohesin, which is loaded onto chromosomes by NIPBL. Once on chromosomes, cohesin binding is stabilized in S phase upon acetylation by ESCO2. CdLS is caused by heterozygous mutations in NIPBL or cohesin subunits SMC1A and SMC3, and RBS is caused by homozygous mutations in ESCO2. The genetic cause of both CdLS and RBS reside within the chromosome cohesion apparatus, and therefore they are collectively known as "cohesinopathies". However, the two syndromes have distinct phenotypes, with differences not explained by their shared ontology. In this study, we have used the zebrafish model to distinguish between developmental pathways downstream of cohesin itself, or its acetylase ESCO2. Esco2 depleted zebrafish embryos exhibit features that resemble RBS, including mitotic defects, craniofacial abnormalities and limb truncations. A microarray analysis of Esco2-depleted embryos revealed that different subsets of genes are regulated downstream of Esco2 when compared with cohesin subunit Rad21. Genes downstream of Rad21 showed significant enrichment for transcriptional regulators, while Esco2-regulated genes were more likely to be involved the cell cycle or apoptosis. RNA in situ hybridization showed that runx1, which is spatiotemporally regulated by cohesin, is expressed normally in Esco2-depleted embryos. Furthermore, myca, which is downregulated in rad21 mutants, is upregulated in Esco2-depleted embryos. High levels of cell death contributed to the morphology of Esco2-depleted embryos without affecting specific developmental pathways. We propose that cell proliferation defects and apoptosis could be the primary cause of the features of RBS. Our results show that mutations in different elements of the cohesion apparatus have distinct developmental outcomes, and provide insight into why CdLS and RBS are distinct diseases.

Developing T lymphocytes are uniquely sensitive to a lack of topoisomerase III alpha.

All organisms possess at least one type IA DNA topoisomerase. These topoisomerases function as part of a DNA structure-specific "dissolvasome," also known as the RTR complex, which has critical functions in faithful DNA replication, recombination, and chromosome segregation. In humans, the heteromeric RTR complex consists of RMI1, RMI2, the Bloom's syndrome gene product (BLM), and topoisomerase 3A (TOP3A) proteins. Here, we describe the identification and characterization of two deleterious mutations in the zebrafish top3a gene that reveal an unexpected tissue-specific requirement of top3a function in developing thymocytes. Deficiency in top3a activates a p53-dependent check-point but does not affect VDJ recombination. Our results suggest that TOP3A could be a candidate gene involved in human primary immunodeficiency syndromes.

Expression of cohesin and condensin genes during zebrafish development supports a non-proliferative role for cohesin.

Cohesin and condensin are similar, but distinct multi-subunit protein complexes that have well-described roles in sister chromatid cohesion and chromosome condensation, respectively. Recently it has emerged that cohesin, and proteins that regulate cohesin function have additional developmental roles. To further understand the role of cohesin in development, we analyzed the expression of genes encoding cohesin and condensin subunits in developing zebrafish embryos and juvenile brain. We found that cohesin subunits are expressed in a pattern that is similar (but not quite identical) to the expression of condensin subunits. Cohesin genes smc1a, rad21, pds5b and smc3 were expressed in the forebrain ventricular zone, the tectum, the mid-hindbrain boundary, the fourth ventricle, branchial arches, the otic vesicle, the eye and faintly in the developing pectoral fins. Condensin genes smc2 and smc4 were expressed in the forebrain ventricular zone, the tectum, the mid-hindbrain boundary, the fourth ventricle, branchial arches, eye and pectoral fins. Condensin genes were additionally expressed in the hindbrain proliferative zone, an area in which cohesin genes were not detected. A comparison with pcna expression and BrdU incorporation revealed that the expression of cohesins and condensins closely overlap with zones of proliferation. Interestingly, cohesin genes were expressed in non-proliferating cells flanking rhombomere boundaries in the developing brain. In mature brain and eye, cohesin was expressed in both proliferating cells and in broad zones of post-mitotic cells. The distribution of cohesin and condensin mRNAs supports existing evidence for a non-cell cycle role for cohesin in the developing brain.

Outer dense fibre protein 2 (ODF2) is a self-interacting centrosomal protein with affinity for microtubules.

Outer dense fibre protein 2 (ODF2) is a major protein of sperm tail outer dense fibres which are prominent sperm tail-specific cytoskeletal structures. Moreover, ODF2 was also identified as a widespread component of the centrosomal scaffold and was found to associate preferentially with the appendages of the mother centriole [Nakagawa, Y., Yamane, Y., Okanoue, T., Tsukita, S. and Tsukita, S. (2001) Mol. Biol. Cell 12, 1687-1697]. Secondary structure predictions indicated ODF2 as an overall coiled-coil protein with a putative fibre forming capacity. To investigate its potential functions in generating the centrosomal scaffold and in microtubule nucleation we asked whether ODF2 is able to form a fibrillar structure by self-association in vivo and if it interacts with microtubules. By cytological investigation of transfected mammalian cells expressing ODF2-GFP fusion proteins and in vitro coprecipitation assays we could demonstrate that ODF2 is a self-interacting protein that forms a fibrillar structure partially linked to the microtubule network. Microtubule cosedimentation and coprecipitation assays indicated ODF2 as a microtubule-associated protein. However, we could not demonstrate a direct interaction of ODF2 with tubulin, suggesting that binding of endogenous ODF2 to the axonemal as well as to centrosomal microtubules may be mediated by, as yet, unknown proteins.