In vivo knockdown of Brachyury results in skeletal defects and urorectal malformations resembling caudal regression syndrome

The T-box transcription factor BRACHYURY (T) is a key regulator of mesoderm formation during early development. Complete loss of T has been shown to lead to embryonic lethality around E10.0. Here we characterize an inducible miRNA-based in vivo knockdown mouse model of T, termed KD3-T, which exhibits a hypomorphic phenotype. KD3-T embryos display axial skeletal defects caused by apoptosis of paraxial mesoderm, which is accompanied by urorectal malformations resembling the murine uro-recto-caudal syndrome and human caudal regression syndrome phenotypes. We show that there is a reduction of T in the notochord of KD3-T embryos which results in impaired notochord differentiation and its subsequent loss, whereas levels of T in the tailbud are sufficient for axis extension and patterning. Furthermore, the notochord in KD3-T embryos adopts a neural character and loses its ability to act as a signaling center. Since KD3-T animals survive until birth, they are useful for examining later roles for T in the development of urorectal tissues

Murine expression and mutation analyses of the prostate androgen-regulated mucin-like protein 1 (Parm1) gene, a candidate for human epispadias.

Background Epispadias is the mildest phenotype of the human bladder exstrophy–epispadias complex (BEEC), and presents with varying degrees of severity. This urogenital birth defect results from a disturbance in the septation process, during which separate urogenital and anorectal components are formed through division of the cloaca. This process is reported to be influenced by androgen signaling. The human PARM1 gene encodes the prostate androgen-regulated mucin-like protein 1, which is expressed in heart, kidney, and placenta. Methods We performed whole mount in situ hybridization analysis of Parm1 expression in mouse embryos between gestational days (GD) 9.5 and 12.5, which are equivalent to human gestational weeks 4–6. Since the spatio-temporal localization of Parm1 corresponded to tissues which are affected in human epispadias, we sequenced PARM1 in 24 affected patients. Results We found Parm1 specifically expressed in the region of the developing cloaca, the umbilical cord, bladder anlage, and the urethral component of the genital tubercle. Additionally, Parm1 expression was detected in the muscle progenitor cells of the somites and head mesenchyme. PARM1 gene analysis revealed no alterations in the coding region of any of the investigated patients. Conclusions These findings suggest that PARM1 does not play a major role in the development of human epispadias. However, we cannot rule out the possibility that a larger sample size would enable detection of rare mutations in this gene

Isolated bladder exstrophy associated with a de novo 0.9 Mb microduplication on chromosome 19p13.12.

The exstrophy-epispadias complex (BEEC) is a urogenital birth defect of varying severity. The causes of the BEEC are likely to be heterogeneous, with individual environmental or genetic risk factors still being largely unknown. In this study, we aimed to identify de novo causative copy number variations (CNVs) that contribute to the BEEC. METHODS Array-based molecular karyotyping was performed to screen 110 individuals with BEEC. Promising CNVs were tested for de novo occurrence by investigating parental DNAs. Genes located in regions of rearrangements were prioritized through expression analysis in mice to be sequenced in the complete cohort, to identify high-penetrance mutations involving small sequence changes. RESULTS A de novo 0.9 Mb microduplication involving chromosomal region 19p13.12 was identified in a single patient. This region harbors 20 validated RefSeq genes, and in situ hybridization data showed specific expression of the Wiz gene in regions surrounding the cloaca and the rectum between GD 9.5 and 13.5. Sanger sequencing of the complete cohort did not reveal any pathogenic alterations affecting the coding region of WIZ. CONCLUSIONS The present study suggests chromosomal region 19p13.12 as possibly involved in the development of CBE, but further studies are needed to prove a causal relation. The spatiotemporal expression patterns determined for the genes encompassed suggest a role for Wiz in the development of the phenotype. Our mutation screening, however, could not confirm that WIZ mutations are a frequent cause of CBE, although rare mutations might be detectable in larger patient samples

Profilaxis de la salmonelosis de los terneros por inmunización materna

Thirty cows were vaccinated with formalin-Killed Salmonella dublin, an aluminium hydroxide adjuvated, within the last 60 days of gestation. The protection obtained from colostrum was determined by means of the experimental infection of their calves. The immune response of antibodies against flagellar (H) and somatic (O) antigens, in cows as well as in calves, were measured using the method of thin-microagglutination.The results showed significant differences (p < 0.001) when compared to the control group. Three treated and three control calves were administered orally with 1010 Salmonella dublin as a total dosis. Calves from vaccinated cows did not show either clinical symtoms nor mortality, but those from the control group evidenced diarrhea, fever, dullness and loss of appetite. Deaths were not recorded, but they eliminated microorganisms by feces for 10 or more days. On the basis of the present results, it is suggested the maternal vaccination in order to protect calves from Salmonellosis at the moment of great vulnerability.Se inmunizaron 30 vacas en los últimos 60 días de gestación con una bacterina de Salmonella dublin, determinando la protección pasiva conferida por el calostro, mediante la infección experimental de sus terneros. La respuesta inmune mediada por anticuerpos contra antígenos flagelares (H) y somáticos (O), tanto en las vacas como en los terneros, se evaluó por la técnica de microaglutinación en placa. Los resultados arrojaron diferencias significativas (p < 0,001) sobre el lote testigo. Tres terneros inmunizados pasivamente y 3 testigos fueron desafiados con 1010 S. dublin como dosis total, por vía oral; los procedentes de vacas vacunadas no presentaron mortalidad ni signos clínicos de salmonelosis; en cambio, el lote testigo evidenció diarreas, fiebre, anorexia y decaimiento, no se observaron muertes pero eliminaron microorganismos por materia fecal durante más de 10 días. Los resultados sugieren el valor de la inmunización materna para la protección de los terneros contra la salmonelosis en el momento en que son más vulnerables a la infección.

BRACHYURY directs histone acetylation to target loci during mesoderm development

T-box transcription factors play essential roles in multiple aspects of vertebrate development. Here, we show that cooperative function of BRACHYURY (T) with histone-modifying enzymes is essential for mouse embryogenesis. A single point mutation (TY88A) results in decreased histone 3 lysine 27 acetylation (H3K27ac) at T target sites, including the T locus, suggesting that T autoregulates the maintenance of its expression and functions by recruiting permissive chromatin modifications to putative enhancers during mesoderm specification. Our data indicate that T mediates H3K27ac recruitment through a physical interaction with p300. In addition, we determine that T plays a prominent role in the specification of hematopoietic and endothelial cell types. Hematopoietic and endothelial gene expression programs are disrupted in TY88A mutant embryos, leading to a defect in the differentiation of hematopoietic progenitors. We show that this role of T is mediated, at least in part, through activation of a distal Lmo2 enhancer

Profilaxis de la salmonelosis de los terneros por inmunización materna

Thirty cows were vaccinated with formalin-Killed Salmonella dublin, an aluminium hydroxide adjuvated, within the last 60 days of gestation. The protection obtained from colostrum was determined by means of the experimental infection of their calves. The immune response of antibodies against flagellar (H) and somatic (O) antigens, in cows as well as in calves, were measured using the method of thin-microagglutination.The results showed significant differences (p < 0.001) when compared to the control group. Three treated and three control calves were administered orally with 1010 Salmonella dublin as a total dosis. Calves from vaccinated cows did not show either clinical symtoms nor mortality, but those from the control group evidenced diarrhea, fever, dullness and loss of appetite. Deaths were not recorded, but they eliminated microorganisms by feces for 10 or more days. On the basis of the present results, it is suggested the maternal vaccination in order to protect calves from Salmonellosis at the moment of great vulnerability.Se inmunizaron 30 vacas en los últimos 60 días de gestación con una bacterina de Salmonella dublin, determinando la protección pasiva conferida por el calostro, mediante la infección experimental de sus terneros. La respuesta inmune mediada por anticuerpos contra antígenos flagelares (H) y somáticos (O), tanto en las vacas como en los terneros, se evaluó por la técnica de microaglutinación en placa. Los resultados arrojaron diferencias significativas (p < 0,001) sobre el lote testigo. Tres terneros inmunizados pasivamente y 3 testigos fueron desafiados con 1010 S. dublin como dosis total, por vía oral; los procedentes de vacas vacunadas no presentaron mortalidad ni signos clínicos de salmonelosis; en cambio, el lote testigo evidenció diarreas, fiebre, anorexia y decaimiento, no se observaron muertes pero eliminaron microorganismos por materia fecal durante más de 10 días. Los resultados sugieren el valor de la inmunización materna para la protección de los terneros contra la salmonelosis en el momento en que son más vulnerables a la infección.

Genome-wide association study and mouse expression data identify a highly conserved 32 kb intergenic region between WNT3 and WNT9b as possible susceptibility locus for isolated classic exstrophy of the bladder

Item does not contain fulltextBladder exstrophy-epispadias complex (BEEC), the severe end of the urorectal malformation spectrum, has a profound impact on continence as well as sexual and renal functions. It is widely accepted that for the majority of cases the genetic basis appears to be multifactorial. Here, we report the first study which utilizes genome-wide association methods to analyze a cohort comprising patients presenting the most common BEEC form, classic bladder exstrophy (CBE), to identify common variation associated with risk for isolated CBE. We employed discovery and follow-up samples comprising 218 cases/865 controls and 78 trios in total, all of European descent. Our discovery sample identified a marker near SALL1, showing genome-wide significant association with CBE. However, analyses performed on follow-up samples did not add further support to these findings. We were also able to identify an association with CBE across our study samples (discovery: P = 8.88 x 10(-5); follow-up: P = 0.0025; combined: 1.09 x 10(-6)) in a highly conserved 32 kb intergenic region containing regulatory elements between WNT3 and WNT9B. Subsequent analyses in mice revealed expression for both genes in the genital region during stages relevant to the development of CBE in humans. Unfortunately, we were not able to replicate the suggestive signal for WNT3 and WNT9B in a sample that was enriched for non-CBE BEEC cases (P = 0.51). Our suggestive findings support the hypothesis that larger samples are warranted to identify association of common variation with CBE

De novo microduplications at 1q41, 2q37.3, and 8q24.3 in patients with VATER/VACTERL association

Item does not contain fulltextThe acronym VATER/VACTERL association describes the combination of at least three of the following congenital anomalies: vertebral defects (V), anorectal malformations (A), cardiac defects (C), tracheoesophageal fistula with or without esophageal atresia (TE), renal malformations (R), and limb defects (L). We aimed to identify highly penetrant de novo copy number variations (CNVs) that contribute to VATER/VACTERL association. Array-based molecular karyotyping was performed in a cohort of 41 patients with VATER/VACTERL association and 6 patients with VATER/VACTERL-like phenotype including all of the patients' parents. Three de novo CNVs were identified involving chromosomal regions 1q41, 2q37.3, and 8q24.3 comprising one (SPATA17), two (CAPN10, GPR35), and three (EPPK1, PLEC, PARP10) genes, respectively. Pre-existing data from the literature prompted us to choose GPR35 and EPPK1 for mouse expression studies. Based on these studies, we prioritized GPR35 for sequencing analysis in an extended cohort of 192 patients with VATER/VACTERL association and VATER/VACTERL-like phenotype. Although no disease-causing mutation was identified, our mouse expression studies suggest GPR35 to be involved in the development of the VATER/VACTERL phenotype. Follow-up of GPR35 and the other genes comprising the identified duplications is warranted