Gut lumen formation defect can cause intestinal atresia: evidence from histological studies of human embryos and intestinal atresia septum.

Intestinal atresia (IA), a common cause of neonatal intestinal obstruction, is a developmental defect, which disrupts the luminal continuity of the intestine. Here, we investigated (i) the process of lumen formation in human embryos; and (ii) how a defective lumen formation led to IA. We performed histological and histochemical study on 6-10 gestation week human embryos and on IA septal regions. To investigate the topology of embryonic intestine development, we conducted 3D reconstruction. We showed that a 6-7th gestation week embryonic gut has no lumen, but filled with mesenchyme cells and vacuoles of a monolayer of epithelial cells. A narrow gut lumen was formed by gestation week-9, the gut was filled with numerous vacuoles of different sizes, some vacuoles were merging with the developing embryonic gut wall. At gestation week-10, a prominent lumen was developed, only few vacuoles were present and were merging with the intestine wall. At IA septal regions, vacuoles were located in the submucous layer, covered by a single layer of epithelium without glandular structure, and surrounded with fibrous tissue. The mucosal epithelium was developed with lamina propria and basement membrane, but the submucosa and the longitudinal smooth muscle layers were not properly developed. Hence, the vacuoles in IA septum could represent a remnant of vacuoles of embryonic gut. In conclusion, the fusion of vacuoles with the developing intestine wall associates with the disappearance of vacuoles and gut lumen formation in human embryos, and perturbation of these developmental events could lead to IA.

Early embryo development anomalies identified by time-lapse system: prevalence and impacting factors.

RESEARCH QUESTION: What is the prevalence of embryo abnormal early cleavage (ACL) identified by time lapse and factors related to patients and treatment that explain ACL occurrence? DESIGN: A single-centre, retrospective cohort study. Data were collected on all IVF cycles for which embryos were observed in the EmbryoScope® between December 2015 and August 2017. Only diploid zygotes cleaved on day 2 were included. The study included 318 cycles (250 couples and 1343 embryos). Embryo videos were retrospectively analysed for ACL. The prevalence of each type of ACL was recorded. The influence of clinical factors (whether they were intrinsic to patients or specific to IVF treatment) on ACL occurrence was analysed in multivariate multilevel mixed-effect logistic regression analysis. RESULTS: A high prevalence of ACL was observed: 37.6% (505/1343) of embryos presented at least one ACL, 22.8% (306/1343) a trichotomous mitosis, 25.8% (347/1343) a rapid cleavage, 6.7% (90/1343) a cell fusion and two or more ACL (16.1%). Part of the variation (12-25%) in ACL occurrence could be explained by embryo origin. Trichotomous mitosis and two or more ACL phenotypes were less likely to occur in women with endometriosis or tubal pathology and tubal pathology alone, respectively. No factor related to IVF cycles was found to be statistically associated with ACL occurrence. CONCLUSIONS: Our findings emphasize the importance of considering embryo origin when interpreting studies focusing on embryo characteristics and factors that could affect their quality. The present study is limited by a small sample size of known embryo implantations and monocentric criterion.

Maternal body mass index associates with blastocyst euploidy and live birth rates: the tip of an iceberg?

RESEARCH QUESTION: Does maternal preconceptional body mass index (BMI) associate with mean blastocyst euploidy rate (m-ER) per patient and live birth rate (LBR) after vitrified-warmed euploid single embryo transfer (SET)? DESIGN: Observational study conducted between April 2013 and March 2020 at a private IVF clinic, involving 1811 Caucasian women undergoing trophectoderm biopsy and comprehensive chromosome testing. The outcomes of 1125 first vitrified-warmed euploid SET were also analysed. Patients were clustered as normal weight (BMI 18.5-25; n = 1392 performing 859 SET), underweight (BMI <18.5; n = 160 performing 112 SET) and overweight (BMI >25; n = 259 performing 154 SET). m-ER per patient was the primary outcome. The secondary outcomes were all clinical outcomes per euploid SET. All data were adjusted for confounders through regression analyses. RESULTS: The m-ER per patient decreases as maternal BMI increases from 17 up to 22-23 before reaching a plateau. A linear regression adjusted for maternal age confirmed this moderate association (unstandardized coefficient B: -0.6%, 95% confidence interval [CI]: -1.1 to -0.1%, P = 0.02). All clinical outcomes were similar between normal weight and underweight women. Overweight women, instead, showed higher miscarriage rate per clinical pregnancy (n = 20/75, 26.7% versus n = 67/461, 14.5%; odds ratio [OR] adjusted for blastocyst quality and day of full blastulation: 2.0, 95% CI: 1.1-3.6, P = 0.01) and lower LBR per SET (n = 55/154, 35.7% versus n = 388/859, 45.2%; OR adjusted for blastocyst quality and day of full blastulation: 0.67, 95% CI: 0.46-0.96, P = 0.03). CONCLUSION: These data indicate a need for future research on more sensitive metrics to assess body fat mass and distribution, as well as on the mechanisms leading to lipotoxicity, thereby impairing embryo competence and/or endometrial receptivity. Overweight women should be informed of their higher risk for miscarriage and, whenever possible, encouraged to lose weight, especially before transfer.

Maternal iron deficiency perturbs embryonic cardiovascular development in mice.

Congenital heart disease (CHD) is the most common class of human birth defects, with a prevalence of 0.9% of births. However, two-thirds of cases have an unknown cause, and many of these are thought to be caused by in utero exposure to environmental teratogens. Here we identify a potential teratogen causing CHD in mice: maternal iron deficiency (ID). We show that maternal ID in mice causes severe cardiovascular defects in the offspring. These defects likely arise from increased retinoic acid signalling in ID embryos. The defects can be prevented by iron administration in early pregnancy. It has also been proposed that teratogen exposure may potentiate the effects of genetic predisposition to CHD through gene-environment interaction. Here we show that maternal ID increases the severity of heart and craniofacial defects in a mouse model of Down syndrome. It will be important to understand if the effects of maternal ID seen here in mice may have clinical implications for women.

Comparison of single euploid blastocyst transfer cycle outcome derived from embryos with normal or abnormal cleavage patterns.

RESEARCH QUESTION: To assess incidence of abnormal cleavage among biopsied blastocysts; to compare euploidy rates of the blastocysts with abnormal and normal cleavage; and to compare single euploid blastocyst transfer (SEBT) outcome derived from embryos with normal or abnormal cleavage. DESIGN: Retrospective analysis of prospectively collected data in a private IVF clinic. Consecutive 554 patients (749 cycles) undergoing preimplantation genetic testing for aneuploidy (n = 497; 671 cycles) or monogenic diseases (n = 57; 78 cycles) were included. All assessments for abnormal cleavage were carried out retrospectively; presence of abnormal cleavage was not a factor in deciding which euploid embryo to transfer. A total of 1015 blastocysts were biopsied and 295 SEBT procedures were carried out. Main outcome measure was live birth rate (LBR). RESULTS: Incidence of reverse cleavage, direct cleavage, and reverse plus direct cleavage, were 7.7%, 6.4% and 2.3%, respectively. Of the 1015 biopsied blastocysts, 35.0% were euploid. Blastocysts with abnormal cleavage, in total, had a significantly higher euploidy rate compared with blastocysts with normal cleavage (44.6% [74/166] versus 33.1% [281/849]; P = 0.017). The LBR after SEBT with normal, reverse and direct cleavage, and direct cleavage plus reverse cleavage, was 133/238 (55.9%), 6/26 (23.1%), 8/24 (33.3%) and 0/3 (0.0%) (P < 0.001). Generalized estimating equation analysis showed that the presence of abnormal cleavage pattern was the only independent predictor of LBR (OR 0.316; 95% CI 0.115 to 0.867; P = 0.013). CONCLUSIONS: Blastocysts with direct or reverse cleavage should be biopsied in preimplantation genetic testing cycles if they are morphologically eligible. Euploid blastocysts with abnormal cleavage, however, have approximately half the LBR of those euploid blastocyst with normal cleavage, hence, blastocysts with abnormal cleavage should have lower priority for transfer.

Centrosomal protein FOR20 knockout mice display embryonic lethality and left-right patterning defects.

Centrosomal protein FOR20 has been reported to be crucial for essential cellular processes, including ciliogenesis, cell migration, and cell cycle in vertebrates. However, the function of FOR20 during mammalian embryonic development remains unknown. To investigate the in vivo function of the For20 gene in mammals, we generated For20 homozygous knockout mice by gene targeting. Our data reveal that homozygous knockout of For20 results in significant embryonic growth arrest and lethality during gestation, while the heterozygotes show no obvious defects. The absence of For20 leads to impaired left-right patterning of embryos and reduced cilia in the embryonic node. Deletion of For20 also disrupts angiogenesis in yolk sacs and embryos. These results highlight a critical role of For20 in early mammalian embryogenesis.

Effects of three pro-nuclei (3PN) incidence on laboratory and clinical outcomes after early rescue intracytoplasmic sperm injection (rescue-ICSI): an analysis of a 5-year period.

OBJECTIVES: We aimed to explore the effect of three pro-nuclei (3PN) incidence on laboratory and clinical outcomes after early rescue intracytoplasmic sperm injection (rescue-ICSI). METHODS: This study included 509 early rescue-ICSI cycles from February 2014 to February 2019. The patients were divided into 3PN = 0% (394 cycles) and 3PN > 0% (115 cycles) group. Main outcomes compared were good quality embryo, available embryo, implantation (IR), clinical pregnancy (CPR), abortion (AR) and live birth rates (LBR). RESULTS: There were no significant differences in the basal characteristics between two groups (p > .05). We observed that 3PN = 0% and 3PN > 0% groups had similar good quality embryo (47.02 versus 46.80%; p = .917) and available embryo (83.28 versus 81.37%; p = .247) rates. Our results showed that the IR (47.26 versus 51.05%; p = .357), CPR (61.17 versus 66.08%; p = .338) and LBR (52.80 versus 50.43%; p = .656) were comparable between 3PN = 0% and 3PN > 0% groups. The 3PN = 0% group showed significantly lower AR than that in the 3PN > 0% group (11.20 versus 21.05%; p = .029; OR 2.114; 95% CI 1.069-4.178). CONCLUSIONS: 3PN incidence made negative effects on the clinical outcomes after early rescue-ICSI.

Relationship between male age, semen parameters and assisted reproductive technology outcomes.

BACKGROUND: Low semen quality often obligates the use of assisted reproductive technology; however, the association between semen quality and assisted reproductive technology outcomes is uncertain. OBJECTIVES: To further assess the impact of semen quality on assisted reproductive technology outcomes. MATERIALS AND METHODS: A retrospective cohort study was carried out at a single academic reproductive medicine center (January 2012-December 2018). Patients undergoing at least one assisted reproductive technology cycle utilizing freshly ejaculated spermatozoa from the male partner were included. We assessed the association between semen quality (as stratified based on WHO 5th edition criteria), paternal age (< or ≥40), and reproductive/perinatal outcomes. To evaluate the differences in assisted reproductive technology outcomes by semen parameters and age, generalized estimating equations were applied for rates of fertilization, pregnancy, implantation, miscarriage, live birth, blast formation, gestational age, and normal embryo biopsy. RESULTS: A total of 2063 couples were identified who underwent 4517 assisted reproductive technology cycles. Average ages of the male and female partners were 39.8 and 37.7, respectively. Lower pregnancy rates were observed in cycles with lower sperm motility (ie <40%; 39.9% vs 44.1%) and total motile count (ie <9 million; 38.3% vs 43.5%). When examining only cycles utilizing Intracytoplasmic Sperm Injection, only a lower motility count was associated with a decline in pregnancy rate (39.1% vs 44.9%). No association was identified between semen quality and gestational age or birth weight. Paternal age was not associated with ART outcomes. However, among assisted reproductive technology cycles in women <40, aneuploidy rate was higher for older men (P < .001). In cycles with women >40, no association between aneuploidy and male age was identified. DISCUSSION: Sperm motility is associated with pregnancy rates, while other semen parameters are not. In cycles in women <40, paternal age is associated with embryo aneuploidy rate. CONCLUSION: Paternal factors are associated with assisted reproductive technology outcomes, and future studies should explore mechanisms by which semen quality is associated with assisted reproductive technology outcomes.

Altered BMP-Smad4 signaling causes complete cleft palate by disturbing osteogenesis in palatal mesenchyme.

As the major receptor mediated BMP signaling in craniofacial development, Bmpr1a expression was detected in the anterior palatal shelves from E13.5 and the posterior palatal shelves from E14.5. However, inactivating BMP receptor in the mesenchyme only leads to anterior cleft palate or submucous cleft palate. The role of BMP signaling in posterior palatal mesenchyme and palatal osteogenesis is still unknown. In this study, a secreted BMP antagonist, Noggin was over-expressed by Osr2-creKI to suppress BMP signaling intensively in mouse palatal mesenchyme, which made the newborn mouse displaying complete cleft palate, a phenotype much severer than the anterior or submucous cleft palate. Immunohistochemical analysis indicated that in the anterior and posterior palatal mesenchyme, the canonical BMP-Smad4 signaling was dramatically down-regulated, while the non-canonical BMP signaling pathways were altered little. Although cell proliferation was reduced only in the anterior palatal mesenchyme, the osteogenic condensation and Osterix distribution were remarkably repressed in the posterior palatal mesenchyme by Noggin over-expression. These findings suggested that BMP-Smad4 signaling was essential for the cell proliferation in the anterior palatal mesenchyme, and for the osteogenesis in the posterior palatal mesenchyme. Interestingly, the constitutive activation of Bmpr1a in palatal mesenchyme also caused the complete cleft palate, in which the enhanced BMP-Smad4 signaling resulted in the premature osteogenic differentiation in palatal mesenchyme. Moreover, neither the Noggin over-expression nor Bmpr1a activation disrupted the elevation of palatal shelves. Our study not only suggested that BMP signaling played the differential roles in the anterior and posterior palatal mesenchyme, but also indicated that BMP-Smad4 signaling was required to be finely tuned for the osteogenesis of palatal mesenchyme.

Functionally distinct roles for T and Tbx6 during mouse development.

The mouse T-box transcription factors T and Tbx6 are co-expressed in the primitive streak and have unique domains of expression; T is expressed in the notochord, while Tbx6 is expressed in the presomitic mesoderm. T-box factors are related through a shared DNA binding domain, the T-domain, and can therefore bind to similar DNA sequences at least in vitro We investigated the functional similarities and differences of T and Tbx6 DNA binding and transcriptional activity in vitro and their interaction genetically in vivo We show that at one target, Dll1, the T-domains of T and Tbx6 have different affinities for the binding sites present in the mesoderm enhancer. We further show using in vitro assays that T and Tbx6 differentially affect transcription with Tbx6 activating expression tenfold higher than T, that T and Tbx6 can compete at target gene enhancers, and that this competition requires a functional DNA binding domain. Next, we addressed whether T and Tbx6 can compete in vivo First, we generated embryos that express Tbx6 at greater than wild-type levels embryos and show that these embryos have short tails, resembling the T heterozygous phenotype. Next, using the dominant-negative TWis allele, we show that Tbx6+/- TWis/+ embryos share similarities with embryos homozygous for the Tbx6 hypomorphic allele rib-vertebrae, specifically fusions of several ribs and malformation of some vertebrae. Finally, we tested whether Tbx6 can functionally replace T using a knockin approach, which resulted in severe T null-like phenotypes in chimeric embryos generated with ES cells heterozygous for a Tbx6 knockin at the T locus. Altogether, our results of differences in affinity for DNA binding sites and transcriptional activity for T and Tbx6 provide a potential mechanism for the failure of Tbx6 to functionally replace T and possible competition phenotypes in vivo.

Analysis of Congenital Heart Defects in Mouse Embryos Using Qualitative and Quantitative Histological Methods.

Congenital heart defects (CHD) are the most common type of birth defect in humans, affecting up to 1% of all live births. However, the underlying causes for CHD are still poorly understood. The developing mouse constitutes a valuable model for the study of CHD, because cardiac developmental programs between mice and humans are highly conserved. The protocol describes in detail how to produce mouse embryos of the desired gestational stage, methods to isolate and preserve the heart for downstream processing, quantitative methods to identify common types of CHD by histology (e.g., ventricular septal defects, atrial septal defects, patent ductus arteriosus), and quantitative histomorphometry methods to measure common muscular compaction phenotypes. These methods articulate all the steps involved in sample preparation, collection, and analysis, allowing scientists to correctly and reproducibly measure CHD.

PRDM15 loss of function links NOTCH and WNT/PCP signaling to patterning defects in holoprosencephaly.

Holoprosencephaly (HPE) is a congenital forebrain defect often associated with embryonic lethality and lifelong disabilities. Currently, therapeutic and diagnostic options are limited by lack of knowledge of potential disease-causing mutations. We have identified a new mutation in the PRDM15 gene (C844Y) associated with a syndromic form of HPE in multiple families. We demonstrate that C844Y is a loss-of-function mutation impairing PRDM15 transcriptional activity. Genetic deletion of murine Prdm15 causes anterior/posterior (A/P) patterning defects and recapitulates the brain malformations observed in patients. Mechanistically, PRDM15 regulates the transcription of key effectors of the NOTCH and WNT/PCP pathways to preserve early midline structures in the developing embryo. Analysis of a large cohort of patients with HPE revealed potentially damaging mutations in several regulators of both pathways. Our findings uncover an unexpected link between NOTCH and WNT/PCP signaling and A/P patterning and set the stage for the identification of new HPE candidate genes.

Lineage-specific roles of hedgehog-GLI signaling during mammalian kidney development.

Aberrant hedgehog (Hh) signaling during embryogenesis results in various severe congenital abnormalities, including renal malformations. The molecular mechanisms that underlie congenital renal malformations remain poorly understood. Here, we review the current understanding of the lineage-specific roles of Hh signaling during renal morphogenesis and how aberrant Hh signaling during embryonic kidney development contributes to renal malformation.

Importance of gestational hypoglycaemia for foetal malformations and skeletal development in rats.

The aim was to investigate embryo-foetal effects of continuous maternal insulin-induced hypoglycaemia extending throughout gestation or until gestation day (GD)17 (typical last day of dosing during pre-clinical evaluation) providing comparator data for safety assessment of longer-acting insulin analogues in non-diabetic rats. Pregnant rats received human insulin (HI)-infusion during gestation until either GD20 or GD17 (HI-GD20; HI-GD17). On GD20, foetal abnormalities and skeletal ossification/mineralisation were evaluated. HI-infusion induced continuous hypoglycaemia. Foetal skeletal and eye malformations (e.g. bent ribs, microphthalmia) were common in both groups. Foetal size and skeletal ossification/mineralisation decreased, particularly with infusion throughout gestation. Concluding, insulin-induced hypoglycaemia during gestation in non-diabetic rats is damaging to embryo-foetal growth and skeletal development, particularly after GD17. Three days without HI-infusion after GD17 allows for some developmental catch-up. Eye development is sensitive to HI-infusion before GD17. These results should serve as a benchmark during pre-clinical safety assessment of longer-acting insulin analogues tested in rats.

Developmental abnormalities in cortical GABAergic system in mice lacking mGlu3 metabotropic glutamate receptors.

Polymorphic variants of the gene encoding for metabotropic glutamate receptor 3 (mGlu3) are linked to schizophrenia. Because abnormalities of cortical GABAergic interneurons lie at the core of the pathophysiology of schizophrenia, we examined whether mGlu3 receptors influence the developmental trajectory of cortical GABAergic transmission in the postnatal life. mGlu3-/- mice showed robust changes in the expression of interneuron-related genes in the prefrontal cortex (PFC), including large reductions in the expression of parvalbumin (PV) and the GluN1 subunit of NMDA receptors. The number of cortical cells enwrapped by perineuronal nets was increased in mGlu3-/- mice, suggesting that mGlu3 receptors shape the temporal window of plasticity of PV+ interneurons. Electrophysiological measurements of GABAA receptor-mediated responses revealed a more depolarized reversal potential of GABA currents in the somata of PFC pyramidal neurons in mGlu3-/- mice at postnatal d 9 associated with a reduced expression of the K+/Cl- symporter. Finally, adult mGlu3-/- mice showed lower power in electroencephalographic rhythms at 1-45 Hz in quiet wakefulness as compared with their wild-type counterparts. These findings suggest that mGlu3 receptors have a strong impact on the development of cortical GABAergic transmission and cortical neural synchronization mechanisms corroborating the concept that genetic variants of mGlu3 receptors may predispose to psychiatric disorders.-Imbriglio, T., Verhaeghe, R., Martinello, K., Pascarelli, M. T., Chece, G., Bucci, D., Notartomaso, S., Quattromani, M., Mascio, G., Scalabrì, F., Simeone, A., Maccari, S., Del Percio, C., Wieloch, T., Fucile, S., Babiloni, C., Battaglia, G., Limatola, C., Nicoletti, F., Cannella, M. Developmental abnormalities in cortical GABAergic system in mice lacking mGlu3 metabotropic glutamate receptors.

Activation of sonic hedgehog signaling by a Smoothened agonist restores congenital defects in mouse models of endocrine-cerebro-osteodysplasia syndrome.

BACKGROUND: Endocrine-cerebro-osteodysplasia (ECO) syndrome is a genetic disorder associated with congenital defects of the endocrine, cerebral, and skeletal systems in humans. ECO syndrome is caused by mutations of the intestinal cell kinase (ICK) gene, which encodes a mitogen-activated protein (MAP) kinase-related kinase that plays a critical role in controlling the length of primary cilia. Lack of ICK function disrupts transduction of sonic hedgehog (SHH) signaling, which is important for development and homeostasis in humans and mice. Craniofacial structure abnormalities, such as cleft palate, are one of the most common defects observed in ECO syndrome patients, but the role of ICK in palatal development has not been studied. METHODS: Using Ick-mutant mice, we investigated the mechanisms by which ICK function loss causes cleft palate and examined pharmacological rescue of the congenital defects. FINDINGS: SHH signaling was compromised with abnormally elongated primary cilia in the developing palate of Ick-mutant mice. Cell proliferation was significantly decreased, resulting in failure of palatal outgrowth, although palatal adhesion and fusion occurred normally. We thus attempted to rescue the congenital palatal defects of Ick mutants by pharmacological activation of SHH signaling. Treatment of Ick-mutant mice with an agonist for Smoothened (SAG) rescued several congenital defects, including cleft palate. INTERPRETATIONS: The recovery of congenital defects by pharmacological intervention in the mouse models for ECO syndrome highlights prenatal SHH signaling modulation as a potential therapeutic measure to overcome congenital defects of ciliopathies.

Associations between embryo grading and congenital malformations in IVF/ICSI pregnancies.

RESEARCH QUESTION: Does the quality of transferred embryos have an impact on the rate of congenital malformations in IVF/intracytoplasmic sperm injection (ICSI)-conceived babies? DESIGN: Retrospective cohort study involving 6637 pregnancies of ≥20 weeks' gestation from women undergoing embryo transfer with a single Day 5 embryo at a private multisite IVF clinic between 2005 and 2015. Embryos were classified as good quality (n = 5537) or poor quality (n = 1100) based on an internal grading system of morphological parameters; malformation rates were compared. RESULTS: In pregnancies proceeding to delivery (≥20 weeks' gestation), poor quality embryos were associated with increased odds of at least one anomaly (adjusted odds ratio [OR] 1.33, 95% confidence interval [CI] 1.03-1.71), major anomalies (adjusted OR 1.42, 95% CI 1.05-1.91), musculoskeletal anomalies (adjusted OR 2.09, 95% CI 1.35-3.22), particularly talipes (adjusted OR 2.88, 95% CI 1.33-6.25), and the International Classification of Diseases (ICD) classification 'Other congenital malformations' (adjusted OR 2.34, 95% CI 1.13-4.34). Furthermore, for pregnancies ≥9 weeks' gestation, poor embryos had more than double the odds of chromosomal anomalies than good embryos (adjusted OR 2.33, 95% CI 1.30-4.18, P = 0.005). CONCLUSIONS: This is the first study to compare the rates of individual congenital malformations for good and poor quality embryos. It provides insight into potential risks of transferring poor quality embryos. In pregnancies ≥20 weeks' gestation, poor quality Day 5 embryos are associated with major malformations, at least one anomaly, musculoskeletal anomalies, talipes and the ICD classification 'Other congenital malformations'. In pregnancies ≥9 weeks' gestation, poor quality Day 5 embryos are associated with chromosomal anomalies.

Pax9 is required for cardiovascular development and interacts with Tbx1 in the pharyngeal endoderm to control 4th pharyngeal arch artery morphogenesis.

Developmental defects affecting the heart and aortic arch arteries are a significant phenotype observed in individuals with 22q11 deletion syndrome and are caused by a microdeletion on chromosome 22q11. TBX1, one of the deleted genes, is expressed throughout the pharyngeal arches and is considered a key gene, when mutated, for the arch artery defects. Pax9 is expressed in the pharyngeal endoderm and is downregulated in Tbx1 mutant mice. We show here that Pax9-deficient mice are born with complex cardiovascular malformations that affect the outflow tract and aortic arch arteries with failure of the 3rd and 4th pharyngeal arch arteries to form correctly. Transcriptome analysis indicated that Pax9 and Tbx1 may function together, and mice double heterozygous for Tbx1/Pax9 presented with a significantly increased incidence of interrupted aortic arch when compared with Tbx1 heterozygous mice. Using a novel Pax9Cre allele, we demonstrated that the site of this Tbx1-Pax9 genetic interaction is the pharyngeal endoderm, therefore revealing that a Tbx1-Pax9-controlled signalling mechanism emanating from the pharyngeal endoderm is required for crucial tissue interactions during normal morphogenesis of the pharyngeal arch artery system.

Molecular determinants in Frizzled, Reck, and Wnt7a for ligand-specific signaling in neurovascular development.

The molecular basis of Wnt-Frizzled specificity is a central question in developmental biology. Reck, a multi-domain and multi-functional glycosylphosphatidylinositol-anchored protein, specifically enhances beta-catenin signaling by Wnt7a and Wnt7b in cooperation with the 7-transmembrane protein Gpr124. Among amino acids that distinguish Wnt7a and Wnt7b from other Wnts, two clusters are essential for signaling in a Reck- and Gpr124-dependent manner. Both clusters are far from the site of Frizzled binding: one resides at the amino terminus and the second resides in a protruding loop. Within Reck, the fourth of five tandem repeats of an unusual domain with six-cysteines (the CC domain) is essential for Wnt7a stimulation: substitutions P256A and W261A in CC4 eliminate this activity without changing protein abundance or surface localization. Mouse embryos carrying ReckP256A,W261A have severe defects in forebrain angiogenesis, providing the strongest evidence to date that Reck promotes CNS angiogenesis by specifically stimulating Wnt7a and Wnt7b signaling.

The Hinrichsen Embryology Collection: Digitization of Historical Histological Human Embryonic Slides and MRI of Whole Fetuses.

The number of human embryology collections is very limited worldwide. Some of these comprise the Carnegie Collection, Kyoto Collection, and the Blechschmidt Collection. One further embryonic collection is the Hinrichsen Collection of the Ruhr University Bochum, Germany, which also contains very well-preserved embryos/fetuses, along with approximately 16,000 histological sections. The digitization of this collection is indispensable to enable conservation of the collection for the future and to provide a large group of embryologists, researchers, and physicians access to these histological slides. A small selection of these scans is available at the website of the Digital Embryology Consortium [https://-human-embryology.org/wiki/Main_Page].