eNOS plays essential roles in the developing heart and aorta linked to disruption of Notch signalling.

eNOS (NOS3) is the enzyme that generates nitric oxide, a signalling molecule and regulator of vascular tone. Loss of eNOS function is associated with increased susceptibility to atherosclerosis, hypertension, thrombosis and stroke. Aortopathy and cardiac hypertrophy have also been found in eNOS null mice, but their aetiology is unclear. We evaluated eNOS nulls before and around birth for cardiac defects, revealing severe abnormalities in the ventricular myocardium and pharyngeal arch arteries. Moreover, in the aortic arch, there were fewer baroreceptors, which sense changes in blood pressure. Adult eNOS null survivors showed evidence of cardiac hypertrophy, aortopathy and cartilaginous metaplasia in the periductal region of the aortic arch. Notch1 and neuregulin were dysregulated in the forming pharyngeal arch arteries and ventricles, suggesting that these pathways may be relevant to the defects observed. Dysregulation of eNOS leads to embryonic and perinatal death, suggesting mutations in eNOS are candidates for causing congenital heart defects in humans. Surviving eNOS mutants have a deficiency of baroreceptors that likely contributes to high blood pressure and may have relevance to human patients who suffer from hypertension associated with aortic arch abnormalities.

Yolk sac cell atlas reveals multiorgan functions during human early development.

The extraembryonic yolk sac (YS) ensures delivery of nutritional support and oxygen to the developing embryo but remains ill-defined in humans. We therefore assembled a comprehensive multiomic reference of the human YS from 3 to 8 postconception weeks by integrating single-cell protein and gene expression data. Beyond its recognized role as a site of hematopoiesis, we highlight roles in metabolism, coagulation, vascular development, and hematopoietic regulation. We reconstructed the emergence and decline of YS hematopoietic stem and progenitor cells from hemogenic endothelium and revealed a YS-specific accelerated route to macrophage production that seeds developing organs. The multiorgan functions of the YS are superseded as intraembryonic organs develop, effecting a multifaceted relay of vital functions as pregnancy proceeds.

VANGL2 regulates luminal epithelial organization and cell turnover in the mammary gland.

The VANGL family of planar cell polarity proteins is implicated in breast cancer however its function in mammary gland biology is unknown. Here, we utilized a panel of Vang1 and Vangl2 mouse alleles to examine the requirement of VANGL family members in the murine mammary gland. We show that Vang1CKOΔ/Δ glands display normal branching while Vangl2flox/flox and Vangl2Lp/Lp tissue exhibit several phenotypes. In MMTV-Cre;Vangl2flox/flox glands, cell turnover is reduced and lumens are narrowed. A Vangl2 missense mutation in the Vangl2Lp/Lp tissue leads to mammary anlage sprouting defects and deficient outgrowth with transplantation of anlage or secondary tissue fragments. In successful Vangl2Lp/Lp outgrowths, three morphological phenotypes are observed: distended ducts, supernumerary end buds, and ectopic acini. Layer specific defects are observed with loss of Vangl2 selectively in either basal or luminal layers of mammary cysts. Loss in the basal compartment inhibits cyst formation, but has the opposite effect in the luminal compartment. Candidate gene analysis on MMTV-Cre;Vangl2flox/flox and Vangl2Lp/Lp tissue reveals a significant reduction in Bmi1 expression, with overexpression of Bmi1 rescuing defects in Vangl2 knockdown cysts. Our results demonstrate that VANGL2 is necessary for normal mammary gland development and indicate differential functional requirements in basal versus luminal mammary compartments.

A novel source of arterial valve cells linked to bicuspid aortic valve without raphe in mice.

Abnormalities of the arterial valve leaflets, predominantly bicuspid aortic valve, are the commonest congenital malformations. Although many studies have investigated the development of the arterial valves, it has been assumed that, as with the atrioventricular valves, endocardial to mesenchymal transition (EndMT) is the predominant mechanism. We show that arterial is distinctly different from atrioventricular valve formation. Whilst the four septal valve leaflets are dominated by NCC and EndMT-derived cells, the intercalated leaflets differentiate directly from Tnnt2-Cre+/Isl1+ progenitors in the outflow wall, via a Notch-Jag dependent mechanism. Further, when this novel group of progenitors are disrupted, development of the intercalated leaflets is disrupted, resulting in leaflet dysplasia and bicuspid valves without raphe, most commonly affecting the aortic valve. This study thus overturns the dogma that heart valves are formed principally by EndMT, identifies a new source of valve interstitial cells, and provides a novel mechanism for causation of bicuspid aortic valves without raphe.

Scrib:Rac1 interactions are required for the morphogenesis of the ventricular myocardium.

AIMS: The organization and maturation of ventricular cardiomyocytes from the embryonic to the adult form is crucial for normal cardiac function. We have shown that a polarity protein, Scrib, may be involved in regulating the early stages of this process. Our goal was to establish whether Scrib plays a cell autonomous role in the ventricular myocardium, and whether this involves well-known polarity pathways. METHODS AND RESULTS: Deletion of Scrib in cardiac precursors utilizing Scrib(flox) mice together with the Nkx2.5-Cre driver resulted in disruption of the cytoarchitecture of the forming trabeculae and ventricular septal defects. Although the majority of mice lacking Scrib in the myocardium survived to adulthood, they developed marked cardiac fibrosis. Scrib did not physically interact with the planar cell polarity (PCP) protein, Vangl2, in early cardiomyocytes as it does in other tissues, suggesting that the anomalies did not result from disruption of PCP signalling. However, Scrib interacted with Rac1 physically in embryonic cardiomyocytes and genetically to result in ventricular abnormalities, suggesting that this interaction is crucial for the development of the early myocardium. CONCLUSIONS: The Scrib-Rac1 interaction plays a crucial role in the organization of developing cardiomyocytes and formation of the ventricular myocardium. Thus, we have identified a novel signalling pathway in the early, functioning, heart muscle. These data also show that the foetus can recover from relatively severe abnormalities in prenatal ventricular development, although cardiac fibrosis can be a long-term consequence.

Calcium-alginate hydrogel-encapsulated fibroblasts provide sustained release of vascular endothelial growth factor.

Vascularization of engineered or damaged tissues is essential to maintain cell viability and proper tissue function. Revascularization of the left ventricle (LV) of the heart after myocardial infarction is particularly important, since hypoxia can give rise to chronic heart failure due to inappropriate remodeling of the LV after death of cardiomyocytes (CMs). Fibroblasts can express vascular endothelial growth factor (VEGF), which plays a major role in angiogenesis and also acts as a chemoattractant and survival factor for CMs and cardiac progenitors. In this in vitro model study, mouse NIH 3T3 fibroblasts encapsulated in 2% w/v Ca-alginate were shown to remain viable for 150 days. Semiquantitative reverse transcription-polymerase chain reaction and immunohistochemistry demonstrated that over 21 days of encapsulation, fibroblasts continued to express VEGF, while enzyme-linked immunosorbent assay showed that there was sustained release of VEGF from the Ca-alginate during this period. The scaffold degraded gradually over the 21 days, without reduction in volume. Cells released from the Ca-alginate at 7 and 21 days as a result of scaffold degradation were shown to retain viability, to adhere to fibronectin in a normal manner, and continue to express VEGF, demonstrating their potential to further contribute to maintenance of cardiac function after scaffold degradation. This model in vitro study therefore demonstrates that fibroblasts encapsulated in Ca-alginate provide sustained release of VEGF.

Neural crest cell survival is dependent on Rho kinase and is required for development of the mid face in mouse embryos.

Neural crest cells (NCC) give rise to much of the tissue that forms the vertebrate head and face, including cartilage and bone, cranial ganglia and teeth. In this study we show that conditional expression of a dominant-negative (DN) form of Rho kinase (Rock) in mouse NCC results in severe hypoplasia of the frontonasal processes and first pharyngeal arch, ultimately resulting in reduction of the maxilla and nasal bones and severe craniofacial clefting affecting the nose, palate and lip. These defects resemble frontonasal dysplasia in humans. Disruption of the actin cytoskeleton, which leads to abnormalities in cell-matrix attachment, is seen in the RockDN;Wnt1-cre mutant embryos. This leads to elevated cell death, resulting in NCC deficiency and hypoplastic NCC-derived craniofacial structures. Rock is thus essential for survival of NCC that form the craniofacial region. We propose that reduced NCC numbers in the frontonasal processes and first pharyngeal arch, resulting from exacerbated cell death, may be the common mechanism underlying frontonasal dysplasia.

The novel mouse mutant, chuzhoi, has disruption of Ptk7 protein and exhibits defects in neural tube, heart and lung development and abnormal planar cell polarity in the ear.

BACKGROUND: The planar cell polarity (PCP) signalling pathway is fundamental to a number of key developmental events, including initiation of neural tube closure. Disruption of the PCP pathway causes the severe neural tube defect of craniorachischisis, in which almost the entire brain and spinal cord fails to close. Identification of mouse mutants with craniorachischisis has proven a powerful way of identifying molecules that are components or regulators of the PCP pathway. In addition, identification of an allelic series of mutants, including hypomorphs and neomorphs in addition to complete nulls, can provide novel genetic tools to help elucidate the function of the PCP proteins. RESULTS: We report the identification of a new N-ethyl-N-nitrosourea (ENU)-induced mutant with craniorachischisis, which we have named chuzhoi (chz). We demonstrate that chuzhoi mutant embryos fail to undergo initiation of neural tube closure, and have characteristics consistent with defective convergent extension. These characteristics include a broadened midline and reduced rate of increase of their length-to-width ratio. In addition, we demonstrate disruption in the orientation of outer hair cells in the inner ear, and defects in heart and lung development in chuzhoi mutants. We demonstrate a genetic interaction between chuzhoi mutants and both Vangl2Lp and Celsr1Crsh mutants, strengthening the hypothesis that chuzhoi is involved in regulating the PCP pathway. We demonstrate that chuzhoi maps to Chromosome 17 and carries a splice site mutation in Ptk7. This mutation results in the insertion of three amino acids into the Ptk7 protein and causes disruption of Ptk7 protein expression in chuzhoi mutants. CONCLUSIONS: The chuzhoi mutant provides an additional genetic resource to help investigate the developmental basis of several congenital abnormalities including neural tube, heart and lung defects and their relationship to disruption of PCP. The chuzhoi mutation differentially affects the expression levels of the two Ptk7 protein isoforms and, while some Ptk7 protein can still be detected at the membrane, chuzhoi mutants demonstrate a significant reduction in membrane localization of Ptk7 protein. This mutant provides a useful tool to allow future studies aimed at understanding the molecular function of Ptk7.

Cells migrating from the neural crest contribute to the innervation of the venous pole of the heart.

Cells migrating from the neural crest are known to septate the outflow tract of the developing heart, and to contribute to the formation of the arterial valves, their supporting sinuses, the coronary arteries and cardiac neural ganglia. Neural crest cells have also been suggested to contribute to development of the venous pole of the heart, but the extent and fate of such cells remains unclear. In this study, in the mouse, it is shown that cells from the neural crest contribute to the parasympathetic and, to a lesser extent, the sympathetic innervation of the venous pole of the heart. Nerves within the venous pole of the heart are shown to be of mixed origin, with some being derived from the neural crest, while others have an alternative origin, presumably placodal. The neurons innervating the nodal tissue, which can exert chronotropic effects on cardiac conduction, are shown not to be derived from the neural crest. In particular, no evidence was found to support previous suggestions that cells from the neural crest make a direct contribution to the myocardial atrioventricular conduction axis, although a small subset of these cells do co-localize with the developing left bundle branch. We have therefore confirmed that cells from the neural crest migrate to the venous pole of the heart, and that their major role is in the development of the parasympathetic innervation. In addition, in some embryos, a population of cells derived from the neural crest persist in the leaflets of the atrioventricular valves, but their role in subsequent development remains unknown.

Disruption of Bardet-Biedl syndrome ciliary proteins perturbs planar cell polarity in vertebrates.

The evolutionarily conserved planar cell polarity (PCP) pathway (or noncanonical Wnt pathway) drives several important cellular processes, including epithelial cell polarization, cell migration and mitotic spindle orientation. In vertebrates, PCP genes have a vital role in polarized convergent extension movements during gastrulation and neurulation. Here we show that mice with mutations in genes involved in Bardet-Biedl syndrome (BBS), a disorder associated with ciliary dysfunction, share phenotypes with PCP mutants including open eyelids, neural tube defects and disrupted cochlear stereociliary bundles. Furthermore, we identify genetic interactions between BBS genes and a PCP gene in both mouse (Ltap, also called Vangl2) and zebrafish (vangl2). In zebrafish, the augmented phenotype results from enhanced defective convergent extension movements. We also show that Vangl2 localizes to the basal body and axoneme of ciliated cells, a pattern reminiscent of that of the BBS proteins. These data suggest that cilia are intrinsically involved in PCP processes.

Zic4, a zinc-finger transcription factor, is expressed in the developing mouse nervous system.

Zic genes comprise a family of transcription factors, characterized by the presence of a zinc-finger domain containing two cysteines and two histidines (C2-H2). Whereas the embryonic expression patterns of Zic1, 2, 3, and 5 have been described in detail, Zic4 has not yet received close attention. We studied the expression of Zic4 by in situ hybridization during mouse embryogenesis. Zic4 mRNA was first detected at low intensity at embryonic day (E) 9 and, by E10.5, expression was up-regulated in the dorsal midline of the forebrain with a strong, expanded expression domain at the boundary between the diencephalon and telencephalon, the septum, and the lamina terminalis. The choroid plexus of the third ventricle expresses Zic4, as does the dorsal part of the spinal neural tube, excluding the roof plate. The dorsal sclerotome and the dorsomedial lip of the dermomyotome also express Zic4 whereas dorsal root ganglia are negative. At E12.5, Zic4 continues to be expressed in the midline of the forebrain and in the dorsal spinal neural tube. Postnatally, Zic4 is expressed in the granule cells of the postnatal day 2 cerebellum, and in the periventricular thalamus and anterior end of the superior colliculus. We conclude that Zic4 has an expression pattern distinct from, but partly overlapping with, other members of the Zic gene family.

Spatiotemporal analysis of programmed cell death during mouse cardiac septation.

Cell death is thought to play an important role in mammalian cardiogenesis, although a precise map of its distribution during the crucial period of cardiac septation has so far been lacking. In this study, the spatiotemporal distribution of programmed cell death (PCD) during mouse cardiac septation is described between embryonic days 10.5 and 13.5. Two types of foci of cell death can be demonstrated in the developing heart. Those with high-intensity, with a PCD index greater than 1%, are clearly visible on individual TUNEL-assayed sections. Low-intensity foci, with a PCD index of less than 1%, become visible only following summation of data. High-intensity foci occur exclusively within the endocardial cushions of the outflow tract and atrioventricular region, appearing at the 52-54 somite stage (late E11.5), concomitant with the formation of the central mesenchymal mass. Low-intensity foci are present throughout the period of cardiac development from E10.5 to E13.5 and are frequently localized to regions of septation, such as the muscular ventricular septum and the mesenchymal cap of the primary atrial septum. Expression of Fas and FasL corresponds to these low-intensity foci, but not those with high-intensity, suggesting that activation of this death receptor may be specifically involved in molecular control of the low-intensity foci.

Mutation of Celsr1 disrupts planar polarity of inner ear hair cells and causes severe neural tube defects in the mouse.

We identified two novel mouse mutants with abnormal head-shaking behavior and neural tube defects during the course of independent ENU mutagenesis experiments. The heterozygous and homozygous mutants exhibit defects in the orientation of sensory hair cells in the organ of Corti, indicating a defect in planar cell polarity. The homozygous mutants exhibit severe neural tube defects as a result of failure to initiate neural tube closure. We show that these mutants, spin cycle and crash, carry independent missense mutations within the coding region of Celsr1, encoding a large protocadherin molecule [1]. Celsr1 is one of three mammalian homologs of Drosophila flamingo/starry night, which is essential for the planar cell polarity pathway in Drosophila together with frizzled, dishevelled, prickle, strabismus/van gogh, and rhoA. The identification of mouse mutants of Celsr1 provides the first evidence for the function of the Celsr family in planar cell polarity in mammals and further supports the involvement of a planar cell polarity pathway in vertebrate neurulation.

Role of Sonic hedgehog in the development of the trachea and oesophagus.

BACKGROUND/PURPOSE: The secreted glycoprotein, Sonic hedgehog (Shh) plays an important patterning role in the development of many organ systems. The authors aimed to study the temporal and spatial pattern of expression of Shh and its receptor Ptc1 during the development of the anterior foregut and to test the hypothesis that the Shh expression pattern is disturbed during the development of oesophageal atresia (OA) and tracheo-oesophageal fistula (TOF) in Adriamycin-treated mouse embryos. METHODS: Saline and Adriamycin-treated (4 mg/kg) CBA/Ca embryos were harvested between embryonic days (E) 10.5 and 12.5, and Shh and Ptc1 expression was studied by whole-mount and section in situ hybridisation using digoxygenin-labelled riboprobes. RESULTS: At E10.5, saline-treated embryos had an undivided foregut in which the ventrally placed prospective tracheal epithelium was positive for Shh, whereas the dorsal part was negative. At E11.5, this pattern had reversed with the separated trachea becoming negative and the oesophagus gaining expression of Shh. Ptc1 was expressed in the mesoderm adjacent to Shh expressing endoderm at both stages. Affected Adriamycin-treated embryos had an undivided foregut at E11.5, the epithelium of which showed diffuse Shh staining that lacked the dorso-ventral patterning seen in controls. CONCLUSIONS: The reversal in the dorso-ventral pattern of Shh expression during the narrow embryologic window in which tracheo-oesophageal separation is initiated suggests that Shh may play an important role in this process. Transient disturbance of this pattern may underlie the abnormal organogenesis in the Adriamycin model.

Disruption of scribble (Scrb1) causes severe neural tube defects in the circletail mouse.

Circletail is one of only two mouse mutants that exhibit the most severe form of neural tube defect (NTD), termed craniorachischisis. In this disorder, almost the entire brain and spinal cord is affected, owing to a failure to initiate neural tube closure. Craniorachischisis is a significant cause of lethality in humans, yet the molecular mechanisms involved remain poorly understood. Here, we report the identification of the gene mutated in circletail (Crc), using a positional cloning approach. This gene, Scrb1, encodes a member of the LAP protein family related to Drosophila scribble, with 16 leucine rich repeats and four PDZ domains. The Crc mutant contains a single base insertion that creates a frame shift and leads to premature termination of the Scrb1 protein. We report the expression pattern of Scrb1 during embryonic and fetal development, and show that Scrb1 expression closely mirrors the phenotypic defects observed in Crc/Crc mutants. In addition, circletail genetically interacts with the loop-tail mutant, and we reveal overlapping expression of Scrb1 with Vangl2, the gene mutated in loop-tail. The identification of the Crc gene further defines the nature of the genetic pathway required for the initiation of neural tube closure and provides an important new candidate that may be implicated in the aetiology of human NTDs.

Dorsoventral patterning in oesophageal atresia with tracheo-oesophageal fistula: Evidence from a new mouse model.

BACKGROUND/PURPOSE: The well-established Adriamycin rat model of oesophageal atresia (OA) and tracheo-oesophageal fistula (TOF) complements recently described mouse genetic models in which loss of function mutations in foregut patterning genes, such as Nkx2.1 (Ttf 1), lead to OA/TOF. The authors aimed to integrate the 2 systems by adapting the Adriamycin model to the mouse to study molecular aspects of tracheo-oesophageal development. METHODS: Pregnant CBA/Ca mice were injected intraperitoneally with 4 mg/kg of Adriamycin on embryonic days 7.5 and 8.5. Embryos and fetuses of various gestational ages were subjected to morphologic or histologic examination. Sections were stained with H & E or processed for immunohistochemistry using an antibody specific for Nkx2.1. RESULTS: Tracheo-oesophageal malformations were observed in 47% of Adriamycin-treated embryos. Early foregut development was similar in Adriamycin-exposed and control embryos but, by E11.5, many treated embryos had an undivided oesophago-trachea, which gave rise to the lung buds and a fistula to the stomach. The fistula originated from the dorsal aspect of the undivided tube and was negative for Nkx2.1, or showed only transient Nkx2.1 expression, compared to the strongly positive bronchi ventrally. CONCLUSIONS: The Adriamycin model of OA is adaptable to the mouse. In the absence of tracheo-oesophageal separation, the dorsal fistula retains its nonrespiratory commitment suggesting that dorsoventral patterning of foregut development is undisturbed by Adriamycin exposure.