4D formation of human embryonic forelimb musculature.

The size, shape and insertion sites of muscles enable them to carry out their precise functions in moving and supporting the skeleton. Although forelimb anatomy is well described, much less is known about the embryonic events that ensure individual muscles reach their mature form. A description of human forelimb muscle development is needed to understand the events that control normal muscle formation and to identify what events are disrupted in congenital abnormalities in which muscles fail to form normally. We provide a new, 4D anatomical characterisation of the developing human upper limb muscles between Carnegie stages 18 and 22 using optical projection tomography. We show that muscles develop in a progressive wave, from proximal to distal and from superficial to deep. We show that some muscle bundles undergo splitting events to form individual muscles, whereas others translocate to reach their correct position within the forelimb. Finally, we show that palmaris longus fails to form from early in development. Our study reveals the timings of, and suggests mechanisms for, crucial events that enable nascent muscle bundles to reach their mature form and position within the human forelimb.

Revisiting the anatomy of the right ventricle in the light of knowledge of its development.

Controversies continue regarding several aspects of the anatomy of the morphologically right ventricle. There is disagreement as to whether the ventricle should be assessed in bipartite or tripartite fashion, and the number of leaflets to be found in the tricuspid valve. In particular, there is no agreement as to whether a muscular outlet septum is present in the normally constructed heart, nor how many septal components are to be found during normal development. Resolving these issues is of potential significance to those investigating and treating children with congenitally malformed hearts. With all these issues in mind, we have revisited our own experience in investigating the development and morphology of the normal right ventricle. To assess development, we have examined a large number of datasets, prepared by both standard and episcopic microscopy, from human and murine embryos. In terms of gross anatomy, we have compared dissections of normal autopsied hearts with virtual dissections of datasets prepared using computed tomography. Our developmental and postnatal studies, taken together, confirm that the ventricle is best assessed in tripartite fashion, with the three parts representing its inlet, apical trabecular, and outlet components. The ventricular septum, however, has only muscular and membranous components. The muscular part incorporates a small component derived from the muscularised fused proximal outflow cushions, but this part cannot be distinguished from the much larger part that is incorporated within the free-standing muscular infundibular sleeve. We confirm that the tricuspid valve itself has three components, which are located inferiorly, septally, and antero-superiorly.

The changing morphology of the ventricular walls of mouse and human with increasing gestation.

That the highly trabeculated ventricular walls of the developing embryos transform to the arrangement during the fetal stages, when the mural architecture is dominated by the thickness of the compact myocardium, has been explained by the coalescence of trabeculations, often erroneously described as 'compaction'. Recent data, however, support differential rates of growth of the trabecular and compact layers as the major driver of change. Here, these processes were assessed quantitatively and visualized in standardized views. We used a larger dataset than has previously been available of mouse hearts, covering the period from embryonic day 10.5 to postnatal day 3, supported by images from human hearts. The volume of the trabecular layer increased throughout development, in contrast to what would be expected had there been 'compaction'. During the transition from embryonic to fetal life, the rapid growth of the compact layer diminished the proportion of trabeculations. Similarly, great expansion of the central cavity reduced the proportion of the total cavity made up of intertrabecular recesses. Illustrations of the hearts with the median value of left ventricular trabeculation confirm a pronounced growth of the compact wall, with prominence of the central cavity. This corresponds, in morphological terms, to a reduction in the extent of the trabecular layer. Similar observations were made in the human hearts. We conclude that it is a period of comparatively slow growth of the trabecular layer, rather than so-called compaction, that is the major determinant of the changing morphology of the ventricular walls of both mouse and human hearts.

Cardiac development demystified by use of the HDBR atlas.

Much has been learned over the last half century regarding the molecular and genetic changes that take place during cardiac development. As yet, however, these advances have not been translated into knowledge regarding the marked changes that take place in the anatomical arrangements of the different cardiac components. As such, therefore, many aspects of cardiac development are still described on the basis of speculation rather than evidence. In this review, we show how controversial aspects of development can readily be arbitrated by the interested spectator by taking advantage of the material now gathered together in the Human Developmental Biology Resource; HDBR. We use the material to demonstrate the changes taking place during the formation of the ventricular loop, the expansion of the atrioventricular canal, the incorporation of the systemic venous sinus, the formation of the pulmonary vein, the process of atrial septation, the remodelling of the pharyngeal arches, the major changes occurring during formation of the outflow tract, the closure of the embryonic interventricular communication, and the formation of the ventricular walls. We suggest that access to the resource makes it possible for the interested observer to arbitrate, for themselves, the ongoing controversies that continue to plague the understanding of cardiac development.

Development of the arterial roots and ventricular outflow tracts.

The separation of the outflow tract of the developing heart into the systemic and pulmonary arterial channels remains controversial and poorly understood. The definitive outflow tracts have three components. The developing outflow tract, in contrast, has usually been described in two parts. When the tract has exclusively myocardial walls, such bipartite description is justified, with an obvious dogleg bend separating proximal and distal components. With the addition of non-myocardial walls distally, it becomes possible to recognise three parts. The middle part, which initially still has myocardial walls, contains within its lumen a pair of intercalated valvar swellings. The swellings interdigitate with the distal ends of major outflow cushions, formed by the remodelling of cardiac jelly, to form the primordiums of the arterial roots. The proximal parts of the major cushions, occupying the proximal part of the outflow tract, which also has myocardial walls, themselves fuse and muscularise. The myocardial shelf thus formed remodels to become the free-standing subpulmonary infundibulum. Details of all these processes are currently lacking. In this account, we describe the anatomical changes seen during the overall remodelling. Our interpretations are based on the interrogation of serially sectioned histological and high-resolution episcopic microscopy datasets prepared from developing human and mouse embryos, with some of the datasets processed and reconstructed to reveal the specific nature of the tissues contributing to the separation of the outflow channels. Our findings confirm that the tripartite postnatal arrangement can be correlated with the changes occurring during development.

Revisiting the anatomy of the left ventricle in the light of knowledge of its development.

Despite centuries of investigation, certain aspects of left ventricular anatomy remain either controversial or uncertain. We make no claims to have resolved these issues, but our review, based on our current knowledge of development, hopefully identifies the issues requiring further investigation. When first formed, the left ventricle had only inlet and apical components. With the expansion of the atrioventricular canal, the developing ventricle cedes part of its inlet to the right ventricle whilst retaining the larger parts of the cushions dividing the atrioventricular canal. Further remodelling of the interventricular communication provides the ventricle with its outlet, with the aortic root being transferred to the left ventricle along with the newly formed myocardium supporting its leaflets. The definitive ventricle possesses inlet, apical and outlet parts. The inlet component is guarded by the mitral valve, with its leaflets, in the normal heart, supported by papillary muscles located infero-septally and supero-laterally. There is but a solitary zone of apposition between the leaflets, which we suggest are best described as being aortic and mural. The trabeculated component extends beyond the inlet to the apex and is confluent with the outlet part, which supports the aortic root. The leaflets of the aortic valve are supported in semilunar fashion within the root, with the ventricular cavity extending to the sinutubular junction. The myocardial-arterial junction, however, stops well short of the sinutubular junction, with myocardium found only at the bases of the sinuses, giving rise to the coronary arteries. We argue that the relationships between the various components should now be described using attitudinally appropriate terms rather than describing them as if the heart is removed from the body and positioned on its apex.

Placentation defects are highly prevalent in embryonic lethal mouse mutants.

Large-scale phenotyping efforts have demonstrated that approximately 25-30% of mouse gene knockouts cause intrauterine lethality. Analysis of these mutants has largely focused on the embryo and not the placenta, despite the crucial role of this extraembryonic organ for developmental progression. Here we screened 103 embryonic lethal and sub-viable mouse knockout lines from the Deciphering the Mechanisms of Developmental Disorders program for placental phenotypes. We found that 68% of knockout lines that are lethal at or after mid-gestation exhibited placental dysmorphologies. Early lethality (embryonic days 9.5-14.5) is almost always associated with severe placental malformations. Placental defects correlate strongly with abnormal brain, heart and vascular development. Analysis of mutant trophoblast stem cells and conditional knockouts suggests that a considerable number of factors that cause embryonic lethality when ablated have primary gene function in trophoblast cells. Our data highlight the hugely under-appreciated importance of placental defects in contributing to abnormal embryo development and suggest key molecular nodes that govern placenta formation.

Control of skeletal morphogenesis by the Hippo-YAP/TAZ pathway.

The Hippo-YAP/TAZ pathway is an important regulator of tissue growth, but can also control cell fate or tissue morphogenesis. Here, we investigate the function of the Hippo pathway during the development of cartilage, which forms the majority of the skeleton. Previously, YAP was proposed to inhibit skeletal size by repressing chondrocyte proliferation and differentiation. We find that, in vitro, Yap/Taz double knockout impairs murine chondrocyte proliferation, whereas constitutively nuclear nls-YAP5SA accelerates proliferation, in line with the canonical role of this pathway in most tissues. However, in vivo, cartilage-specific knockout of Yap/Taz does not prevent chondrocyte proliferation, differentiation or skeletal growth, but rather results in various skeletal deformities including cleft palate. Cartilage-specific expression of nls-YAP5SA or knockout of Lats1/2 do not increase cartilage growth, but instead lead to catastrophic malformations resembling chondrodysplasia or achondrogenesis. Physiological YAP target genes in cartilage include Ctgf, Cyr61 and several matrix remodelling enzymes. Thus, YAP/TAZ activity controls chondrocyte proliferation in vitro, possibly reflecting a regenerative response, but is dispensable for chondrocyte proliferation in vivo, and instead functions to control cartilage morphogenesis via regulation of the extracellular matrix.

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.

The venous system of E14.5 mouse embryos-reference data and examples for diagnosing malformations in embryos with gene deletions.

Approximately one-third of randomly produced knockout mouse lines produce homozygous offspring, which fail to survive the perinatal period. The majority of these die around or after embryonic day (E)14.5, presumably from cardiovascular insufficiency. For diagnosing structural abnormalities underlying death and diseases and for researching gene function, the phenotype of these individuals has to be analysed. This makes the creation of reference data, which define normal anatomy and normal variations the highest priority. While such data do exist for the heart and arteries, they are still missing for the venous system. Here we provide high-quality descriptive and metric information on the normal anatomy of the venous system of E14.5 embryos. Using high-resolution digital volume data and 3D models from 206 genetically normal embryos, bred on the C57BL/6N background, we present precise descriptive and metric information of the venous system as it presents itself in each of the six developmental stages of E14.5. The resulting data shed new light on the maturation and remodelling of the venous system at transition of embryo to foetal life and provide a reference that can be used for detecting venous abnormalities in mutants. To explore this capacity, we analysed the venous phenotype of embryos from 7 knockout lines (Atp11a, Morc2a, 1700067K01Rik, B9d2, Oaz1, Celf4 and Coro1c). Careful comparisons enabled the diagnosis of not only simple malformations, such as dual inferior vena cava, but also complex and subtle abnormalities, which would have escaped diagnosis in the absence of detailed, stage-specific referenced data.

Miniseries 1-Part II: the comparative anatomy of the atrioventricular conduction axis.

AIMS: The arrangement of the conduction axis is markedly different in various mammalian species. Knowledge of such variation may serve to question the validity of using animals as prospective models for design of systems for clinical use. METHODS AND RESULTS: We compared the arrangement of the atrioventricular conduction axis in human, murine, canine, porcine, and bovine hearts, examining serially sectioned datasets from 20 human, 16 murine, 3 porcine, 5 canine, and 1 bovine hearts. We also analysed computed tomographic datasets obtained from bovines and one human heart. Unlike the situation in the human heart, there is no formation of an atrioventricular fibrous membranous septum in the murine, canine, porcine, nor bovine hearts. Canine, porcine, and bovine hearts also lack an infero-septal recess, when defined as a fibrous plate supporting the buttress of the atrial septum. In these species, half of the non-coronary leaflet is directly opposed to the ventricular septal surface. CONCLUSION: There is a long right-sided non-branching component of the axis, which skirts the attachment of the non-coronary sinus of the aortic root. In the bovine heart, moreover, the left bundle branch usually extends intramyocardially as a solitary tape before surfacing and ramifying on the left ventricular septal surface. The difference in the atrioventricular conduction axis between species may influence the anatomical substrates for atrioventricular re-entry tachycardia, as well as providing inferences for assessing the risks of transcatheter implantation of the aortic valve. Further studies are now needed to assess these possibilities.

Miniseries 1-Part I: the Development of the atrioventricular conduction axis.

Despite years of research, many details of the formation of the atrioventricular conduction axis remain uncertain. In this study, we aimed to clarify the situation. We studied three-dimensional reconstructions of serial histological sections and episcopic datasets of human embryos, supplementing these findings with assessment of material housed at the Human Developmental Biological Resource. We also examined serially sectioned human foetal hearts between 10 and 30 weeks of gestation. The conduction axis originates from the primary interventricular ring, which is initially at right angles to the plane of the atrioventricular canal, with which it co-localizes in the lesser curvature of the heart loop. With rightward expansion of the atrioventricular canal, the primary ring bends rightward, encircling the newly forming right atrioventricular junction. Subsequent to remodelling of the outflow tract, part of the primary ring remains localized on the crest of the muscular ventricular septum. By 7 weeks, its atrioventricular part has extended perpendicular to the septal parts. The atrioventricular node is formed at the inferior transition between the ventricular and atrial parts, with the transition itself marking the site of the penetrating atrioventricular bundle. Only subsequent to muscularization of the true second atrial septum does it become possible to recognize the definitive node. The conversion of the developmental arrangement into the definitive situation as seen postnatally requires additional remodelling in the first month of foetal development, concomitant with formation of the inferior pyramidal space and the infero-septal recess of the subaortic outflow tract.

Msx1 haploinsufficiency modifies the Pax9-deficient cardiovascular phenotype.

BACKGROUND: Successful embryogenesis relies on the coordinated interaction between genes and tissues. The transcription factors Pax9 and Msx1 genetically interact during mouse craniofacial morphogenesis, and mice deficient for either gene display abnormal tooth and palate development. Pax9 is expressed specifically in the pharyngeal endoderm at mid-embryogenesis, and mice deficient for Pax9 on a C57Bl/6 genetic background also have cardiovascular defects affecting the outflow tract and aortic arch arteries giving double-outlet right ventricle, absent common carotid arteries and interruption of the aortic arch. RESULTS: In this study we have investigated both the effect of a different genetic background and Msx1 haploinsufficiency on the presentation of the Pax9-deficient cardiovascular phenotype. Compared to mice on a C57Bl/6 background, congenic CD1-Pax9-/- mice displayed a significantly reduced incidence of outflow tract defects but aortic arch defects were unchanged. Pax9-/- mice with Msx1 haploinsufficiency, however, have a reduced incidence of interrupted aortic arch, but more cases with cervical origins of the right subclavian artery and aortic arch, than seen in Pax9-/- mice. This alteration in arch artery defects was accompanied by a rescue in third pharyngeal arch neural crest cell migration and smooth muscle cell coverage of the third pharyngeal arch arteries. Although this change in phenotype could theoretically be compatible with post-natal survival, using tissue-specific inactivation of Pax9 to maintain correct palate development whilst inducing the cardiovascular defects was unable to prevent postnatal death in the mutant mice. Hyoid bone and thyroid cartilage formation were abnormal in Pax9-/- mice. CONCLUSIONS: Msx1 haploinsufficiency mitigates the arch artery defects in Pax9-/- mice, potentially by maintaining the survival of the 3rd arch artery through unimpaired migration of neural crest cells to the third pharyngeal arches. With the neural crest cell derived hyoid bone and thyroid cartilage also being defective in Pax9-/- mice, we speculate that the pharyngeal endoderm is a key signalling centre that impacts on neural crest cell behaviour highlighting the ability of cells in different tissues to act synergistically or antagonistically during embryo development.

WDR11-mediated Hedgehog signalling defects underlie a new ciliopathy related to Kallmann syndrome.

WDR11 has been implicated in congenital hypogonadotropic hypogonadism (CHH) and Kallmann syndrome (KS), human developmental genetic disorders defined by delayed puberty and infertility. However, WDR11's role in development is poorly understood. Here, we report that WDR11 modulates the Hedgehog (Hh) signalling pathway and is essential for ciliogenesis. Disruption of WDR11 expression in mouse and zebrafish results in phenotypic characteristics associated with defective Hh signalling, accompanied by dysgenesis of ciliated tissues. Wdr11-null mice also exhibit early-onset obesity. We find that WDR11 shuttles from the cilium to the nucleus in response to Hh signalling. WDR11 regulates the proteolytic processing of GLI3 and cooperates with the transcription factor EMX1 in the induction of downstream Hh pathway gene expression and gonadotrophin-releasing hormone production. The CHH/KS-associated human mutations result in loss of function of WDR11. Treatment with the Hh agonist purmorphamine partially rescues the WDR11 haploinsufficiency phenotypes. Our study reveals a novel class of ciliopathy caused by WDR11 mutations and suggests that CHH/KS may be a part of the human ciliopathy spectrum.

Myoarchitectural disarray of hypertrophic cardiomyopathy begins pre-birth.

Myoarchitectural disarray - the multiscalar disorganisation of myocytes, is a recognised histopathological hallmark of adult human hypertrophic cardiomyopathy (HCM). It occurs before the establishment of left ventricular hypertrophy (LVH) but its early origins and evolution around the time of birth are unknown. Our aim is to investigate whether myoarchitectural abnormalities in HCM are present in the fetal heart. We used wild-type, heterozygous and homozygous hearts (n = 56) from a Mybpc3-targeted knock-out HCM mouse model and imaged the 3D micro-structure by high-resolution episcopic microscopy. We developed a novel structure tensor approach to extract, display and quantify myocyte orientation and its local angular uniformity by helical angle, angle of intrusion and myoarchitectural disarray index, respectively, immediately before and after birth. In wild-type, we demonstrate uniformity of orientation of cardiomyocytes with smooth transitions of helical angle transmurally both before and after birth but with traces of disarray at the septal insertion points of the right ventricle. In comparison, heterozygous mice free of LVH, and homozygous mice showed not only loss of the normal linear helical angulation transmural profiles observed in wild-type but also fewer circumferentially arranged myocytes at birth. Heterozygous and homozygous showed more disarray with a wider distribution than in wild-type before birth. In heterozygous mice, disarray was seen in the anterior, septal and inferior walls irrespective of stage, whereas in homozygous mice it extended to the whole LV circumference including the lateral wall. In conclusion, myoarchitectural disarray is detectable in the fetal heart of an HCM mouse model before the development of LVH.

Spatiotemporal regulation of enhancers during cardiogenesis.

With the advance in chromatin immunoprecipitation followed by high-throughput sequencing, there has been a dramatic increase in our understanding of distal enhancer function. In the developing heart, the identification and characterisation of such enhancers have deepened our knowledge of the mechanisms of transcriptional regulation that drives cardiac differentiation. With next-generation sequencing techniques becoming widely accessible, the quantity of data describing the genome-wide distribution of cardiac-specific transcription factor and chromatin modifiers has rapidly increased and it is now becoming clear that the usage of enhancers is highly dynamic and complex, both during the development and in the adult. The identification of those enhancers has revealed new insights into the transcriptional mechanisms of how tissue-specific gene expression patterns are established, maintained, and change dynamically during development and upon physiological stress.

Deciphering the mechanisms of developmental disorders: phenotype analysis of embryos from mutant mouse lines.

The Deciphering the Mechanisms of Developmental Disorders (DMDD) consortium is a research programme set up to identify genes in the mouse, which if mutated (or knocked-out) result in embryonic lethality when homozygous, and initiate the study of why disruption of their function has such profound effects on embryo development and survival. The project uses a combination of comprehensive high resolution 3D imaging and tissue histology to identify abnormalities in embryo and placental structures of embryonic lethal lines. The image data we have collected and the phenotypes scored are freely available through the project website (http://dmdd.org.uk). In this article we describe the web interface to the images that allows the embryo data to be viewed at full resolution in different planes, discuss how to search the database for a phenotype, and our approach to organising the data for an embryo and a mutant line so it is easy to comprehend and intuitive to navigate.

iASPP, a previously unidentified regulator of desmosomes, prevents arrhythmogenic right ventricular cardiomyopathy (ARVC)-induced sudden death.

Desmosomes are anchoring junctions that exist in cells that endure physical stress such as cardiac myocytes. The importance of desmosomes in maintaining the homeostasis of the myocardium is underscored by frequent mutations of desmosome components found in human patients and animal models. Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a phenotype caused by mutations in desmosomal components in ∼ 50% of patients, however, the causes in the remaining 50% of patients still remain unknown. A deficiency of inhibitor of apoptosis-stimulating protein of p53 (iASPP), an evolutionarily conserved inhibitor of p53, caused by spontaneous mutation recently has been associated with a lethal autosomal recessive cardiomyopathy in Poll Hereford calves and Wa3 mice. However, the molecular mechanisms that mediate this putative function of iASPP are completely unknown. Here, we show that iASPP is expressed at intercalated discs in human and mouse postmitotic cardiomyocytes. iASPP interacts with desmoplakin and desmin in cardiomyocytes to maintain the integrity of desmosomes and intermediate filament networks in vitro and in vivo. iASPP deficiency specifically induces right ventricular dilatation in mouse embryos at embryonic day 16.5. iASPP-deficient mice with exon 8 deletion (Ppp1r13l(Δ8/Δ8)) die of sudden cardiac death, displaying features of ARVC. Intercalated discs in cardiomyocytes from four of six human ARVC cases show reduced or loss of iASPP. ARVC-derived desmoplakin mutants DSP-1-V30M and DSP-1-S299R exhibit weaker binding to iASPP. These data demonstrate that by interacting with desmoplakin and desmin, iASPP is an important regulator of desmosomal function both in vitro and in vivo. This newly identified property of iASPP may provide new molecular insight into the pathogenesis of ARVC.

The cardiac-restricted protein ADP-ribosylhydrolase-like 1 is essential for heart chamber outgrowth and acts on muscle actin filament assembly.

Adprhl1, a member of the ADP-ribosylhydrolase protein family, is expressed exclusively in the developing heart of all vertebrates. In the amphibian Xenopus laevis, distribution of its mRNA is biased towards actively growing chamber myocardium. Morpholino oligonucleotide-mediated knockdown of all Adprhl1 variants inhibits striated myofibril assembly and prevents outgrowth of the ventricle. The resulting ventricles retain normal electrical conduction and express markers of chamber muscle differentiation but are functionally inert. Using a cardiac-specific Gal4 binary expression system, we show that the abundance of Adprhl1 protein in tadpole hearts is tightly controlled through a negative regulatory mechanism targeting the 5'-coding sequence of Xenopus adprhl1. Over-expression of full length (40kDa) Adprhl1 variants modified to escape such repression, also disrupts cardiac myofibrillogenesis. Disarrayed myofibrils persist that show extensive branching, with sarcomere division occurring at the actin-Z-disc boundary. Ultimately, Adprhl1-positive cells contain thin actin threads, connected to numerous circular branch points. Recombinant Adprhl1 can localize to stripes adjacent to the Z-disc, suggesting a direct role for Adprhl1 in modifying Z-disc and actin dynamics as heart chambers grow. Modelling the structure of Adprhl1 suggests this cardiac-specific protein is a pseudoenzyme, lacking key residues necessary for ADP-ribosylhydrolase catalytic activity.