Morphogenetic processes in the development and evolution of the arteries of the pharyngeal arches: their relations to congenital cardiovascular malformations.

The heart and aortic arch arteries in amniotes form a double circulation, taking oxygenated blood from the heart to the body and deoxygenated blood to the lungs. These major vessels are formed in embryonic development from a series of paired and symmetrical arteries that undergo a complex remodelling process to form the asymmetric arch arteries in the adult. These embryonic arteries form in the pharyngeal arches, which are symmetrical bulges on the lateral surface of the head. The pharyngeal arches, and their associated arteries, are found in all classes of vertebrates, but the number varies, typically with the number of arches reducing through evolution. For example, jawed vertebrates have six pairs of pharyngeal arch arteries but amniotes, a clade of tetrapod vertebrates, have five pairs. This had led to the unusual numbering system attributed to each of the pharyngeal arch arteries in amniotes (1, 2, 3, 4, and 6). We, therefore, propose that these instead be given names to reflect the vessel: mandibular (1st), hyoid (2nd), carotid (3rd), aortic (4th) and pulmonary (most caudal). Aberrant arch artery formation or remodelling leads to life-threatening congenital cardiovascular malformations, such as interruption of the aortic arch, cervical origin of arteries, and vascular rings. We discuss why an alleged fifth arch artery has erroneously been used to interpret congenital cardiac lesions, which are better explained as abnormal collateral channels, or remodelling of the aortic sac.

The advantages of naming rather than numbering the arteries of the pharyngeal arches.

Controversies continue as to how many pharyngeal arches, with their contained arteries, are to be found in the developing human. Resolving these controversies is of significance to paediatric cardiologists since many investigating abnormalities of the extrapericardial arterial pathways interpret their findings on the basis of persistence of a fifth set of such arteries within an overall complement of six sets. The evidence supporting such an interpretation is open to question. In this review, we present the history of the existence of six such arteries, emphasising that the initial accounts of human development had provided evidence for the existence of only five sets. We summarise the current evidence that substantiates these initial findings. We then show that the lesions interpreted on the basis of persistence of the non-existing fifth arch arteries are well described on the basis of the persistence of collateral channels, known to exist during normal development, or alternatively due to remodelling of the aortic sac.

A revised terminology for the pharyngeal arches and the arch arteries.

The pharyngeal arches are a series of bulges found on the lateral surface of the head of vertebrate embryos. In humans, and other amniotes, there are five pharyngeal arches and traditionally these have been labelled from cranial to caudal-1, 2, 3, 4 and 6. This numbering is odd-there is no '5'. Two reasons have been given for this. One is that during development, a 'fifth' arch forms transiently but is not fully realised. The second is that this numbering fits with the evolutionary history of the pharyngeal arches. Recent studies, however, have shown that neither of these justifications have basis. The traditional labelling is problematic as it causes confusion to those trying to understand the development of the pharyngeal arches. In particular, it creates difficulties in the field of congenital cardiac malformations, where it is common to find congenital cardiac lesions interpreted on the basis of persistence of the postulated arteries of the fifth arch. To resolve these problems and to take account of the recent studies that have clarified pharyngeal arch development, we propose a new terminology for the pharyngeal arches. In this revised scheme, the pharyngeal arches are to be labelled as follows-the first, most cranial, the mandibular (M), the second, the hyoid (H), the third, the carotid (C), the fourth, the aortic (A) and the last, most caudal, the pulmonary (P).

Key separable events in the remodelling of the pharyngeal arches.

The pharyngeal arches are a series of bulges on the lateral surface of the embryonic head. They are a defining feature of the most conserved, the phylotypic, stage of vertebrate development. In many vertebrate clades, the segmental arrangement of the pharyngeal arches is translated into the iterative anatomy of the gill arches. However, in amniotes the pharyngeal arches undergo a rearrangement during development and the segmental organisation of the pharynx is lost. This remodelling involves the expansion of the second arch which comes to overlie the more posterior arches. A transient sinus forms between the expanded second arch and the posterior arches, that is then lost, and the posterior arches are internalised. The morphogenesis of the second arch has been viewed as being central to this remodelling. Yet little is known about this process. Therefore, in this study, we have characterised the development of the second arch. We show that as the second arch expands, its posterior margin forms a leading edge and that the mesenchymal cells subjacent to this are in an elevated proliferative state. We further show that the posterior marginal epithelium is the site of expression of three key developmental signalling molecules: BMP7, FGF8 and SHH, and that their expression continues throughout the period of expansion. Using a novel approach, we have been able to simultaneously inhibit these three pathways, and we find that when this is done the second arch fails to establish its caudal projection and that there is a loss of proliferation in the posterior mesenchymal cells of the second arch. We have further used this manipulation to ask if the internalisation of the posterior arches is dependent upon the expansion of the second arch. We find that it is not-the posterior arches are still internalised when the expansion of the second arch is curtailed. We further show that while the collapse of the sinus is dependent upon thyroid hormone signalling, that this is not the case for the internalisation of the posterior pouches. Thus, the internalisation of the posterior arches is not dependent on the expansion of the second arch or on the collapse of the sinus. Finally, we show that the termination of expansion of the second arch correlates with a burst of morphogenetic cell death suggesting a mechanism for ending this. Thus, while it has long been thought that it is the morphogenesis of the second arch that drives the remodelling of the pharyngeal arches, we show that this is not the case. Rather the remodelling of the pharyngeal arches is a composite process that can split into contemporaneous but separate events: the expansion of the second arch, the internalisation of the posterior arches and the collapse of the sinus.

Syndesmosis injuries in professional rugby players: associated injuries and complications can lead to an unpredictable time to return to play.

OBJECTIVES: Syndesmosis injuries are common and increasing in contact sports with a marked impact on players and teams alike. They can result in an unpredictable and often prolonged return to pre-injury level. We aim to evaluate the time to return to play (RTP) after syndesmosis injuries in professional male rugby players. METHODS: A cohort study including all professional rugby players with syndesmosis injuries, treated both operatively and non-operatively by the senior author was performed. The follow up period was a minimum of 12 months or until RTP. Players with previous ankle injuries or associated ankle fractures were excluded. Outcome measures included players age, body mass index (BMI), field position, seven-a-side or 15-a-side match, mechanism of injury, clinical findings, radiological findings, return to training (RTT), and RTP dates. RESULTS: For the period July 2015 to July 2019, a total of 13 professional male rugby players were included in the study. The leading mechanism of syndesmosis injury was in contact during a tackle. Six players had a grade 3 injury (40%), 4 players had a grade 2B injury (27%), 2 players had a grade 2A injury (13%) and 3 players had a grade 1 injury (20%). Two of the aforementioned players presented with new contralateral syndesmosis injuries during the study period. The median time for RTT and RTP was 97 days (IQR: 36) and 112 days (IQR: 54), respectively. All players with syndesmosis injuries were able to return to play. No discrepancy was found between seven-a-side and 15-a-side players regarding injury mechanism, injury severity, and RTP. CONCLUSION: Most syndesmosis injuries in professional rugby players are acquired during a tackle. These injuries are often unstable, requiring surgical intervention, with an unpredictable recovery period. Injury severity, surgical complications, delayed diagnosis, and associated injuries can prolong this period. The subtype of rugby (seven-a-side and 15-a-side) does not affect the injury severity or return to play. LEVEL OF EVIDENCE: Level 4.

Development of the mammalian main olfactory bulb.

The mammalian main olfactory bulb is a crucial processing centre for the sense of smell. The olfactory bulb forms early during development and is functional from birth. However, the olfactory system continues to mature and change throughout life as a target of constitutive adult neurogenesis. Our Review synthesises current knowledge of prenatal, postnatal and adult olfactory bulb development, focusing on the maturation, morphology, functions and interactions of its diverse constituent glutamatergic and GABAergic cell types. We highlight not only the great advances in the understanding of olfactory bulb development made in recent years, but also the gaps in our present knowledge that most urgently require addressing.

A developmental basis for the anatomical diversity of dermis in homeostasis and wound repair.

The dermis has disparate embryonic origins; abdominal dermis develops from lateral plate mesoderm, dorsal dermis from paraxial mesoderm and facial dermis from neural crest. However, the cell and molecular differences and their functional implications have not been described. We hypothesise that the embryonic origin of the dermis underpins regional characteristics of skin, including its response to wounding. We have compared abdomen, back and cheek, three anatomical sites representing the distinct embryonic tissues from which the dermis can arise, during homeostasis and wound repair using RNA sequencing, histology and fibroblast cultures. Our transcriptional analyses demonstrate differences between body sites that reflect their diverse origins. Moreover, we report histological and transcriptional variations during a wound response, including site differences in ECM composition, cell migration and proliferation, and re-enactment of distinct developmental programmes. These findings reveal profound regional variation in the mechanisms of tissue repair. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

The zebrafish Znt1asa17 mutant reveals roles of zinc transporter-1a in embryonic development.

BACKGROUND: Zinc is one of the vital micronutrients required through various developmental stages in animals. Zinc transporter-1 (ZnT1; Slc30a1) is essential in vertebrates for nutritional zinc uptake and cellular zinc extrusion. Knockout of ZnT1 is lethal in vertebrates and there are therefore few functional studies of this protein in vivo. METHODS: In the present study we characterised the embryonic development in a zebrafish Znt1a mutant (Znt1asa17) which is lacking the last 40 amino acids of Znt1a as generated by TILLING. In parallel experiments, we compared the development of a zebrafish embryo Znt1a morphant (Znt1aMO) which was generated by knockdown of Znt1a using morpholino-modified oligonucliotides. RESULTS: The homozygous Znt1asa17 embryo is viable, but displays a subtle phenotype informing on the biological roles of Znt1a. The Znt1asa17 fish have delayed development, including attenuated epiboly. They further show a decrease in phosphorylated extracellular signal-regulated kinases 1 and 2 (pERK1/2), retarded yolk resorption, and impaired clearance of free Zn2+ from the vitelline fluid and its storage in hatching gland cells. All these aberrations are milder versions of those observed upon knockdown of Znt1a by morpholinos. Interestingly, the phenotype could be rescued by addition of the cell-permeable zinc chelator, N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN) to the incubation medium and was aggravated by addition of zinc(II). Thus, the Znt1asa17 mutant has a reduced ability to handle zinc and can be characterised as a hypomorph. CONCLUSION: This study is the first to show that the last 40 amino acids of Znt1a are of importance for its role in zinc homeostasis and ability to activate the MAPK/ERK pathway contrary to what was previously thought.

A reappraisal and revision of the numbering of the pharyngeal arches.

The pharyngeal arches are a prominent and significant feature of vertebrate embryos. These are visible as a series of bulges on the lateral surface of the embryonic head. In humans, and other amniotes, there are five pharyngeal arches numbered 1, 2, 3, 4 and 6; note the missing '5'. This is the standard scheme for the numbering of these structures, and it is a feature of modern anatomy textbooks. In this article, we discuss the rationale behind this odd numbering, and consider its origins. One reason given is that there is a transient 5th arch that is never fully realized, while another is that this numbering reflects considerations from comparative anatomy. We show here, however, that neither of these reasons has substance. There is no evidence from embryology for a '5th' arch, and the comparative argument does not hold as it does not apply across the vertebrates. We conclude that there is no justification for this strange numbering. We suggest that the pharyngeal arches should simply be numbered 1, 2, 3, 4 and 5 as this would be in keeping with the embryology and with the general numbering of the pharyngeal arches across the vertebrates.

Extracellular matrix protein anosmin-1 modulates olfactory ensheathing cell maturation in chick olfactory bulb development.

Olfactory ensheathing cells (OECs) are a specialized class of glia, wrapping around olfactory sensory axons that target the olfactory bulb (OB) and cross the peripheral nervous system/central nervous system boundary during development and continue to do so post-natally. OEC subpopulations perform distinct subtype-specific functions dependent on their maturity status. Disrupted OEC development is thought to be associated with abnormal OB morphogenesis, leading to anosmia, a defining characteristic of Kallmann syndrome. Hence, anosmin-1 encoded by Kallmann syndrome gene (KAL-1) might modulate OEC differentiation/maturation in the OB. We performed in ovo electroporation of shRNA in the olfactory placode to knock-down kal in chick embryos, resulting in abnormal OB morphogenesis and loss of olfactory sensory axonal innervation into OB. BLBP-expressing OECs appeared to form a thinner and poorly organized outmost OB layer where SOX10 expressing OECs were completely absent with emergence of GFAP-expressing OECs. Furthermore, in embryonic day 10 chick OB explant cultures, GFAP expression in OECs accumulating along the OB nerve layers was dramatically reduced by recombinant anosmin-1. We then purified immature OECs from embryonic day 10 chick OB. These cells express GFAP after 7 days in vitro, exhibiting a multipolar morphology. Overexpression of chick anosmin, exogenous anosmin-1 or FGF2 could inhibit GFAP expression with cells presenting elongated morphology, which was blocked by the FGF receptor inhibitor Su5402. These data demonstrate that anosmin-1 functions via FGF signalling in regulating OEC maturation, thereby providing a permissive glial environment for axonal innervation into the OB during development.

Diminution of pharyngeal segmentation and the evolution of the amniotes.

BACKGROUND: The pharyngeal arches are a series of bulges found on the lateral surface of the head of vertebrate embryos, and it is within these segments that components of the later anatomy are laid down. In most vertebrates, the post-otic pharyngeal arches will form the branchial apparatus, while in amniotes these segments are believed to generate the larynx. It has been unclear how the development of these segments has been altered with the emergence of the amniotes. RESULTS: In this study, we examined the development of pharyngeal arches in amniotes and show that the post-otic pharyngeal arches in this clade are greatly diminished. We find that the post-otic segments do not undergo myogenesis or skeletogenesis, but are remodelled before these processes occur. We also find that nested DLX expression, which is a feature of all the pharyngeal arches in anamniotes, is associated with the anterior segments but less so with the posterior arches in amniotes. We further show that the posterior arches of the mouse embryo fail to properly delineate, which demonstrates the lack of function of these posterior segments in later development. CONCLUSION: In amniotes, there has been a loss of the ancestral "branchial" developmental programme that is a general feature of gnathostomes; myogenesis and skeletogenesis This is likely to have facilitated the emergence of the larynx as a new structure not constrained by the segmental organisation of the posterior pharyngeal region.

The emergence of mesencephalic trigeminal neurons.

BACKGROUND: The cells of the mesencephalic trigeminal nucleus (MTN) are the proprioceptive sensory neurons that innervate the jaw closing muscles. These cells differentiate close to the two key signalling centres that influence the dorsal midbrain, the isthmus, which mediates its effects via FGF and WNT signalling and the roof plate, which is a major source of BMP signalling as well as WNT signalling. METHODS: In this study, we have set out to analyse the importance of FGF, WNT and BMP signalling for the development of the MTN. We have employed pharmacological inhibitors of these pathways in explant cultures as well as utilising the electroporation of inhibitory constructs in vivo in the chick embryo. RESULTS: We find that interfering with either FGF or WNT signalling has pronounced effects on MTN development whilst abrogation of BMP signalling has no effect. We show that treatment of explants with either FGF or WNT antagonists results in the generation of fewer MTN neurons and affects MTN axon extension and that inhibition of both these pathways has an additive effect. To complement these studies, we have used in vivo electroporation to inhibit BMP, FGF and WNT signalling within dorsal midbrain cells prior to, and during, their differentiation as MTN neurons. Again, we find that inhibition of BMP signalling has no effect on the development of MTN neurons. We additionally find that cells electroporated with inhibitory constructs for either FGF or WNT signalling can differentiate as MTN neurons suggesting that these pathways are not required cell intrinsically for the emergence of these neurons. Indeed, we also show that explants of dorsal mesencephalon lacking both the isthmus and roof plate can generate MTN neurons. However, we did find that inhibiting FGF or WNT signalling had consequences for MTN differentiation. CONCLUSIONS: Our results suggest that the emergence of MTN neurons is an intrinsic property of the dorsal mesencephalon of gnathostomes, and that this population undergoes expansion, and maturation, along with the rest of the dorsal midbrain under the influence of FGF and WNT signalling.

Serotonin Receptor 1A (HTR1A), a Novel Regulator of GnRH Neuronal Migration in Chick Embryo.

The hypothalamic GnRH neurons are a small group of cells that regulate the reproductive axis. These neurons are specified within the olfactory placode, delaminate from this structure, and then migrate to enter the forebrain before populating the hypothalamus. We have used microarray technology to analyze the transcriptome of the olfactory placode at a number of key time points for GnRH ontogeny using the chick embryo. This resulted in the identification of a large number of genes whose expression levels change significantly over this period. This repertoire includes those genes that are known to be important for GnRH neuronal development as well as many novel genes, such as the serotonin receptor 1A, HTR1A. We find that HTR1A is expressed in the region of the olfactory placode that generates GnRH neurons. We further show that when this receptor is inactivated using a selective HTR1A antagonist as well as a gene knockdown approach using RNAi, this resulted in delayed migration causing the GnRH neurons to stall just outside the forebrain. These findings implicate HTR1A as being important for GnRH neuronal migration from the olfactory placode to the forebrain. Our study thus extends the repertoire of genes involved in GnRH neuron biology and thus identifies new candidate genes that can be screened for in patients who do not show mutations in any of the previously identified hypogonadotrophic hypogonadism/Kallmann syndrome genes.

Mode of reduction in the number of pharyngeal segments within the sarcopterygians.

BACKGROUND: Pharyngeal segmentation is a defining feature of vertebrate embryos and is apparent as a series of bulges found on the lateral surface of the embryonic head, the pharyngeal arches. The ancestral condition for gnathostomes is to have seven pharyngeal segments: jaw, hyoid, and five posterior branchial arches. However, within the sarcopterygians, the pharyngeal region has undergone extensive remodelling that resulted in a reduction in the number of pharyngeal segments, such that amniotes have only five pharyngeal arches. The aim of this study is to probe the developmental basis of this loss of pharyngeal segments. RESULTS: We have therefore compared the development of the pharyngeal arches in an amniote, the chick, which has five segments, with those of a chondrichthyan, the catshark, which has seven segments. We have analysed the early phase of pharyngeal segmentation and we find that in both the most anterior segments form first with the posterior segments being added sequentially. We also documented the patterns of innervation of the pharynx in several vertebrates and note that the three most anterior segments receive distinct innervation: the first arch being innervated by the Vth nerve, the second by the VIIth and the third by the IXth. Finally, we have analysed Hox gene expression, and show that the anterior limit of Hoxa2 aligns with the second pouch and arch in both chick and catshark, while Hoxa3 is transiently associated with the third arch and pouch. Surprisingly, we have found that Hoxb1 expression is spatially and temporally dynamic and that it is always associated with the last most recently formed pouch and that this domains moves caudally as additional pouches are generated. CONCLUSION: We propose that the first three pharyngeal segments are homologous, as is the posterior limit of the pharynx, and that the loss of segments occurred between these two points. We suggest that this loss results from a curtailment of the posterior expansion of the pharyngeal endoderm in amniotes at relatively earlier time point, and thus the generation of fewer segments.

Differential Cellular Responses to Hedgehog Signalling in Vertebrates-What is the Role of Competence?

A surprisingly small number of signalling pathways generate a plethora of cellular responses ranging from the acquisition of multiple cell fates to proliferation, differentiation, morphogenesis and cell death. These diverse responses may be due to the dose-dependent activities of signalling factors, or to intrinsic differences in the response of cells to a given signal-a phenomenon called differential cellular competence. In this review, we focus on temporal and spatial differences in competence for Hedgehog (HH) signalling, a signalling pathway that is reiteratively employed in embryos and adult organisms. We discuss the upstream signals and mechanisms that may establish differential competence for HHs in a range of different tissues. We argue that the changing competence for HH signalling provides a four-dimensional framework for the interpretation of the signal that is essential for the emergence of functional anatomy. A number of diseases-including several types of cancer-are caused by malfunctions of the HH pathway. A better understanding of what provides differential competence for this signal may reveal HH-related disease mechanisms and equip us with more specific tools to manipulate HH signalling in the clinic.