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Purpose The aim of this study is to describe the incidence of Acute Kidney Injury (AKI) amongst patients admitted to the Intensive Care Unit (ICU) with COVID-19. In addition we aim to detail the range of Renal Replacement Therapy (RRT) modalities offered to these patients (including peritoneal dialysis - PD - and intermittent haemodialysis - IHD) in order to meet demand during pandemic conditions. Materials and methods Single-centre retrospective case note review of adult patients with confirmed COVID-19 admitted to ICU. Results Amongst 136 patients without a prior history of End Stage Kidney Disease (ESKD), 108 (79%) developed AKI and 63% of admitted patients received RRT. Due to resource limitations the range of RRT options were expanded from solely Continuous Veno-Venous HaemoDiaFiltration (CVVHDF - our usual standard of care) to include PD (in 35 patients) and IHD (in 15 patients). During the study period the proportion of RRT provided within ICU as CVVHDF fell from 100% to a nadir of 39%. There were no significant complications of either PD or IHD. Conclusions During periods of resource limitations PD and IHD can safely be used to reduce dependence on CVVHDF in select patients with AKI secondary to COVID-19., Highlights • AKI frequently complicates COVID-19 in patients admitted to ICU. • Large numbers of patients and high rates of AKI leads to great demand for RRT. • In selected patients PD and IHD can supplement CRRT in meeting this demand. • PD and IHD can be implemented rapidly and safely through a collaborative approach.

Using the zebrafish neural tube as a model, we uncover thein vivomechanisms allowing the generation of two opposing apical epithelial surfaces within the centre of an initially unpolarised, solid organ. We show that NOK/Pals1/Mpp5a and Rab11a play a dual role in coordinating the generation of ipsilateral junctional belts whilst simultaneously releasing contralateral adhesions across the centre of the tissue. We show that Nok and Rab11a mediated resolution of cell-cell adhesions are both necessary for midline lumen opening and contribute to later maintenance of epithelial organisation. We propose these roles for both Nok and Rab11a operate through the transmembrane protein Crumbs. In light of a recent conflicting publication, we also clarify that the junction remodelling role of Nok is not specific to dividing cells.

Using the zebrafish neural tube as a model, we uncover the in vivo mechanisms allowing the generation of two opposing apical epithelial surfaces within the centre of an initially unpolarised, solid organ. We show that Mpp5a and Rab11a play a dual role in coordinating the generation of ipsilateral junctional belts whilst simultaneously releasing contralateral adhesions across the centre of the tissue. We show that Mpp5a- and Rab11a-mediated resolution of cell-cell adhesions are both necessary for midline lumen opening and contribute to later maintenance of epithelial organisation. We propose that these roles for both Mpp5a and Rab11a operate through the transmembrane protein Crumbs. In light of a recent conflicting publication, we also clarify that the junction-remodelling role of Mpp5a is not specific to dividing cells.

In vitro, developing neurons progress through well-defined stages to form an axon and multiple dendrites.In vivo, neurons are derived from progenitors within a polarised neuroepithelium and it is not clear how axon initiation observedin vitrorelates to what occurs in a complex, three-dimensionalin vivoenvironment. Here we show that the position of axon initiation in embryonic zebrafish spinal neurons is extremely consistent across neuronal sub-types. We investigated what mechanisms may regulate axon positioningin vivoand found that microtubule organising centres are located distant from the site of axon initiation in contrast to that observedin vitro, and that microtubule plus-ends are not enriched in the axon during axon initiation. F-actin accumulation precedes axon formation and nascent axons form but are not stabilised in the absence of microtubules. Laminin depletion removes a spatial cue for axon initiation but axon initiation remains robust.

Tissue internalisation is a key morphogenetic mechanism by which embryonic tissues generate complex internal organs and a number of studies of epithelia have outlined a general view of tissue internalisation. Here we have used quantitative live imaging and mutant analysis to determine whether similar mechanisms are responsible for internalisation in a tissue that apparently does not have a typical epithelial organisation - the zebrafish neural plate. We found that although zebrafish embryos begin neurulation without a conventional epithelium, medially located neural plate cells adopt strategies typical of epithelia in order to constrict their dorsal surface membrane during cell internalisation. Furthermore, we show that Myosin-II activity is a significant driver of this transient cell remodeling which also depends on Cdh2 (N-cadherin). Abrogation of Cdh2 results in defective Myosin-II distribution, mislocalised internalisation events and defective neural plate morphogenesis. Our work suggests Cdh2 coordinates Myosin-II dependent internalisation of the zebrafish neural plate.

Tissue internalisation is a key morphogenetic mechanism by which embryonic tissues generate complex internal organs and a number of studies of epithelia have outlined a general view of tissue internalisation. Here we have used quantitative live imaging and mutant analysis to determine whether similar mechanisms are responsible for internalisation in a tissue that apparently does not have a typical epithelial organisation – the zebrafish neural plate. We found that although zebrafish embryos begin neurulation without a conventional epithelium, medially located neural plate cells adopt strategies typical of epithelia in order to constrict their dorsal surface membrane during cell internalisation. Furthermore, we show that Myosin-II activity is a significant driver of this transient cell remodeling which also depends on Cdh2 (N-cadherin). Abrogation of Cdh2 results in defective Myosin-II distribution, mislocalised internalisation events and defective neural plate morphogenesis. Our work suggests Cdh2 coordinates Myosin-II dependent internalisation of the zebrafish neural plate.

Background During the initial stages zebrafish neurulation, neural plate cells undergo highly coordinated movements before they assemble into a multicellular solid neural rod. We have previously identified that the underlying mesoderm is critical to ensure such coordination and generate correct neural tube organization. However, how intertissue coordination is achieved in vivo during zebrafish neural tube morphogenesis is unknown. Results In this work, we use quantitative live imaging to study the coordinated movements of neural ectoderm and mesoderm during dorsal tissue convergence. We show the extracellular matrix components laminin and fibronectin that lie between mesoderm and neural plate act to couple the movements of neural plate and mesoderm during early stages of neurulation and to maintain the close apposition of these two tissues. Conclusions Our study highlights the importance of the extracellular matrix proteins laminin and libronectin in coupling the movements and spatial proximity of mesoderm and neuroectoderm during the morphogenetic movements of neurulation. Developmental Dynamics 245:580-589, 2016. © 2016 Wiley Periodicals, Inc.

SummaryWe use the transparency of zebrafish embryos to reveal thede novogeneration of a simple squamous epithelium and identify the cellular architecture in the epithelial transition zone that ties this squamous epithelium to the columnar neuroepithelium within the embryo’s brain. The simple squamous epithelium of the rhombencephalic roof plate is pioneered by distinct mesenchymal cells at the dorsal midline of the neural tube. Subsequently a progenitor zone is established at the interface between columnar epithelium of the rhombic lip and the expanding squamous epithelium of the roof plate. Surprisingly this interface consists of a single progenitor cell type that we have named the veil cell. Veil cells expressgdf6aand constitute a lineage restricted stem zone that generates the squamous roof plate by direct transformation and asymmetrically fated divisions. Experimental restriction of roof plate expansion leads to extrusion of veil cell daughters and squamous cells, suggesting veil cell fate is regulated by the space available for roof plate growth.

The molecular organization of the zebrafish brain and its relation to neuroanatomical divisions are still largely unknown. In this study we have analyzed the expression of a small transcript encoding for the IQ containing polypeptide Pcp4a in developing and juvenile zebrafish. The transcript is exclusively expressed in neural structures with a pattern that is highly specific for restricted domains and cell populations throughout development, and it allows us to follow the development of these structures at different times. The expression of pcp4a characterizes the dorsocaudal telencephalon, dorsal habenula, pretectal nuclei, preglomerular complex, mammillary bodies, and deep layers of the optic tectum and is a hallmark of a subpopulation of reticulospinal neurons. In the telencephalon, comparison of the expression of pcp4a with other pallial markers showed a rostrocaudal gradient in the expression of these genes, which suggests that the dorsal telencephalon of zebrafish may be organized in distinct areas with different molecular natures. Pcp4 has been involved in modulating calcium signals and in binding to calmodulin, but its precise role in neuronal functions is not known. The analysis of pcp4a expression and localization in the zebrafish brain suggests that pcp4a may be a useful marker for sensory and some motor neuronal circuitries and for telencephalic areas processing sensory inputs.

By using fluorescent tracers, we have investigated the origin of the cells that form the regenerating spinal cord after tail amputation in urodele amphibians. We show that spinal cord cells immediately adjacent to the amputation plane die and are removed by phagocytic cells. Spinal cells just anterior to these dying cells are destined to make the majority of the regenerating cord. The largest contribution is likely to come from the radial ependymal cells, but we also demonstrate that postmitotic neurons in this location can translocate into the regenerating cord. These neurons integrate into the regenerate structure and survive for at least 4 weeks. We find no evidence that these translocated neurons dedifferentiate and divide during this regeneration process. We discuss the possibility that these neurons survive long term in the regenerate cord and become part of the functional neuronal circuitry.

Fate mapping was once the province of classical experimental embryologists. Now a battery of new and sophisticated methods can be used to trace where cells go and what they do in embryos. Here we use examples from gastrulating fish and amphibian embryos and from the chick limb bud and central nervous system to show how this information has contributed to our understanding of developmental processes. This knowledge will become increasingly important in interpreting the complex patterns of gene expression that are being discovered during development, as well as in understanding the effects of genetic manipulations and in directing experimental interventions.

The organizer at the midbrain-hindbrain boundary (MHB organizer) has been proposed to induce and polarize the midbrain during development. We investigate the requirement for the MHB organizer in acerebellar mutants, which lack a MHB and cerebellum, but retain a tectum, and are mutant for fgf8, a candidate inducer and polarizer. We examine the retinotectal projection in the mutants to assay polarity in the tectum. In mutant tecta, retinal ganglion cell (RGC) axons form overlapping termination fields, especially in the ventral tectum, and along both the anterior-posterior and dorsal-ventral axis of the tectum, consistent with a MHB requirement in generating midbrain polarity. However, polarity is not completely lost in the mutant tecta, in spite of the absence of the MHB. Moreover, graded expression of the ephrin family ligand Ephrin-A5b is eliminated, whereas Ephrin-A2 and Ephrin-A5a expression is leveled in acerebellar mutant tecta, showing that ephrins are differentially affected by the absence of the MHB. Some RGC axons overshoot beyond the mutant tectum, suggesting that the MHB also serves a barrier function for axonal growth. By transplanting whole eye primordia, we show that mapping defects and overshooting largely, but not exclusively, depend on tectal, but not retinal genotype, and thus demonstrate an independent function for Fgf8 in retinal development. The MHB organizer, possibly via Fgf8 itself, is thus required for midbrain polarisation and for restricting axonal growth, but other cell populations may also influence midbrain polarity.

Responses to extracellular nucleotides (e.g., ATP, ADP, etc.) have been demonstrated in a number of embryonic cell types suggesting they may be important signalling molecules during embryonic development. Here the authors describe for the first time the expression of a G-protein–coupled receptor for extracellular ATP, chick P2Y1 (cP2Y1), during embryonic development of the chick. During the first 10 days of embryonic development, cP2Y1 is expressed in a developmentally regulated manner in the limb buds, mesonephros, brain, somites, and facial primordia, suggesting that this receptor may have a role in the development of each of these systems. Dev Dyn 1999;214:152–158. © 1999 Wiley-Liss, Inc.

The extent to which the spatial organisation of craniofacial development is due to intrinsic properties of the neural crest is at present unclear. There is some experimental evidence supporting the concept of a prepattern established within crest while contiguous with the neural plate. In experiments in which the neural tube and premigratory crest are relocated within the branchial region, crest cells retain patterns of gene expression appropriate for their position of origin after migration into the branchial arches, resulting in skeletal abnormalities. But in apparent conflict with these findings, when crest is rerouted by late deletion of adjacent crest, infilling crest alters its pattern of gene expression to match its new location, and a normal facial skeleton results. In order to reconcile these findings and thus identify processes of relevance to the course of normal development, we have performed a series of neural tube and crest rotations producing a more extensive reorganisation of cephalic crest than has been previously described. Lineage analysis using DiI labelling of crest derived from the rotated hindbrain reveals that crest does not migrate into the branchial arch it would have colonised in normal development, rather it simply populates the nearest available branchial arches. We also find that crest adjacent to the grafted region contributes to a greater number of branchial arches than it would in normal development, resulting in branchial arches containing mixed cell populations not occurring in normal development. We find that after exchange of first and third arch crest by rotation of r1–7, crest alters its expression ofhoxa-2andhoxa-3to match its new location within the embryo resulting in the reestablishment of the normal branchial arch Hox code. A facial skeleton in which all the normal components are present, with some additional ectopic first arch structures, is formed in this situation. In contrast, when second and third arch crest are exchanged by rotation of r3 to 7, ectopic Hox gene expression is stable, resulting in the persistence of an abnormal branchial arch Hox code and extensive defects in the hyoid skeleton. We suggest that the intrinsic properties of crest have an effect on the spatial organisation of structures derived from the branchial arches, but that exposure to increasingly novel environments within the branchial region or “community effects” within mixed populations of cells can result in alterations to crest Hox code and morphogenetic fate. In both classes of operation we find that there is a tight link between the resulting branchial arch Hox code and a particular skeletal morphology.

Developing facial primordia change shape substantially in stages leading up to primary palate formation. We investigated expansion of cell populations within each of the four facial primordia of chick embryos between HH-stages 20 and 28, by using DiI labelling. Populations of cells centred around the nasal pits in the upper face, the midline of the paired mandibular primordia in the lower face, and at sites of fusion contribute most to overall expansion. Abundant Msx-1 transcripts are found in regions of high expansion, and Fgf-8 transcripts are seen in ectoderm associated with some of these regions. Many cell populations display preferential expansion along one axis. Maxillary and mandibular primordia cell populations expand along the proximodistal axis, whereas at the distal tip of the frontonasal mass, cell populations expand mediolaterally. Thus outgrowth occurs at the tips of mandibular and maxillary primordia, but at the base of the frontonasal mass. At regions where adjacent primordia abut each other, we found bidirectional movement of cells between primordia, unidirectional movement or could detect no movement at all. Regions of highest expansion in each primordium have the highest percentage of S phase labelled cells. Cell death occurs in some regions of low expansion but it seems likely that cell rearrangements and intercalations also contribute to shaping. These rearrangements could be associated with stretching of the primordia by neighbouring tissues. Treatment of chick embryos with retinoic acid causes clefts of the primary palate (Tamarin et al. [1984] J. Embryol. Exp. Morphol. 84:105-123). We found a decrease in expansion of cell populations that normally contribute to primary palate formation but surprisingly little ectopic cell death. Expansion of other cell populations in the treated upper face was more even rather than directed. This further supports the idea that tension exerted by neighbouring tissues plays a major role in global shaping of the upper face.

We exposed st.10 chicks to retinoic acid (RA), both globally, and locally to individual rhombomeres, to look at its role in specification of various aspects of hindbrain derived morphology. Previous studies have looked at RA exposure at earlier stages, during axial specification. Stage 10 is the time of morphological segmentation of the hindbrain and is just prior to neural crest migration. Rhombomere 4 localised RA injections result in specific alterations of pathways some crest cells that normally migrate to sites of differentiation of neurogenic derivatives. The r4 crest cells that give rise to mesenchymal derivatives are unaffected. In addition, r4 gene expression is also partially altered by RA; within 6 hours of r4 exposure to RA, ectopic expression of Krox-20 is seen in r4 and Hoxb-1 expression is lost while Hoxa-2 expression continues normally. When we examined these RA-treated animals later in development, they showed an anterior displacement of the facial ganglion in addition to a mis-direction of the extensions of its distal axons and a dramatic decrease in the number of contralateral vestibuloacoustic neurons normally seen in r4. Only this r4-specific neuronal type is affected in r4; the motor neuron projections seem normal in experimental animals. The specificity of this result, combined with the loss of Hoxb-1 expression in r4 and the work by Krumlauf and co-workers showing gain of contralateral neurons co-localised with ectopic Hoxb-1 expression, indicates a role for Hoxb-1 and RA in the specification of this cell type in normal development. These results suggest that RA, at st.10, is able to affect some aspects of segment identity while leaving others unchanged.

Considerable evidence has demonstrated that retinoic acid influences the formation of the primary body axis in vertebrates and that this may occur through the regulation of Hox gene expression. In this study, we show that the phenotype induced by exogenous retinoic acid in the zebrafish can also be generated by the overexpression of Hoxa-1 following injection of synthetic RNA into the fertilised egg. The isolation, sequence and expression pattern of the zebrafish Hoxa-1 gene is described. We show that exogenously applied retinoic acid causes the ectopic accumulation of Hoxa-1 message during gastrulation in the hypoblast in the head region. Overexpression of Hoxa-1 following injection of RNA causes abnormal growth of the anterior hindbrain, duplication of Mauthner neurons in rhombomere (r) 2 and fate changes of r2 mesenchymal and neurogenic neural crest. These results are discussed in terms of the role of Hoxa-1 in controlling anterior hindbrain patterning and the relationship between expression of Hoxa-1 and retinoic acid.

By analysing the cellular and subcellular events that occur in the centre of the developing zebrafish neural rod, we have uncovered a novel mechanism of cell polarisation during lumen formation. Cells from each side of the neural rod interdigitate across the tissue midline. This is necessary for localisation of apical junctional proteins to the region where cells intersect the tissue midline. Cells assemble a mirror-symmetric microtubule cytoskeleton around the tissue midline, which is necessary for the trafficking of proteins required for normal lumen formation, such as partitioning defective 3 and Rab11a to this point. This occurs in advance and is independent of the midline cell division that has been shown to have a powerful role in lumen organisation. To our knowledge, this is the first example of the initiation of apical polarisation part way along the length of a cell, rather than at a cell extremity. Although the midline division is not necessary for apical polarisation, it confers a morphogenetic advantage by efficiently eliminating cellular processes that would otherwise bridge the developing lumen.

Dystrophin is normally expressed in a number of tissues including muscle, brain and the outer plexiform layer of the retina. In Duchenne and Becker muscular dystrophy abnormal or deficient dystrophin expression leads to muscle degeneration and has been implicated in mental retardation and a form of night blindness. We have examined the expression of dystrophin immunoreactivity in cochlear tissues of normal guinea-pig and mouse, and whether expression is perturbed in the cochlea of the dystrophic MDX mouse. A single band of approximately 427 kDa, corresponding to a full-length isoform of dystrophin was detected in guinea-pig and normal mouse but was absent from the MDX mouse. Cochleae from guinea-pig, normal and MDX mouse also showed a second dystrophin isoform of 116 kDa molecular weight with the C-terminal specific antibody. Immunostained guinea pig cochlear half turns were examined by laser scanning confocal microscopy. Dystrophin was localized in both inner and outer hair cells with staining patterns which were qualitatively similar with both antibodies. In the outer hair cells labelling of the lateral wall was especially distinctive. The synaptic region of both hair cell types was also strongly labelled.

Exogenously applied retinoic acid given at the early stages of gastrulation causes abnormal development of the caudal midbrain and anterior hindbrain in vertebrate embryos. We describe the limits of the brain regions that are affected using neuroanatomical criteria in the zebrafish embryo. Analysis of the reticulospinal complex shows that the Mauthner cell, which normally differentiates in rhombomere 4, is duplicated either in this rhombomere or in rhombomere 2. Using probes for zebrafish krx20 and pax2, it is demonstrated that retinoic acid affects the expression domains of these regulatory genes in a manner that is consistent with the neuroanatomical data. Expression of the goosecoid gene, which expressed in the prospective anterior mesoderm from the onset of gastrulation, is unaffected by the doses of retinoic acid used in this study, reflecting the normal development of the anterior end of the embryo.

Most of the mammalian heart is formed from mesodermal progenitors in the first and second heart fields (FHF and SHF), whereby the FHF gives rise to the left ventricle and parts of the atria and the SHF to the right ventricle, outflow tract and parts of the atria. Whereas SHF progenitors have been characterized in detail, using specific molecular markers, comprehensive studies on the FHF have been hampered by the lack of exclusive markers. Here, we present Hcn4 (hyperpolarization-activated cyclic nucleotide-gated channel 4) as an FHF marker. Lineage-traced Hcn4+/FHF cells delineate FHF-derived structures in the heart and primarily contribute to cardiomyogenic cell lineages, thereby identifying an early cardiomyogenic progenitor pool. As a surface marker, HCN4 also allowed the isolation of cardiomyogenic Hcn4+/FHF progenitors from human embryonic stem cells. We conclude that a primary purpose of the FHF is to generate cardiac muscle and support the contractile activity of the primitive heart tube, whereas SHF-derived progenitors contribute to heart cell lineage diversification.

The mechanisms that generate diverse neuronal phenotypes within the central nervous system are thought to involve local cues or cell-cell interactions acting late in neurogenesis, perhaps as late as the last precursor cell division. We describe here a clonal analysis of neuronal development in the chick hindbrain, using an intracellular tracer to mark single precursor cells, that suggests the operation of an alternative strategy. The majority of clones, ranging from 1 to 46 cells, contained neurons of only one of several possible phenotypes. These single-phenotype clones were not positionally restricted within a rhombomere but were interspersed with other clones containing distinct phenotypes. The assignment of neuronal phenotype in this brain region may, therefore, be made in early precursors and remembered through several rounds of mitotic expansion and dispersal.

The neurons within the segmented hindbrain of the early chick embryo have been mapped with the neuronal tracers HRP and fluorescent lysinated dextran. We have categorised neurons according to their axonal pathways and have then compared rhombomeres with respect to the number and class of neurons present. The results indicate that most rhombomeres are similar in that they contain the same set of basic neuronal types but differ in that particular neuronal types are more abundant in some rhombomeres than others. The data support the concept that the hindbrain develops according to ‘variations on a segmental theme’ rather than ‘each segment is unique’. Many of the cell types occupy distinct mediolateral domains that are probably established by both the differential migration of some neuronal classes and the spatial segregation of distinct precursors. The caudal rhombomeres 7 and 8 are exceptional in that they do not have the full set of basic neuronal types and also contain two additional medial cell types that are not present rostrally. The mechanisms that may generate the regional diversity apparent in the more mature hindbrain are discussed.

A method is described that allows the introduction by electroporation of either small dyes or larger RNA, DNA, or morpholino constructs into single cells or small groups of cells in zebrafish embryos or larvae. The dye or construct is delivered to cells via a patch-like microelectrode that also delivers the electroporation stimulus train. This technique allows the experimenter to target cells of their choice at a particular time of development and at a particular location in the embryo, and is useful for fate mapping, analysing neuronal organisation, ectopic expression and gene knockdown experiments.

In the developing neural tube of the zebrafish, a newly discovered kind of cell division occurs that produces two daughter cells with mirror symmetry. Cells are polarized in mirror symmetry on either side of the neural midline, and Pard3, a protein known to be involved in polarized cell divisions, is deposited within the cleavage furrow of the midline. The development of cell polarity is an essential prerequisite for tissue morphogenesis during embryogenesis, particularly in the development of epithelia1,2. In addition, oriented cell division can have a powerful influence on tissue morphogenesis3. Here we identify a novel mode of polarized cell division that generates pairs of neural progenitors with mirror-symmetric polarity in the developing zebrafish neural tube and has dramatic consequences for the organization of embryonic tissue. We show that during neural rod formation the polarity protein Pard3 is localized to the cleavage furrow of dividing progenitors, and then mirror-symmetrically inherited by the two daughter cells. This allows the daughter cells to integrate into opposite sides of the developing neural tube. Furthermore, these mirror-symmetric divisions have powerful morphogenetic influence: when forced to occur in ectopic locations during neurulation, they orchestrate the development of mirror-image pattern formation and the consequent generation of ectopic neural tubes.

During early formation of the eye, the optic vesicle becomes partitioned into a proximal domain that forms the optic nerve and a distal domain that forms the retina. In this study, we investigate the activity of Nodal,Hedgehog (Hh) and Fgf signals and Vax family homeodomain proteins in this patterning event. We show that zebrafish vax1 and vax2 are expressed in overlapping domains encompassing the ventral retina, optic stalks and preoptic area. Abrogation of Vax1 and Vax2 activity leads to a failure to close the choroid fissure and progressive expansion of retinal tissue into the optic nerve, finally resulting in a fusion of retinal neurons and pigment epithelium with forebrain tissue.We show that Hh signals acting through Smoothened act downstream of the Nodal pathway to promote Vax gene expression. However, in the absence of both Nodal and Hh signals, Vax genes are expressed revealing that other signals,which we show include Fgfs, contribute to Vax gene regulation. Finally, we show that Pax2.1 and Vax1/Vax2 are likely to act in parallel downstream of Hh activity and that the bel locus (yet to be cloned) mediates the ability of Hh-, and perhaps Fgf-, signals to induce Vax expression in the preoptic area. Taking all these results together, we present a model of the partitioning of the optic vesicle along its proximo-distal axis.

Various studies have suggested that the rate of adult skin healing may be in some way dependent on signals emanating from cutaneous nerves. Further, it appears that adult wounds become hyperinnervated by sensory nerves during the process of healing. In order to investigate this reciprocal relationship further, we have used a simple embryonic model to look at the effect of wounds on nerves, and conversely, the effect of nerves on wounds. We find that wounds made to the dorsum of the chick wing bud, at a stage prior to normal innervation (at E4), or soon after the normal establishment of cutaneous innervation (at E7), subtly alter the pattern of branching by perturbing developmental guidance cues, but do not cause hyperinnervation, whereas wounding at E14 does cause hyperinnervation. By creating chicks with nerveless wings, we show that from E7, wound healing in the absence of nerves is significantly impaired. These observations suggest that, from the earliest stages of skin innervation, the presence of nerves is beneficial to the healing process, but that, in contrast to neonatal and adult tissues, wound healing in the embryo and early foetus does not trigger hyperinnervation.

We have investigated the function of the G protein-coupled receptor for extracellular ATP, chick P2Y1 (cP2Y1) during development of the chick limb. cP2Y1 is strongly expressed in undifferentiated limb mesenchyme cells but appears to be lost from cells as they differentiate, raising the possibility that the function of this receptor may be to inhibit cell differentiation. This pattern of expression was particularly striking surrounding areas of cartilage formation. We tested whether cP2Y1 was able to regulate cartilage formation by using an in-vitro micromass model of chondrogenesis. Because limb cells in micromass culture lose expression of cP2Y1, we have used a gain-of-function approach to demonstrate that cP2Y1 expression can inhibit cartilage differentiation. We also demonstrate that early limb mesenchyme cells release ATP into the extracellular medium and have mechanisms to breakdown extracellular ATP. These results suggest that extracellular ATP, signaling through cP2Y1, can modulate the differentiation of limb mesenchyme cells in vitro, and the expression pattern of cP2Y1 suggests that this type of signaling could play a similar role in ovo. © 2001 Wiley-Liss, Inc.

During tail regeneration in urodele amphibians such as axolotls, all of the tissue types, including muscle, dermis, spinal cord, and cartilage, are regenerated. It is not known how this diversity of cell types is reformed with such precision. In particular, the number and variety of mature cell types in the remaining stump that contribute to the blastema is unclear. Using Nomarski imaging, we followed the process of regeneration in the larval axolotl tail. Combining this with in vivo fluorescent labeling of single muscle fibers, we show that mature muscle dedifferentiates. Muscle dedifferentiation occurs by the synchronous fragmentation of the multinucleate muscle fiber into mononucleate cells followed by rapid cell proliferation and the extension of cell processes. We further show that direct clipping of the muscle fiber and severe tissue damage around the fiber are both required to initiate dedifferentiation. Our observations also make it possible to estimate for the first time how many of the blastema cells arise specifically from muscle dedifferentiation. Calculations based on our data suggest that up to 29% of nondermal-derived cells in the blastema come from dedifferentiation of mature muscle fibers. Overall, these results show that endogenous multinucleate muscle fibers can dedifferentiate into mononucleate cells and contribute significantly to the blastema.

Ventral midline cells in the neural tube have distinct properties at different rostrocaudal levels, apparently in response to differential signalling by axial mesoderm. Floor plate cells are induced by sonic hedgehog (SHH) secreted from the notochord whereas ventral midline cells of the rostral diencephalon (RDVM cells) appear to be induced by the dual actions of SHH and bone morphogenetic protein 7 (BMP7) from prechordal mesoderm. We have examined the cellular and molecular events that govern the program of differentiation of RDVM cells under the influence of the axial mesoderm. By fate mapping, we show that prospective RDVM cells migrate rostrally within the neural plate, passing over rostral notochord before establishing register with prechordal mesoderm at stage 7. Despite the co-expression of SHH and BMP7 by rostral notochord, prospective RDVM cells appear to be specified initially as caudal ventral midline neurectodermal cells and to acquire RDVM properties only at stage 7. We provide evidence that the signalling properties of axial mesoderm over this period are regulated by the BMP antagonist, chordin. Chordin is expressed throughout the axial mesoderm as it extends, but is downregulated in prechordal mesoderm coincident with the onset of RDVM cell differentiation. Addition of chordin to conjugate explant cultures of prechordal mesoderm and neural tissue prevents the rostralization of ventral midline cells by prechordal mesoderm. Chordin may thus act to refine the patterning of the ventral midline along the rostrocaudal axis.

Clonal analysis of the chick embryo hindbrain has shown that during the first 48 hr of neurogenesis the large majority of neural progenitor cells generate clones of neurons of only a single major phenotype or of only closely related phenotypes. This is despite considerable spatial intermixing of diverse neuronal phenotypes at these stages of development and suggests that phenotype may be decided early in mitotic precursors and remembered through several subsequent rounds of division and dispersal (Lumsden et al. [1994] Development 120:1581-1589). Here we have used fate-mapping and clonal analysis to study neuroepithelial cell dispersal and mixing in the early hindbrain and discuss this data in relation to the generation of single phenotype neuronal clones. We find that dispersal is not uniform throughout the dorsoventral axis of the neural tube, but is highly dependent on position along that axis. Neuronal identity is related to the spatial origin and, hence, environment of the cell, and the spatial intermixing of diverse neuronal phenotypes at HH stage 20 is largely the result of circumferential neuronal migration as medially born branchial motor neurons migrate laterally while the more laterally born mlf neurons migrate medially. Constraints on the dispersal of clonally related progenitors, in particular those that lie adjacent to the floor plate, may serve to restrict the fate of these cells to the generation of only one major neuronal phenotype, i.e., motor neurons.

The early development of the vertebrate head is dependent on the formation of two series of segmented structures, the rhombomeres of the hindbrain and the branchial arch series. The initial formation of these two systems is closely linked, as the principal source of branchial arch mesenchyme is the neural crest, which derives from the lateral edge of the neural plate at the time of rhombomere formation. The subsequent development of the two systems maintains a close level of integration, as specific spatial relationships between skeletal, muscle and neural elements arising from the same axial level are established. Given the level of conservation of these anatomical relationships in vertebrates, it is likely that they are a reflection of a key mechanism in early facial and pharyngeal development. One model, in part based on these findings, proposed that the neural crest acquires an axial-level specific combination of gene expression while part of the neural plate. This prepattern is then maintained throughout the crest's subsequent development. In the model, this combination of gene expression would then specify the form of the facial and pharyngeal structures that the crest would give rise to. In this review we evaluate recent evidence on whether early facial development involves a crest prespecification of this type, and conclude that it is not the case.

We wish to understand how limbs are positioned with respect to the dorso-ventral axis of the body in vertebrate embryos, and how different regions of limb bud ectoderm, i.e. dorsal ectoderm, apical ridge and ventral ectoderm, originate. Signals from dorsal and ventral ectoderm control dorso-ventral patterning while the apical ectodermal ridge (AER) controls bud outgrowth and patterning along the proximo-distal axis. We show, using cell-fate tracers, the existence of two distinct ectodermal compartments, dorsal versus ventral, in both presumptive limb and flank of early chick embryos. This organisation of limb ectoderm is the first direct evidence, in vertebrates, of compartments in non-neural ectoderm. Since the apical ridge appears to be confined to this compartment boundary, this positions the limb. The mesoderm, unlike the ectoderm, does not contain two separate dorsal and ventral cell lineages, suggesting that dorsal and ventral ectoderm compartments may be important to ensure appropriate control of mesodermal cell fate. Surprisingly, we also show that cells which form the apical ridge are initially scattered in a wide region of early ectoderm and that both dorsal and ventral ectoderm cells contribute to the apical ridge, intermingling to some extent within it.

Anteroposterior polarity in the vertebrate limb is thought to be regulated in response to signals derived from a specialized region of distal posterior mesenchyme, the zone of polarizing activity. Sonic Hedgehog (Shh) is expressed in the zone of polarizing activity and appears to mediate the action of the zone of polarizing activity. Here we have manipulated Shh signal in the limb to assess whether it acts as a longrange signal to directly pattern all the digits. Firstly, we demonstrate that alterations in digit development are dependent upon the dose of Shh applied. DiI-labeling experiments indicate that cells giving rise to the extra digits lie within a 300 μm radius of a Shh bead and that the most posterior digits come from cells that lie very close to the bead. A response to Shh involves a 12-16 hour period in which no irreversible changes in digit pattern occur. Increasing the time of exposure to Shh leads to specification of additional digits, firstly digit 2, then 3, then 4. Cell marking experiments demonstrate that cells giving rise to posterior digits are first specified as anterior digits and later adopt a more posterior character. To monitor the direct range of Shh signalling, we developed sensitive assays for localizing Shh by attaching alkaline phosphatase to Shh and introducing cells expressing these forms into the limb bud. These experiments demonstrate that long-range diffusion across the anteroposterior axis of the limb is possible. However, despite a dramatic difference in their diffusibility in the limb mesenchyme, the two forms of alkaline phosphatase-tagged Shh proteins share similar polarizing activity. Moreover, Shh-N (aminoterminal peptide of Shh)-coated beads and Shh-expressing cells also exhibit similar patterning activity despite a significant difference in the diffusibility of Shh from these two sources. Finally, we demonstrate that when Shh-N is attached to an integral membrane protein, cells transfected with this anchored signal also induce mirror-image pattern duplications in a dose-dependent fashion similar to the zone of polarizing activity itself. These data suggest that it is unlikely that Shh itself signals digit formation at a distance. Beads soaked in Shh-N do not induce Shh in anterior limb mesenchyme ruling out direct propagation of a Shh signal. However, Shh induces dose-dependent expression of Bmp genes in anterior mesenchyme at the start of the promotion phase. Taken together, these results argue that the dose-dependent effects of Shh in the regulation of anteroposterior pattern in the limb may be mediated by some other signal(s). BMPs are plausible candidates.

Rhombomeres are segmental units of the hindbrain that are separated from each other by a specialised zone of boundary cells. Retinoic acid application to a recently segmented hindbrain leads to disappearance of posterior rhombomere boundaries. Boundary loss is preceded by changes in segmental expression of Krox-20 and Cek-8 and followed by alterations in Hox gene expression. The characteristic morphology of boundary cells, their expression of follistatin and the periodic accumulation of axons normally associated with boundaries are all lost. In the absence of boundaries, we detect no change in anteroposterior dispersal of precursor cells and, in most cases, no substantial cell mixing between former rhombomeric units. This is consistent with the idea that lineage restriction can be maintained by processes other than a mechanical barrier composed of boundary cells. Much of the early organisation of the motor nuclei appears normal despite the loss of boundaries and altered Hox expression.

Retinoic acid is a very potent teratogen and has also been implicated as an endogenous developmental signalling molecule in vertebrate embryos. One of the regions of the embryo reliably affected by exogenously applied RA is the hindbrain. In this paper, we describe in detail the hindbrain of Xenopus laevis embryos briefly treated with various levels of RA at gastrula stages. Such treatments lead to development of embryos with loss of anterior structures. In addition, RA has a general effect on rhombomere morphology and specific effects on the development of the anterior rhombomeres. This effect is demonstrated using neurofilament antibodies, HRP staining and in situ hybridisation using a probe for expression of the Xenopus Krox-20 gene. Anatomically it is evident that the development of the hindbrain normally anterior to the otocyst (rhombomeres 1-4) is abnormal following RA treatment. Sensory and motor axons of cranial nerves V and Vll form a single root and the peripheral paths of V and VH and IX and X are also abnormal, as is the more anterior location of the otocyst. These anatomical changes are accompanied by changes in the pattern of expression for the gene XKrox-20, which normally expresses in rhombomeres 3 and 5, but is found in a single band in the anterior hindbrain of treated embryos which standardly fail to generate the normal external segmental appearance. The results are discussed in terms of both the teratogenic and possible endogenous roles of RA during normal development of the central nervous system. We conclude that low doses of RA applied during gastrulation have specific effects on the anterior Xenopus hindbrain which appear to be evolutionarily conserved in the light of similar recent findings in zebrafish.

Notochordless Xenopus embryos were produced by u.v. irradiation of the uncleaved fertilized egg. The spinal cords were examined using intermediate filament staining for glial cells, retrograde HRP staining for neuronal morphology and an anti-glycinergic antibody to reveal commissural cells and axons. The floorplate cells of the normal cord appear to be absent and their position along the ventral midline of the cord is occupied by motor neurones, Kolmer-Agduhr cells, radial glial cells and a ventrally placed marginal zone containing the longitudinal axons. Motor neurone number is reduced to 15 % of control values, and the sensory extramedullary cell number is increased twentyfold. Commissural axons are still able to cross the ventral cord but do so at abnormal angles and some commissural axons continue to grow circumferentially up the contralateral side of the cord rather than turning to grow longitudinally. Extracellular electrophysiological recordings from motor axons reveal that the normal alternation of locomotor activity on the left and right side of the embryo is lost in notochordless animals. These results suggest that the notochord and/or the normal floor plate structure are important for the development of the laterality of spinal cord connections and may influence motor neurone proliferation or differentiation.

During growth of the axolotl, motor neurons, and muscle fibres are added to the motor system. By double labelling neurons with tritiated thymidine and retrogradely transported HRP, we show that some motor neurons are born at postembryonic stages. Further analysis of motor neurons with the aid of HRP reveals this population of newly born cells relatively frequently in small (5-7 cm long) axolotls, but only rarely in large (7-13 cm long) axolotls. Evidence is presented that suggests that these immature cells are in the process of migrating from close to the ependyma out to the ventral horn. HRP transport also reveals growth cones of advancing axons within spinal nerves in animals up to 6 cm in length. Cell counts by light and electron microscopic methods show that muscle fibres are generated throughout larval life in the iliotibialis, a typical limb muscle. This analysis provides data consistent with the notion that new muscle fibres are added from a localised growth zone situated at the superficial edge of the muscle. These results are discussed in terms of the correlation between continuous growth of the motor system and the ability of the axolotl to functionally repair lesions to the peripheral nervous system.

Bio-electrosprays are rapidly emerging as a viable protocol for directly engineering living cells. This communication reports the bio-electrospraying of multicellular organisms, namely zebrafish embryos. The results demonstrate that the bio-electrospray protocol fails to induce any embryological perturbations. In addition to analysing overall embryo morphology, we use transgenic embryos that express green fluorescent protein in specific brain neurons to determine that neuronal numbers and organization are completely normal. These results demonstrate that the bio-electrospraying protocol does not interfere with the complex gene regulation and cell movements required for the development of a multicellular organism.

Horseradish peroxidase was used to describe the positions and approximate numbers of neurones with axons that descend to the lumbar spinal cord in normal axolotls and axolotls whose spinal cord had been transected 3–23 months previously. Three to 4 months after the transection approximately 10% of the axons had grown across the cut and returned to the lumbar spinal cord whereas 23 months after the transection the number and distribution of these cells were approaching those of the controls.

Directed axon regeneration following damage to the central or peripheral nervous system is possible in several vertebrates (Table 1). In most cases, the regenerating axons can be shown to be part of the nervous system displaying some aspect of neuronal development, such as the neurogenesis associated with larval or neonatal development or the more unusual event of neurogenesis and axon outgrowth in adult tissues. This correlation may offer some clues as to why successful axon regeneration occurs in some systems but not in others.

We have produced detailed fate maps for mesenchyme and apical ridge of a stage 20 chick wing bud. The fate maps of the mesenchyme show that most of the wing arises from the posterior half of the bud. Subapical mesenchyme gives rise to digits. Cell populations beneath the ridge in the mid apical region fan out into the anterior tip of the handplate, while posterior cell populations extend right along the posterior margin. Subapical mesenchyme of the leg bud behaves similarly. The absence of anterior bending of posterior cell populations has implications when considering models of vertebrate limb evolution. The fatemaps of the apical ridge show that there is also a marked anterior expansion and cells that were in anterior apical ridge later become incorporated into non-ridge ectoderm along the margin of the bud. Mesenchyme and apical ridge do not expand in concert - the apical ridge extends more anteriorly. We used the fatemaps to investigate the relation-ship between cell lineage and elaboration of Hoxd-13 and Fgf-4 domains. Hoxd-13 and Fgf-4 are initially expressed posteriorly until about the mid-point of the early wing bud in mesenchyme and apical ridge respectively. Later in development, the genes come to be expressed throughout most of the handplate and apical ridge respectively. We found that at the proximal edge of the Hoxd-13 domain, cell populations stopped expressing the gene as development proceeded and found no evidence that the changes in extent of the domains were due to initiation of gene expression in anterior cells. Instead the changes in extent of expression fit with the fate maps and can be attributed to expansion and fanning out of cell populations initially expressing the genes.