Experimental analyses of the rearrangement of ectodermal cells during gastrulation and neurulation in avian embryos

The rearrangement of ectodermal cells was studied in chimeras in which grafts were transplanted during late gastrula and early neurula stages to heterotopic locations in avian embryos. Three types of experiments were done. In all experiments, Hensen's node was extirpated completely and replaced with an epithelial plug derived from 1 of 3 regions of the prospective ectoderm. In type-1 experiments, Hensen's node was replaced with a plug consisting of precursor cells of the floor plate of the neural tube. In type-2 experiments, Hensen's node was replaced with a plug consisting of precursor cells of the lateral wall of the neural tube. In type-3 experiments, Hensen's node was replaced with a plug consisting of precursor cells of the epidermal ectoderm. In all experiments, the amount and direction of cell rearrangement that occurred in the transplanted ectodermal plug was essentially typical for prospective ectodermal cells normally residing within Hensen's node. That is, transplanted ectodermal cells underwent lateralto-medial cell-cell intercalation and contributed to the ventral midline of the neural tube along its entire rostrocaudal extent. In most embryos, a notochord was reconstituted from host cells, despite the fact that Hensen's node — the prime source of prospective notochordal cells in intact embryos — was extirpated completely; however, a few embryos had long notochordal gaps. In such essentially notochordless embryos, the ventral midline of the neural tube still derived from grafted cells, but it failed to form a floor plate, providing further confirmation of the results of several previous studies that the notochord is required to induce the floor plate. Collectively, our results provide evidence that the rearrangement of ectodermal cells does not require the presence of a trail of prospective floor plate cells (laid down by the regressing Hensen's node), or of a notochordal substrate, and that the continued presence of an organizer per se, ostensibly Hensen's node, is not required. In addition, our results demonstrate that the rearrangement of cells still occurs in the absence of boundaries between ectodermal cells of different phenotypes (e.g., between cells of the floor plate and lateral walls of the neural tube). Finally, our results reveal further that the amount and direction of cellular rearrangement is not regulated in a cell-autonomous fashion, but rather it is determined by the overall magnitude and vector of the displacement of the community of rearranging cells within a developmental field.     

3D reconstructions of quail–chick chimeras provide a new fate map of the avian scapula

Limbed vertebrates have functionally integrated postcranial axial and appendicular systems derived from two distinct populations of embryonic mesoderm. The axial skeletal elements arise from the paraxial somites, the appendicular skeleton and sternum arise from the somatic lateral plate mesoderm, and all of the muscles for both systems arise from the somites. Recent studies in amniotes demonstrate that the scapula has a mixed mesodermal origin. Here we determine the relative contribution of somitic and lateral plate mesoderm to the avian scapula from quail-chick chimeras. We generate 3D reconstructions of the grafted tissue in the host revealing a very different distribution of somitic cells in the scapula than previously reported. This novel 3D visualization of the cryptic border between somitic and lateral plate populations reveals the dynamics of musculoskeletal morphogenesis and demonstrates the importance of 3D visualization of chimera data. Reconstructions of chimeras make clear three significant contrasts with existing models of scapular development. First, the majority of the avian scapula is lateral plate derived and the somitic contribution to the scapular blade is significantly smaller than in previous models. Second, the segmentation of the somitic component of the blade is partially lost; and third, there are striking differences in growth rates between different tissues derived from the same somites that contribute to the structures of the cervical thoracic transition, including the scapula. These data call for the reassessment of theories on the development, homology, and evolution of the vertebrate scapula.     

Developmental Morphology of the Head Mesoderm and Reevaluation of Segmental Theories of the Vertebrate Head: Evidence from Embryos of an Agnathan Vertebrate, Lampetra japonica

Due to the peculiar morphology of its preotic head, lampreys have long been treated as an intermediate animal which links amphioxus and gnathostomes. To reevaluate the segmental theory of classical comparative embryology, mesodermal development was observed in embryos of a lamprey, Lampetra japonica, by scanning electron microscopy and immunohistochemistry. Signs of segmentation are visible in future postotic somites at an early neurula stage, whereas the rostral mesoderm is unsegmented and rostromedially confluent with the prechordal plate. The premandibular and mandibular mesoderm develop from the prechordal plate in a caudal to rostral direction and can be called the preaxial mesoderm as opposed to the caudally developing gastral mesoderm. With the exception of the premandibular mesoderm, the head mesodermal sheet is secondarily regionalized by the otocyst and pharyngeal pouches into the mandibular mesoderm, hyoid mesoderm, and somite 0. The head mesodermal components never develop into cephalic myotomes, but the latter develop only from postotic somites. These results show that the lamprey embryo shows a typical vertebrate phylotype and that the basic mesodermal configuration of vertebrates already existed prior to the split of agnatha-gnathostomata; lamprey does not represent an intermediate state between amphioxus and gnathostomes. Unlike interpretations of theories of head segmentation that the mesodermal segments are primarily equivalent along the axis, there is no evidence in vertebrate embryos for the presence of preotic myotomes. We conclude that mesomere-based theories of head metamerism are inappropriate and that the formulated vertebrate head should possess the distinction between primarily unsegmented head mesoderm which includes preaxial components at least in part and somites in the trunk which are shared in all the known vertebrate embryos as the vertebrate phylotype.     

Macrophages of hemangioblastic lineage invade the lens vesicle-ectoderm interspace during closure and detachment of the avian embryonic lens

Summary An area of cell death is apparent in the lens vesicle margin and the lens stalk during closure and detachment of the lens anlage from the cephalic ectoderm. Free phagocytic cells closely associated with this area of cell death have been interpreted as cells migrating from the lens epithelium. Scanning and transmission electron microscopy, light-microscopic histochemical staining for acid phosphatase and immunostaining using MB1 (a monoclonal antibody specific for quail endothelial and hemopoietic cells) of chimeras of chick embryo and quail yolk sac were used to analyze these lens vesicle-associated free phagocytic cells. The cells have morphological features identical to those of macrophages in other embryonic tissues. In contrast to epithelial cells phagocytosing cell debris, they exhibit strong acid phosphatase activity, a feature typical of macrophages. In addition, free phagocytic cells are MB1 positive in chick embryo-quail yolk sac chimeras, hence they proceed from cells of hemangioblastic lineage originating in the yolk sac. These results indicate that the lens vesicle-associated free phagocytic cells are macrophages. Observations of MB1 positive amoeboid cells in the juxta-retinal mesenchyme and on the borders of the optic cup suggest that these macrophages migrate through the mesenchyme surrounding the eye primordium. Macrophages are seen in both the interspace between lens vesicle and ectoderm and in the lumen of the lens as well as within both the ectoderm and the lens epithelium. In these locations they remove cell debris, and thereby contribute to the complete disappearance of the area of cell death. Macrophages remain in the lens vesicle-ectoderm interspace until developmental stages at which it is invaded by corneal endothelial cells.     

Loss of TJP1 disrupts gastrulation patterning and increases differentiation toward the germ cell lineage in human pluripotent stem cells

1. Download : Download high-res image (185KB)
2. Download : Download full-size image

     

Tangential migration of glutamatergic neurons and cortical patterning during development: Lessons from Cajal‐Retzius cells

Tangential migration is a mode of cell movement, which in the developing cerebral cortex, is defined by displacement parallel to the ventricular surface and orthogonal to the radial glial fibers. This mode of long‐range migration is a strategy by which distinct neuronal classes generated from spatially and molecularly distinct origins can integrate to form appropriate neural circuits within the cortical plate. While it was previously believed that only GABAergic cortical interneurons migrate tangentially from their origins in the subpallial ganglionic eminences to integrate in the cortical plate, it is now known that transient populations of glutamatergic neurons also adopt this mode of migration. These include Cajal‐Retzius cells (CRs), subplate neurons (SPs), and cortical plate transient neurons (CPTs), which have crucial roles in orchestrating the radial and tangential development of the embryonic cerebral cortex in a noncell‐autonomous manner. While CRs have been extensively studied, it is only in the last decade that the molecular mechanisms governing their tangential migration have begun to be elucidated. To date, the mechanisms of SPs and CPTs tangential migration remain unknown. We therefore review the known signaling pathways, which regulate parameters of CRs migration including their motility, contact‐redistribution and adhesion to the pial surface, and discuss this in the context of how CR migration may regulate their signaling activity in a spatial and temporal manner. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 847–881, 2016     

Molecular effects of lithium exposure during mouse and chick gastrulation and subsequent valve dysmorphogenesis

BACKGROUND: Lithium (Li) has been associated with cardiac teratogenicity in the developing fetus. We took advantage of the association of therapeutic administration of Li with an increase in heart defects to gain insight into both normal and pathological heart and valve development with GSK‐3 inhibition. The objective of this study was to define whether Li mimicry of canonical Wnt/β‐catenin signaling induces cardiac valve defects. METHODS: Li was administered by a single intraperitoneal injection to the pregnant mouse on embryonic day E6.75, much earlier than heretofore analyzed. On E15.5 developing heart defects were defined by Doppler ultrasound. The embryonic hearts were analyzed for changes in patterning of active canonical Wnt expression and nuclear factor of the activated T cells‐c1 (NFATc1), both key regulators of valve development. Li‐exposed chick embryos were used to define the early cell populations during gastrulation that are susceptible to GSK‐3 inhibition and may relate to valve formation. RESULTS: Li exposure during gastrulation decreased the number of prechordal plate (PP) cells that reached the anterior intestinal portal, a region associated with valve development. Li decreased expression of Hex, an endoderm cardiac inducing molecule, normally also expressed by the PP cells, and of Sox 4 at the anterior intestinal portal and NFAT, critical factors in valvulogenesis. CONCLUSIONS: Cells existing already during gastrulation are associated with valve formation days later. The Wnt/β‐catenin signaling in PP cells is normally repressed by Wnt antagonists and Hex is up‐regulated. The antagonism occurring at the receptor level is bypassed by Li exposure by its intracellular inactivation of GSK‐3 directly to augment Wnt signaling. Birth Defects Research (Part A), 2008. © 2008 Wiley‐Liss, Inc.     

Cranial cartilages: Players in the evolution of the cranium during evolution of the chordates in general and of the vertebrates in particular

The present contribution is chiefly a review, augmented by some new results on amphioxus and lamprey anatomy, that draws on paleontological and developmental data to suggest a scenario for cranial cartilage evolution in the phylum chordata. Consideration is given to the cartilage-related tissues of invertebrate chordates (amphioxus and some fossil groups like vetulicolians) as well as in the two major divisions of the subphylum Vertebrata (namely, agnathans, and gnathostomes). In the invertebrate chordates, which can be considered plausible proxy ancestors of the vertebrates, only a viscerocranium is present, whereas a neurocranium is absent. For this situation, we examine how cartilage-related tissues of this head region prefigure the cellular cartilage types in the vertebrates. We then focus on the vertebrate neurocranium, where cyclostomes evidently lack neural-crest derived trabecular cartilage (although this point needs to be established more firmly). In the more complex gnathostome, several neural-crest derived cartilage types are present: namely, the trabecular cartilages of the prechordal region and the parachordal cartilage the chordal region. In sum, we present an evolutionary framework for cranial cartilage evolution in chordates and suggest aspects of the subject that should profit from additional study.     

Fibronectin in early avian embryos: Synthesis and distribution along the migration pathways of neural crest cells

Summary Immunoperoxidase labelling for fibronectin (FN) in chick embryos showed FN-positive basement membranes surrounding the neural crest cell population prior to crest-cell migration. At cranial levels, crest cells migrated laterally into a large cell-free space. Initially they moved as a tongue of cells contacting the FN-positive basement membrane of the ectoderm, but later the crest cell population expanded into space further from the ectoderm, until eventually the entire cranial cell-free space was occupied by mesenchyme cells. This was accompanied by the appearance of FN among the crest cells. At trunk levels, crest cells entered a relatively small space already containing FN-positive extracellular material. At later stages the migration of trunk crest cells broadly matched the distribution of FN. In vitro, chick and quail embryo ectoderm, endoderm, somites, notochord and neural tube synthesized and organized fibrous FN-matrices, as shown by immunofluorescence. Ectoderm and endoderm deposited this matrix only on the substrate face. The FN content of endoderm and neural tube matrices was transient, the immunofluorescence intensity declining after 1–2 days in culture. Some crest cells of cranial and sacral axial levels synthesized FN. Our data suggests that these were the earliest crest cells to migrate from these levels. This ability may be the first expression of mesenchymal differentiation in these crest cells, and in vivo enable them to occupy a large space. Almost all crest cells from cervico-lumbar axial levels were unable to synthesize FN. In vivo, this inability may magnify the response of these crest cells to FN provided by the neighbouring embryonic tissues.     

Derivation of neural precursor cells from human ES cells at 3% O(2) is efficient, enhances survival and presents no barrier to regional specification and functional differentiation

In vitro stem cell systems traditionally employ oxygen levels that are far removed from the in vivo situation. This study investigates whether an ambient environment containing a physiological oxygen level of 3% (normoxia) enables the generation of neural precursor cells (NPCs) from human embryonic stem cells (hESCs) and whether the resultant NPCs can undergo regional specification and functional maturation. We report robust and efficient neural conversion at 3% O(2), demonstration of tri-lineage potential of resultant NPCs and the subsequent electrophysiological maturation of neurons. We also show that NPCs derived under 3% O(2) can be differentiated long term in the absence of neurotrophins and can be readily specified into both spinal motor neurons and midbrain dopaminergic neurons. Finally, modelling the oxygen stress that occurs during transplantation, we demonstrate that in vitro transfer of NPCs from a 20 to 3% O(2) environment results in significant cell death, while maintenance in 3% O(2) is protective. Together these findings support 3% O(2) as a physiologically relevant system to study stem cell-derived neuronal differentiation and function as well as to model neuronal injury.     

Apoptosis of premigratory neural crest cells in rhombomeres 3 and 5: consequences for patterning of the branchial region

In the avian hindbrain, premigratory neural crest cells undergo programmed cell death (apoptosis) in rhombomeres 3 and 5 (r3, r5). Here, we have attempted to analyze the significance of the loss of neural crest cells from these odd-numbered rhombomeres. When apoptosis is prevented in r3 and r5, r3 crest migrate into the first arch and r5 into the third arch. Interestingly, these extra neural crest cells contributed to the formation of ectopic muscle attachment sites that are also found in those species in which r3 and r5 neural crest cells do not undergo apoptosis. Thus, apoptosis in the odd-numbered rhombomeres appears to be an evolutionarily derived mechanism that is required to eliminate r3 and r5 crest migration into first and third arches and thereby remove these muscle attachment sites.     

Face off against ROS: Tcof1/Treacle safeguards neuroepithelial cells and progenitor neural crest cells from oxidative stress during craniofacial development

One‐third of all congenital birth defects affect the head and face, and most craniofacial anomalies are considered to arise through defects in the development of cranial neural crest cells. Cranial neural crest cells give rise to the majority of craniofacial bones, cartilages and connective tissues. Therefore, understanding the events that control normal cranial neural crest and subsequent craniofacial development is important for elucidating the pathogenetic mechanisms of craniofacial anomalies and for the exploring potential therapeutic avenues for their prevention. Treacher Collins syndrome (TCS) is a congenital disorder characterized by severe craniofacial anomalies. An animal model of TCS, generated through mutation of Tcof1, the mouse (Mus musculus) homologue of the gene primarily mutated in association with TCS in humans, has recently revealed significant insights into the pathogenesis of TCS. Apoptotic elimination of neuroepithelial cells including neural crest cells is the primary cause of craniofacial defects in Tcof1 mutant embryos. However, our understanding of the mechanisms that induce tissue‐specific apoptosis remains incomplete. In this review, we describe recent advances in our understanding of the pathogenesis TCS. Furthermore, we discuss the role of Tcof1 in normal embryonic development, the correlation between genetic and environmental factors on the severity of craniofacial abnormalities, and the prospect for prenatal prevention of craniofacial anomalies.     

Uracil nucleotides stimulate human neural precursor cell proliferation and dopaminergic differentiation: involvement of MEK/ERK signalling

Isolation and propagation of neural stem cells derived from human brain tissue uniquely enables the study of human neurogenesis in vitro. In addition, ex vivo-expanded human neural stem/precursor cells (NPCs) may offer novel therapeutic strategies. We investigated the effects of extracellular nucleotides on the proliferation and differentiation of human mesencephalic neural stem/precursor cells (hmNPCs). When combined with the mitogens epidermal growth factor and fibroblast growth factor 2, UTP (1 microm) boosted proliferation of hmNPCs as shown by increased expression of the proliferation marker proliferating cell nuclear antigen (330%). UTP-induced proliferation was abrogated by the preferential P2Y receptor blocker pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). UTP also stimulated dopaminergic differentiation. Treatment with UTP (100 microm) increased the number of tyrosine hydroxylase (TH)-positive cells and TH protein by 267 and 319% respectively. UTP-stimulated dopaminergic differentiation of hmNPCs was blocked by the P2 receptor antagonists suramin (10 microm) and PPADS (100 microm). In addition, UDP (1 microm) enhanced TH protein expression by 194%. During differentiation, treatment with UTP stimulated the extracellular signal-regulated kinase (ERK) pathway. Both ERK1/2 phosphorylation and dopaminergic differentiation were inhibited by U0126, a selective ERK kinase inhibitor, as well as by suramin. When other P2 receptor agonists (ATP, ADP and adenosine 5'-O-(2-thiophosphate) (ADPbetaS); all 100 microm) were applied, both proliferation and dopaminergic differentiation of NPCs were compromised. We conclude that uracil nucleotides exert specific P2 receptor-mediated effects on midbrain-derived human NPCs, and may be used to enhance both proliferation and dopaminergic differentiation.     

Regeneration of a chimeric retina from single cells in vitro: cell-lineage-dependent formation of radial cell columns by segregated chick and quail cells

Summary We report here that similar to E6-chicken retinal cells, dissociated cells from 5.5-day-old (E5.5) quail retinae reaggregate in rotary culture, multiply about tenfold and reestablish histotypical areas. These cellular aggregates include all nuclear layers either with inversed or correct laminar polarity, depending on the local origin of the cells (called rosetted and laminar in-vitro-retinae (IVR), respectively; Layer and Willbold 1989). In combined cultures, chick and quail cells are evenly mixed only during the first two days of culture. Along with the assembly of single cells into rosettes and then into discrete laminae, sectors of chick and quail cells begin to segregate. They are delineated by borders running radially through all three nuclear layers. Thus, interspecies migration of cells at this advanced stage of differentiation is strongly inhibited. Concomitant with this segregation, coherent radial columns spanning all three layers but containing cells from either species only, can be traced histologically. We conclude that a weak segregation of chick and quail retinal cells takes place already at the single cell level, but that the permanent segregation of entire tissue parts must be due to clonal cellular proliferation within the IVR in conjunction with some developmental-structural mechanism retaining clonal progenies within a columnar order.Abbreviations ECM extracellular matrix - E5.5 days of embryonic age - GCL ganglion cell layer - GC's ganglion cells - i.c. in culture - INL inner nuclear layer - rosetted in-vitro-retina retinal cell organoid aggregated from single cells of the central retina - IPL inner plexiform layer - MRE marginal retinal epithelium - ONL outer nuclear layer - OPL outer plexiform layer - OS ora serrate - PR photoreceptor cell - laminated in-vitro-retina fully laminated retinal cellorganoid resembling an E15-retina aggregated from cells of the eye periphery including RPE - RPE retinal pigment epithelium