Induction of mirror-image supernumerary jaws in chicken mandibular mesenchyme by Sonic Hedgehog-producing cells

Previous studies have shown that Sonic Hedgehog (Shh) signaling is crucial for the development of the first branchial arch (BA1) into a lower-jaw in avian and mammalian embryos. We have already shown that if Shh expression is precociously inhibited in pharyngeal endoderm, neural crest cells migrate to BA1 but fail to survive, and Meckel's cartilage and associated structures do not develop. This phenotype can be rescued by addition of an exogenous source of Shh. To decipher the role of Shh, we explored the consequences of providing an extra source of Shh to the presumptive BA1 territory. Grafting quail fibroblasts engineered to produce Shh (QT6-Shh), at the 5- to 8-somite stage, resulted in the induction of mirror-image extra lower jaws, caudolateral to the normal one. It turns out that the oral opening epithelium, in which Shh, Fgf8 and Bmp4 are expressed in a definite pattern, functions as an organizing center for lower-jaw development. In our experimental design, the extra source of Shh activates Fgf8, Bmp4 and Shh genes in caudal BA1 ectoderm in a spatial pattern similar to that of the oral epithelium, and regularly leads to the formation of two extra lower-jaw-organizing centers with opposite rostrocaudal polarities. These results emphasize the similarities between the developmental processes of the limb and mandibular buds, and show that in both cases Shh-producing cells create a zone of polarizing activity for the structures deriving from them.     

The occluding junctions of mouse duodenal enterocytes during development

Summary The architecture of occluding junctions during the differentiation of the mouse duodenum was studied in freeze-fractured material. Irregular zonulae occludentes (ZO) (Type I) are numerous during fetal life, and are characterized by their irregular width, and by the presence of basal open-ended extensions fused with the discontinuous basal strand of the ZO. Regular ZOs (Type II), typical of the adult villous epithelium, appear after Type I junctions by day 16 of gestation. Two patterns are distinguishable: in the first, parallel strands of ridges and furrows are found without crossing branches; in the second pattern, the junction zone is organized like a network of short branches forming various types of polygons. In fetal and adult mice fasciae occludentes (FO) (Type III) are present on the lateral cell membranes; in unfixed specimens particles are found in the furrows of the E-face and pits on the ridges of the P-face. In fixed tissues, the particles are aligned on the ridges of the P-face. These results indicate that fixation with glutaraldehyde modifies considerably the affinity of junctional particles toward the P-face during the fracture process. Moreover, the presence of numerous large FOs on the lateral cell membranes of enterocytes during late fetal life and in the adult, is possibly related to cell movement along the intestinal villi.     

Relationship between Neural Crest Cells and Cranial Mesoderm during Head Muscle Development

Background

In vertebrates, the skeletal elements of the jaw, together with the connective tissues and tendons, originate from neural crest cells, while the associated muscles derive mainly from cranial mesoderm. Previous studies have shown that neural crest cells migrate in close association with cranial mesoderm and then circumscribe but do not penetrate the core of muscle precursor cells of the branchial arches at early stages of development, thus defining a sharp boundary between neural crest cells and mesodermal muscle progenitor cells. Tendons constitute one of the neural crest derivatives likely to interact with muscle formation. However, head tendon formation has not been studied, nor have tendon and muscle interactions in the head.

Methodology/Principal Findings

Reinvestigation of the relationship between cranial neural crest cells and muscle precursor cells during development of the first branchial arch, using quail/chick chimeras and molecular markers revealed several novel features concerning the interface between neural crest cells and mesoderm. We observed that neural crest cells migrate into the cephalic mesoderm containing myogenic precursor cells, leading to the presence of neural crest cells inside the mesodermal core of the first branchial arch. We have also established that all the forming tendons associated with branchiomeric and eye muscles are of neural crest origin and express the Scleraxis marker in chick and mouse embryos. Moreover, analysis of Scleraxis expression in the absence of branchiomeric muscles in Tbx1^−/− mutant mice, showed that muscles are not necessary for the initiation of tendon formation but are required for further tendon development.

Conclusions/Significance

This results show that neural crest cells and muscle progenitor cells are more extensively mixed than previously believed during arch development. In addition, our results show that interactions between muscles and tendons during craniofacial development are similar to those observed in the limb, despite the distinct embryological origin of these cell types in the head.     

A modelling approach to explore some hypotheses of the failure of neuroprotective trials in ischemic stroke patients

Ischemic stroke is the third cause of death in industrialised countries, but no satisfactory treatment is currently available. The hundreds of neuroprotective drugs developed to block the ischemic cascade gave very promising results in animal models but the clinical trials performed with these drugs showed no beneficial effects in stroke patients. Many hypotheses were advanced to explain this discrepancy, among which the morphological and functional differences between human and rodent brains. This discrepancy could be partly due to the differences in white matter and glial cell proportions between human and rodent brains. In order to test this hypothesis, we built a mathematical model of the main early pathophysiological mechanisms of stroke in rodent and in human brains. This model is a two-scale model and relies on a set of ordinary differential equations. We built two versions of this model (for human and rodent brains) differing in their white matter and glial cell proportions. Then, we carried out in silico experiments with various neuroprotective drugs. The simulation results obtained with a sodium channel blocker show that the proportion of penumbra recovery is much higher in rodent than in human brain and the results are similar with some other neuroprotective drugs tested during phase III trials. This in silico investigation suggests that the proportions of glial cells and white matter have an influence on neuroprotective drug efficacy. It reinforces the hypothesis that histological and morphological differences between rodent and human brains can partly explain the failure of these agents in clinical trials.     

Dual origin of the floor plate in the avian embryo

Molecular analysis carried out on quail-chick chimeras, in which quail Hensen's node was substituted for its chick counterpart at the five- to six-somite stage (ss), showed that the floor plate of the avian neural tube is composed of distinct areas: (1) a median one (medial floor plate or MFP) derived from Hensen's node and characterised by the same gene expression pattern as the node cells (i.e. expression of HNF3beta and Shh to the exclusion of genes early expressed in the neural ectoderm such as CSox1); and (2) lateral regions that are differentiated from the neuralised ectoderm (CSox1 positive) and form the lateral floor plate (LFP). LFP cells are induced by the MFP to express HNF3beta transiently, Shh continuously and other floor-plate characteristic genes such as NETRIN: In contrast to MFP cells, LFP cells also express neural markers such as Nkx2.2 and Sim1. This pattern of avian floor-plate development presents some similarities to floor-plate formation in zebrafish embryos. We also demonstrate that, although MFP and LFP have different embryonic origins in normal development, one can experimentally obtain a complete floor plate in the neural epithelium by the inductive action of either a notochord or a MFP. The competence of the neuroepithelium to respond to notochord or MFP signals is restricted to a short time window, as only the posterior-most region of the neural plate of embryos younger than 15 ss is able to differentiate a complete floor plate comprising MFP and LFP. Moreover, MFP differentiation requires between 4 and 5 days of exposure to the inducing tissues. Under the same conditions LFP and SHH-producing cells only induce LFP-type cells. These results show that the capacity to induce a complete floor plate is restricted to node-derived tissues and probably involves a still unknown factor that is not SHH, the latter being able to induce only LFP characteristics in neuralised epithelium.     

Cellular dynamics and molecular control of the development of organizer-derived cells in quail-chick chimeras

Malformations affecting the nervous system in humans are numerous and various in etiology. Many are due to genetic deficiencies or mechanical accidents occurring at early stages of development. It is thus of interest to reproduce such human malformations in animal models. The avian embryo is particularly suitable for researching the role of morphogenetic movements and genetic signaling during early neurogenesis. The last ten years of research with Nicole Le Douarin in the Nogent Institut have brought answers to questions formulated by Etienne Wolff at the beginning of his career, by showing that Hensen's node, the avian organizer, is at the source of all the midline cells of the embryo and ensures cell survival, growth and differentiation of neural and mesodermal tissues.     

Transfer of an avian genetic reflex epilepsy by embryonic brain graft: a tissue autonomous process?

Electroencephalographic characteristics and clinical symptoms of an avian genetic reflex epilepsy have been transferred from Fayoumi epileptic (Fepi) chickens to non-epileptic chickens by embryonic homotopic grafts of brain neuroepithelium. Transplanted tissues belonging to the prosencephalic vesicle transferred epileptic electrical features while tissues from the mesencephalic vesicle were responsible for seizure motor manifestations of the disease. Thus each of these tissues can express their own specificity when grafted separately in a normal host, but they co-operate to produce the complete epileptic phenotype when grafted together.     

Modelling methodology in physiopathology

Diseases are complex systems. Modelling them, i.e. systems physiopathology, is a quite demanding, complicated, multidimensional, multiscale process. As such, in order to achieve the goal of the model and further to optimise a rather-time and resource-consuming process, a relevant and easy to practice methodology is required. It includes guidance for validation. Also, the model development should be managed as a complicated process, along a strategy which has been elaborated in the beginning. It should be flexible enough to meet every case. A model is a representation of the available knowledge. All available knowledge does not have the same level of evidence and, further, there is a large variability of the values of all parameters (e.g. affinity constant or ionic current) across the literature. In addition, in a complex biological system there are always values lacking for a few or sometimes many parameters. All these three aspects are sources of uncertainty on the range of validity of the models and raise unsolved problems for designing a relevant model. Tools and techniques for integrating the parameter range of experimental values, level of evidence and missing data are needed.     

From the primitive to the definitive aorta: angioblasts and hemangioblasts during aorta-associated haematopoiesis

Intra-aortic haematopoiesis is a transient phenomenon, present in all the vertebrate species examined. Aorta-associated haematopoiesis produces Haematopoietic Stem Cells (HSC) that emerge from the ventral aortic endothelium through endothelial cells (EC) that switch to HSC. HSC emergence is followed by the colonization of definitive haematopoietic organs. Since intra-aortic haematopoiesis is born from EC of the aortic floor, we wondered how vascular integrity was maintained during haematopoietic production. Transplantation experiments have brought about evidence according to which two distinct endothelial lineages contribute to the embryonic vasculature. One comes from the splanchnic mesoderm and gives rise to EC and haematopoietic cells (HC). The other originates from the somite and is restricted to EC differentiation. We have used interspecific quail/chick grafts to study aortic organogenesis during the course of haematopoiesis. We demonstrate that: 1) before haematopoiesis, the aorta, originally entirely of splanchnic origin, is colonized by EC from the somite. This colonization contributes to create a new roof and sides, which are hence formed by somite-derived EC whereas the floor is contributed by splanchnopleural-derived EC; 2) as haematopoiesis proceeds, somite-derived EC begin to colonize the aortic floor and are found beneath HSC clusters; 3) after haematopoiesis, aortic hemangioblasts disappear from the endothelium and are replaced by somite-derived EC. At this stage, the whole aortic endothelium is derived from somitic cells; 4) we have identified a new cell population from the somite that contributes to the vascular smooth muscle cells (VSMC). This population appears distinct from the somite-derived EC. Using lineage tracing with non-replicative retroviral vectors, we show that EC do not give rise to VSMC as previously thought. Taken together, our results bring about new lights on aorta morphogenesis and the time-restricted production of haematopoiesis.     

A mathematical model of ion movements in grey matter during a stroke

The development of cytotoxic oedema during a stroke consists in cell swelling and shrinking of the extracellular space. This phenomenon is triggered by ion movements through voltage-gated channels, exchangers and pumps. During ischaemia, sodium, calcium and chloride enter the neurons whereas potassium and glutamate are expelled out of the cells. A mathematical model is proposed to represent the long-term dynamics of membrane potentials, cell volumes and ionic concentrations in intracellular and extracellular spaces during a stroke and to study the influence of each ionic current on cell swelling. The model relies on electrophysiological mechanisms and takes into account the behaviour of two types of cells: neurons and also astrocytes known to play a key role in the excitotoxic process in grey matter. The results obtained when a severe or a moderate ischaemia is simulated are consistent with those observed in the in vitro and in vivo experiments. As this model appears to be robust, it is used to perform illustrative simulations aimed at studying the effect of some channel blockers on cell swelling. This approach may help to explore new therapeutic strategies in order to reduce stroke damage.