A positive correlation between permissiveness of mesoderm to neural crest migration and early DRG growth.

The microenvironment created by grafting rostral somitic halves in place of normal somites leads to the formation of nonsegmented peripheral ganglia (Kalcheim and Teillet, 1989; Goldstein and Kalcheim, 1991) and is mitogenic for neural crest (NC) cells that become dorsal root ganglia (DRG) (Goldstein et al., 1990). We have now extended these studies by using three surgical manipulations to determine how additional mesodermal tissues affected DRG growth in chick embryos. The following experimental manipulations were performed: (1) unilateral deletion of epithelial somites, similar deletions followed by replacing the somites with (2) a three-dimensional collagen matrix, or (3) fragments of quail lateral plate mesoderm. When somites were absent or replaced by collagen matrix, ganglia were unsegmented, and their volumes were decreased by 21% and 12%, respectively, compared to contralateral intact DRG. In contrast, when lateral plate mesoderm was transplanted in place of somitic mesoderm, NC cells migrated into the grafted mesoderm and formed unsegmented DRG whose volumes were increased by 62.6% compared to the contralateral ganglia. These results suggest that although DRG precursors do not require sclerotome to begin migration and condensation processes, DRG size is modulated by the properties of the mesoderm. Permissiveness to migration is positively correlated with an increase in DRG volume. This volume increase observed in grafts of lateral plate mesoderm is likely to result from enhanced proliferation of neural crest progenitors, previously demonstrated for DRG cells in rostral somitic grafts.     

Early stages of neural crest ontogeny: formation and regulation of cell delamination

Long standing research of the Neural Crest embodies the most fundamental questions of Developmental Biology. Understanding the mechanisms responsible for specification, delamination, migration and phenotypic differentiation of this highly diversifying group of progenitors has been a challenge for many researchers over the years and continues to attract newcomers into the field. Only a few leaps were more significant than the discovery and successful exploitation of the quail-chick model by Nicole Le Douarin and colleagues from the Institute of Embryology at Nogent-sur-Marne. The accurate fate mapping of the neural crest performed at virtually all axial levels was followed by the determination of its developmental potentialities as initially analysed at a population level and then followed by many other significant findings. Altogether, these results paved the way to innumerable questions which brought us from an organismic view to mechanistic approaches. Among them, elucidation of functions played by identified genes is now rapidly underway. Emerging results lead the way back to an integrated understanding of the nature of interactions between the developing neural crest and neighbouring structures. The Nogent Institute thus performed an authentic "tour de force" in bringing the Neural Crest to the forefront of Developmental Biology. The present review is dedicated to the pivotal contributions of Nicole Le Douarin and her collaborators and to unforgettable memories that one of the authors bears from the time spent in the Nogent Institute. We summarize here recent advances in our understanding of early stages of crest ontogeny that comprise specification of epithelial progenitors to a neural crest fate and the onset of neural crest migration. Particular emphasis is given to signaling by BMP and Wnt molecules, to the role of the cell cycle in generating cell movement and to possible interactions between both mechanisms.     

Cell rearrangements during development of the somite and its derivatives

The generation of somites, and the subsequent formation of their major derivatives, muscle-, cartilage-, dermis- and tendon-cell lineages, is tightly orchestrated and, to different extents, these are also mutually supporting processes. They involve complex and timely reorganizations of the paraxial mesoderm, such as multiple phases of epithelial-mesenchymal rearrangements and vice-versa, cellular movements and migrations, and modifications of both cell shape and cell cycle properties. These morphogenetic changes are triggered by local environmental signals and are tightly associated to a genetic program imparting cell-specific fates. Elucidating these signals and their downstream effectors, in addition to determining the state of specification of responsive cell subsets and that of single progenitors in the various domains, is only beginning.     

The sub-lip domain--a distinct pathway for myotome precursors that demonstrate rostral-caudal migration

We have previously reported that the myotome is formed by a first wave of pioneer cells generated from all along the dorsomedial portion of the epithelial somite and a second wave of cells issued from all four edges of the dermomyotome. Cells from the extreme rostral and caudal edges directly generate myofibers that elongate towards the opposite pole of each segment and along the pre-existing myotomal scaffold. In contrast, cells from the dorsomedial and ventrolateral lips first reach the extreme edges and then contribute to myofiber formation. The mechanism by which these epithelial cells translocate remained unknown and was the goal of the present study. We have found that epithelial cells along the dorsomedial and ventrolateral lips of the dermomyotome first delaminate into the immediate underlayer of the corresponding lips, the sub-lip domain, then migrate longitudinally along this pathway until reaching the extreme edges from which they differentiate into myofibers. Cells of the sub-lip domain are negative for Pax3 and desmin but express MyoD, Myf5 and FREK, suggesting that they are specific myogenic progenitors.     

The third wave of myotome colonization by mitotically competent progenitors: regulating the balance between differentiation and proliferation during muscle development

The myotome is formed by a first wave of pioneer cells originating from the entire dorsomedial region of epithelial somites and a second wave that derives from all four lips of the dermomyotome but generates myofibers from only the rostral and caudal edges. Because the precedent progenitors exit the cell cycle upon myotome colonization, subsequent waves must account for consecutive growth. In this study, double labeling with CM-DiI and BrdU revealed the appearance of a third wave of progenitors that enter the myotome as mitotically active cells from both rostral and caudal dermomyotome edges. These cells express the fibroblast growth factor (FGF) receptor FREK and treatment with FGF4 promotes their proliferation and redistribution towards the center of the myotome. Yet, they are negative for MyoD, Myf5 and FGF4, which are, however, expressed in myofibers. The proliferating progenitors first appear around the 30-somite stage in cervical-level myotomes and their number continuously increases, making up 85% of total muscle nuclei by embryonic day (E)4. By this stage, generation of second-wave myofibers, which also enter from the extreme lips is still under way. Formation of the latter fibers peaks at 30 somites and progressively decreases with age until E4. Thus, cells in these dermomyotome lips generate simultaneously distinct types of muscle progenitors in changing proportions as a function of age. Consistent with a heterogeneity in the cellular composition of the extreme lips, MyoD is normally expressed in only a subset of these epithelial cells. Treatment with Sonic hedgehog drives most of them to become MyoD positive and then to become myofibers, with a concurrent reduction in the proportion of proliferating muscle precursors.     

A rapid in vivo assay system for analyzing the organogenetic capacity of human kidney cells

Transplantation of human kidney-derived cells is a potential therapeutic modality for promoting regeneration of diseased renal tissue. However, assays that determine the ability of candidate populations for renal cell therapy to undergo appropriate differentiation and morphogenesis are limited. We report here a rapid and humane assay for characterizing tubulogenic potency utilizing the well-established chorioallantoic membrane CAM) of the chick embryo. Adult human kidney-derived cells expanded in monolayer were suspended in Matrigel and grafted onto the CAM. After a week, grafts were assessed histologically. Strikingly, many of the renal cells self-organized into tubular structures. Host blood vessels penetrated and presumably fed the grafts. Immuno- and histochemical staining revealed that tubular structures were epithelial, but not blood vessels. Some of the cells both within and outside the tubules were dividing. Analysis for markers of proximal and distal renal tubules revealed that grafts contained individual cells of a proximal tubular phenotype and many tubules of distal tubule character. Our results demonstrate that the chick CAM is a useful xenograft system for screening for differentiation and morphogenesis in cells with potential use in renal regenerative medicine.     

A tale of two subunits: how the neomorphic R132H IDH1 mutation enhances production of αHG

Heterozygously expressed single-point mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2, respectively) render these dimeric enzymes capable of producing the novel metabolite α-hydroxyglutarate (αHG). Accumulation of αHG is used as a biomarker for a number of cancer types, helping to identify tumors with similar IDH mutations. With IDH1, it has been shown that one role of the mutation is to increase the rate of conversion from αKG to αHG. To improve our understanding of the function of this mutation, we have detailed the kinetics of the normal (isocitrate to αKG) and neomorphic (αKG to αHG) reactions, as well as the coupled conversion of isocitrate to αHG. We find that the mutant IDH1 is very efficient in this coupled reaction, with the ability to form αHG from isocitrate and NADP(+). The wild type/wild type IDH1 is also able to catalyze this conversion, though it is much more sensitive to concentrations of isocitrate. This difference in behavior can be attributed to the competitive binding between isocitrate and αKG, which is made more favorable for αKG by the neomorphic mutation at arginine 132. Thus, each partial reaction in the heterodimer is functionally isolated from the other. To test whether there is a cooperative effect resulting from the two subunits being in a dimer, we selectively inactivated each subunit with a secondary mutation in the NADP/H binding site. We observed that the remaining, active subunit was unaffected in its associated activity, reinforcing the notion of each subunit being functionally independent. This was further demonstrated using a monomeric form of IDH from Azotobacter vinelandii, which can be shown to gain the same neomorphic reaction when a homologous mutation is introduced into that protein.     

The role of tyrosine sulfation in the dimerization of the CXCR4:SDF‐1 complex

Oligomerization of G protein‐coupled receptors is a recognized mode of regulation of receptor activities, with alternate oligomeric states resulting in different signaling functions. The CXCR4 chemokine receptor is a G protein‐coupled receptor that is post‐translationally modified by tyrosine sulfation at three sites on its N‐terminus (Y7, Y12, Y21), leading to enhanced affinity for its ligand, stromal cell derived factor (SDF‐1, also called CXCL12). The complex has been implicated in cancer metastasis and is a therapeutic target in cancer treatment. Using molecular dynamics simulation of NMR‐derived structures of the CXCR4 N‐terminus in complex with SDF‐1, and calculations of electrostatic binding energies for these complexes, we address the role of tyrosine sulfation in this complex. Our results show that sulfation stabilizes the dimeric state of the CXCR4:SDF‐1 complex through hydrogen bonding across the dimer interface, conformational changes in residues at the dimer interface, and an enhancement in electrostatic binding energies associated with dimerization. These findings suggest a mechanism through which post‐translational modifications such as tyrosine sulfation might regulate downstream function through modulation of the oligomeric state of the modified system.