Cell polarity in precartilage mouse limb mesenchyme cells

The patterns of orientation of individual mesenchyme cells have been evaluated in the hindlimb of the mouse embryo during the period of transition from early aggregation (Day 12) to cartilage formation (Day 13). Orientation was measured by determining the angular relationship between the Golgi-nucleus axis of each cell relative to either the longitudinal limb axis or the center of the cartilaginous aggregate. Patterns were assessed qualitatively and quantitatively in horizontal, vertical, and transverse sections of the proximal, middle, and distal precartilage mesenchyme. These analyses showed that the mesenchyme cells are oriented predominantly toward the longitudinal axes of both the early (Day 12) and late (Day 13) aggregates.     

Cell lineage in mammalian craniofacial mesenchyme

We have analysed the contributions of neural crest and mesoderm to mammalian craniofacial mesenchyme and its derivatives by cell lineage tracing experiments in mouse embryos, using the permanent genetic markers Wnt1-cre for neural crest and Mesp1-cre for mesoderm, combined with the Rosa26 reporter. At the end of neural crest cell migration (E9.5) the two patterns are reciprocal, with a mutual boundary just posterior to the eye. Mesodermal cells expressing endothelial markers (angioblasts) are found not to respect this boundary; they are associated with the migrating neural crest from the 5-somite stage, and by E9.5 they form a pre-endothelial meshwork throughout the cranial mesenchyme. Mesodermal cells of the myogenic lineage also migrate with neural crest cells, as the branchial arches form. By E17.5 the neural crest-mesoderm boundary in the subectodermal mesenchyme becomes out of register with that of the underlying skeletogenic layer, which is between the frontal and parietal bones. At E13.5 the primordia of these bones lie basolateral to the brain, extending towards the vertex of the skull during the following 4-5 days. We used DiI labelling of the bone primordia in ex-utero E13.5 embryos to distinguish between two possibilities for the origin of the frontal and parietal bones: (1) recruitment from adjacent connective tissue or (2) proliferation of the original primordia. The results clearly demonstrated that the bone primordia extend vertically by intrinsic growth, without detectable recruitment of adjacent mesenchymal cells.     

Direct lineage tracing reveals the ontogeny of pancreatic cell fates during mouse embryogenesis

Lineage tracing follows the progeny of labeled cells through development. This technique identifies precursors of mature cell types in vivo and describes the cell fate restriction steps they undergo in temporal order. In the mouse pancreas, direct cell lineage tracing reveals that Pdx1- expressing progenitors in the early embryo give rise to all pancreatic cells. The progenitors for the mature pancreatic ducts separate from the endocrine/exocrine tissues before E12.5. Expression of Ngn3 and pancreatic polypeptide marks endocrine cell lineages during early embryogenesis, and these cells behave as transient progenitors rather than stem cells. In adults, Ngn3 is expressed within the endocrine islets, and the NGN3+ cells seem to contribute to pancreatic islet renewal. These results indicate the stage at which each progenitor population is restricted to a particular fate and provide markers for isolating progenitors to study their growth, differentiation, and the genes necessary for their development.     

Dynamics of BMP signaling in limb bud mesenchyme and polydactyly

Mutations in the Bone Morphogenetic Protein (BMP) pathway are associated with a range of defects in skeletal formation. Genetic analysis of BMP signaling requirements is complicated by the presence of three partially redundant BMPs that are required for multiple stages of limb development. We generated an inducible allele of a BMP inhibitor, Gremlin, which reduces BMP signaling. We show that BMPs act in a dose and time dependent manner in which early reduction of BMPs result in digit loss, while inhibiting overall BMP signaling between E10.5 and E11.5 allows polydactylous digit formation. During this period, inhibiting BMPs extends the duration of FGF signaling. Sox9 is initially expressed in normal digit ray domains but at reduced levels that correlate with the reduction in BMP signaling. The persistence of elevated FGF signaling likely promotes cell proliferation and survival, inhibiting the activation of Sox9 and secondarily, inhibiting the differentiation of Sox9-expressing chondrocytes. Our results provide new insights into the timing and clarify the mechanisms underlying BMP signaling during digit morphogenesis.     

Effects of ethanol and hydrogen peroxide on mouse limb bud mesenchyme differentiation and cell death

Many of the morphological defects associated with embryonic alcohol exposure are a result of cell death. During limb development, ethanol administration produces cell death in the limb and digital defects, including postaxial ectrodactyly. Because an accumulation of reactive oxygen species (ROS) is produced in adult and embryonic tissues by ethanol exposure, this investigation examines the possibility that ethanol-induced cell death in the limb is a result of ROS. Using an in vitro primary culture of limb mesenchyme, the effects of hydrogen peroxide (H2O2) and ethanol on cell death and differentiation were examined. In addition, a dichlorofluorescein diacetate assay was performed to determine the relative intracellular ROS levels after exposure to several concentrations of ethanol and H2O2. Exposure of 1 to 100 microM H2O2 resulted in a 1.08-1.21 times control increase in cartilage matrix accumulation. Cell death was increased 1.69-2.76 times the untreated control value. Production of ROS ranged from 1.25-1.51 times untreated controls. Ethanol exposure of 0.25 to 1.00% (v/v) did not affect cartilage matrix accumulation but resulted in an increase of cell death (1.45-2.31 times untreated control). Intracellular ROS levels after ethanol exposure increased 1.08-1.15 times control but were lower than that produced by 1 microM H2O2. On the basis of the correlation between ROS level produced by H2O2, it was concluded that ethanol-induced cell death in limb mesenchyme is a result of a non-ROS-mediated mechanism. Therefore, in addition to ethanol-induced cell death mediated by ROS reported in the literature, ethanol-induced cell death can be induced in limb mesenchyme by mechanisms that are not dependent upon ROS.     

A cellular lineage analysis of the chick limb bud

The chick limb bud has been used as a model system for studying pattern formation and tissue development for more than 50 years. However, the lineal relationships among the different cell types and the migrational boundaries of individual cells within the limb mesenchyme have not been explored. We have used a retroviral lineage analysis system to track the fate of single limb bud mesenchymal cells at different times in early limb development. We find that progenitor cells labeled at stage 19-22 can give rise to multiple cell types including clones containing cells of all five of the major lateral plate mesoderm-derived tissues (cartilage, perichondrium, tendon, muscle connective tissue, and dermis). There is a bias, however, such that clones are more likely to contain the cell types of spatially adjacent tissues such as cartilage/perichondrium and tendon/muscle connective tissue. It has been recently proposed that distinct proximodistal segments are established early in limb development; however our analysis suggests that there is not a strict barrier to cellular migration along the proximodistal axis in the early stage 19-22 limb buds. Finally, our data indicate the presence of a dorsal/ventral boundary established by stage 16 that is inhibitory to cellular mixing. This boundary is demarcated by the expression of the LIM-homeodomain factor lmx1b.     

Chondrogenesis of limb bud mesenchyme in vitro: stimulation by cations

To analyze the nature of cell-cell interactions in chondrogenesis, two cations that influence these interactions, calcium and poly-L-lysine (PL), were tested for their effects on chondrogenesis in vitro. High density cultures of chick limb bud mesenchyme (Hamilton-Hamburger stages 23/24), were exposed to culture media containing calcium (0.6-3.3 mM) or PL (1-10 micrograms/ml). Both cations stimulated chondrogenesis in a dose-dependent manner, and also promoted cartilage formation in normally non-chondrogenic, low cell density cultures. Chondrogenesis was assayed based on cartilage nodule number, [35S]sulfate incorporation, and expression of type II collagen as detected by immunohistochemistry. The calcium effect was not mimicked by other divalent cations (Cd, Co, Ni, Mg, Mn, and Sr). The effect of PL was dependent on its Mr (greater than or equal to 14K) and charge, and was mimicked by poly-D-lysine but not by lysine or other analogs of PL or lysine (epsilon-amino caproic acid, lysozyme, poly-L-arginine, and spermidine). Calcium and PL probably act by different mechanisms since their effects were additive, and required their presence on different days of culture: calcium acted on Day 1, and PL on Day 2. It is proposed that calcium may play a role in the cell aggregation phase of chondrogenesis whereas PL, or a naturally occurring polypeptide of similar nature, may promote chondrogenesis by crosslinking specific anionic components of the cell surface or extracellular matrix.     

Abnormal anteroposterior and dorsoventral patterning of the limb bud in the absence of retinoids.

We describe here how the early limb bud of the quail embryo develops in the absence of retinoids, including retinoic acid. Retinoid-deficient embryos develop to about stage 20/21, thus allowing patterns of early gene activity in the limb bud to be readily examined. Genes representing different aspects of limb polarity were analysed. Concerning the anteroposterior axis, Hoxb-8 was up-regulated and its border was shifted anteriorly whereas shh and the mesodermal expression of bmp-2 were down-regulated in the absence of retinoids. Concerning the apical ectodermal genes, fgf-4 was down-regulated whereas fgf-8 and the ectodermal domain of bmp-2 were unaffected. Genes involved in dorsoventral polarity were all disrupted. Wnt-7a, normally confined to the dorsal ectoderm, was ectopically expressed in the ventral ectoderm and the corresponding dorsal mesodermal gene Lmx-1 spread into the ventral mesoderm. En-1 was partially or completely absent from the ventral ectoderm. These dorsoventral patterns of expression resemble those seen in En-1 knockout mouse limb buds. Overall, the patterns of gene expression are also similar to the Japanese limbless mutant. These experiments demonstrate that the retinoid-deficient embryo is a valuable tool for dissecting pathways of gene activity in the limb bud and reveal for the first time a role for retinoic acid in the organisation of the dorsoventral axis.     

Modification of the cell cycle of limb bud mesenchyme during in vitro cartilage differentiation

A consistent chondrogenesis takes place in micro high-density cultures derived from limb mesenchymal cells of chick embryos of stages 23-24. Flow-cytometric measurements of DNA content showed that cells in the phase of G1 or G0 made up 51% of the dispersed cell suspensions. The proportion of these cells increased to 71% by the onset of cartilage differentiation in day-2 cultures. This ratio was 84% when the voluminous matrix formation began on the 4th day of culturing. Thereafter, it increased to 90% by the 6th day, and to 93% by the 14th day. The results suggest that cartilage differentiates from G0 mesenchymal cells of the limb. In our measurements, however, the G0 phase includes all non-proliferative cell population which have identical DNA content with G1 cells. Therefore, the G0 phase contains also an increasing number of chondroblasts and chondrocytes as the chondrogenesis proceeds.     

Cell lineage analysis of the amphipod crustacean Parhyale hawaiensis reveals an early restriction of cell fates

In the amphipod crustacean, Parhyale hawaiensis, the first few embryonic cleavages are total and generate a stereotypical arrangement of cells. In particular, at the eight-cell stage there are four macromeres and four micromeres, and each of these cells is uniquely identifiable. We describe our studies of the cell fate pattern of these eight blastomeres, and find that the eight clones resulting from these cells set up distinct cell lineages that differ in terms of proliferation, migration and cell fate. Remarkably, the cell fate of each blastomere is restricted to a single germ layer. The ectoderm originates from three of the macromeres, while the remaining macromere generates the visceral mesoderm. Two of the micromeres generate the somatic mesoderm, a third micromere generates the endoderm and the fourth micromere generates the germline. These findings demonstrate for the first time a total cleavage pattern in an arthropod which results in an invariant cell fate of the blastomeres, but notably, the cell lineage pattern of Parhyale reported shows no clear resemblance to those found in spiralians, nematodes or deuterostomes. Finally, the techniques we have developed for the analysis of Parhyale development suggest that this arthropod may be particularly useful for future functional analyses of crustacean development.     

Separation of precursor myogenic and chondrogenic cells in early limb bud mesenchyme by a monoclonal antibody

We have addressed the problem of the segregation of cell lineages during the development of cartilage and muscle in the chick limb bud. The following experiments demonstrate that early limb buds consist of at least two independent subpopulations of committed precursor cells-- those in (a) the myogenic and (b) the chondrogenic lineage--which can be physically separated. Cells obtained from stage 20, 21, and 22 limb buds were cultured for 5 h in the presence of a monoclonal antibody that was originally isolated for its ability to detach preferentially myogenic cells from extracellular matrices. The detached limb bud cells were collected and replated in normal medium. Within 2 d nearly all of the replated cells had differentiated into myoblasts and myotubes; no chondroblasts differentiated in these cultures. In contrast, the original adherent population that remained after the antibody-induced detachment of the myogenic cells differentiated largely into cartilage and was devoid of muscle. Rearing the antibody-detached cells (i.e., replicating myogenic precursors and postmitotic myoblasts) in medium known to promote chondrogenesis did not induce these cells to chondrify. Conversely, rearing the attached precursor cells (i.e., chondrogenic precursors) in medium known to promote myogenesis did not induce these cells to undergo myogenesis. The definitive mononucleated myoblasts and multinucleated myotubes were identified by muscle- specific antibodies against light meromyosin or desmin, whereas the definitive chondroblasts were identified by a monoclonal antibody against the keratan sulfate chains of the cartilage-specific sulfated proteoglycan. These findings are interpreted as supporting the lineage hypothesis in which the differentiation program of a cell is determined by means of transit through compartments of a lineage.     

Accelerated maturation of limb mesenchyme by the BrachypodH mouse mutation

Mesenchyme cell populations prepared from proximal and distal halves of stage 20 mouse forelimb buds are shown to behave under in vitro micromass culture conditions like analogous cell populations obtained from chick embryo limb buds. While the distal cells are spontaneously chondrogenic, the proximal cells make aggregates which are only potentially chondrogenic after treatment with dibutyryl cyclic AMP. In addition, stage 20 mouse whole limb bud cells homozygous for the brachypodismH (bpH) mutation are shown to behave similarly to 'normal' proximal cells. Both make fewer aggregates and nodules and both have faster aggregation rates (determined as the rate of disappearance of single cells over time) in rotation cultures than 'normal' distal or whole limb bud cells. These results support the hypothesis that the bpH mutation specifically decreases the proportion of spontaneously chondrogenic mesenchyme cells (that is, distal-like cells) present at certain developmental stages in the limb bud, resulting in a prematurely high proportion of proximal-like cells.     

A re-evaluation of the contributions of Apterous and Notch to the dorsoventral lineage restriction boundary in the Drosophila wing

The Drosophila limb primordia are subdivided into compartments: cell populations that do not mix during development. The wing is subdivided into dorsal (D) and ventral (V) compartments by the activity of the selector gene apterous in D cells. Apterous causes segregation of D and V cell populations by at least two distinct mechanisms. The LRR transmembrane proteins Capricious and Tartan are transiently expressed in D cells and contribute to initial segregation of D and V cells. Signaling between D and V cells mediated by Notch and Fringe contributes to the maintenance of the DV affinity boundary. Given that Notch is activated symmetrically, in D and V cells adjacent to the boundary, its role in boundary formation remains somewhat unclear. We re-examine the roles of Apterous and Fringe activities in DV boundary formation and present evidence that Fringe cannot, by itself, generate an affinity difference between D and V cells. Although not sufficient, Fringe is required via Notch activation for expression of an Apterous-dependent affinity difference. We propose that Apterous controls expression of surface proteins that confer an affinity difference in conjunction with activated Notch. Thus, we view Apterous as instructive and Notch activity as essential, but permissive.     

Speed of pattern appearance in reaction-diffusion models: Implications in the pattern formation of limb bud mesenchyme cells

It has been postulated that fibroblast growth factor (FGF) treatment of cultured limb bud mesenchyme cells reinforces the lateral inhibitory effect, but the cells also show accelerated pattern appearance. In the present study, we analyze how a small change in a specific parameter affects the speed of pattern appearance in a Turing reaction-diffusion system using linear stability analysis. It is shown that the sign of the change in appearance speed is qualitatively decided if the system is under the diffusion-driven instability condition, and this is confirmed by numerical simulations. Numerical simulations also show that a small change in parameter value induced easily detectable differences in the appearance speed of patterns. Analysis of the Gierer-Meinhardt model revealed that a change in a single parameter can explain two effects of FGF on limb mesenchyme cells—reinforcement of lateral inhibition and earlier appearance of pattern. These qualitative properties and easy detectability make this feature a promising tool to elucidate the underlying mechanisms of biological pattern formationwhere the quantitative parameters are difficult to obtain.     

Cell generation times in normal and talpid3 mutant chick limb mesenchyme

A comparative study of the cell cycle and its parameters, and of limb pattern development, was carried out in normal and talpid3 wing buds. The relative duration of the G2 + M phase is the least variable. The change in the length of the G1 phase and the S phase is associated with the appearance of mucopolysaccharides in chondrogenic zones and of myosin in myogenic zones. The effect of the ta3 gene is failure of chondrogenesis-induced lengthening of the G1 phase to appear in the chondrogenic condensation in the limb mesenchyme. The data also raise the question of the possible role of the apical ectodermal ridge in control of the cell division rate in the sub-ridge region.