A Requirement for FGF Signalling in the Formation of Primitive Streak-Like Intermediates from Primitive Ectoderm in Culture

Background

Embryonic stem (ES) cells hold considerable promise as a source of cells with therapeutic potential, including cells that can be used for drug screening and in cell replacement therapies. Differentiation of ES cells into the somatic lineages is a regulated process; before the promise of these cells can be realised robust and rational methods for directing differentiation into normal, functional and safe cells need to be developed. Previous in vivo studies have implicated fibroblast growth factor (FGF) signalling in lineage specification from pluripotent cells. Although FGF signalling has been suggested as essential for specification of mesoderm and endoderm in vivo and in culture, the exact role of this pathway remains unclear.

Methodology/Principal Findings

Using a culture model based on early primitive ectoderm-like (EPL) cells we have investigated the role of FGF signalling in the specification of mesoderm. We were unable to demonstrate any mesoderm inductive capability associated with FGF1, 4 or 8 signalling, even when the factors were present at high concentrations, nor any enhancement in mesoderm formation induced by exogenous BMP4. Furthermore, there was no evidence of alteration of mesoderm sub-type formed with addition of FGF1, 4 or 8. Inhibition of endogenous FGF signalling, however, prevented mesoderm and favoured neural differentiation, suggesting FGF signalling was required but not sufficient for the differentiation of primitive ectoderm into primitive streak-like intermediates. The maintenance of ES cell/early epiblast pluripotent marker expression was also observed in cultures when FGF signalling was inhibited.

Conclusions/Significance

FGF signalling has been shown to be required for the differentiation of primitive ectoderm to neurectoderm. This, coupled with our observations, suggest FGF signalling is required for differentiation of the primitive ectoderm into the germ lineages at gastrulation.     

Inhibition of Rho kinase regulates specification of early differentiation events in P19 embryonal carcinoma stem cells

Background

The Rho kinase pathway plays a key role in many early cell/tissue determination events that take place in embryogenesis. Rho and its downstream effector Rho kinase (ROCK) play pivotal roles in cell migration, apoptosis (membrane blebbing), cell proliferation/cell cycle, cell-cell adhesion and gene regulation. We and others have previously demonstrated that inhibition of ROCK blocks endoderm differentiation in embryonal carcinoma stem cells, however, the effect of ROCK inhibition on mesoderm and ectoderm specification has not been fully examined. In this study, the role of ROCK within the specification and differentiation of all three germ layers was examined.

Methodology/Principal Findings

P19 cells were treated with the specific ROCK inhibitor Y-27623, and increase in differentiation efficiency into neuro-ectodermal and mesodermal lineages was observed. However, as expected a dramatic decrease in early endodermal markers was observed when ROCK was inhibited. Interestingly, within these ROCK-inhibited RA treated cultures, increased levels of mesodermal or ectodermal markers were not observed, instead it was found that the pluripotent markers SSEA-1 and Oct-4 remained up-regulated similar to that seen in undifferentiated cultures. Using standard and widely accepted methods for reproducible P19 differentiation into all three germ layers, an enhancement of mesoderm and ectoderm differentiation with a concurrent loss of endoderm lineage specification was observed with Y-27632 treatment. Evidence would suggest that this effect is in part mediated through TGF-β and SMAD signaling as ROCK-inhibited cells displayed aberrant SMAD activation and did not return to a ‘ground’ state after the inhibition had been removed.

Conclusions/Significance

Given this data and the fact that only a partial rescue of normal differentiation capacity occurred when ROCK inhibition was alleviated, the effect of ROCK inhibition on the differentiation capacity of pluripotent cell populations should be further examined to elucidate the role of the Rho-ROCK pathway in early cellular ‘fate’ decision making processes.     

Eps15R and clathrin regulate EphB2‐mediated cell repulsion

Expression of Eph receptors and their ligands, the ephrins, have important functions in boundary formation and morphogenesis in both adult and embryonic tissue. The EphB receptors and ephrinB ligands are transmembrane proteins that are expressed in different cells and their interaction drives cell repulsion. For cell repulsion to occur, trans‐endocytosis of the inter‐cellular receptor‐ligand EphB‐ephrinB complex is required. The molecular mechanism underlying trans‐endocytosis is poorly defined. Here we show that the process is clathrin‐ and Eps15R‐mediated using Co115 colorectal cell lines stably expressing EphB2 and ephrinB1. Cell repulsion in co‐cultures of EphB2‐ and ephrinB1‐expressing cells is significantly reduced by knockdown of Eps15R but not Eps15. A novel interaction motif in Eps15R, DPFxxLDPF, is shown to bind directly to the clathrin terminal domain in vitro. Moreover, the interaction between Eps15R and clathrin is required for EphB2‐mediated cell repulsion as shown in a rescue experiment in the EphB2 co‐culture assay where wild type Eps15R but not the clathrin‐binding mutant rescues cell repulsion. These results provide the first evidence that Eps15R together with clathrin control EphB/ephrinB trans‐endocytosis and thereby cell repulsion.      

Rab11 Helps Maintain Apical Crumbs and Adherens Junctions in the Drosophila Embryonic Ectoderm

Background

Tissue morphogenesis and organogenesis require that cells retain stable cell-cell adhesion while changing shape and moving. One mechanism to accommodate this plasticity in cell adhesion involves regulated trafficking of junctional proteins.

Methodology/Principal Findings

Here we explored trafficking of junctional proteins in two well-characterized model epithelia, the Drosophila embryonic ectoderm and amnioserosa. We find that DE-cadherin, the transmembrane protein of adherens junctions, is actively trafficked through putative vesicles, and appears to travel through both Rab5-positive and Rab11-positive structures. We manipulated the functions of Rab11 and Rab5 to examine the effects on junctional stability and morphogenesis. Reducing Rab11 function, either using a dominant negative construct or loss of function alleles, disrupts integrity of the ectoderm and leads to loss of adherens junctions. Strikingly, the apical junctional regulator Crumbs is lost before AJs are destabilized, while the basolateral protein Dlg remains cortical. Altering Rab5 function had less dramatic effects, not disrupting adherens junction integrity but affecting dorsal closure.

Conclusions/Significance

We contrast our results with what others saw when disrupting other trafficking regulators, and when disrupting Rab function in other tissues; together these data suggest distinct mechanisms regulate junctional stability and plasticity in different tissues.     

Junctional adhesion molecule 2 mediates the interaction between hatched blastocyst and luminal epithelium: induction by progesterone and LIF

Background

Junctional adhesion molecule 2 (Jam2) is a member of the JAM superfamily. JAMs are localized at intercellular contacts and participated in the assembly and maintenance of junctions, and control of cell permeability. Because Jam2 is highly expressed in the luminal epithelium on day 4 of pregnancy, this study was to determine whether Jam2 plays a role in uterine receptivity and blastocyst attachment in mouse uterus.

Methodology/Principal Findings

Jam2 is highly expressed in the uterine luminal epithelium on days 3 and 4 of pregnancy. Progesterone induces Jam2 expression in ovariectomized mice, which is blocked by progesterone antagonist RU486. Jam2 expression on day 4 of pregnancy is also inhibited by RU486 treatment. Leukemia inhibitory factor (LIF) up-regulates Jam2 protein in isolated luminal epithelium from day 4 uterus, which is blocked by S3I-201, a cell-permeable inhibitor for Stat3 phosphorylation. Under adhesion assay, recombinant Jam2 protein increases the rate of blastocyst adhesion. Both soluble recombinant Jam2 and Jam3 can reverse this process.

Conclusion

Jam2 is highly expressed in the luminal epithelium of receptive uterus and up-regulated by progesterone and LIF via tyrosine phosphorylation of Stat3. Jam2 may play a role in the interaction between hatched blastocyst and receptive uterus.     

Modulation of Hepatocarcinoma Cell Morphology and Activity by Parylene-C Coating on PDMS

Background

The ability to understand and locally control the morphogenesis of mammalian cells is a fundamental objective of cell and developmental biology as well as tissue engineering research. We present parylene-C (ParC) deposited on polydimethylsiloxane (PDMS) as a new substratum for in vitro advanced cell culture in the case of Human Hepatocarcinoma (HepG2) cells.

Principal Findings

Our findings establish that the intrinsic properties of ParC-coated PDMS (ParC/PDMS) influence and modulate initial extracellular matrix (ECM; here, type-I collagen) surface architecture, as compared to non-coated PDMS substratum. Morphological changes induced by the presence of ParC on PDMS were shown to directly affect liver cell metabolic activity and the expression of transmembrane receptors implicated in cell adhesion and cell-cell interaction. These changes were characterized by atomic force microscopy (AFM), which elucidated differences in HepG2 cell adhesion, spreading, and reorganization into two- or three-dimensional structures by neosynthesis of ECM components. Local modulation of cell aggregation was successfully performed using ParC/PDMS micropatterns constructed by simple microfabrication.

Conclusion/Significance

We demonstrated for the first time the modulation of HepG2 cells'' behavior in relation to the intrinsic physical properties of PDMS and ParC, enabling the local modulation of cell spreading in a 2D or 3D manner by simple microfabrication techniques. This work will provide promising insights into the development of cell-based platforms that have many applications in the field of in vitro liver tissue engineering, pharmacology and therapeutics.     

IFN-γ-inducible protein of 10 kDa upregulates the effector functions of eosinophils through β2 integrin and CXCR3

Background

Eosinophils play an important role in the pathogenesis of bronchial asthma and its exacerbation. Recent reports suggest the involvement of IFN-γ-inducible protein of 10 kDa (IP-10) in virus-induced asthma exacerbation. The objective of this study was to examine whether CXCR3 ligands including IP-10 modify the effector functions of eosinophils.

Methods

Eosinophils isolated from the blood of healthy donors were stimulated with CXCR3 ligands and their adhesion to rh-ICAM-1 was then measured using eosinophil peroxidase assays. The generation of eosinophil superoxide anion (O₂^-) was examined based on the superoxide dismutase-inhibitable reduction of cytochrome C. Eosinophil-derived neurotoxin (EDN) release was evaluated to determine whether CXCR3 ligands induced eosinophil degranulation. Cytokine and chemokine production by eosinophils was examined using a Bio-plex assay.

Results

Eosinophil adhesion to ICAM-1 was significantly enhanced by IP-10, which also significantly induced eosinophil O₂^- generation in the presence of ICAM-1. Both the enhanced adhesion and O₂^- generation were inhibited by an anti-β₂ integrin mAb or an anti-CXCR3 mAb. Other CXCR3 ligands, such as monokine induced by IFN-γ (Mig) and IFN-inducible T cell α chemoattractant (I-TAC), also induced eosinophil adhesion and O₂^- generation in the presence of ICAM-1. IP-10, but not Mig or I-TAC, increased the release of EDN. IP-10 increased the production of a number of cytokines and chemokines by eosinophils.

Conclusions

These findings suggest that CXCR3 ligands such as IP-10 can directly upregulate the effector functions of eosinophils. These effects might be involved in the activation and infiltration of eosinophils in the airway of asthma, especially in virus-induced asthma exacerbation.     

Expression of myoferlin in human airway epithelium and its role in cell adhesion and zonula occludens-1 expression

Background

Normal airway epithelial barrier function is maintained by cell-cell contacts which require the translocation of adhesion proteins at the cell surface, through membrane vesicle trafficking and fusion events. Myoferlin and dysferlin, members of the multiple-C2-domain Ferlin superfamily, have been implicated in membrane fusion processes through the induction of membrane curvature. The objectives of this study were to examine the expression of dysferlin and myoferlin within the human airway and determine the roles of these proteins in airway epithelial homeostasis.

Methods

The expression of dysferlin and myoferlin were evaluated in normal human airway sections by immunohistochemistry, and primary human airway epithelial cells and fibroblasts by immuno blot. Localization of dysferlin and myoferlin in epithelial cells were determined using confocal microscopy. Functional outcomes analyzed included cell adhesion, protein expression, and cell detachment following dysferlin and myoferlin siRNA knock-down, using the human bronchial epithelial cell line, 16HBE.

Results

Primary human airway epithelial cells express both dysferlin and myoferlin whereas fibroblasts isolated from bronchi and the parenchyma only express myoferlin. Expression of dysferlin and myoferlin was further localized within the Golgi, cell cytoplasm and plasma membrane of 16HBE cells using confocal micrscopy. Treatment of 16HBE cells with myoferlin siRNA, but not dysferlin siRNA, resulted in a rounded cell morphology and loss of cell adhesion. This cell shedding following myoferlin knockdown was associated with decreased expression of tight junction molecule, zonula occludens-1 (ZO-1) and increased number of cells positive for apoptotic markers Annexin V and propidium iodide. Cell shedding was not associated with release of the innate inflammatory cytokines IL-6 and IL-8.

Conclusions/Significance

This study demonstrates the heterogeneous expression of myoferlin within epithelial cells and fibroblasts of the respiratory airway. The effect of myoferlin on the expression of ZO-1 in airway epithelial cells indicates its role in membrane fusion events that regulate cell detachment and apoptosis within the airway epithelium.     

Plasmodium vivax adherence to placental glycosaminoglycans

Background

Plasmodium vivax infections seldom kill directly but do cause indirect mortality by reducing birth weight and causing abortion. Cytoadherence and sequestration in the microvasculature are central to the pathogenesis of severe Plasmodium falciparum malaria, but the contribution of cytoadherence to pathology in other human malarias is less clear.

Methodology

The adherence properties of P. vivax infected red blood cells (PvIRBC) were evaluated under static and flow conditions.

Principal Findings

P. vivax isolates from 33 patients were studied. None adhered to immobilized CD36, ICAM-1, or thrombospondin, putative ligands for P. falciparum vascular cytoadherence, or umbilical vein endothelial cells, but all adhered to immobilized chondroitin sulphate A (CSA) and hyaluronic acid (HA), the receptors for adhesion of P. falciparum in the placenta. PvIRBC also adhered to fresh placental cells (N = 5). Pre-incubation with chondroitinase prevented PvIRBC adherence to CSA, and reduced binding to HA, whereas preincubation with hyaluronidase prevented adherence to HA, but did not reduce binding to CSA significantly. Pre-incubation of PvIRBC with soluble CSA and HA reduced binding to the immobilized receptors and prevented placental binding. PvIRBC adhesion was prevented by pre-incubation with trypsin, inhibited by heparin, and reduced by EGTA. Under laminar flow conditions the mean (SD) shear stress reducing maximum attachment by 50% was 0.06 (0.02) Pa but, having adhered, the PvIRBC could then resist detachment by stresses up to 5 Pa. At 37°C adherence began approximately 16 hours after red cell invasion with maximal adherence at 30 hours. At 39°C adherence began earlier and peaked at 24 hours.

Significance

Adherence of P. vivax-infected erythrocytes to glycosaminoglycans may contribute to the pathogenesis of vivax malaria and lead to intrauterine growth retardation.     

mNanog Possesses Dorsal Mesoderm-Inducing Ability by Modulating Both BMP and Activin/Nodal Signaling in Xenopus Ectodermal Cells

Background

In Xenopus early embryogenesis, various genes are involved with mesoderm formation. In particular, dorsal mesoderm contains the organizer region and induces neural tissues through the inhibition of bone morphogenetic protein (BMP) signaling. In our initial study to identify novel genes necessary for maintaining the undifferentiated state, we unexpectedly revealed mesoderm-inducing activity for mNanog in Xenopus.

Methodology/Principal Findings

The present series of experiments investigated the effect of mNanog gene expression on Xenopus embryo. Ectopic expression of mNanog induced dorsal mesoderm gene activity, secondary axis formation, and weakly upregulated Activin/nodal signaling. The injection of mNanog also effectively inhibited the target genes of BMP signaling, while Xvent2 injection downregulated the dorsal mesoderm gene expression induced by mNanog injection.

Conclusions/Significance

These results suggested that mNanog expression induces dorsal mesoderm by regulating both Activin/nodal signaling and BMP signaling in Xenopus. This finding highlights the possibly novel function for mNanog in stimulating the endogenous gene network in Xenopus mesoderm formation.     

Proteomic analyses reveal common promiscuous patterns of cell surface proteins on human embryonic stem cells and sperms

Background

It has long been proposed that early embryos and reproductive organs exhibit similar gene expression profiles. However, whether this similarity is propagated to the protein level remains largely unknown. We have previously characterised the promiscuous expression pattern of cell surface proteins on mouse embryonic stem (mES) cells. As cell surface proteins also play critical functions in human embryonic stem (hES) cells and germ cells, it is important to reveal whether a promiscuous pattern of cell surface proteins also exists for these cells.

Methods and Principal Findings

Surface proteins of hES cells and human mature sperms (hSperms) were purified by biotin labelling and subjected to proteomic analyses. More than 1000 transmembrane or secreted cell surface proteins were identified on the two cell types, respectively. Proteins from both cell types covered a large variety of functional categories including signal transduction, adhesion and transporting. Moreover, both cell types promiscuously expressed a wide variety of tissue specific surface proteins, and some surface proteins were heterogeneously expressed.

Conclusions/Significance

Our findings indicate that the promiscuous expression of functional and tissue specific cell surface proteins may be a common pattern in embryonic stem cells and germ cells. The conservation of gene expression patterns between early embryonic cells and reproductive cells is propagated to the protein level. These results have deep implications for the cell surface signature characterisation of pluripotent stem cells and germ cells and may lead the way to a new area of study, i.e., the functional significance of promiscuous gene expression in pluripotent and germ cells.     

Associations between Parent-Adolescent Attachment Relationship Quality,Negative Life Events and Mental Health

Purpose

The aim of this study was to examine the association of negative life events and parent-adolescent attachment relationship quality with mental health problems and to explore an interaction between the parent-adolescent attachment relationship and one or multiple negative life events on the mental health of adolescents.

Methods

A two-year longitudinal study was conducted among first-year secondary school students (N = 3181). The occurrence of life events and the quality of parent-adolescent attachment were assessed at baseline and mental health status at two-year follow-up by means of self-report questionnaires. Binary logistic regression analyses were conducted to assess associations between life events, parent-adolescent attachment and mental health problems. Relative Excess Risk due to Interaction techniques were used to determine the interaction effects on the additive scale.

Results

Life events were related to mental health status, as was parent-adolescent attachment. The combined effect of an unfavourable parent-adolescent attachment with life events on mental health was larger than the sum of the two individual effects. Among adolescents with one life event or multiple life events, an unfavourable parent-adolescent attachment increased the risk of mental health problems at follow-up compared to the group without life events.

Conclusion

Results supported an interaction effect between parent-adolescent attachment and negative life events on mental health. Especially adolescents with one or multiple life events and an unfavourable parent-adolescent attachment seems to be a vulnerable group for mental health problems. Implications for further research are discussed.     

Matrix metalloproteinase-2 Promotes αvβ3 Integrin-Mediated Adhesion and Migration of Human Melanoma Cells by Cleaving Fibronectin

Background

Matrix metalloproteinase-2 (MMP-2) is a key regulator in the migration of tumor cells. αvβ3 integrin has been reported to play a critical role in cell adhesion and regulate the migration of tumor cells by promoting MMP-2 activation. However, little is known about the effects of MMP-2 on αvβ3 integrin activity and αvβ3 integrin-mediated adhesion and migration of tumor cells.

Methodology/Principal Findings

Human melanoma cells were seeded using an agarose drop model and/or subjected to in vitro analysis using immunofluorescence, adhesion, migration and invasion assays to investigate the relationship between active MMP-2 and αvβ3 integrin during the adhesion and migration of the tumor cells. We found that MMP-2 was localized at the leading edge of spreading cells before αvβ3 integrin. αvβ3 integrin-mediated adhesion and migration of the tumor cells were inhibited by a MMP-2 inhibitor. MMP-2 cleaved fibronectin into small fragments, which promoted the adhesion and migration of the tumor cells.

Conclusion/Significance

MMP-2 cleaves fibronectin into small fragments to enhance the adhesion and migration of human melanoma cells mediated by αvβ3 integrin. These results indicate that MMP-2 may guide the direction of the tumor cell migration.     

Persistent Inflammation and Endothelial Activation in HIV-1 Infected Patients after 12 Years of Antiretroviral Therapy

Objective

The study investigated markers of inflammation and endothelial activation in HIV infected patients after 12 years of successful combination antiretroviral treatment (cART).

Methods

Inflammation and endothelial activation were assessed by measuring levels of immunoglobulins, β2-microglobulin, interleukin (IL) 8, tumor necrosis factor α (TNFα), vascular cell adhesion molecule-1 (sVCAM-1), intercellular adhesion molecule-1 (sICAM-1), sE-Selectin, and sP-Selectin.

Results

HIV infected patients had higher levels of β2-microglobulin, IL-8, TNFα, and sICAM-1 than uninfected controls, and HIV infected patients lacked correlation between platelet counts and sP-Selectin levels found in uninfected controls.

Conclusion

Discrete signs of systemic and vascular inflammation persist even after very long term cART.     

Copper deficiency induced emphysema is associated with focal adhesion kinase inactivation

Background

Copper is an important regulator of hypoxia inducible factor 1 alpha (HIF-1α) dependent vascular endothelial growth factor (VEGF) expression, and is also required for the activity of lysyl oxidase (LOX) to effect matrix protein cross-linking. Cell detachment from the extracellular matrix can induce apoptosis (anoikis) via inactivation of focal adhesion kinase (FAK).

Methodology

To examine the molecular mechanisms whereby copper depletion causes the destruction of the normal alveolar architecture via anoikis, Male Sprague-Dawley rats were fed a copper deficient diet for 6 weeks while being treated with the copper chelator, tetrathiomolybdate. Other groups of rats were treated with the inhibitor of auto-phosphorylation of FAK, 1,2,4,5-benzenetetraamine tetrahydrochloride (1,2,4,5-BT) or FAK small interfering RNA (siRNA).

Principal Findings

Copper depletion caused emphysematous changes, decreased HIF-1α activity, and downregulated VEGF expression in the rat lungs. Cleaved caspase-3, caspase-8 and Bcl-2 interacting mediator of cell death (Bim) expression was increased, and the phosphorylation of FAK was decreased in copper depleted rat lungs. Administration of 1,2,4,5-BT and FAK siRNA caused emphysematous lung destruction associated with increased expression of cleaved capase-3, caspase-8 and Bim.

Conclusions

These data indicate that copper-dependent mechanisms contribute to the pathogenesis of emphysema, which may be associated with decreased HIF-1α and FAK activity in the lung.     

Tenascin-C enhances pancreatic cancer cell growth and motility and affects cell adhesion through activation of the integrin pathway

Background

Pancreatic cancer (PDAC) is characterized by an abundant fibrous tissue rich in Tenascin-C (TNC), a large ECM glycoprotein mainly synthesized by pancreatic stellate cells (PSCs). In human pancreatic tissues, TNC expression increases in the progression from low-grade precursor lesions to invasive cancer. Aim of this study was the functional characterization of the effects of TNC on biologic relevant properties of pancreatic cancer cells.

Methods

Proliferation, migration and adhesion assays were performed on pancreatic cancer cell lines treated with TNC or grown on a TNC-rich matrix. Stable transfectants expressing the large TNC splice variant were generated to test the effects of endogenous TNC. TNC-dependent integrin signaling was investigated by immunoblotting, immunofluorescence and pharmacological inhibition.

Results

Endogenous TNC promoted pancreatic cancer cell growth and migration. A TNC-rich matrix also enhanced migration as well as the adhesion to the uncoated growth surface of poorly differentiated cell lines. In contrast, adhesion to fibronectin was significantly decreased in the presence of TNC. The effects of TNC on cell adhesion were paralleled by changes in the activation state of paxillin and Akt.

Conclusion

TNC affects proliferation, migration and adhesion of poorly differentiated pancreatic cancer cell lines and might therefore play a role in PDAC spreading and metastasis in vivo.     

Variation in mesoderm specification across Drosophilids is compensated by different rates of myoblast fusion during body wall musculature development

Background

It has been shown that species separated by relatively short evolutionary distances may have extreme variations in egg size and shape. Those variations are expected to modify the polarized morphogenetic gradients that pattern the dorso-ventral axis of embryos. Currently, little is known about the effects of scaling over the embryonic architecture of organisms. We began examining this problem by asking if changes in embryo size in closely related species of Drosophila modify all three dorso-ventral germ layers or only particular layers, and whether or not tissue patterning would be affected at later stages.

Principal Findings

Here we report that changes in scale affect predominantly the mesodermal layer at early stages, while the neuroectoderm remains constant across the species studied. Next, we examined the fate of somatic myoblast precursor cells that derive from the mesoderm to test whether the assembly of the larval body wall musculature would be affected by the variation in mesoderm specification. Our results show that in all four species analyzed, the stereotyped organization of the body wall musculature is not disrupted and remains the same as in D. melanogaster. Instead, the excess or shortage of myoblast precursors is compensated by the formation of individual muscle fibers containing more or less fused myoblasts.

Conclusions

Our data suggest that changes in embryonic scaling often lead to expansions or retractions of the mesodermal domain across Drosophila species. At later stages, two compensatory cellular mechanisms assure the formation of a highly stereotyped larval somatic musculature: an invariable selection of 30 muscle founder cells per hemisegment, which seed the formation of a complete array of muscle fibers, and a variable rate in myoblast fusion that modifies the number of myoblasts that fuse to individual muscle fibers.