Nidogen in development and disease.

Nidogen, also known as entactin, is a multifunctional glycoprotein that plays a crucial role in the maintenance of the basement membrane (BM), morphogenesis and neuronal plasticity. This review aims to provide an overview of the structural features, molecular interactions and diverse functions associated with Nidogen. As a bridging molecule within the BM, Nidogen acts as a linchpin connecting various extracellular matrix (ECM) components. Its involvement in tissue development, homeostasis, and pathological conditions underscores its biological and medical significance. We discuss the current state of knowledge regarding Nidogen's role in tissue maintenance, cell adhesion, migration, and signaling, shedding light on its intricate contributions to physiological and pathological processes.

Remodeling of the Platynereis Musculature during Sexual Maturation.

BACKGROUND: The external transformations associated with sexual maturation in Platynereis dumerilii (Audouin and Milne Edwards) are well studied, whereas the internal changes along the body axis have not been systematically analyzed. Therefore, we examined muscle morphology in body regions located anterior or posterior to the prospective atokous/epitokous border to generate a structural basis for internal transformations. RESULTS: All dorsal and ventral longitudinal muscles were significantly reduced in size and density after sexual maturation and strongly atrophied, with the greatest decrease in the anterior segments of females. Despite the general reduction in size throughout the longitudinal muscles, we found a specific degradation mechanism for the posterior segments, which were characterized by the formation of secondary bundle-like fibrous structures. In addition, we observed a profound remodeling of the transversal muscles in the posterior segments of both sexes, apparently resulting in excessive thickening of these muscles. Accordingly, the entire transversal muscle complex was severely swollen and ultrastructurally characterized by a greatly increased number of mitochondria. As a possible trigger for this remodeling, we discovered an enormous number of small, blind-ending blood vessels that completely penetrated the longitudinal and transversal muscles in posterior segments. In addition, both the number of visceral muscles as well as their coelothelial covering were reduced during sexual maturation. CONCLUSIONS: We hypothesize that a possible reason for the secondary bundling of the longitudinal fibers, as well as the difference in size of the posterior transversal muscles, could be the high degree of posterior vascularization. The different degree of muscle remodeling thus depends on segmental affiliation and reflects the tasks in the motility of the different body regions after maturation. The strongest atrophy was found in the anterior segments, while signs of redifferentiation were encountered in posterior segments, supported by the vigorous growth of vessels supplying the transformed epitokous parapodia and associated muscles, which allows rapid swimming during swarming and gamete release.

Serpent/dGATAb regulates Laminin B1 and Laminin B2 expression during Drosophila embryogenesis.

Transcriptional regulation of Laminin expression during embryogenesis is a key step required for proper ECM assembly. We show, that in Drosophila the Laminin B1 and Laminin B2 genes share expression patterns in mesodermal cells as well as in endodermal and ectodermal gut primordia, yolk and amnioserosa. In the absence of the GATA transcription factor Serpent, the spatial extend of Laminin reporter gene expression was strongly limited, indicating that Laminin expression in many tissues depends on Serpent activity. We demonstrate a direct binding of Serpent to the intronic enhancers of Laminin B1 and Laminin B2. In addition, ectopically expressed Serpent activated enhancer elements of Laminin B1 and Laminin B2. Our results reveal Serpent as an important regulator of Laminin expression across tissues.

Characterization of Drosophila Nidogen/entactin reveals roles in basement membrane stability, barrier function and nervous system patterning.

Basement membranes (BMs) are specialized layers of extracellular matrix (ECM) mainly composed of Laminin, type IV Collagen, Perlecan and Nidogen/entactin (NDG). Recent in vivo studies challenged the initially proposed role of NDG as a major ECM linker molecule by revealing dispensability for viability and BM formation. Here, we report the characterization of the single Ndg gene in Drosophila. Embryonic Ndg expression was primarily observed in mesodermal tissues and the chordotonal organs, whereas NDG protein localized to all BMs. Although loss of Laminin strongly affected BM localization of NDG, Ndg-null mutants exhibited no overt changes in the distribution of BM components. Although Drosophila Ndg mutants were viable, loss of NDG led to ultrastructural BM defects that compromised barrier function and stability in vivo Moreover, loss of NDG impaired larval crawling behavior and reduced responses to vibrational stimuli. Further morphological analysis revealed accompanying defects in the larval peripheral nervous system, especially in the chordotonal organs and the neuromuscular junction (NMJ). Taken together, our analysis suggests that NDG is not essential for BM assembly but mediates BM stability and ECM-dependent neural plasticity during Drosophila development.

Distinct genetic programs guide Drosophila circular and longitudinal visceral myoblast fusion.

BACKGROUND: The visceral musculature of Drosophila larvae comprises circular visceral muscles tightly interwoven with longitudinal visceral muscles. During myogenesis, the circular muscles arise by one-to-one fusion of a circular visceral founder cell (FC) with a visceral fusion-competent myoblast (FCM) from the trunk visceral mesoderm, and longitudinal muscles arise from FCs of the caudal visceral mesoderm. Longitudinal FCs migrate anteriorly under guidance of fibroblast growth factors during embryogenesis; it is proposed that they fuse with FCMs from the trunk visceral mesoderm to give rise to syncytia containing up to six nuclei. RESULTS: Using fluorescence in situ hybridization and immunochemical analyses, we investigated whether these fusion events during migration use the same molecular repertoire and cellular components as fusion-restricted myogenic adhesive structure (FuRMAS), the adhesive signaling center that mediates myoblast fusion in the somatic mesoderm. Longitudinal muscles were formed by the fusion of one FC with Sns-positive FCMs, and defects in FCM specification led to defects in longitudinal muscle formation. At the fusion sites, Duf/Kirre and the adaptor protein Rols7 accumulated in longitudinal FCs, and Blow and F-actin accumulated in FCMs. The accumulation of these four proteins at the fusion sites argues for FuRMAS-like adhesion and signaling centers. Longitudinal fusion was disturbed in rols and blow single, and scar wip double mutants. Mutants of wasp or its interaction partner wip had no defects in longitudinal fusion. CONCLUSIONS: Our results indicated that all embryonic fusion events depend on the same cell-adhesion molecules, but that the need for Rols7 and regulators of F-actin distinctly differs. Rols7 was required for longitudinal visceral and somatic myoblast fusion but not for circular visceral fusion. Importantly, longitudinal fusion depended on Kette and SCAR/Wave but was independent of WASp-dependent Arp2/3 activation. Thus, the complexity of the players involved in muscle formation increases from binucleated circular muscles to longitudinal visceral muscles to somatic muscles.

The role of LamininB2 (LanB2) during mesoderm differentiation in Drosophila.

In Drosophila, four genes encode for laminin subunits and the formation of two laminin heterotrimers has been postulated. We report the identification of mutations in the Drosophila LamininB2 (LanB2) gene that encodes for the only laminin γ subunit and is found in both heterotrimers. We describe their effects on embryogenesis, in particular the differentiation of visceral tissues with respect to the ECM. Analysis of mesoderm endoderm interaction indicates disrupted basement membranes and defective endoderm migration, which finally interferes with visceral myotube stretching. Extracellular deposition of laminin is blocked due to the loss of the LanB2 subunit, resulting in an abnormal distribution of ECM components. Our data, concerning the different function of both trimers during organogenesis, suggest that these trimers might act in a cumulative way and probably at multiple steps during ECM assembly. We also observed genetic interactions with kon-tiki and thrombospondin, indicating a role for laminin during muscle attachment.

Rumination as a predictor of relapse in mindfulness-based cognitive therapy for depression.

OBJECTIVES: In mindfulness-based cognitive therapy (MBCT), it is proposed that training in mindfulness should reduce the tendency of formerly depressed patients to enter into ruminative thinking, thus reducing their risk of depressive relapse. However, data showing that rumination is associated with depressive relapse are lacking. METHOD: In an uncontrolled study with 24 formerly depressed patients, rumination was assessed with the Ruminative Response Scale. To assess the occurrence of relapse or recurrence, the Structured Clinical Interview for DSM-IV was administered 12 months after the end of the MBCT. RESULTS: Rumination significantly decreased during the MBCT course. Post-treatment levels of rumination predicted the risk of relapse of major depressive disorder in the 12-month follow-up period even after controlling for numbers of previous episodes and residual depressive symptoms. CONCLUSIONS: The results provide preliminary evidence that rumination is important in the process of depressive relapse.

Fusion of circular and longitudinal muscles in Drosophila is independent of the endoderm but further visceral muscle differentiation requires a close contact between mesoderm and endoderm.

In this study we describe the morphological and genetic analysis of the Drosophila mutant gürtelchen (gurt). gurt was identified by screening an EMS collection for novel mutations affecting visceral mesoderm development and was named after the distinct belt shaped visceral phenotype. Interestingly, determination of visceral cell identities and subsequent visceral myoblast fusion is not affected in mutant embryos indicating a later defect in visceral development. gurt is in fact a new huckebein (hkb) allele and as such exhibits nearly complete loss of endodermal derived structures. Targeted ablation of the endodermal primordia produces a phenotype that resembles the visceral defects observed in huckebein(gürtelchen) (hkb(gurt)) mutant embryos. It was shown previously that visceral mesoderm development requires complex interactions between visceral myoblasts and adjacent tissues. Signals from the neighbouring somatic myoblasts play an important role in cell type determination and are a prerequisite for visceral muscle fusion. Furthermore, the visceral mesoderm is known to influence endodermal migration and midgut epithelium formation. Our analyses of the visceral phenotype of hkb(gurt) mutant embryos reveal that the adjacent endoderm plays a critical role in the later stages of visceral muscle development, and is required for visceral muscle elongation and outgrowth after proper myoblast fusion.

WASP and SCAR have distinct roles in activating the Arp2/3 complex during myoblast fusion.

Myoblast fusion takes place in two steps in mammals and in Drosophila. First, founder cells (FCs) and fusion-competent myoblasts (FCMs) fuse to form a trinucleated precursor, which then recruits further FCMs. This process depends on the formation of the fusion-restricted myogenic-adhesive structure (FuRMAS), which contains filamentous actin (F-actin) plugs at the sites of cell contact. Fusion relies on the HEM2 (NAP1) homolog Kette, as well as Blow and WASP, a member of the Wiskott-Aldrich-syndrome protein family. Here, we show the identification and characterization of schwächling--a new Arp3-null allele. Ultrastructural analyses demonstrate that Arp3 schwächling mutants can form a fusion pore, but fail to integrate the fusing FCM. Double-mutant experiments revealed that fusion is blocked completely in Arp3 and wasp double mutants, suggesting the involvement of a further F-actin regulator. Indeed, double-mutant analyses with scar/WAVE and with the WASP-interacting partner vrp1 (sltr, wip)/WIP show that the F-actin regulator scar also controls F-actin formation during myoblast fusion. Furthermore, the synergistic phenotype observed in Arp3 wasp and in scar vrp1 double mutants suggests that WASP and SCAR have distinct roles in controlling F-actin formation. From these findings we derived a new model for actin regulation during myoblast fusion.

The Wiskott-Aldrich syndrome protein (WASP) is essential for myoblast fusion in Drosophila.

In higher organisms, mononucleated myoblasts fuse to form multinucleated myotubes. During this process, myoblasts undergo specific changes in cell morphology and cytoarchitecture. Previously, we have shown that the actin regulator Kette (Hem-2/Nap-1) is essential for myoblast fusion. In this study, we describe the role of the evolutionary conserved Wiskott-Aldrich syndrome protein that serves as a regulator for the Arp2/3 complex for myoblast fusion. By screening an EMS mutagenesis collection, we discovered a new wasp allele that does not complete fusion during myogenesis. Interestingly, this new wasp3D3-035 allele is characterized by a disruption of fusion after precursor formation. The molecular lesion in this wasp allele leads to a stop codon preventing translation of the CA domain. Usually, the WASP protein exerts its function through the Arp2/3-interacting CA domain. Accordingly, a waspDeltaCA that is expressed in a wild-type background acts as dominant-negative during the fusion process. Furthermore, we show that the myoblast fusion phenotype of kette mutant embryos can be suppressed by reducing the gene dose of wasp3D3-035. Thus, Kette antagonizes WASP function during myoblast fusion.

Blown fuse regulates stretching and outgrowth but not myoblast fusion of the circular visceral muscles in Drosophila.

Circular visceral muscles of Drosophila are binuclear syncytia arising from fusion of two different kinds of myoblasts: a circular visceral founder cell and one visceral fusion-competent myoblast. In contrast to fusion leading to the somatic body-wall musculature, myoblast fusion for the circular visceral muscles does not result in massive syncytia but instead in syncytia interconnected with multiple cytoplasmic bridges, which differentiate into large web-shaped muscles. Here, we show that these syncytial circular visceral muscles build a gut-enclosing network with the interwoven longitudinal visceral muscles. At the ultrastructural level, during circular visceral myoblast fusion and the first step of somatic myoblast fusion prefusion complexes and electron-dense plaques were not detectable which was surprising as these structures are characteristic for the second step of somatic myoblast fusion. Moreover, we demonstrate that Blown fuse (Blow), a cytoplasmic protein essential for the second step of somatic myoblast fusion, plays a different role in circular visceral myogenesis. Blow is known to be essential for progression beyond the prefusion complex in the somatic mesoderm; however, analysis of blow mutants established that it has a restricted role in stretching and outgrowth of the syncytia in the circular visceral muscles. Furthermore, we also found that in the visceral mesoderm, Blow is expressed in both the fusion-competent myoblasts and circular visceral founders, while expression in the somatic mesoderm is initially restricted to fusion-competent myoblasts. We also demonstrate that different enhancer elements in the first intron of blow are responsible for this distinct expression pattern. Thus, we propose a model for Blow in which this protein is involved in at least two clearly differing processes during Drosophila muscle formation, namely somatic myoblast fusion on the one hand and stretching and outgrowth of circular visceral muscles on the other.

The ADAM metalloprotease Kuzbanian is crucial for proper heart formation in Drosophila melanogaster.

We have screened a collection of EMS mutagenized fly lines in order to identify genes involved in cardiogenesis. In the present work, we have studied a group of alleles exhibiting a hypertrophic heart. Our analysis revealed that the ADAM protein (A Disintegrin And Metalloprotease) Kuzbanian, which is the functional homologue of the vertebrate ADAM10, is crucial for proper heart formation. ADAMs are a family of transmembrane proteins that play a critical role during the proteolytic conversion (shedding) of membrane bound proteins to soluble forms. Enzymes harboring a sheddase function recently became candidates for causing several congenital diseases, like distinct forms of the Alzheimer disease. ADAMs play also a pivotal role during heart formation and vascularisation in vertebrates, therefore mutations in ADAM genes potentially could cause congenital heart defects in humans. In Drosophila, the zygotic loss of an active form of the Kuzbanian protein results in a dramatic excess of cardiomyocytes, accompanied by a loss of pericardial cells. Our data presented herein suggest that Kuzbanian acts during lateral inhibition within the cardiac primordium. Furthermore we discuss a second function of Kuzbanian in heart cell morphogenesis.

kette and blown fuse interact genetically during the second fusion step of myogenesis in Drosophila.

Drosophila myoblast fusion proceeds in two steps. The first one gives rise to small syncytia, the muscle precursor cells, which then recruit further fusion competent myoblasts to reach the final muscle size. We have identified Kette as an essential component for myoblast fusion. In kette mutants, founder cells and fusion-competent myoblasts are determined correctly and overcome the very first fusion. But then, at the precursor cell stage, fusion is interrupted. At the ultrastructural level, fusion is characterised by cell-cell recognition, alignment, formation of prefusion complexes, electron dense plaques and membrane breakdown. In kette mutants, electron dense plaques of aberrant length accumulate and fusion is interrupted owing to a complete failure of membrane breakdown. Furthermore, we show that kette interacts genetically with blown fuse (blow) which is known to be required to proceed from prefusion complexes to the formation of the electron dense plaques. Interestingly, a surplus of Kette can replace Blow function during myogenesis. We propose a model in which Dumbfounded/Sticks and stones-dependent cell adhesion is mediated over Rolling Pebbles, Myoblast city, Crk, Blown fuse and Kette, and thus induces membrane fusion.

Myoblast determination in the somatic and visceral mesoderm depends on Notch signalling as well as on milliways(mili(Alk)) as receptor for Jeb signalling.

The visceral muscles of the Drosophila midgut consist of syncytia and arise by fusion of founder and fusion-competent myoblasts, as described for the somatic muscles. A single-step fusion results in the formation of binucleate circular midgut muscles, whereas a multiple-step fusion process produces the longitudinal muscles. A prerequisite for muscle fusion is the establishment of myoblast diversity in the mesoderm prior to the fusion process itself. We provide evidence for a role of Notch signalling during establishment of the different cell types in the visceral mesoderm, demonstrating that the basic mechanism underlying the segregation of somatic muscle founder cells is also conserved during visceral founder cell determination. Searching for genes involved in the determination and differentiation of the different visceral cell types, we identified two independent mutations causing loss of visceral midgut muscles. In both of these mutants visceral muscle founder cells are missing and the visceral mesoderm consists of fusion-competent myoblasts only. Thus, no fusion occurs resulting in a complete disruption of visceral myogenesis. Subsequent characterisation of the mutations revealed that they are novel alleles of jelly belly (jeb) and the Drosophila Alk homologue named milliways (mili(Alk)). We show that the process of founder cell determination in the visceral mesoderm depends on Jeb signalling via the Milliways/Alk receptor. Moreover, we demonstrate that in the somatic mesoderm determination of the opposite cell type, the fusion-competent myoblasts, also depends on Jeb and Alk, revealing different roles for Jeb signalling in specifying myoblast diversity. This novel mechanism uncovers a crosstalk between somatic and visceral mesoderm leading not only to the determination of different cell types but also maintains the separation of mesodermal tissues, the somatic and splanchnic mesoderm.

The two origins of hemocytes in Drosophila.

As in many other organisms, the blood of Drosophila consists of several types of hemocytes, which originate from the mesoderm. By lineage analyses of transplanted cells, we specified two separate anlagen that give rise to different populations of hemocytes: embryonic hemocytes and lymph gland hemocytes. The anlage of the embryonic hemocytes is restricted to a region within the head mesoderm between 70 and 80% egg length. In contrast to all other mesodermal cells, the cells of this anlage are already determined as hemocytes at the blastoderm stage. Unexpectedly, these hemocytes do not degenerate during late larval stages, but have the capacity to persist through metamorphosis and are still detectable in the adult fly. A second anlage, which gives rise to additional hemocytes at the onset of metamorphosis, is located within the thoracic mesoderm at 50 to 53% egg length. After transplantation within this region, clones were detected in the larval lymph glands. Labeled hemocytes are released by the lymph glands not before the late third larval instar. The anlage of these lymph gland-derived hemocytes is not determined at the blastoderm stage, as indicated by the overlap of clones with other tissues. Our analyses reveal that the hemocytes of pupae and adult flies consist of a mixture of embryonic hemocytes and lymph gland-derived hemocytes, originating from two distinct anlagen that are determined at different stages of development.

The formation of syncytia within the visceral musculature of the Drosophila midgut is dependent on duf, sns and mbc.

The visceral musculature of the Drosophila midgut consists of an inner layer of circular and an outer layer of longitudinal muscles. Here, we show that the circular muscles are organised as binucleate syncytia that persist through metamorphosis. At stage 11, prior to the onset of the fusion processes, we detected two classes of myoblasts within the visceral trunk mesoderm. One class expresses the founder-cell marker rP298-LacZ in a one- to two-cells-wide strip along the ventralmost part of the visceral mesoderm, whereas the adjacent two to three cell rows are characterised by the expression of Sticks-and-stones (SNS). During the process of cell fusion at stage 12 SNS expression decreases within the newly formed syncytia that spread out dorsally over the midgut. At both margins of the visceral band several cells remain unfused and continue to express SNS. Additional rP298-LacZ-expressing cells arise from the posterior tip of the mesoderm, migrate anteriorly and eventually fuse with the remaining SNS-expressing cells, generating the longitudinal muscles. Thus, although previous studies proposed a separate primordium for the longitudinal musculature located at the posteriormost part of the mesoderm anlage, our cell lineage analyses as well as our morphological observations reveal that a second population of cells originates from the trunk mesoderm. Mutations of genes that are involved in somatic myoblast fusion, such as sns, dumbfounded (duf) or myoblast city (mbc), also cause severe defects within the visceral musculature. The circular muscles are highly unorganised while the longitudinal muscles are almost absent. Thus the fusion process seems to be essential for a proper visceral myogenesis. Our results provide strong evidence that the founder-cell hypothesis also applies to visceral myogenesis, employing the same genetic components as are used in the somatic myoblast fusion processes.