Marcio Fontenele

Position matters: variability in the spatial pattern of BMP modulators generates functional diversity.

Bone morphogenetic proteins (BMPs) perform a variety of functions during development. Considering a single BMP, what enables its multiple roles in tissues of varied sizes and shapes? What regulates the spatial distribution and activity patterns of the BMP in these different developmental contexts? Some BMP functions require controlling spread of the BMP morphogen, while others require formation of localized, high concentration peaks of BMP activity. Here we review work in Drosophila that describes spatial regulation of the BMP encoded by decapentaplegic (dpp) indifferent developmental contexts. We concentrate on extracellular modulation of BMP function and discuss the mechanisms that generate concentrated peaks of Dpp activity, subdivide territories of different activity levels or regulate spread of the Dpp morphogen from a point source. We compare these findings with data from vertebrates and non‐model organisms to discuss how changes in the regulation of Dpp distribution by extracellular modulators may lead to variability in dpp function in different species. genesis 49:698–718, 2011.

The embryogenesis of the Tick Rhipicephalus (Boophilus) microplus: The establishment of a new chelicerate model system

Summary: Chelicerates, which include spiders, ticks, mites, scorpions, and horseshoe crabs, are members of the phylum Arthropoda. In recent years, several molecular experimental studies of chelicerates have examined the embryology of spiders; however, the embryology of other groups, such as ticks (Acari: Parasitiformes), has been largely neglected. Ticks and mites are believed to constitute a monophyletic group, the Acari. Due to their blood‐sucking activities, ticks are also known to be vectors of several diseases. In this study, we analyzed the embryonic development of the cattle tick, Rhipicephalus (Boophilus) microplus (Acari: Ixodidae). First, we developed an embryonic staging system consisting of 14 embryonic stages. Second, histological analysis and antibody staining unexpectedly revealed the presence of a population of tick cells with similar characteristics to the spider cumulus. Cumulus cell populations also exist in other chelicerates; these cells are responsible for the breaking of radial symmetry through bone morphogenetic protein signaling. Third, it was determined that the posterior (opisthosomal) embryonic region of R. microplus is segmented. Finally, we identified the presence of a transient ventral midline furrow and the formation and regression of a fourth leg pair; these features may be regarded as hallmarks of late tick embryogenesis. Importantly, most of the aforementioned features are absent from mite embryos, suggesting that mites and ticks do not constitute a monophyletic group or that mites have lost these features. Taken together, our findings provide fundamental common ground for improving knowledge regarding tick embryonic development, thereby facilitating the establishment of a new chelicerate model system. genesis 51:803–818.

The Ca2+-dependent protease Calpain A regulates Cactus/IκB levels during Drosophila development in response to maternal Dpp signals

Regulation of NF kappaB activity is central to many processes during development and disease. Activation of NF kappaB family members depends on degradation of inhibitory I kappaB proteins. In Drosophila, a nuclear gradient of the NF kappaB/c-rel protein Dorsal subdivides the embryonic dorsal-ventral axis, defining the extent and location of mesodermal and ectodermal territories. Activation of the Toll pathway directs Dorsal nuclear translocation by inducing proteosomal degradation of the I kappaB homologue Cactus. Another mechanism that impacts on Dorsal activation involves the Toll-independent pathway, which regulates constitutive Cactus degradation. We have shown that the BMP protein Decapentaplegic (Dpp) inhibits Cactus degradation independent of Toll. Here we report on a novel element of this pathway: the calcium-dependent protease Calpain A. Calpain A knockdowns increase Cactus levels, shifting the Dorsal gradient and dorsal-ventral patterning. As shown for mammalian I kappaB, this effect requires PEST sequences in the Cactus C-terminus, implying a conserved role for calpains. Alteration of Calpain A or dpp results in similar effects on Dorsal target genes. Epistatic analysis confirms Calpain A activity is regulated by Dpp, indicating that Dpp signals increase Cactus levels through Calpain A inhibition, thereby interfering with Dorsal activation. This mechanism may allow coordination of Toll, BMP and Ca(2+) signals, conferring precision to Dorsal-target expression domains.

Toll signals regulate dorsal–ventral patterning and anterior–posterior placement of the embryo in the hemipteran Rhodnius prolixus

BackgroundInsect embryonic dorso-ventral patterning depends greatly on two pathways: the Toll pathway and the Bone Morphogenetic Protein pathway. While the relative contribution of each pathway has been investigated in holometabolous insects, their role has not been explored in insects with a hemimetabolous type of development. The hemimetabolous insect Rhodnius prolixus, an important vector of Chagas disease in the Americas, develops from an intermediate germ band and displays complex movements during katatrepsis that are not observed in other orders. However, little is known about the molecular events that regulate its embryogenesis. Here we investigate the expression and function of genes potentially involved in the initial patterning events that establish the embryonic dorso-ventral axis in this hemipteran.ResultsWe establish a staging system for early embryogenesis that allows us to correlate embryo morphology with gene expression profiles. Using this system, we investigate the role of Toll pathway genes during embryogenesis. Detailed analyses of gene expression throughout development, coupled with functional analyses using parental RNA interference, revealed that maternal Toll is required to establish germ layers along the dorso-ventral axis and for embryo placement along the anterior-posterior axis. Interestingly, knockdown of the Toll pathway effector Rp-dorsal appears to regulate the expression of the Bone Morphogenetic Protein antagonist Rp-short-gastrulation.ConclusionsOur results indicate that Toll signals are the initiating event in dorso-ventral patterning during Rhodnius embryogenesis, and this is the first report of a conserved role for Toll in a hemipteran. Furthermore, as Rp-dorsal RNA interference generates anteriorly misplaced embryos, our results indicate a novel role for Toll signals in establishment of the anterior-posterior axis in Rhodnius.

Calpain A modulates Toll responses by limited Cactus/IκB proteolysis

The calcium-dependent protease Calpain A targets Cactus/IκB for proteolysis. Calpain A generates Cactus fragments deleted of Toll-responsive N-terminal sequences that are incorporated into signaling complexes with NFκB/c-Rel. It is proposed that Calpain A regulates free Cactus and modulates Toll signals in the Drosophila embryo and immune system.

αPS1βPS integrin receptors regulate the differential distribution of Sog fragments in polarized epithelia

Bone morphogenetic proteins (BMPs) have important functions during epithelial development. In Drosophila, extracellular Short gastrulation (Sog) limits the action of the BMP family member Decapentaplegic (Dpp). We have shown that Integrin receptors regulate Sog activity and distribution during pupal wing development to direct placement of wing veins. Here, we show that Integrins perform a similar function in the follicular epithelium, impacting Dpp function during oogenesis and embryonic development. As reported for the wing, this effect is specific to mew, which codes for αPS1 integrin. Sog is subject to cleavage by metalloproteases, generating fragments with different properties. We also show that Integrins regulate the distribution of C‐ and N‐terminal Sog fragments in both epithelia, suggesting they may regulate the quality of BMP outputs. Our data indicate that αPS1βPS integrin receptors regulate the amount and type of Sog fragments available for diffusion in the extracellular space during oogenesis and pupal wing development. genesis 48:31–43, 2010.

Knockdown of Lmo7 inhibits chick myogenesis

The multifunctional protein Lmo7 has been implicated in some aspects of myogenesis in mammals. Here we studied the distribution and expression of Lmo7 and the effects of Lmo7 knockdown in primary cultures of chick skeletal muscle cells. Lmo7 was localized within the nuclei of myoblasts and at the perinuclear region of myotubes. Knockdown of Lmo7 using siRNA specific to chick reduces the number and width of myotubes and the number of MyoD positive‐myoblasts. Both Wnt3a enriched medium and Bio, activators of the Wnt/beta‐catenin pathway, could rescue the effects of the Lmo7 knockdown suggesting a crosstalk between the Wnt/beta‐catenin and Lmo7‐mediated signaling pathways. Our data shows a role of Lmo7 during the initial events of chick skeletal myogenesis, particularly in myoblast survival.

A novel function for the IκB inhibitor Cactus in promoting Dorsal nuclear localization and activity in the Drosophila embryo

ABSTRACT The evolutionarily conserved Toll signaling pathway controls innate immunity across phyla and embryonic patterning in insects. In the Drosophila embryo, Toll is required to establish gene expression domains along the dorsal-ventral axis. Pathway activation induces degradation of the IκB inhibitor Cactus, resulting in a ventral-to-dorsal nuclear gradient of the NFκB effector Dorsal. Here, we investigate how cactus modulates Toll signals through its effects on the Dorsal gradient and on Dorsal target genes. Quantitative analysis using a series of loss- and gain-of-function conditions shows that the ventral and lateral aspects of the Dorsal gradient can behave differently with respect to Cactus fluctuations. In lateral and dorsal embryo domains, loss of Cactus allows more Dorsal to translocate to the nucleus. Unexpectedly, cactus loss-of-function alleles decrease Dorsal nuclear localization ventrally, where Toll signals are high. Overexpression analysis suggests that this ability of Cactus to enhance Toll stems from the mobilization of a free Cactus pool induced by the Calpain A protease. These results indicate that Cactus acts to bolster Dorsal activation, in addition to its role as a NFκB inhibitor, ensuring a correct response to Toll signals. Summary: During dorsoventral patterning of the Drosophila embryo, ventral and lateral aspects of the embryonic Dorsal/NFκB gradient can behave differently in response to variations in Cactus concentration.

Balance between S-nitrosylation and denitrosylation modulates myoblast proliferation independently of soluble guanylyl cyclase activation

Nitric oxide (NO) contributes to myogenesis by regulating the transition between myoblast proliferation and fusion through cGMP signaling. NO can form S-nitrosothiols (RSNO), which control signaling pathways in many different cell types. However, neither the role of RSNO content nor its regulation by the denitrosylase activity of S-nitrosoglutathione reductase (GSNOR) during myogenesis is understood. Here, we used primary cultures of chick embryonic skeletal muscle cells to investigate whether changes in intracellular RSNO alter proliferation and fusion of myoblasts in the presence and absence of cGMP. Cultures were grown to fuse most of the myoblasts into myotubes, with and without S-nitrosocysteine (CysNO), 8-Br-cGMP, DETA-NO, or inhibitors for NO synthase (NOS), GSNOR, soluble guanylyl cyclase (sGC), or a combination of these, followed by analysis of GSNOR activity, protein expression, RSNO, cGMP, and cell morphology. Although the activity of GSNOR increased progressively over 72 h, inhibiting GSNOR (by GSNOR inhibitor - GSNORi - or by knocking down GSNOR with siRNA) produced an increase in RSNO and in the number of myoblasts and fibroblasts, accompanied by a decrease in myoblast fusion index. This was also detected with CysNO supplementation. Enhanced myoblast number was proportional to GSNOR inhibition. Effects of the GSNORi and GSNOR knockdown were blunted by NOS inhibition, suggesting their dependence on NO synthesis. Interestingly, GSNORi and GSNOR knockdown reversed the attenuated proliferation obtained with sGC inhibition in myoblasts, but not in fibroblasts. Hence myoblast proliferation is enhanced by increasing RSNO, and regulated by GSNOR activity, independently of cGMP production and signaling.

γ-Secretase Inhibition Induces Muscle Hypertrophy in a Notch-Independent Mechanism

A wide variety of cellular processes and signaling events are regulated by the proteolytic enzyme γ‐secretase. Notch‐1 is one of the substrates of γ‐secretase and its role in the regulation of muscle differentiation has been well described. Importantly, besides Notch‐1, a number of proteins have been identified to undergo proteolysis by γ‐secretase. To date, the specific role of γ‐secretase during embryonic skeletal muscle differentiation has not been studied. Therefore, we address this question through the analysis of in vitro grown chick myogenic cells during the formation of multinucleated myotubes. The γ‐secretase inhibitor DAPT (N‐N[‐(3,5‐Difluorophenacetyl‐l‐alanyl)]‐S‐328 phenylglycine‐t‐butyl‐ester) induces muscle hypertrophy. Knockdown of Notch‐1 using siRNA specific to chick shows no significant effect in myotube size, suggesting that γ‐secretase‐dependent effects on muscle hypertrophy in chick myogenic cells are Notch‐1‐independent. We also investigate the effects of γ‐secretase inhibition in the whole proteomic profile of chick myogenic cells. We identified 276 differentially expressed proteins from Label‐free proteomic approach. Data overview of interaction network obtained from STRING show that after γ‐secretase inhibition cells exhibited imbalance in protein metabolism, cytoskeleton/adhesion, and Sonic Hedgehog signaling. The collection of these results provides new insights into the role of γ‐secretase in skeletal muscle hypertrophy.