Zebrafish bmp4 functions during late gastrulation to specify ventroposterior cell fates

Bone morphogenetic proteins (BMPs) are key mediators of dorsoventral patterning in vertebrates and are required for the induction of ventral fates in fish and frogs. A widely accepted model of dorsoventral patterning postulates that a morphogenetic BMP activity gradient patterns cell fates along the dorsoventral axis. Recent work in zebrafish suggests that the role of BMP signaling changes over time, with BMPs required for global dorsoventral patterning during early gastrulation and for tail patterning during late gastrulation and early somitogenesis. Key questions remain about the late phase, including which BMP ligands are required and how the functions of BMPs differ during the early and late gastrula stages. In a screen for dominant enhancers of mutations in the homeobox genes vox and vent, which function in parallel to bmp signaling, we identified an insertion mutation in bmp4. We then performed a reverse genetic screen to isolate a null allele of bmp4. We report the characterization of these two alleles and demonstrate that BMP4 is required during the later phase of BMP signaling for the specification of ventroposterior cell fates. Our results indicate that different bmp genes are essential at different stages. In addition, we present genetic evidence supporting a role for a morphogenetic BMP gradient in establishing mesodermal fates during the later phase of BMP signaling.     

Dynamic control of Dps protein levels by ClpXP and ClpAP proteases in Escherichia coli

The Escherichia coli starvation-induced DNA protection protein Dps was observed to be degraded rapidly during exponential growth. This turnover is dependent on the clpP and clpX genes. The clpA gene is not required for Dps proteolysis, suggesting that Dps is a substrate for ClpXP protease but not for ClpAP protease. Dps proteolysis was found to be highly regulated. Upon carbon starvation, Dps is stabilized, which together with increased Dps synthesis allows strong accumulation of Dps in the stationary phase. The addition of glucose to starving cells results in rapid resumption of Dps proteolysis by ClpXP. Oxidative stress also leads to efficient stabilization of Dps. After hyperosmotic shift, however, proteolysis remains unaffected. Thus, regulated proteolysis of Dps strongly contributes to controlling Dps levels under very specific stress conditions. In contrast to the regulated degradation of RpoS by ClpXP, Dps proteolysis is independent of the recognition factor RssB. In addition, during starvation, clpP and, to a somewhat lesser extent, clpA are involved in maintaining ongoing Dps synthesis (acting at the level of Dps translation), which is required for strong Dps accumulation in long-term stationary phase cells. In summary, both ClpXP and ClpAP exert significant control of Dps levels by affecting log phase stability and stationary phase synthesis of Dps respectively.     

The Thick Veins Gene of Drosophila Is Required for Dorsoventral Polarity of the Embryo

We have discovered a new member of the class of genes controlling embryonic dorsoventral patterning. Mutants of the thick veins (tkv) gene have been described previously (as slater alleles) as embryonic lethal, lacking dorsal epidermis, but not as showing a recognizable dorsoventral phenotype. We show here that maternal alteration of function coupled with zygotic reduction of function of tkv is strongly ventralizing. In addition, in double heterozygous combinations in the mother, tkv mutations increase the ventralizing effect of dominant, weakly ventralizing alleles of the maternal effect, dorsoventral genes easter and cactus. An interaction is also seen with zygotic dorsoventral genes: tkv interacts maternally and zygotically in double heterozygotes with decapentaplegic and zygotically with screw in double homozygotes. We conclude that both maternally and zygotically supplied wild-type tkv product can play a role in dorsoventral patterning of the early embryo. On the basis of the phenotype of trans-heterozygous adult escapers, we propose that tkv might act by potentiating the activity of the zygotically acting decapentaplegic gene.     

Regulated proteolysis of a transcription factor complex is critical to cell cycle progression in Caulobacter crescentus

Cell cycle transitions are often triggered by the proteolysis of key regulatory proteins. In Caulobacter crescentus, the G1‐S transition involves the degradation of an essential DNA‐binding response regulator, CtrA, by the ClpXP protease. Here, we show that another critical cell cycle regulator, SciP, is also degraded during the G1‐S transition, but by the Lon protease. SciP is a small protein that binds directly to CtrA and prevents it from activating target genes during G1. We demonstrate that SciP must be degraded during the G1‐S transition so that cells can properly activate CtrA‐dependent genes following DNA replication initiation and the reaccumulation of CtrA. These results indicate that like CtrA, SciP levels are tightly regulated during the Caulobacter cell cycle. In addition, we show that formation of a complex between CtrA and SciP at target promoters protects both proteins from their respective proteases. Degradation of either protein thus helps trigger the destruction of the other, facilitating a cooperative disassembly of the complex. Collectively, our results indicate that ClpXP and Lon each degrade an important cell cycle regulator, helping to trigger the onset of S phase and prepare cells for the subsequent programmes of gene expression critical to polar morphogenesis and cell division.     

Genes that control dorsoventral polarity affect gene expression along the anteroposterior axis of the Drosophila embryo

At least 13 genes control the establishment of dorsoventral polarity in the Drosophila embryo and more than 30 genes control the anteroposterior pattern of body segments. Each group of genes is thought to control pattern formation along one body axis, independently of the other group. We have used the expression of the fushi tarazu (ftz) segmentation gene as a positional marker to investigate the relationship between the dorsoventral and anteroposterior axes. The ftz gene is normally expressed in seven transverse stripes. Changes in the striped pattern in embryos mutant for other genes (or progeny of females homozygous for maternal-effect mutations) can reveal alterations of cell fate resulting from such mutations. We show that in the absence of any of ten maternal-effect dorsoventral polarity gene functions, the characteristic stripes of ftz protein are altered. Normally there is a difference between ftz stripe spacing on the dorsal and ventral sides of the embryo; in dorsalized mutant embryos the ftz stripes appear to be altered so that dorsal-type spacing occurs on all sides of the embryo. These results indicate that cells respond to dorsoventral positional information in establishing early patterns of gene expression along the anteroposterior axis and that there may be more significant interactions between the different axes of positional information than previously determined.     

Sepsis upregulates the gene expression of multiple ubiquitin ligases in skeletal muscle

Muscle wasting during sepsis reflects increased expression and activity of the ubiquitin-proteasome proteolytic pathway and is at least in part mediated by glucocorticoids. The ubiquitination of proteins destined to be degraded by the proteasome is regulated by multiple enzymes, including ubiquitin ligases. We tested the hypothesis that sepsis upregulates the gene expression of the newly described ubiquitin ligases, MuRF1 and atrogin-1/MAFbx. Sepsis was induced in rats by cecal ligation and puncture. Control rats were sham-operated. In some experiments, rats were treated with the glucocorticoid receptor antagonist RU 38486 before induction of sepsis. At various time points after induction of sepsis, mRNA levels for MuRF1 and atrogin-1/MAFbx were determined in extensor digitorum longus muscles by real-time PCR. Sepsis resulted in a 10-16-fold increase in gene expression of the ubiquitin ligases studied here. These changes were much greater than those observed previously for another ubiquitin ligase, E3alpha, in muscle during sepsis. Treatment of rats with RU 38486 prevented the sepsis-induced increase in mRNA levels for MuRF1 and atrogin-1/MAFbx, suggesting that glucocorticoids participate in the upregulation of these genes in muscle during sepsis. The present results lend further support to the concept that the ubiquitin-proteasome pathway plays an important role in sepsis-induced muscle proteolysis and suggest that multiple ubiquitin ligases may participate in the development of muscle wasting during sepsis.     

A gradient of BMP activity specifies dorsal-ventral fates in early Xenopus embryos.

BMP-4 is an extracellular signalling molecule belonging to the TGF-beta superfamily that plays a central role in dorsoventral patterning in vertebrate gastrulae. We review the evidence indicating that BMP-4 acts as a morphogen, specifying dorsoventral positional values in a concentration-dependent manner. An activity gradient of BMP-4 is established not by simple diffusion from a localised source, but by diffusion of inhibitory binding proteins that act on a uniform level of BMP-4 protein. These in turn are regulated by the activity of tolloid-related metalloproteases, such as Xenopus xolloid and zebrafish tolloid.     

The deubiquitylating enzyme Cops6 regulates different developmental processes during early zebrafish embryogenesis

Zebrafish cops6 encodes a putative deubiquitylating enzyme (DUB) that belongs to the JAMM family. It consists of 297 amino acids and includes the Mov34/MPN/PAD-1 (PF01398) domain. Ubiquitylation is involved in many cellular processes and deconjugation of ubiquitin-modified substrates is important to maintain a sufficient amount of free ubiquitin in the cell. Here, we report our findings regarding the general function of the cops6 gene, as a continuation of our previous studies involving DUB knockdown screening. We have found that cops6 plays different roles in early embryonic development in the zebrafish, including dorsoventral patterning, convergent extension movement and brain formation. In addition, our findings indicate that cops6 plays an anti-apoptotic role during segmentation. Overall, the present study that consolidates our previous work on zebrafish DUB genes, corroborates the hypothesis of multi-functional roles for DUB genes during development.     

Mediolateral patterning of somites: multiple axial signals, including Sonic hedgehog, regulate Nkx-3.1 expression

The axial structures, the notochord and the neural tube, play an essential role in the dorsoventral patterning of somites and in the differentiation of their many cell lineages. Here, we investigated the role of the axial structures in the mediolateral patterning of the somite by using a newly identified murine homeobox gene, Nkx-3.1, as a medial somitic marker in explant in vitro assays. Nkx-3.1 is dynamically expressed during somitogenesis only in the youngest, most newly-formed somites at the caudal end of the embryo. We found that the expression of Nkx-3.1 in pre-somitic tissue explants is induced by the notochord and maintained in newly-differentiated somites by the notochord and both ventral and dorsal parts of the neural tube. We showed that Sonic hedgehog (Shh) is one of the signaling molecules that can reproduce the effect of the axial structures by exposing explants to either COS cells transfected with a Shh expression construct or to recombinant SHH. Shh could induce and maintain Nkx-3.1 expression in pre-somitic mesoderm and young somites but not in more mature, differentiated ones. The effects of Shh on Nkx-3.1 expression were antagonized by a forskolin-induced increase in the activity of cyclic AMP-dependent protein kinase A. Additionally, we confirmed that the expression of the earliest expressed murine myogenic marker, myf 5, is also regulated by the axial strucutres but that Shh by itself is not capable of inducing or maintaining it. We suggest that the establishment of somitic medial and lateral compartments and the early events in myogenesis are governed by a combination of positive and inhibitory signals derived from the neighboring structures, as has previously been proposed for the dorsoventral patterning of somites.     

Temporal regulation of apterous activity during development of the Drosophila wing

Dorsoventral axis formation in the Drosophila wing depends on the activity of the selector gene apterous. Although selector genes are usually thought of as binary developmental switches, we find that Apterous activity is negatively regulated during wing development by its target gene dLMO. Apterous-dependent expression of Serrate and fringe in dorsal cells leads to the restricted activation of Notch along the dorsoventral compartment boundary. We present evidence that the ability of cells to participate in this Apterous-dependent cell-interaction is under spatial and temporal control. Apterous-dependent expression of dLMO causes downregulation of Serrate and fringe and allows expression of delta in dorsal cells. This limits the time window during which dorsoventral cell interactions can lead to localized activation of Notch and induction of the dorsoventral organizer. Overactivation of Apterous in the absence of dLMO leads to overexpression of Serrate, reduced expression of delta and concomitant defects in differentiation and cell survival in the wing primordium. Thus, downregulation of Apterous activity is needed to allow normal wing development.     

New Global Insights on the Regulation of the Biphasic Life Cycle and Virulence Via ClpP-Dependent Proteolysis in Legionella pneumophila

Legionella pneumophila, an environmental bacterium that parasitizes protozoa, causes Legionnaires’ disease in humans that is characterized by severe pneumonia. This bacterium adopts a distinct biphasic life cycle consisting of a nonvirulent replicative phase and a virulent transmissive phase in response to different environmental conditions. Hence, the timely and fine-tuned expression of growth and virulence factors in a life cycle–dependent manner is crucial for survival and replication. Here, we report that the completion of the biphasic life cycle and bacterial pathogenesis is greatly dependent on the protein homeostasis regulated by caseinolytic protease P (ClpP)-dependent proteolysis. We characterized the ClpP-dependent dynamic profiles of the regulatory and substrate proteins during the biphasic life cycle of L. pneumophila using proteomic approaches and discovered that ClpP-dependent proteolysis specifically and conditionally degraded the substrate proteins, thereby directly playing a regulatory role or indirectly controlling cellular events via the regulatory proteins. We further observed that ClpP-dependent proteolysis is required to monitor the abundance of fatty acid biosynthesis–related protein Lpg0102/Lpg0361/Lpg0362 and SpoT for the normal regulation of L. pneumophila differentiation. We also found that the control of the biphasic life cycle and bacterial virulence is independent. Furthermore, the ClpP-dependent proteolysis of Dot/Icm (defect in organelle trafficking/intracellular multiplication) type IVB secretion system and effector proteins at a specific phase of the life cycle is essential for bacterial pathogenesis. Therefore, our findings provide novel insights on ClpP-dependent proteolysis, which spans a broad physiological spectrum involving key metabolic pathways that regulate the transition of the biphasic life cycle and bacterial virulence of L. pneumophila, facilitating adaptation to aquatic and intracellular niches.     

Proteolysis and its regulation at the surface of Streptococcus pyogenes

Pathogenic bacteria often produce proteinases that are believed to be involved in virulence. Moreover, several host defence systems depend on proteolysis, demonstrating that proteolysis and its regulation play an important role during bacterial infections. Here, we discuss how proteolytical events are regulated at the surface of Streptococcus pyogenes during infection with this important human pathogen. Streptococcus pyogenes produces proteinases, and host proteinases are produced and released as a result of the infection. Streptococcus pyogenes also recruits host proteinase inhibitors to its surface, suggesting that proteolysis is tightly regulated at the bacterial surface. We propose that the initial phase of a S. pyogenes infection is characterized by inhibition of proteolysis and complement activity at the bacterial surface. This is achieved mainly through binding of host proteinase inhibitors and complement regulatory proteins to bacterial surface proteins. In a later phase of the infection, massive proteolytic activity will release bacterial surface proteins and degrade human tissues, thus facilitating bacterial spread. These proteolytic events are regulated both temporally and spatially, and should influence virulence and the outcome of S. pyogenes infections.