Digital development and morphogenesis.

Signalling interactions between the polarizing region, which produces SHH, and the apical ectodermal ridge, which produces FGFs, are essential for outgrowth and patterning of vertebrate limbs. However, mechanisms that mediate translation of early positional information of cells into anatomy remain largely unknown. In particular, the molecular and cellular basis of digit morphogenesis are not fully understood, either in terms of the formation of the different digits along the antero-posterior axis or in the way digits stop growing once pattern formation has been completed. Here we will review recent data about digit development. Manipulation of morphogenetic signals during digit formation, including application of SHH interdigitally, has shown that digit primordia possess a certain plasticity, and that digit anatomy becomes irreversibly fixed during morphogenesis. The process of generation of joints and thus segmentation and formation of digit tips is also discussed.

PA subunit from influenza virus polymerase complex interacts with a cellular protein with homology to a family of transcriptional activators.

The PA subunit of the influenza virus polymerase complex is a phosphoprotein that induces proteolytic degradation of coexpressed proteins. Point mutants with reduced proteolysis induction reconstitute viral ribonucleoproteins defective in replication but not in transcriptional activity. To look for cellular factors that could associate with PA protein, we have carried out a yeast two-hybrid screen. Using a human kidney cDNA library, we identified two different interacting clones. One of them was identified as the human homologue of a previously described cDNA clone from Gallus gallus called CLE. The human gene encodes a protein of 36 kDa (hCLE) and is expressed ubiquitously in all human organs tested. The interaction of PA and hCLE was also observed with purified proteins in vitro by using pull-down and pep-spot experiments. Mapping of the interaction showed that hCLE interacts with PA subunit at two regions (positions 493 to 512 and 557 to 574) in the PA protein sequence. Immunofluorescence studies showed that the hCLE protein localizes in both the nucleus and the cytosol, although with a predominantly cytosolic distribution. hCLE was found associated with active, highly purified virus ribonucleoproteins reconstituted in vivo from cloned cDNAs, suggesting that PA-hCLE interaction is functionally relevant. Searches in the databases showed that hCLE has 38% sequence homology to the central region of the yeast factor Cdc68, which modulates transcription by interaction with transactivators. Similar homologies were found with the other members of the Cdc68 homologue family of transcriptional activators, including the human FACT protein.

Role of FGFs in the control of programmed cell death during limb development.

We have investigated the role of FGFs in the control of programmed cell death during limb development by analyzing the effects of increasing and blocking FGF signaling in the avian limb bud. BMPs are currently considered as the signals responsible for cell death. Here we show that FGF signaling is also necessary for apoptosis and that the establishment of the areas of cell death is regulated by the convergence of FGF- and BMP-mediated signaling pathways. As previously demonstrated, cell death is inhibited for short intervals (12 hours) after administration of FGFs. However, this initial inhibition is followed (24 hours) by a dramatic increase in cell death, which can be abolished by treatments with a BMP antagonist (Noggin or Gremlin). Conversely, blockage of FGF signaling by applying a specific FGF-inhibitor (SU5402) into the interdigital regions inhibits both physiological cell death and that mediated by exogenous BMPs. Furthermore, FGF receptors 1, 2 and 3 are expressed in the autopodial mesoderm during the regression of the interdigital tissue, and the expression of FGFR3 in the interdigital regions is regulated by FGFs and BMPs in the same fashion as apopotosis. Together our findings indicate that, in the absence of FGF signaling BMPs are not sufficient to trigger apoptosis in the developing limb. Although we provide evidence for a positive influence of FGFs on BMP gene expression, the physiological implication of FGFs in apoptosis appears to result from their requirement for the expression of genes of the apoptotic cascade. We have identified MSX2 and Snail as candidate genes associated with apoptosis the expression of which requires the combined action of FGFs and BMPs.

"Fingering" the vertebrate limb.

A detailed and precise picture is being pieced together about how the pattern of digits develops in vertebrate limbs. What is particularly exciting is that it will soon be possible to trace the process all the way from establishment of a signalling centre in a small bud of undifferentiated cells right through to final limb anatomy. The development of the vertebrate limb is a traditional model in which to explore mechanisms involved in pattern formation, and there is accelerating knowledge about the genes involved. One reason why the limb is holding its place in the post-genomic age is that it is rich in pre-genomic embryology. Here, we will focus on recent findings about the aspect of vertebrate limb development concerned with digit pattern across the anteroposterior axis of the limb. This process is controlled by a signalling region in the early limb bud known as the polarizing region. Interactions between polarizing region cells and other cells in the limb bud ensure that a thumb develops at one edge of the hand (anterior) and a little finger at the other (posterior).

Autoregulation of Shh expression and Shh induction of cell death suggest a mechanism for modulating polarising activity during chick limb development.

The polarising region expresses the signalling molecule sonic hedgehog (Shh), and is an embryonic signalling centre essential for outgrowth and patterning of the vertebrate limb. Previous work has suggested that there is a buffering mechanism that regulates polarising activity. Little is known about how the number of Shh-expressing cells is controlled but, paradoxically, the polarising region appears to overlap with the posterior necrotic zone, a region of programmed cell death. We have investigated how Shh expression and cell death respond when levels of polarising activity are altered, and show an autoregulatory effect of Shh on Shh expression and that Shh affects cell death in the posterior necrotic zone. When we increased Shh signalling, by grafting polarising region cells or applying Shh protein beads, this led to a reduction in the endogenous Shh domain and an increase in posterior cell death. In contrast, cells in other necrotic regions of the limb bud, including the interdigital areas, were rescued from death by Shh protein. Application of Shh protein to late limb buds also caused alterations in digit morphogenesis. When we reduced the number of Shh-expressing cells in the polarising region by surgery or drug-induced killing, this led to an expansion of the Shh domain and a decrease in the number of dead cells. Furthermore, direct prevention of cell death using a retroviral vector expressing Bcl2 led to an increase in Shh expression. Finally, we provide evidence that the fate of some of the Shh-expressing cells in the polarising region is to undergo apoptosis and contribute to the posterior necrotic zone during normal limb development. Taken together, these results show that there is a buffering system that regulates the number of Shh-expressing cells and thus polarising activity during limb development. They also suggest that cell death induced by Shh could be the cellular mechanism involved. Such an autoregulatory process based on cell death could represent a general way for regulating patterning signals in embryos.

A model for anteroposterior patterning of the vertebrate limb based on sequential long- and short-range Shh signalling and Bmp signalling.

It has been proposed that digit identity in chick limb bud is specified in a dose-dependent fashion by a long-range morphogen, produced by the polarising region. One candidate is Sonic hedgehog (Shh) protein, but it is not clear whether Shh acts long or short range or via Bmps. Here we dissect the relationship between Shh and Bmp signalling. We show that Shh is necessary not only for initiating bmp2 expression but also for sustaining its expression during the period when additional digits are being specified. We also show that we can reproduce much of the effect of Shh during this period by applying only Bmp2. We further demonstrate that it is Bmps that are responsible for digit specification by transiently adding Noggin or Bmp antibodies to limbs treated with Shh. In such limbs, multiple additional digits still form but they all have the same identity. We also explored time dependency and range of Shh signalling by examining ptc expression. We show that high-level ptc expression is induced rapidly when either Shh beads or polarising regions are grafted to a host limb. Furthermore, we find that high-level ptc expression is first widespread but later more restricted. All these data lead us to propose a new model for digit patterning. We suggest that Shh initially acts long range to prime the region of the limb competent to form digits and thus control digit number. Then later, Shh acts short range to induce expression of Bmps, whose morphogenetic action specifies digit identity.

The replication activity of influenza virus polymerase is linked to the capacity of the PA subunit to induce proteolysis.

The PA subunit of the influenza virus polymerase complex is a phosphorylated protein that induces a proteolytic process that decreases its own accumulation levels and those of coexpressed proteins. The amino-terminal third of the protein is responsible for the induction of proteolysis. We mutated five potential casein kinase II phosphorylation sites located in the amino-terminal third of the protein. Mutations affecting position 157 almost completely abrogated proteolysis induction, whereas a mutation at position 162 produced a moderate decrease and mutations at positions 151, 200, and 224 did not affect proteolysis induction. Reconstitution of the influenza virus polymerase in vivo with viral model RNA containing the chloramphenicol acetyltransferase (CAT) gene indicated that the CAT activity obtained correlated with the capacity of each PA mutant to induce proteolysis. RNA protection assays of the products obtained with viral polymerase, reconstituted in vivo with model RNAs, indicated that mutations at position 157 led to a selective loss of the ability to synthesize cRNA from the viral RNA template but not to transcribe viral RNA, while a mutation affecting position 162 showed an intermediate phenotype. Collectively, these data provide a link between PA-mediated induction of proteolysis and the replication activity of the polymerase.

The PA influenza virus polymerase subunit is a phosphorylated protein.

The induction of proteolysis by expression of the influenza virus PA polymerase subunit is the only biochemical activity ascribed to this protein. In the course of studying viral protein synthesis by two-dimensional gel electrophoresis, we observed the existence of several PA isoforms with different isoelectric points. These isoforms were also present when the PA gene was singly expressed in three different expression systems, indicating that a cellular activity is responsible for its post-translational modification. In vivo labelling with [32P]orthophosphate, followed by two-dimensional gel electrophoresis, clearly demonstrated the incorporation of phosphate into the PA molecule. Phosphoserine and phosphothreonine epitopes were present in PA, while phosphotyrosine residues were absent, as tested by immunoblotting with specific antibodies. These facts, as well as the presence of multiple consensus sites for casein kinase II (CKII) phosphorylation, prompted us to test the involvement of this kinase in PA covalent modification. PA protein purified by immunoprecipitation could be specifically labelled by the catalytic alpha subunit of human CKII, which was expressed and purified from bacteria. Collectively, these data demonstrate that the PA subunit of the influenza virus RNA polymerase is a phosphoprotein.

Mutational analysis of the influenza virus A/Victoria/3/75 PA protein: studies of interaction with PB1 protein and identification of a dominant negative mutant.

The RNA polymerase activity and PB1 binding of influenza virus PA mutants were studied using an in vivo-reconstituted polymerase assay and a two hybrid system. Deletions covering the whole PA protein abolished polymerase activity, but the deletion of the 154 N-terminal amino acids allowed PB1 binding, indicating that the PA protein N terminus is not absolutely required for this interaction. Further internal or C-terminal deletions abolished PB1 interaction, suggesting that most of the protein is involved in this association. As a novel finding we showed that a single amino acid insertion mutant, PAI672, was responsible for a temperature-sensitive phenotype. Mutant PAS509, which had a serine insertion at position 509, bound to PB1 like wild-type PA but did not show any polymerase activity. Over-expression of PAS509 interfered with the polymerase activity of wild-type PA, identifying PAS509 as a dominant negative mutant.

The amino-terminal one-third of the influenza virus PA protein is responsible for the induction of proteolysis.

We have previously described the fact that the individual expression of influenza virus PA protein induced a generalized proteolysis (J.J. Sanz-Ezquerro, S. de la Luna, Ortin, and A. Nieto, J. Virol. 69:2420-2426, 1995). In this study, we have further characterized this effect by mapping the regions of PA protein required and have found by deletion analysis that the first 247 amino acids are sufficient to bring about this activity. PA mutants that were able to decrease the accumulation levels of coexpressed proteins also presented lower steady-state levels due to a reduction in their half-lives. Furthermore, the PA wild type produced a decrease in the stationary levels of different PA versions, indicating that is itself a target for its induced proteolytic process. All of the PA proteins that induced proteolysis presented nuclear localization, being the sequences responsible for nuclear transport located inside the first 247 amino acids of the molecule. To distinguish between the regions involved in nuclear localization and those involved in induction of proteolysis, we fused the nuclear localization signal of the simian virus 40 T antigen to the carboxy terminus of the cytosolic versions of PA. None of the cytosolic PA versions affected in the first 247-amino-acid part of PA, which were now located in the nucleus, were able to induce proteolysis, suggesting that conservation of a particular conformation in this region of the molecule is required for the effect observed. The fact that all of the PA proteins able to induce proteolysis presented nuclear localization, together with the observation that this activity is shared by influenza virus PA proteins from two different type A viruses, suggests a physiological role for this PA protein activity in viral infection.

Individual expression of influenza virus PA protein induces degradation of coexpressed proteins.

In the process of in vivo reconstitution of influenza virus transcriptase-replicase complex, an inhibitory effect was observed when the level of PA protein expression was increased. This inhibition was paralleled by a decrease in the accumulation of the other influenza virus core proteins. The sole expression of PA protein was sufficient to reduce the accumulation level of the proteins encoded by the coexpressed genes. The PA effect was observed upon influenza virus and non-influenza virus proteins and independently of the expression system chosen and the origin of cell line used. The expression of PA protein did not induce variations in the translation of the target proteins but did induce variations on their half-lives, which were clearly reduced. A functional PA subunit seems to be necessary to induce this negative effect, because an inactive point mutant was unable to decrease the steady-state levels or the half-lives of the reporter proteins. The PA effect was observed as early as 5 h after its expression, and continuous synthesis of proteins was not required for performance of its biological activity. The results presented represent the first biological activity of individually expressed PA polymerase subunit.