The formation of a complex multicellular organism from a single cell is one of the most amazing processes of biology. Embryonic development is characterised by the careful regulation of cellular ...behaviours such that cells proliferate, migrate, differentiate and form tissues at the correct place and time. These processes are genetically controlled and depend both on the history of cells, their lineage, and on the activities of signalling pathways, which coordinate the cell interactions leading to organogenesis. The aim of the Frontiers research topic “Signalling pathways in embryonic development” has been to provide a forum for experts in cell and developmental biology to share recent advances in the field of signalling during embryonic development. Sixteen articles in a variety of formats are united in this Topic, offering a valuable collection for researchers looking for an update in the knowledge of signalling pathways operating during embryogenesis. The works, focused mainly on vertebrates, explore different aspects of this theme from cell communication to organ formation and have implications for areas as distant as evolution or pathology. Understanding developmental signalling pathways is important for several reasons. It gives us information about basic mechanisms of cell function and interactions needed for morphogenesis and organogenesis. It uncovers the basis of congenital malformations, since errors at any step of cell signalling during development are a major cause of defects. This fundamental insight gives us clues to understand the mechanisms operating in evolution that explain diversity in form and function. And finally, it allows the identification of possible causes of disease in the adult organism (such as cancer or degenerative diseases) pinpointing possible targets for therapeutic approaches.
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.
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.
Birds have played a central role in many biological disciplines, particularly ecology, evolution, and behavior. The chicken, as a model vertebrate, also represents an important experimental system ...for developmental biologists, immunologists, cell biologists, and geneticists. However, genomic resources for the chicken have lagged behind those for other model organisms, with only 1845 nonredundant full-length chicken cDNA sequences currently deposited in the EMBL databank. We describe a large-scale expressed-sequence-tag (EST) project aimed at gene discovery in chickens (http://www.chick.umist.ac.uk). In total, 339,314 ESTs have been sequenced from 64 cDNA libraries generated from 21 different embryonic and adult tissues. These were clustered and assembled into 85,486 contiguous sequences (contigs). We find that a minimum of 38% of the contigs have orthologs in other organisms and define an upper limit of 13,000 new chicken genes. The remaining contigs may include novel avian specific or rapidly evolving genes. Comparison of the contigs with known chicken genes and orthologs indicates that 30% include cDNAs that contain the start codon and 20% of the contigs represent full-length cDNA sequences. Using this dataset, we estimate that chickens have approximately 35,000 genes in total, suggesting that this number may be a characteristic feature of vertebrates.
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.
Sonic Hedgehog (Shh) signaling by the polarizing region, at the posterior of the vertebrate limb bud, is pivotal in determining digit number and identity. Shh establishes a gradient of the ...bifunctional transcriptional effector, Gli3, with high levels of full-length activator (Gli3A) in the posterior bud, where digits form, and high levels of shorter repressor (Gli3R) in the anterior. Repressor formation depends on protein kinase A (PKA), but in
Drosophila, PKA also plays a role in activator function. Increasing PKA levels in chick limb development using Forskolin had no effect on posterior polarizing activity but weak polarizing activity, based on ligand-independent Shh signaling, was induced in anterior limb bud cells resulting in extra digits. Manipulating PKA activity levels directly with a retrovirus expressing activated PKA induced extra digits similar to those induced by Forskolin treatment suggesting that PKA may have a previously unrecognized positive role in Shh signaling in vertebrate limbs. Expressing dominant negative PKA also induced extra, sometimes multiple digits, from anterior limb bud demonstrating the negative role in Shh signaling. PKA levels in the limb bud are high posteriorly and low anteriorly, suggesting that PKA activity may influence the outcome of Shh signaling in normal development.
“Fingering” the vertebrate limb Sanz-Ezquerro, Juan Jose; Tickle, Cheryll
Differentiation (London),
December 2001, Volume:
69, Issue:
2
Journal Article
Peer reviewed
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 antero-posterior 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).
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