In 1976, a major symposium on mammalian development, ‘Embryogenesis in Mammals’, was held at the Ciba Foundation in London. The meeting marked a decade of progress in probing the cellular basis of mouse development; yet, as remarked by Dorothea Bennett at the time, the field had hardly been touched by molecular biology. Fifteen years later, (3-5 June, 1991), another Ciba Symposium, ‘Postimplantation Development in the Mouse’, chaired by Dr Anne McLaren (University College London), took place. In contrast to 1976, it soon became apparent that molecular approaches now dominate the field. Nevertheless, the conference began with a couple of non-molecular talks on two related, long standing problems that have stubbornly resisted definitive solutions, to date: gastrulation in the mouse embryo and the origin of the three germ layers of the fetus. Gastrulation takes place on the inside of the cup-shaped egg cylinder, and little has been known about it except that mesoderm formation involves movement of cells from the epiblast or primitive ectoderm out through the proximal-posterior ridge known as the primitive streak. K. Lawson (Hubrecht Laboratory, Utrecht) described the tracing of epiblast cell fates by cell marking techniques and showed the similarity of fate maps of the mouse, the chick and Ambystornu, despite obvious differences of shape and size in these embryos. P. Tam (Children’s Medical Research Foundation, Sydney) described other cell marking experiments, which show that the majority of the ectodermal and endodermal cells of the embryo arise by recruitment rather than movement through the primitive streak. R. Beddington (AFRC, Edinburgh) pointed out that definitive answers to questions of germ layer origin await the development of distinctive tissue-specific markers for the early embryo. The following two talks concentrated on a gene known to be crucial for early mesoderm formation, namely Brachyury ( T ) , first detected by its haploinsufficient phenotype of short-tailedness. T/ T homozygotes die as embryos, thc primary deficiency being an inability to form mesoderm, which leads to deficiencies in formation of the notochord, allantois and somites. Beddington presented a clonal analysis which suggests that T is cell autonomous. B. Hermann (Tubingen) presented molecular data on T , which shows high
conservation of the N-terminal region of the protein in mouse. Xenopus and zebrafish (and lesser conservation in the C-terminal region). The protein localizes to the nucleus but its precise function is still not known. Herrmann discussed visual evidence on abnormalities in the primitive streak that suggest that mesoderm formation is a two-step process, in which T is necessary for the second step, reception of a signal that ‘instructs’ pre-mesodermal cells to differentiate as mesoderm. Other talks focussed on genetic control of the determination and differentiation of later events in mesodermal tissues. M. Buckingham (Pasteur, Paris) described a family of helix-loop-helix (HLH) proteins (MyoD1, Myogenin, Myf5 and Myf6) involved in myogenic determination. These can form homodimers or heterodimers amongst themselves or with the inhibitory HLH protein, I d . The sequence of activations of these regulatory proteins, and of the muscle 3tructural genes. is different between the myotoines and limb bud and Buckingham hypothesized that the activations of the structural genes reflect different threshold requirements for different homo- and heterodimeric combinations of HLH proteins. R. Balling (Max Planck Institute, Gottingen) described the role of Pax-1, a homeobox-containing gene, identical to the classical locus undulated (un) and its role in skeletal development. Although uniformly expressed in the sclerotomes, mutations in un create their most severe defects in the lumbar region. The absence of skull defects may reflect the skeletogenic effects of the neural crest cells in the head region (the trunk neural crest cells not having this activity) and the relatively milder effects in the trunk may reflect partial functional redundancy by other genes of the Pcrx family. Kidney is a third mesodcrmal tissue whose developmental control wa5 addressed. L. Saxen (Helsinki) described data on syndecan and other adhesion molecules in the mesenchymal condensation reaction, while N. Hastie (Edinburgh) described thc Wilm’s tumor gene, which encodes a zinc-finger protein. This gene may be required in mesenchymal cells that participate in mesenchymal-epithelial cell interactions. Another key regulatory gene that was described is the male sex-determining gene, Sry. R. Lovell-Badge (Mill Hill, London) described experiments showing that Sry, related to several known transcriptional factors and DNA binding proteins, is a key gene in testis determination in the mouse. It is, thus, almost certainly, the long-postulated sex-determining gene of the Y chromosome ( T d y , for testis-determining locus of the Y, in mouse: TDF, for testis-determining factor in man.). A small genomic fragment containing Sry, and no other ORFs, can produce male development in chromosomally female (XX) mice, though not all wch transgenics develop as males (possibly because of position effects associated with certain insertion sites). Furthermore, and surprisingly, the human homolog, S R Y , cannot substitute for it in mice although it is
expressed (suggesting some mismatch in downstream regulation). The remaining group of talks were, broadly, on the subject of pattern-determining genes and genetic approaches to analyzing the function of these genes. One group of such genes are those that encode the secreted factors of the TGF-/3 family, described by B. Hogan (Vanderbilt, Tennessee). These may be involved in various cross-tissue interactions; some direct genetic evidence for such an interaction has been obtained in Drosophila for the decapentaplegic (dpp) gene, while in situs for several members of this gene family in the mouse are consistent with such activity. BMP-4 (bone rnorphogenetic protein-4) may be involved in specifying posterior and ventral mesoderm. Other pattern-determining genes, but of the transcriptional factor category, that were discussed included the Krox and Hox genes, the wnt genes, and the engrailed homologues. D. Wilkinson (Mill Hill, London) described segmentation in the vertebrate hindbrain, and the expression patterns of several Hox genes and Krox-20 in a segmental pattern within the rhombomeres (a pattern that is at least analogous to the metameric patterning of expression of the Antennapedia-like homeobox genes of Drosophila, a family to which the Hox genes belong). A. McMahon (Roche, NJ) described the Wnt family, and the results of inactivation of Wnt-l . These include elimination of the midbrain and cerebellum, and loss of expression of the two engrailed homologues, En-1 and En-2, in the areas that would normally express Wnt-I. The latter effect is, in some respects, reminiscent of the interaction in expression of wg and en in Drosophilu. The final three talks concentrated on mutant hunting and gene targetting experiments. E. Robertson (Columbia) described insertion mutations (generated by either retroviruses or transgenes) in embryonic stem (ES) cells, including one that produces its initial defect at the primitive streak stage. A. Bradley (Houston) described gene targetting experiments in ES cells and various strategies for obtaining ‘subtle’ mutations as well as knock-outs, for studying gene function. A. Joyner (Mt Sinai, Toronto) described both ‘gene trap’ and ‘enhancer trap’ strategies for identifying genes of developmental interest. In the former, expression is only obtained if the ZacZ construct is inserted directly into a reading frame; not surprisingly, it is highly
mutagenic. Enhancer traps are less mutagenic but surprisingly, only 30% of those obtained so far show regionally restricted expression. Perhaps the most unusual aspect of the meeting was the amount of time devoted to discussion of basic questions of procedure and philosophy. One set of recurring questions concerned the nature and ubiquity of functional redundancy and how such might be selected in evolution. A. McLaren pointed out that it is important to distinguish between ‘redundancy’ in the sociological sense (as in ‘I’ve just been made redundant’) versus that in the informational sense. In the latter, some degree of redundancy is essential for the accurate transmission of the message. With respect to selectability, even small advantages associated with having ‘back up’ functions (particularly if there are some unique features of the otherwise redundant functions) can undoubtedly be selected for in evolution. A second question which came up at several points was how one determines which genes are most ‘interesting’ for study. Since bizarre phenotypes can be associated with partial inactivation of mundane (housekeeping) functions, it is clear that phenotypes can only be a partial guide. A third issue, of practical import, is how workers in the field of mouse development can keep up with the growing number of expression patterns for all sorts of different genes. This kind of information is often essential in deciding which experiments to do next and, at present, there is no systematic way of keeping track of the patterns. Various suggestions for different kinds of data bases were discussed. While the 1976 meeting may have marked a culmination of sorts for the older. classical and descriptive field of mammalian development, this meeting may later be seen as a transitional one between the earlier field and the one that is coming into being. Molecular methods now dominate mouse developmental studies but the answers to many of the basic questions still remain to be found. One may confidently expect that the next Ciba Symposium on mammalian development (in 1996 or 2001?) will be as strong on the answers as on the questions and techniques. Adam S. Wilkins is with the Company of Biologists, Dept of Zoology, Downing St, Cambridge CB2 3EJ, CJK. I
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conservation of the N-terminal region of the protein in mouse. Xenopus and zebrafish (and lesser conservation in the C-terminal region). The protein localizes to the nucleus but its precise function is still not known. Herrmann discussed visual evidence on abnormalities in the primitive streak that suggest that mesoderm formation is a two-step process, in which T is necessary for the second step, reception of a signal that ‘instructs’ pre-mesodermal cells to differentiate as mesoderm. Other talks focussed on genetic control of the determination and differentiation of later events in mesodermal tissues. M. Buckingham (Pasteur, Paris) described a family of helix-loop-helix (HLH) proteins (MyoD1, Myogenin, Myf5 and Myf6) involved in myogenic determination. These can form homodimers or heterodimers amongst themselves or with the inhibitory HLH protein, I d . The sequence of activations of these regulatory proteins, and of the muscle 3tructural genes. is different between the myotoines and limb bud and Buckingham hypothesized that the activations of the structural genes reflect different threshold requirements for different homo- and heterodimeric combinations of HLH proteins. R. Balling (Max Planck Institute, Gottingen) described the role of Pax-1, a homeobox-containing gene, identical to the classical locus undulated (un) and its role in skeletal development. Although uniformly expressed in the sclerotomes, mutations in un create their most severe defects in the lumbar region. The absence of skull defects may reflect the skeletogenic effects of the neural crest cells in the head region (the trunk neural crest cells not having this activity) and the relatively milder effects in the trunk may reflect partial functional redundancy by other genes of the Pcrx family. Kidney is a third mesodcrmal tissue whose developmental control wa5 addressed. L. Saxen (Helsinki) described data on syndecan and other adhesion molecules in the mesenchymal condensation reaction, while N. Hastie (Edinburgh) described thc Wilm’s tumor gene, which encodes a zinc-finger protein. This gene may be required in mesenchymal cells that participate in mesenchymal-epithelial cell interactions. Another key regulatory gene that was described is the male sex-determining gene, Sry. R. Lovell-Badge (Mill Hill, London) described experiments showing that Sry, related to several known transcriptional factors and DNA binding proteins, is a key gene in testis determination in the mouse. It is, thus, almost certainly, the long-postulated sex-determining gene of the Y chromosome ( T d y , for testis-determining locus of the Y, in mouse: TDF, for testis-determining factor in man.). A small genomic fragment containing Sry, and no other ORFs, can produce male development in chromosomally female (XX) mice, though not all wch transgenics develop as males (possibly because of position effects associated with certain insertion sites). Furthermore, and surprisingly, the human homolog, S R Y , cannot substitute for it in mice although it is
expressed (suggesting some mismatch in downstream regulation). The remaining group of talks were, broadly, on the subject of pattern-determining genes and genetic approaches to analyzing the function of these genes. One group of such genes are those that encode the secreted factors of the TGF-/3 family, described by B. Hogan (Vanderbilt, Tennessee). These may be involved in various cross-tissue interactions; some direct genetic evidence for such an interaction has been obtained in Drosophila for the decapentaplegic (dpp) gene, while in situs for several members of this gene family in the mouse are consistent with such activity. BMP-4 (bone rnorphogenetic protein-4) may be involved in specifying posterior and ventral mesoderm. Other pattern-determining genes, but of the transcriptional factor category, that were discussed included the Krox and Hox genes, the wnt genes, and the engrailed homologues. D. Wilkinson (Mill Hill, London) described segmentation in the vertebrate hindbrain, and the expression patterns of several Hox genes and Krox-20 in a segmental pattern within the rhombomeres (a pattern that is at least analogous to the metameric patterning of expression of the Antennapedia-like homeobox genes of Drosophila, a family to which the Hox genes belong). A. McMahon (Roche, NJ) described the Wnt family, and the results of inactivation of Wnt-l . These include elimination of the midbrain and cerebellum, and loss of expression of the two engrailed homologues, En-1 and En-2, in the areas that would normally express Wnt-I. The latter effect is, in some respects, reminiscent of the interaction in expression of wg and en in Drosophilu. The final three talks concentrated on mutant hunting and gene targetting experiments. E. Robertson (Columbia) described insertion mutations (generated by either retroviruses or transgenes) in embryonic stem (ES) cells, including one that produces its initial defect at the primitive streak stage. A. Bradley (Houston) described gene targetting experiments in ES cells and various strategies for obtaining ‘subtle’ mutations as well as knock-outs, for studying gene function. A. Joyner (Mt Sinai, Toronto) described both ‘gene trap’ and ‘enhancer trap’ strategies for identifying genes of developmental interest. In the former, expression is only obtained if the ZacZ construct is inserted directly into a reading frame; not surprisingly, it is highly
mutagenic. Enhancer traps are less mutagenic but surprisingly, only 30% of those obtained so far show regionally restricted expression. Perhaps the most unusual aspect of the meeting was the amount of time devoted to discussion of basic questions of procedure and philosophy. One set of recurring questions concerned the nature and ubiquity of functional redundancy and how such might be selected in evolution. A. McLaren pointed out that it is important to distinguish between ‘redundancy’ in the sociological sense (as in ‘I’ve just been made redundant’) versus that in the informational sense. In the latter, some degree of redundancy is essential for the accurate transmission of the message. With respect to selectability, even small advantages associated with having ‘back up’ functions (particularly if there are some unique features of the otherwise redundant functions) can undoubtedly be selected for in evolution. A second question which came up at several points was how one determines which genes are most ‘interesting’ for study. Since bizarre phenotypes can be associated with partial inactivation of mundane (housekeeping) functions, it is clear that phenotypes can only be a partial guide. A third issue, of practical import, is how workers in the field of mouse development can keep up with the growing number of expression patterns for all sorts of different genes. This kind of information is often essential in deciding which experiments to do next and, at present, there is no systematic way of keeping track of the patterns. Various suggestions for different kinds of data bases were discussed. While the 1976 meeting may have marked a culmination of sorts for the older. classical and descriptive field of mammalian development, this meeting may later be seen as a transitional one between the earlier field and the one that is coming into being. Molecular methods now dominate mouse developmental studies but the answers to many of the basic questions still remain to be found. One may confidently expect that the next Ciba Symposium on mammalian development (in 1996 or 2001?) will be as strong on the answers as on the questions and techniques. Adam S. Wilkins is with the Company of Biologists, Dept of Zoology, Downing St, Cambridge CB2 3EJ, CJK. I
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