John Gerhart - US grants

There are two projects described in this proposal. The first concerns purification and characterization of "maturation-promoting factor" (MPF), a cytoplasmic agent appearing at the G2-M transition of the cell cycle of mitotic and meiotic cells. MPF is probably a trigger or central effector of the transition and might better be called "M-phase promoting factor". The cell also contains systems to activate and inactivate MPF at the beginning and end of M phase. We have purified MPF 80-100 fold from unfertilized eggs of Xenopus laevis, a naturally synchronous source of M-phase cells. During further purification,, we will determine whether MPF continues to behave as a single component of 110 20 Kd, and whether the MPF-inactivating agent can be removed. In collaborative work with Dr. M Kirschner, we will attempt to prepare an antibody to MPF. MPF has long been suspected to have protein kinase activity, and we will test this by inactivation of MPF with kinase affinity probes. In addition, the MPF-inactivation system will be partially purified and characterized with regard to its Ca[unreadable]++ and ATP requirement in and vitro. These studies generally concern health problems of cell proliferation and non-proliferation. The second project concerns the formation and function of axial determinants in fertilized eggs of X. laevis. These determinants are thought to control the development of embryonic dorsal structures by cells cleaved from the part of the egg. The formation and function of these agents probably occurs long before gene expression begins; their identity is poorly known, although their importance is clear. We can control their position and extent of formation by experimental means, and will study their formation under controlled conditions. Also, we will study the action of these determinants in causing certain vegetal cells to induce their equatorial neighboring cells to form dorsal mesoderm (the Spemann organizer). These studies concern health problems of vertebrate embryonic development.

My long-term objectives are 1) to identify the developmental processes transforming the unicellular organization of the egg of the vertebrate Xenopus laevis is into the multicellular organization of the embryo, particularly into the anteroposterior array of dorsal structures of the body axis; and 2) to understand the cell biological/molecular basis of these processes. We have examined three stage-specific processes for their roles in dorsal development: 1) cortical rotation of the egg in its first cell cycle, which changes the egg from cylindrical to bilateral symmetry, 2) formation of the Spemann organizer by inductions from vegetal and marginal zone cells in the blastula stages ("organizing the organizer"), and 3) functioning of the Spemann organizer in neural induction in the gastrula stage. Inhibition of any of these processes leads to the same syndrome of final developmental defects: truncation of the body aids from the anterior end, at a level defined by the extent of inhibition. Complete failure of any process results in radially ventralized embryos, with no body axis, a default pathway of development. Dorsalized embryos arise when these processes are exaggerated. The spectrum of dorsalized-ventralized anatomies, which may be general for vertebrates, contains forms similar to those of well known human birth defects such as cyclopia, microcephaly, and acephaly. In the request period, we will study each process further: For rotation, we will characterize the anchoring and directionality of the microtubules serving as tracks, and ask about the immediate local effect of rotation on the dorsal quadrant of the egg. For the formation of the organizer, we will study the role of the late blastula organizer in inducing the Spemann (gastrula) organizer, and the differentiation of the latter into anterior and posterior parts during gastrulation, using explants and molecular markers. We will also ask whether the entire spectrum of dorsalized- ventralized anatomies can be related quantitatively to one variable, the width (0 degrees - 360 degrees circumference) of the late blastula organizer. And finally for the functioning of the Spemann organizer itself, we will ask about the steps of planar induction of anteroposterior neural markers, the dependence of neural pattern on the age, size, and parts of the organizer, and the progressive acquisition of anterior fates by neural cells, again using explants and markers.

Dr. Gerhart is requesting funds for the travel of speakers invited to the 49th annual symposium of the Society for Developmental Biology, which will be held June 28-30, 1990 at Georgetown University in Washington D.C. The subject of the symposium is "Cell-cell interactions in early development". Speakers will describe developmentally important interactions in a variety of embryos (Drosophila, C. elegans, sea urchins, vertebrates), in slime molds (Dictyostelium), and in plants, as analyzed by molecular, genetic, and embryological methods. Other speakers will discuss cell biological and molecular aspects of receptor function (for growth factors) and signal transduction as studied in cell culture model systems, which are likely to have relevance to embryological studies. The meeting is usually of medium size (300-400 registrants) with a single platform session held at a time (25-30 speakers), and with 60-80 posters. It is attended by graduate students, postdoctoral fellows, and senior researchers. Most speakers have agreed to prepare summaries of their presentations for publication in the annual symposium volume. %%% Progress in the area of cell-cell interactions in early development have been substantial in the last few years and it is therefore valuable to hold such a symposium to discuss and disseminate information on the status of the field.

Origins of the chordate bodyplan and mode of development will be sought in the hemichordates, the little studied sister taxon (with echinoderms) of chordates. Hemichordates possess: 1) a short stomacord which forms from the gut roof and contains vacuolated cells like a chordate notochord or prechordal plate, 2) a dorsal nerve cord formed by neurulation like a chordate dorsal hollow neural tube, 3) a long array of chordate-like gill slits, and 4) a transient post-anal tail in the juvenile of some species, like a chordate tail. They may also have left-right asymmetries like those of chordates. Except for the gill slits, none of these traits has been convincingly identified as chordate-like by anatomical scrutiny, and hemichordates also share traits with annelids and arthropods, such as a ventral nerve cord besides their dorsal cord. We are obtaining cDNA sequences from Saccoglossus kowalevskii, an enteropneust hemichordate, for in situ hybridization to assess hemichordate- chordate similarities more incisively. We find a set of chordate-homologous genes expressed in the dorsal midline of the hemichordate embryo (bmp2/4, twg) where the dorsal nerve cord forms and in the ventral midline (netrin, admp) where the ventral nerve cord forms. The animal's entire body wall contains an intraepidermal nerve net from which axons collect at the two midlines. It is like a chordate neural tube with bmp in the roofplate and netrin in the floorplate. We will continue to compare nervous systems and other traits. The three-part organization of hemichordates corresponds to the chordate anteroposterior axis in terms of expression domains of various homeobox genes (rtx, six3, pitx, otx, Hox3,4,9). The prosome is like the anterior head/ventral forebrain, the mesosome like the dorsal forebrain, midbrain and first branchial arch, and the metasome like the rest of the chordate body. These similarities are presumably conserved from a three-part common ancestor. Hemi-chordate development will be studied for the presence of one or more inductive parts of Spemann's organizer, which in chordates is involved in the development of all four specific traits. The stomochord region expresses otx and dkk genes, we find, as does the chordate head organizer. Knowledge of hemichordates will help to explain why chordates have certain aspects of their body plan and development, and by difference, what additions and modifications have been made only within the chordate line.

DESCRIPTION (provided by applicant): The origin of the body plan, major traits, and modes of development of chordates will be sought in the next grant period by our making a molecular developmental analysis of hemichordates, the phylum of bilateral animals that last shared an ancestor with chordates. Traits possessed by modern members of both phyla were probably possessed by the common ancestor and just brought forward in the chordate lineage, not evolved anew by chordates. Hemichordates have long beer thought to possess chordate-like features, but anatomical comparisons of the 2 groups, while suggestive, have not allowed definitive conclusions. We have found that hemichordates are remarkably Iike chordates in the anteroposterior dimension in terms of their map of 28 domains of ectodermal expression of orthologs of transcription factors which are important for neural patterning in chordates. Furthermore, hemichordates possess 4 signaling centers (secreted Wnts, Fgfs, Hh) along this dimension, similar to those in chordates, such as the anterior neural ridge and the isthmic organizer. The anteroposterior similarities prevail despite the fact that the hemichordate nervous system is diffuse throughout the ectoderm, not centralized as in chordates. In the dorsoventral dimension hemichordates possess 2 opposed signaling centers, 1 for Bmps and 1 for Chordin, as do chordates, but the orientation is more like that of protostomes such as Drosophila (Bmp dorsal; Chordin ventral). We propose to analyze hemichordate development further to determine the formation and function of the 4 anteroposterior signaling centers and 2 dorsoventral signaling centers in patterning the hemichordate embryo and to compare our findings with those on chordates to further elucidate ancestral developmental modes. Furthermore, the development of isolated ectoderm will be studied to see if it has a neural default circuitry or whether it is autonomously specified for neurogenesis, independent of Bmp. Then, the mesendoderm will be examined for the expression of orthologs of genes expressed in Spemann's organizer, which is a major developmental trait of chordates. siRNA interventions will be used to evaluate the role of the encoded gene products in development.