Cellular and Molecular Events in Early Leech Development

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0091261WeisblatGlossiphoniid leeches such as Helobdella robusta, are useful for studying developmental processes for two reasons: first, they are relatively simple animals with large, accessible embryos that are suited for combined cellular and molecular techniques. Second, since leeches belong to the phylum Annelida, an otherwise poorly studied group, the results obtained are useful for making interphyletic comparisons. Such comparisons are essential to distinguish general developmental processes from phylum-specific ones, and also to learn how evolutionary modification of developmental processes leads to the appearance of diverse animal types. For example, annelids and arthropods resemble each other (and differ from mollusks) in that they both have segmented body plans. But the early Helobdella embryo is strikingly different from the early Drosophila embryo and similar to molluscan embryos. Thus, the question of how cell fates are determined in the early leech embryo is of considerable interest. The experiments to be undertaken fall into two main areas. Most of the proposed effort will go into continuing investigations of cell fate determination and differentiation, combining reverse genetic approaches with embryological techniques for which the Helobdella embryo is well suited. Specific topics to be investigated include: 1. The mesoderm-ectoderm fate decision. Dr. Weisblat and his research group will test the hypothesis that maternally inherited transcripts of Hro-nos, the nanos homolog in Helobdella, are translated only at the animal pole of the embryo, and that inheriting high levels of HRO-NOS protein causes the animal daughter of macromere D' to assume an ectodermal fate. 2. The origins of the germline. Hro-nos is also transcribed zygotically. The hypothesis that Hro-nos is associated with primordial germ cells in leech, as in other bilaterians will also be tested. 3. Signalling by HRO-HH, a hedgehog-class gene product in gut formation. Preliminary results indicate that HRO-HH signaling from endoderm is critical for gut formation in leech as in vertebrate. Experiments will be undertaken to confirm this result and look for downstream targets of HRO-NOS signaling.The second part of the work entails investigations of the dynamics of cells and cellular processes. Specific topics to be investigated include: 1. The regulation of cell-cell fusion. An early step in leech midgut formation entails the fusion of macromeres A''' and B''', a process which is regulated by signals emanating from the D quadrant of the embryo. Blastomere isolation and recombination experiments will be performed to pinpoint the timing and source of the signal. 2. The dynamics of early WNT signalling. Dr. Weisblat's group has recently discovered that a WNT signalling pathway is operative in the 2-cell embryo of the leech, and serves in part to regulate cell-cell adhesion following the first cell division. This is the earliest possible example of intercellular embryonic signalling. The experiments proposed are designed to determine whether WNT expression is regulated at the level of transcription, translation, or post-translational processing.