The events that occur immediately after fertilization of an egg are critical to fertility and development. During this short time, the genomes of two parents are combined to create a new genome for the embryo, and complex macromolecular events are activated in previously specialized and rather quiescent egg cells. Nuclei, arrested in meiosis, have to reinitiate and complete meiosis, and then switch from a meiotic cell cycle, with its unique molecular constituents, to a mitotic cell cycle. Pronuclei have to form (or reorganize/decondense in the case of the sperm nucleus), fuse, and/or coordinate their cell cycles. Yet despite its importance, this initial stage of development is poorly understood at the molecular level. We use the molecular biology and excellent genetics of Drosophila melanogaster to dissect the molecules and pathways necessary for egg activation and the first embryonic mitosis.

1. Egg Activation:
The mature oocyte that is poised to begin embryogenesis differs in critical ways from a fertilized egg.  The oocyte’s outer coverings are specialized to allow sperm binding and entry.  The oocyte is stocked with maternal RNAs and proteins to support embryogenesis, but is itself not highly transcriptionally active; instead its nucleus is arrested at a specific stage of meiosis (metaphase I in Drosophila).  All of these features must be modified to allow development to proceed.  The process that accomplishes this is called egg activation.  Since egg activation is rapid and often occurs inside the female, many of its molecular features are not known.  The research in our lab focuses on the following:

a. Delineating egg activation pathways in Drosophila.
Calcium signaling pathways are major transducers of egg activation in vertebrates and marine invertebrates, and we and our colleagues have shown that at least one calcium signal transduction pathway is necessary for egg activation in Drosophila.  Our work has focused on the sarah gene (the Drosophila homolog of calcipressin).  We have found that sarah is required for several aspects of egg activation, including progression through meiosis, polyadenylation and translation of mRNAs, and full remodeling of the sperm nucleus (Horner et al., 2006) (Figure 1).  Calcipressin can act as either an activator or inhibitor of the calcium and calmodulin-dependent phosphatase, Calcineurin.  This is the first instance in which the Calcipressin/Calcineurin pathway has been shown to be involved in egg activation in any organism.  We are now examining whether other mediators of calcium signaling, such as CaMKII and calmodulin, are also necessary for egg activation in Drosophila.  Drosophila as a model organism provides the ability to use its excellent genetics to discover new molecules important for a biological process.  We are currently investigating the molecular identities of two genes, wispy and prage, that show egg activation defects.  Knowing the nature of these molecules will also allow elucidation and dissection of the molecular pathways that connect the calcium trigger of activation to the downstream outcomes of this process (cell cycle changes, translation, etc).

b. Determining the molecular targets of activation.
The activating trigger must exert its effects on existing proteins that have been pre-loaded into the egg; therefore, we believe there may be a global change to proteins on a post-translational level to transduce the activating signal.  Two proteins critical for early mitotic divisions in fertilized eggs (Ya and Gnu) are dephosphorylated during activation.  We are determining the extent to which phosphorylation changes occur to other proteins during activation, with the goal of identifying new proteins that are candidates for mediating post-activational functions.

2. The first embryonic mitosis:

            After activation, egg nuclei complete meiosis to become haploid, and the sperm nucleus has to reorganize before the haploid parental genomes combine to begin the rapid mitotic divisions of early embryogenesis.  One of the few known molecular targets of activation is the chromatin- and lamin-associated protein YA (“Young Arrest”).  Mutations in the YA gene prevent the fertilized egg from beginning mitosis, and cause an abnormally condensed chromatin state in the haploid male and female pronuclei (Lin and Wolfner, 1991).  Dephosphorylation of YA upon activation correlates with its ability to enter nuclei.  We have shown that YA mediates nuclear organization and coordination to initiate the mitotic phase of embryogenesis. In wild type Drosophila embryos, YA protein is located in the nuclear lamina and nucleoplasm (Figure 2). The nuclear lamina is a cross-linked proteinaceous layer that underlies the inner nuclear membrane, and has been suggested to have various functions such as the maintenance of nuclear shape and organization of nuclear pores as well as regulating transcription or DNA replication. It is important for the structure of all higher eukayotic cells, and mutations in lamina proteins result in a cariety of human disease syndromes. We have found that YA interacts with lamin Dm0 (the major constituent of the nuclear lamina in Drosophila), histone H2B, DNA, and itself (Goldberg et al., 1998; Liu and Wolfner, 1998; Yu and Wolfner, 2002). Our model for YA function is that it is targeted to the lamina by binding to assembled lamin and interacts with chromatin to promote a chromatin condensation state appropriate for the initial zygotic mitosis.