In a 4-cell C. elegans embryo the ventral blastomere EMS requires polarty -signaling from its posterior sister cell , P2. This signaling event enables EMS to orient its division spindle along the anterior-posterior (Al) axis and to specify the endoderm fate of its posterior daughter cell, E. Wnt pathway components have been implicated in mediating P2/EMS signaling. However, no single mutants or varous mutant combinations of the Wnt pathway components disrupt EMS polarty completely. Here we describe the identification of a pathway that is defined by two tyosine kinase related proteins , SRCl and MES, which function in parallel with Wnt signaling to specify endoderm and to orient the division axis of EMS. We show that SRC, a C. elegans homolog of c-Src , functions downstream of MESl to specifically enhance phosphotyrosine accumulation at the P2/EMS junction in order to control cell fate and mitotic spindle orientation in both the P2 and EMS cells. In the canonical Wnt pathway, GSK-3 is conserved across species and acts as a negative regulator. However, in C. elegans we find that GSK-3 functions in a positive manner and in parallel with other components in the Wnt pathway to specify endoderm during embryogenesis. In addition, we also show that GSK3 regulates C. elegans germine development, a function of GSK-3 that is not associated with Wnt signalng-. It is required for the dierentiation of somatic gonadal cells as well as the regulation of meiotic cell cycle in germ cells. Our results indicate that GSK-3 modulates multiple signaling pathways to regulate both embryogenesis and germline development in C. elegans. CHAPTER I GENERAL INTRODUCTION Overview Multicellular organsms, no matter how complicated, all begin as a single cell , the zygote. How does the zygote generate cell diversity to form different tissues and eventually develop into a functional organism? The simple answer to this question is asymetrc cell division, in which a cell divides into two cells with dierent developmental potentials. Asymmetrc cell divisions may involve intrnsic or extrnsic factors. With intrnsic factors, unequal amounts of cell-fate determnants are paritioned into the two daughter cells. With extrnsic factors, daughter cells are intialy equivalent but adopt diferent fates as the result of the interactions of the daughter cells with each other or with their environment, referred to as cell-cell signaling events. Signaling events in C. elegans occur as early as the 4-cell stage. One of the earliest signaling events occurs between P2 and ABp which involves the conserved Deltaotch lie signaling pathway, APXl/GLPl signaling. This P2-ABp signaling event renders ABp dierent from its sister cell, ABa, that has the same development potential (Mello et aI., 1994). The other signaling event is P2/EMS signaling, which enables EMS divide asymmetrcally along the anterior-posterior (Al) axis to give rise to two daughters with different cell fates. The anterior daughter, MS, is a mesodermal precursor and develops into body wall muscle and pharngeal muscle. The posterior daughter, E, is the only source of intestie (endoderm). P2/EMS signaling was best demonstrated by culturng isolated blastomeres from wild-tye embryos in vitro. EMS isolated early in its cell cycle and cultured alone produces two MS-like daughters that make only mesodermal tissue consisting of body wall muscle and pharngeal muscle. When P2, and P2 only, is placed in contact with EMS, EMS makes endoderm (E fate) in addition to mesoderm (MS fate) (Goldstein , 1992). Moreover, when EMS is cultured alone, its mitotic spindle elongates along the axis established by centrosome migration without any rotation. However, when EMS is put in contact with P2 early in its cell cycle, it rotates its centrosomes following centrosome migration and reorients its division axis in accordance to its contact site with P2. Therefore, P2 signaling polarzes EMS in such a way that a posterior daughter (E) is born next to P2 and produces endoderm (Goldstein, 1995a; Goldstein, 1995b). EMS remains competent to E cell fate specification potential in response to P2 signaling for a longer time than it remains competent to orient its mitotic spindle (Goldstein , 1995a; Goldstein, 1995b), suggesting that the signals or their responses may be different. Genetic and molecular insights into the nature of P2/EMS signaling have come , in par, from the identification of five mom (more mesoderm) genes required for endoderm specification. Homozygous mothers for any of the mom genes produce dead embryos that are nicely differentiated but lack gut and have extra pharngeal tissue. This occurs because both EMS daughters adopt an MS like fate (Rocheleau et al. , 1997; Thorp et aI. , 1997). Another maternal effect lethal mutat popl (posterior pharynx defective), has the opposite phenotype to the mom mutants. popmutat mothers lay dead embryos with extra gut at the expense of pharnx (Lin et al., 1995) Molecular cloning of the mom and popgenes has identified them as components of the conserved Wnt signaling pathway. momencodes a homolog of porcupine (pore) and mom2 and momare similar to Wnt/wingless (wg) and frizzled (tz) respectively. poplis related to Drosophila pangolin/dTCF (pan) and mamalian TCF/LEF family of transcription factors that are regula.ted by Wnt signaling pathway. Reverse genetics using RNAi (RNA interference) also identifed other conserved components involved in Wnt signalng including dishevelled homologs dsh(Dishevelled) and mig(migration defective), apr(APC related) and wrm(worm armadillo, also known as f3-catenin), as genes required for E cell fate specifcation (Rocheleau , 1999; Rocheleau et aI. , 1997). Therefore, the widely conserved Wnt signal transduction pathway is required for endoderm induction during embryogenesis in C. elegans. Genetic studies also indicate that other signaling mechanisms contrbute to endoderm specification. These mechansms include components related to MAPK (mitogen activated protein kinase) signaling factors. momis a TAKI (transformng growth factor f3--activated kinaseI) MAP Kinase , kinase kinase homolog, and lit-l (loss of intestine) encodes a Nemo, MAP Kiase related protein (Ishita et al. , 1999; Meneghini et al., 1999; Rocheleau et al. , 1999; Shi et aI. , 1999). Genetic data suggest that several parallel signaling inputs converging on the WRMl/LITl protein complex to downregulate POPl activity in the E cell to specify E cell fate (Rocheleau et al. , 1997; Rocheleau et al. , 1999; Shin et al., 1999). Several of the genetically defined signaling components for E cell fate specification also exhibit a skewed Al orientation of the EMS division axis. However, withn the intact embryo, no single or multiple mutant combinations among the above mentioned mutants have a complete loss of EMS centrosome rotation resulting in a default left-right (UR) division axis (Schlesinger et al. , 1999). These findings indicate that an as-yet-unidentified factor(s) must direct EMS spindle orientation and possibly determne E cell fate specification during P2/EMS signaling. Figure 1-1 summarzes the genetic pathways involved in P2/EMS signaling.