INTRODUCTION A central question in the fields of cell biology and develop- mental genetics concerns how a cell chooses to adopt a partic- ular cell fate and differentiate into a specific cell type. While the mechanisms that contribute to the decision process are still emerging, it is known that one pathway to cellular differen- tiation involves asymmetric cell division in which cell fate determinants are partitioned unequally between daughter cells. The orientation of the mitotic spindle has long been known to be important in directing asymmetric cell divisions during embryogenesis (reviewed by Strome, 1993). More recently, molecular genetic analyses in C. elegans (Kemphues et al., 1988; Cheng et al, 1995; Guo and Kemphues, 1995; Etemad- Moghadam et al., 1995) and Drosophila (Kraut et al., 1996) have identified molecules that couple spindle orientation to the asymmetric localization of cell fate determinants (reviewed by Rhyu and Knoblich, 1995; White and Strome, 1996; Doe, 1996). Members of the myosin family of actin-based molecular motors have been implicated in the asymmetric segregation of factors necessary for cell fate determination in yeast (Jansen et al., 1996; Bobola et al., 1996) and C. elegans (Guo and Kemphues, 1996). Less is known about the pathways which establish the initial cellular asymmetry that directs spindle ori- entation and polarized transport. Given the importance of the microtubule cytoskeleton in cytoplasmic organization and intracellular transport in polarized cell types, microtubules and their associated motor molecules may be part of the primary mechanism that establishes this cytoplasmic asymmetry. Oogenesis in Drosophila melanogaster is an attractive system for studying the role of microtubule motors in cellular differentiation (for a comprehensive review of oogenesis, see Spradling, 1993). Cytological and pharmacological studies have suggested that the microtubule cytoskeleton is essential for oocyte differentiation through its roles in intercellular transport and in the asymmetric localization of determinants within the developing oocyte that influence axis specification (reviewed by Theurkauf, 1994; St Johnston, 1995). Oocyte development occurs in a cyst of 16 interconnected cells that arises from a single cystoblast which undergoes 4 synchronous rounds of division with incomplete cytokinesis. One cell dif- ferentiates as the oocyte, while the remaining 15 become highly polyploid nurse cells. The nurse cells synthesize materials which are transferred through the cytoplasmic con- nections (ring canals) into the differentiating oocyte (reviewed by Mahajan-Miklos and Cooley, 1994). The asymmetric transport of materials is thought to occur along a polarized microtubule array established shortly after the 16-cell cyst is formed (Mahowald and Strassheim, 1970; Theurkauf et al., 1993). Microtubules originate in the pro-oocyte and extend their plus ends through the ring canals into the nurse cells, creating a polarized transport system on which a minus-end directed microtubule motor could function. Within the germline cyst a polarity must be established in 2409 Development 124, 2409-2419 (1997) Printed in Great Britain © The Company of Biologists Limited 1997 DEV8417 During animal development cellular differentiation is often preceded by an asymmetric cell division whose polarity is determined by the orientation of the mitotic spindle. In the fruit fly, Drosophila melanogaster, the oocyte differentiates in a 16-cell syncytium that arises from a cystoblast which undergoes 4 synchronous divisions with incomplete cytoki- nesis. During these divisions, spindle orientation is highly ordered and is thought to impart a polarity to the cyst that is necessary for the subsequent differentiation of the oocyte. Using mutations in the Drosophila cytoplasmic dynein heavy chain gene, Dhc64C, we show that cytoplasmic dynein is required at two stages of oogenesis. Early in oogenesis, dynein mutations disrupt spindle orientation in dividing cysts and block oocyte determination. The local- ization of dynein in mitotic cysts suggests spindle orienta- tion is mediated by the microtubule motor cytoplasmic dynein. Later in oogenesis, dynein function is necessary for proper differentiation, but does not appear to participate in morphogen localization within the oocyte. These results provide evidence for a novel developmental role for the cytoplasmic dynein motor in cellular determination and differentiation. Key words: dynein, cell fate, asymmetric division, Drosophila, spindle orientation, oogenesis SUMMARY The microtubule motor cytoplasmic dynein is required for spindle orientation during germline cell divisions and oocyte differentiation in Drosophila Maura McGrail † and Thomas S. Hays* Department of Genetics and Cell Biology, University of Minnesota, 1445 Gortner Avenue, St. Paul, MN 55108, USA † Present address: Howard Hughes Medical Institute, Division of Cellular and Molecular Medicine, UCSD School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093, USA *Author for correspondence (e-mail: tom-h@biosci.cbs.umn.edu)