Development 129, 387-397 (2002) 387 Printed in Great Britain © The Company of Biologists Limited 2002 DEV8905 Cell polarity and gastrulation in C. elegans Jeremy Nance1,2 and James R. Priess1,2,* 1Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA 2Howard Hughes Medical Institute, Seattle, Washington 98109, USA *Author for correspondence (e-mail: [email protected]) Accepted 17 October 2001 SUMMARY Gastrulation in C. elegans embryos involves formation of a apical flattening associated with an apical concentration of blastocoel and the ingression of surface cells into the non-muscle myosin. We provide evidence that ingression blastocoel. Mutations in the par-3 gene cause abnormal times are determined by genes that control cell fate, though separations between embryonic cells, suggesting that the interactions with neighboring cells can prevent ingression. PAR-3 protein has a role in blastocoel formation. In normal development, PAR proteins localize to either the apical or basal surfaces of cells prior to blastocoel formation; we Key words: Blastocoel, Apical-basal polarity, Gastrulation, demonstrate that this localization is determined by cell Ingression, Morphogenesis, Cell fate, Cell shape, Non-muscle contacts. Cells that ingress into the blastocoel undergo an myosin, LIT-1, HMR-1, NMY-2, PAR-2, PAR-3, PAR-6, C. elegans INTRODUCTION 1992; Lin et al., 1995; Rocheleau et al., 1997). These endodermal and mesodermal precursors must then move from Numerous studies on C. elegans embryos have revealed the the outer surface of the embryo into the interior for the basic strategies that establish the anterior-posterior and dorsal- formation of a functional intestine and muscular system. ventral axes. Axis specification in C. elegans embryos begins Animal embryos use several strategies to position with fertilization of the egg, where the point of sperm entry endodermal and mesodermal precursors into their interior, a defines the posterior pole (Goldstein and Hird, 1996). process called gastrulation. In many embryos, such as those of Fertilization induces the association of a group of proteins, sea urchins and amphibians, the early embryonic cleavages collectively called PAR proteins, to either the anterior or generate a cluster of cells (the blastula), and a central cavity posterior cortex. For example, PAR-3 and PAR-6 associate (the blastocoel) develops within this cluster. Certain cells with the anterior pole while PAR-2 and PAR-1 associate with detach from their neighbors on the cell surface and enter the the posterior pole (Boyd et al., 1996; Etemad-Moghadam et al., blastocoel (ingression), or large groups of cells fold into the 1995; Guo and Kemphues, 1995; Hung and Kemphues, 1999). blastocoel (invagination and involution) (Gilbert and Raunio, The localization of the PAR proteins is interdependent; 1997). In some animals, gastrulation occurs without formation mutations in par-3 cause the anterior mislocalization of PAR- of a blastocoel. In ctenophores, for example, ectodermal cells 2, and mutations in par-2 cause the posterior mislocalization spread over and internalize endodermal cells (epiboly) (Komai, of PAR-3 (Boyd et al., 1996; Etemad-Moghadam et al., 1995). 1968; Martindale and Henry, 1999). The dorsal-ventral axis is determined as the embryo divides Gastrulation in C. elegans begins at the 26-cell stage, when from two cells to four (reviewed by Schnabel and Priess, 1997). two endodermal precursors ingress from the surface of the The division of the anterior cell generates two initially embryo into the interior. Shortly thereafter, mesodermal equivalent daughters that express a receptor related to the precursors and germline precursors follow the endodermal Notch protein. The division of the posterior cell generates non- precursors into the interior of the embryo (Sulston et al., 1983). equivalent daughters, one of which expresses a ligand for the Very little is known about the cellular or molecular basis for receptor. In an apparently random manner, only one of the these events. Several mutations have been identified that receptor-expressing cells contacts the ligand-expressing cell; prevent or delay ingression of the endodermal precursors, most this interaction leads to the specification of dorsal cell types of which cause the endodermal precursors to divide such as hypodermis (skin). prematurely (Denich et al., 1984; Knight and Wood, 1998). In addition to anterior-posterior and dorsal-ventral axes, the Inhibiting embryonic transcription also causes the endodermal early embryo must establish an outer-inner polarity. Cells on precursors to divide precociously and prevents ingression the ventral surface of the embryo become committed to (Powell-Coffman et al., 1996). Ingression of the endodermal endodermal and mesodermal fates through a combination of precursors is likely to require embryonic transcription of one cell signaling events and asymmetrically localized factors or more genes in a chromosomal region called the endoderm (Bowerman et al., 1993; Bowerman et al., 1992; Goldstein, determining region, or EDR; a chromosomal deficiency that 388 J. Nance and J. R. Priess deletes the EDR delays or prevents ingression (Zhu et al., Bowerman, 1996). Embryo combination experiments were performed 1997). There are at least two genes in the EDR that are involved at 15°C. To inhibit transcription, embryonic culture medium was in specification of the endodermal cell fate, suggesting a supplemented with 50 µg/ml α-amanitin (Sigma). Laser ablations possible link between cell fate and ingression (Zhu et al., were performed as described elsewhere (Mello et al., 1992). 1997). 4D images were acquired as described (Thomas et al., 1996) using In this report we investigate the cellular basis for gastrulation 4D Grabber v1.32 software (C. Thomas, Integrated Microscopy Resource, University of Wisconsin-Madison, USA). Movies were in C. elegans. PAR proteins that show anterior-posterior analyzed using 4D Viewer v4.11 (C. Thomas, Integrated asymmetry in 1-cell and 2-cell embryos subsequently develop Microscopy Resource, University of Wisconsin-Madison, USA) outer-inner, or apical-basal, polarity as the blastula forms. We and Nematode Navigator software (kindly provided by J. Pitt, demonstrate that the apical-basal localization of the PAR http://www.fhcrc.org/labs/priess/nn.html). The volume of the proteins is dependent on cell-cell contacts by generating double blastocoel and embryo (± s.d.) were calculated using NIH Image1.62 embryos with abnormal patterns of cell contact. We show that (Wayne Rasband, National Institutes of Health, USA). ingression is associated with changes in the shape of the Ingressions were scored when a cell sank permanently from the ingressing cells and a redistribution of non-muscle myosin. surface of the embryo. Comparisons of ingression times were based Finally, we present evidence that cell fate, rather than cell on the analysis of 4D videorecordings of three embryos; all cells that position, is the predominant factor regulating ingression, ingressed during gastrulation were followed in each embryo. Ingression times were normalized to cell division timings presented although steric interactions can play an important role. by Sulston et al. (Sulston et al., 1983) using the interval between the MSa and MSaaa divisions as a measure of developmental time. Descriptions of wild-type gastrulation were based on observations of MATERIALS AND METHODS these and additional 4D videorecordings. Ectopic ingressions in mex- 1 mutants were scored in the ABpr lineage. lit-1 temperature shifts Nematode culture and strains were from 15°C to 25°C at the 24-cell stage. Because of the variable Nematodes were cultured and manipulated as described (Brenner, expressivity of the lit-1 mutant phenotype, analysis of MS ingression 1974). Unless otherwise indicated, experiments were performed on was restricted to the 7/11 embryos showing simultaneous ingression the wild-type N2 (var. Bristol). The following mutants were utilized: of all wishbone cells. In the E ablation experiments, analysis was chromosome II LG II: unc-4(e120) (Brenner, 1974), mex-1(zu120) restricted to the anterior MS central cells that do not contact the E (Mello et al., 1992); unc-32(e189) (Brenner, 1974), lit-1(t1512ts) corpse. (Kaletta et al., 1997), unc-45(e286ts) (Brenner, 1974), par-2(lw32) (Cheng et al., 1995), par-3(it71) (Cheng et al., 1995), lon-1(e185) Electron microscopy (Brenner, 1974); LGV: zuDf2 (Zhu et al., 1997). The following Mixed-stage embryos were fixed, embedded, sectioned and stained for integrated transgenes containing green fluorescent protein (GFP) transmission electron microscopy (TEM) as described (Priess and reporters were used: zuIs3 (end-1::GFP) (J. Nance, unpublished), Hirsh, 1986). Several thick sections were cut from each block to ruIs32 (pie-1::GFP::HIS-11) (Pratis et al., 2001), pxIs6 (pha- determine the precise stage and orientation of specific embryos. 4::GFP::HIS-11) (Portereiko and Mango, 2001), itIs153 (pie-1::PAR- Sections were analyzed from >50 embryos embedded in three 2::GFP) (Wallenfang and Seydoux, 2000). Mex-1 embryos were mex- different blocks. Embryos for scanning electron microscopy were 1 unc-4. Par-2 embryos were par-2 unc-45. Par-3 embryos were par-3 fixed as for TEM, omitting the tannic acid treatment. Embryos were lon-1; zuIs3. transferred by mouth pipet to a poly-L-lysine coated coverslip, then dehydrated and dried as described (Braet et al., 1997). dsRNA-mediated interference (RNAi) Standard techniques were used to synthesize double-stranded RNA Immunostaining and fluorescence microscopy (dsRNA) from T7 promoter-tagged, PCR-amplified cDNA or Embryos were fixed and processed for overnight immunostaining at genomic DNA. For analysis of ama-1(RNAi) embryos (2-4 µg/µl 4°C as described (Leung et al., 1999). Cultured blastomeres were dsRNA made from cDNA region 2876-3450), young adult fixed in –20°C methanol for 15 minutes, then immunostained at 37°C hermaphrodites carrying both a maternal (pie-1::GFP::HIS-11) and for 1 hour. A strain containing the itIs153 reporter was used for zygotic (end-1::GFP) GFP reporter were injected in the gonad or analysis of PAR-2 in cultured blastomeres.