Polarization of the C. elegans embryo by RhoGAP-mediated exclusion of PAR-6 from cell contacts

Abstract

Early embryos of some metazoans polarize radially to facilitate critical patterning events such as gastrulation and asymmetric cell division; however, little is known about how radial polarity is established. Early embryos of Caenorhabditis elegans polarize radially when cell contacts restrict the polarity protein PAR-6 to contact-free cell surfaces, where PAR-6 regulates gastrulation movements. We have identified a Rho guanosine triphosphatase activating protein (RhoGAP), PAC-1, which mediates C. elegans radial polarity and gastrulation by excluding PAR-6 from contacted cell surfaces. We show that PAC-1 is recruited to cell contacts, and we suggest that PAC-1 controls radial polarity by restricting active CDC-42 to contact-free surfaces, where CDC-42 binds and recruits PAR-6. Thus, PAC-1 provides a dynamic molecular link between cell contacts and PAR proteins that polarizes embryos radially.

Fig. 1

Inner-outer PAR asymmetry. In all figures anterior is left, nuclei are blue, and asterisk indicates germline precursor cell. Genotypes (italicized) and proteins shown (capitalized) are indicated. Bar in first panel applies to all panels unless indicated. (A, B) Live 8-cell embryos. (C-H) Immunostained 8-cell embryos. Arrows indicate PAR proteins. Bar, 5μm.

Fig. 2

Gastrulation and myosin localization. (A, B) Live 44-cell embryos. EPC descendants (‘E’, colored green) have ingressed in wild type (A) but not in the pac-1 embryo (B). (C, D) mid-26-cell embryos. EPCs (Ea, Ep) are indicated. Arrows, p-rMLC. (C’,D’) Cell outlines. Bar, 5μm.

Fig. 3

PAC-1 localization and time of function. (A) Predicted PAC-1 protein showing domains and mutations. (B–C) Coimmunostained 7-cell embryo; arrow, GFP-PAC-1. (D) 8-cell embryo; arrow, GFP-PAC-1. (E) Double embryo with ectopic contacts (arrowheads); cells from each embryo are numbered differently. GFP-PAC-1 is present at endogenous and ectopic contacts. (F,G) Coimmunostained 8-cell embryo. GFP-ZF1-PAC-1 has degraded from somatic cells except C cell (labeled). PAR-6 (arrows) localizes symmetrically in cells lacking GFP-ZF1-PAC-1. (H,I) Coimmunostained 26-cell embryo; cells expressing GFP-PAC-1 (arrow) are outlined (H). PAR-6 is absent from inner surfaces (arrowhead) of cells expressing GFP-PAC-1 (I). Bar, 5μm.

Fig. 4

PAR-6 localization by the PAC-1 RhoGAP domain and CDC-42. (A,B) Coimmunostained 8-cell embryo. GFP-PAC-1(R984A) localizes to inner surfaces (arrow in A) but PAR-6 localizes symmetrically (arrows in B). (C,D) Coimmunostained 8-cell embryo; restriction of P granules to germline precursor cell indicates normal A/P polarity. HA-ZF1-CDC-42 has mostly degraded from cells marked ‘x’ and PAR-6 is cytoplasmic in these cells. (E,F) Coimmunostained 12-cell embryo expressing HA-CDC-42(CA). PAR-6 localizes to inner and outer surfaces (arrows). (G,H) Live 8-cell embryos. GFP-PAR-6 is enriched at the outer cortex (arrows); GFP-PAR-6(CM2) localizes to the cytoplasm. Bar, 5μm.