Extracellular control of PAR protein localization during asymmetric cell division in the C. elegans embryo

Abstract

The axis of asymmetric cell division is controlled to determine the future position of differentiated cells during animal development. The asymmetric localization of PAR proteins in the Drosophila neuroblast and C. elegans embryo are aligned with the axes of the embryo. However, whether extracellular or intracellular signals determine the orientation of the localization of PAR proteins remains controversial. In C. elegans, the P0 zygote and germline cells (P1, P2, and P3) undergo a series of asymmetric cell divisions. Interestingly, the axis of the P0 and P1 divisions is opposite to that of the P2 and P3 divisions. PAR-2, a ring-finger protein, and PAR-1, a kinase, relocalize to the anterior side of the P2 and P3 germline precursors at the site of contact with endodermal precursors. Using an in vitro method, we have found that the PAR-2 protein is distributed asymmetrically in the absence of extracellular signals, but the orientation of the protein localization in the P2 and P3 cells is determined by contact with endodermal precursor cells. Our mutant analyses suggest that mes-1 and src-1, which respectively encode a transmembrane protein and a tyrosine kinase, were not required to establish the asymmetric distribution of PAR-2, but were required to determine its orientation at the site of contact with the endodermal precursors. The PAR-2 localization during the asymmetric P2 and P3 divisions is controlled by extracellular signals via MES-1/SRC-1 signaling. Our findings suggest that Src functions as an evolutionarily conserved molecular link that coordinates extrinsic cues with PAR protein localization.

Fig. 1.

Extracellular control of the asymmetric cell division of germline precursors P2 and P3. (A) Cell lineage in early embryonic C. elegans development. The left-right position of the cells reflects the anterior-posterior position in the embryo. The P0 zygote and germline cells (P1, P2, P3 and P4) are indicated in red. (B,C) DIC images of germline and somatic daughters born after the P2 and P3 divisions. Germline daughters (P3 and P4, red) were in contact with an endodermal precursor (E or E.x, green), whereas the somatic daughters (C cell, yellow in B; D cell, blue in C) were distal to the endodermal precursors in intact embryos. Anterior is leftwards and ventral is downwards. (D,E) DIC images of the progeny of an isolated P1 (D) and P2 cell (E) cultured in vitro. In the isolated P1 cell cultures, the germline daughter (P4, red) was in contact with the endodermal precursor (E.x, green), whereas one somatic daughter (D, blue) was in contact with the somatic C daughter cell (C.x, yellow). In P2 isolates, the P4 cell was in contact with the C daughter cell, and the somatic daughter D was not. ‘x’ indicates a daughter cell (e.g. ‘E.x’ indicates a daughter of the E cell). (F) Summary of the position of the P4 cell born after the P3 division in intact embryos, P1 isolates and P2 isolates. (G) Diagram showing the measured angle of the P2 and P3 divisions. (H,I) DIC images of germline daughters (P3 or P4, red), somatic daughters (C cell, yellow; D cell, blue) born after the P2 and P3 division, and attached endodermal precursors (E or E.x, green). (J-M) Histograms of the angle of P2 and P3 division when P2 was attached to EMS (J) or AB.x (L), or when P3 was attached to E (K) or MS (M). Scale bars: 10 μm.

Fig. 2.

Asymmetric cell division of the germline precursor P2 cell is highly cell-autonomous and responsive to extracellular signals in C. elegans. (A) Experimental procedure for examining the time window for P2 or EMS to send or receive signals. P2 and EMS were isolated just after birth, cultured in isolation for various times and then attached to a newly born P2 or EMS cell isolated from another embryo. The results are shown in Table 1. (B) Schematic procedure for examining whether the axis of the asymmetric P2 division is oriented to a second EMS. A second EMS was placed against a P2 cell on the opposite side of the original EMS in a P2-EMS pair generated from a P1 isolate. The axis was defined as having a ‘normal’ orientation if the daughter P3 cell was generated on the original EMS side, and a ‘reversed’ orientation if it was generated on the second EMS side. The germline P1, P2 and P3 cells are red, the somatic daughter C born after the P2 division is yellow, and the endodermal precursors (EMS) are green. (C) Dissecting microscopy images of the germline daughter P3 (red) and somatic daughter C (yellow) after the P2 division in the presence of a second EMS. In the upper panel, the axis of the P2 division was in the normal orientation; in the lower panel, the axis was reversed. (D) Summary of the axis of P2 division in the presence of a second EMS. The normal, reversed or perpendicular orientation is shown by a gray, black or white bar, respectively. ‘C-P1-D’ (completion of P1 division) was defined as the period 4-6 minutes after the mother P1 cell initiated cleavage. ‘BEFORE’ and ‘AFTER’ were the periods 3 minutes before and after ‘C-P1-D’, respectively.

Fig. 3.

PAR-2 localization and spindle orientation are guided by extracellular signals. (A-F) Fluorescence microscopy images of GFP-fused PAR-2 (A,B), GFP-fused PAR-6 (C,D) and GFP-fused TBG-1 (γ-tubulin) (E,F) in the P2 or P3 cell in intact embryos. The paired arrows indicate the cell boundary between the P cell (P2 or P3) and the endodermal precursor (EMS, E or E.p). Throughout this figure, the arrowheads indicate the PAR protein or centrosome positions. The arrowheads with an asterisk indicate PAR protein at the apical cortex or non-cell-contact area. Cell boundaries without the cortical GFP signal are marked by broken white lines (A,D-F). Anterior is leftwards and ventral is downwards. (G,H) Series of fluorescence microscopy images of GFP-fused PAR-2 (G) or GFP-fused PAR-6 (H) in P2 isolates. (I-P) Series of fluorescence microscopy images of GFP-fused PAR-2 (I-L) or GFP-fused tubulin [TBG-1 (γ-tubulin) (M,N) or β-tubulin (O,P)] in the P2 or P3 cell attached to various cells in vitro. Minute 0 is the time at which observation started. Scale bars: 10 μm.

Fig. 4.

MES-1/SRC-1 signaling is required to orient the asymmetric cell division of the germline precursor P2 cell. (A,B) DIC images of the P2 division in mes-1 or src-1 mutants. Germline precursor P2 and its germline daughter P3 (red) and somatic daughter C (yellow). Endodermal precursors (EMS and E) are green. (C-E) Fluorescence microscopy images of GFP-fused PAR-2 in wild-type (C), src-1(RNAi) (D) and mes-1(bn7) (E) embryos. Paired arrows indicate the cell boundary between the P2 cell and endodermal precursor (EMS and E). Green arrowheads indicate PAR-2 protein at the contact site with endodermal precursors, and green arrowheads with an asterisk indicate PAR-2 protein localized to the apical cortex. Cell boundaries without PAR-2 signals are marked by broken white lines. Minute 0 is the P2 birth time (at the completion of the mother P1 division). Anterior is leftwards and ventral is downwards. (F) Fluorescence microscopy images of GFP-fused PAR-2 protein in a P2 cell isolated from a mes-1(bn7) embryo. (G) Schematic diagram for making chimeric P2-EMS pairs. The P2 and EMS were isolated, and one was replaced with a cell isolated from a mutant embryo, producing a chimeric P2-EMS pair. (H-L) Histogram of the axis of P2 division in a chimeric P2-EMS pair. (H) A wild-type cell was replaced with a wild-type cell from another embryo. (I-L) A wild-type cell was replaced with a mutant cell, resulting in pairs with a wild-type P2 and mes-1 mutant EMS (I), mes-1 mutant P2 and wild-type EMS (J), wild-type P2 and src-1 mutant EMS (K), or src-1 mutant P2 and wild-type EMS (L). The angle of the P2 division was estimated at 22.5° intervals on a dissecting microscope. Scale bars 10 μm.