Dual roles for the trimeric G protein Go in asymmetric cell division in Drosophila

Abstract

During asymmetric division, a cell polarizes and differentially distributes components to its opposite ends. The subsequent division differentially segregates the two component pools to the daughters, which thereby inherit different developmental directives. In Drosophila sensory organ precursor cells, the localization of Numb protein to the cell's anterior cortex is a key patterning event and is achieved by the combined action of many proteins, including Pins, which itself is localized anteriorly. Here, a role is described for the trimeric G protein Go in the anterior localization of Numb and daughter cell fate specification. Go is shown to interact with Pins. In addition to a role in recruiting Numb to an asymmetric location in the cell's cortex, Go transduces a signal from the Frizzled receptor that directs the position in which the complex forms. Thus, Go likely integrates the signaling that directs the formation of the complex with the signaling that directs where the complex forms.

Fig. 1.

Schematic of SOP divisions and resulting sense organs. Double-headed arrows indicate axes of division.

Fig. 2.

Loss of function and overexpression of Go cause orientation and asymmetric division defects. (a) Adult WT thorax. Macrochaetae and microchaetae show stereotypical posterior orientation. (b) Thorax with Go mutant clones show bald regions (arrows), and surviving bristles are small and misoriented. (c) Thorax of a pnr-Gal4, UAS-Go^GTP fly. Bristle orientation defects occur, as well as bald regions (arrows). pnr-Gal4 drives in eight thoracic macrochaetae; on average, 15% of these macrochaetae were lost per thorax, and all thoraces lost at least one macrochaeta. (d) A WT thoracic bristle (microchaeta) at high resolution, showing external socket and a hair. (e–h) Bristle structures resulting from overexpression of Go^GTP (e and g) or loss of Go (f and h) (late clones). Defective bristles may contain two sockets/two hairs (e), one socket/two hairs (f), or one socket/no hair (g and h). Three percent of pnr-Gal4, UAS-Go^GTP bristles and 8% of Go bristles had hair/socket duplications. Socket-only phenotype occurred at rates of 24% and 5%, respectively. (i–p) Wing margin stout bristles: WT (i), Go (j and l), or UAS-Go^WT or UAS-Go^GTP (k and m–p). Defective bristles may contain two sockets/two hairs (j), one socket/three hairs (k), one socket/two hairs (l), two sockets/one hair (m), no socket/two hairs (n), no socket/one hair (o), or one or two sockets/no hair (p). MS1096-Gal4 driving a single copy of UAS-Go^WT or UAS-Go^GTP affected 1.5% and 3% of margin bristles, respectively. All wings had at least one defective margin bristle. Wing margin defects of Go clones (16) prevented an assessment of frequency of defects.

Fig. 3.

Loss and overexpression of Go causes aberrant SOP Numb crescents and aberrant cell specification. (a–d) Dorsocentral SOPs in metaphase (a and c) and early telophase (b and d) stained for DNA (blue), Sens (green), and Numb (red) in WT (a and b) and UAS-Go^WT (c and d) cells. Anterior is to the right. UAS-Go^WT SOPs show Numb crescents in variable positions. (e and f) Overexpression of Go^GTP in mitotic SOPs (α-Sens, blue) leads to severe defects in Numb localization (red), such as near-ubiquitous plasma membrane staining (e) or loss from the plasma membrane (f), sometimes with intracellular vesicular staining (arrow). (g–j) Four-cell bristle clusters resulting from SOP divisions stained with α-Sens (blue) at ≈25 h APF. (g) WT contains two internal cells, neuron (α-Elav, green) and a sheath cell (α-Pros, red), and two external cells. (h) In UAS-Go^GTP, internal cells often duplicate at the expense of external cells, forming two neuronal (green) and two sheath (red) cells. In sca-Gal4, UAS-Go^GTP this transformation occurred in ≈10% of bristles. (i) Rarely (≈1%), four neurons form. (j) UAS-Go^GTP can induce duplication of external cells, resulting in clustered Sens-positive cells (blue) not expressing internal cell markers. α-Sens staining begins to decay at this stage, preventing an assessment of the frequency of this occurrence. (k and l) Shown is ≈35-h APF staining with α-Su(H) (socket cell, red) and α-Elav (neuron, green). WT clusters have a single socket and a single neuron cell (k), whereas ≈4% of UAS-Go^GTP clusters lose neurons and duplicate the socket cells (l).

Fig. 4.

Localization and biochemical/genetic interactions of Go in SOPs. (a) In a mitotic SOP (α-Sens, blue), Go protein (green) can form an anterior cortical crescent (arrow) overlapping with the Numb (red). (b) (Upper) Go-GTP resin and Go-GDP resin, but not GST resin, precipitate full-length Pins (≈70 kDa) from Drosophila extracts. Go-GDP (more effectively than Go-GTP) binds a slower migrating, phosphatase-sensitive form of Pins (≈75 kDa). (Lower) Go-GDP binds purified MBP-Pins; Go-GTP binds much less. Equal amounts of GST and GST-Go protein were loaded in each lane. (c) In WT (Left), Pins (red) localizes in an anterior crescent in mitotic SOPs (α-Sens, blue), colocalizing with Numb (green; yellow in merge). Overexpression of Go (UAS-Go^WT, Right) decreases plasma membrane localization of Pins. Numb crescent formation is disturbed less. (d and e) WT (Left) and sca-Gal4; UAS-Go^GTP (Right) neuroblasts stained for Numb (d, red) or Pins (e, red) and DNA (blue); apical is up. (f) Quantification of stout bristle asymmetric division defects upon overexpression of different forms of Go and Gi and in Gi homozygotes. Go^GTP, but not Gi^GTP, induces asymmetric division defects. Removal of Gi produces phenotypes only in Go heterozygotes. Mean values are shown as bars; numbers indicate the number of wings analyzed for each genotype. Statistical significance was assessed by unpaired t test. ∗∗∗, P < 0.005; ∗∗, P < 0.01; ∗, P < 0.05; n.s., P > 0.1. (g) Overexpressed Go-induced division defects are attenuated by removal of one copy of Gγ1, strongly enhanced by pins, and unaffected by loco. Removal of pins produces frequent bristle defects, rescued by a hsp70-pins transgene.

Fig. 5.

Genetic interactions of Go with Fz receptors. (a–d) Removal of fz rescues asymmetric cell division defects of UAS-Go^WT (a and c) but not UAS-Go^GTP (b and d). (e, f, and h–j) Coexpression of UAS-fz enhances both phenotypes (e and f), whereas UAS-fz alone does not produce any asymmetric division defects (h). Expression of Dfz2 produces ectopic margin bristles (i), whereas co-overexpression of Go and Dfz2 results in mutual enhancement of both division and ectopic bristle phenotypes (j, arrows). (g) Division defects are quantified, normalizing UAS-Go^WT and UAS-Go^GTP as 100%. Data are presented as in Fig. 4.

Fig. 6.

Schematic comparison of the fz and Go phenotypes in SOP divisions (a) and the PCP mechanism (b). (Left) In the WT SOP (a) and wing epithelial cell (b), a gradient of an extracellular signal (schematized by the light-green triangle) is likely decoded by Fz (originally equally distributed around the cell surface, schematized by the green shading). This decoding results in a posterior (SOP) or distal (PCP) enrichment of Fz (schematized by the dark-green accumulations), followed by anterior localization of the Numb crescent (red in a) and distal localization of the hair growth site (black in b). (Center) In the absence of Fz, cell polarization still occurs because of the intrinsic cell polarization machinery, but at random locations; this phenomenon is illustrated by the improper positioning of the Numb crescent (a) or hair growth site (b). (Right) In Go mutants, Fz signaling fails, resulting in failure of Fz to redistribute (illustrated by the persistent green shading). Also, the intrinsic cell polarization machinery is faulty, leading to diffuse or multifocal Numb staining (red in a) or formation of multiple hair growth sites (black in b).