RhoL controls invasion and Rap1 localization during immune cell transmigration in Drosophila

Abstract

Human immune cells have to penetrate an endothelial barrier during their beneficial pursuit of infection and their destructive infiltration of tissues in autoimmune diseases. This transmigration requires Rap1 GTPase to activate integrin affinity. We define a new model system for this process by demonstrating, with live imaging and genetics, that during embryonic development Drosophila melanogaster immune cells penetrate an epithelial, Drosophila E-cadherin (DE-cadherin)-based tissue barrier. A mutant in RhoL, a GTPase homologue that is specifically expressed in haemocytes, blocks this invasive step but not other aspects of guided migration. RhoL mediates integrin adhesion caused by Drosophila Rap1 overexpression and moves Rap1 away from a concentration in the cytoplasm to the leading edge during invasive migration. These findings indicate that a programmed migratory step during Drosophila development bears striking molecular similarities to vertebrate immune cell transmigration during inflammation, and identify RhoL as a new regulator of invasion, adhesion and Rap1 localization. Our work establishes the utility of Drosophila for identifying novel components of immune cell transmigration and for understanding the in vivo interplay of immune cells with the barriers they penetrate.

FIGURE 1. Drosophila immune cells move into the tail during development by penetrating an epithelial barrier and carrying out chain migration

Schematic drawings of embryos from a dorsal perspective with anterior to the left, showing hemocytes (green) migrating into the tail during st 11-12; boxed area corresponds to pictures below. Dashed box in rightmost schematic corresponds to (e). (a-c’) Dorsal confocal images of fixed wild type srpHemoGAL4 UAS-GFP embryos which express GFP in hemocytes. Embryos are from successively later stages as indicated above and were stained with anti-Cadherin (red) and anti-GFP (green) antibodies as well as DAPI to visualize nuclei (blue). Hemocytes moving into the tail are indicated with white arrows; examining these locations in the ‘ panels which show only the Cadherin channel reveals that a breach in the Cadherin barrier appears as hemocytes move into the tail. This is particularly evident when one compares the side of the hindgut (upper white arrow in b) where hemocytes have not yet entered and there is no break in the Cadherin staining (upper white arrow in b’) to the other side of the hindgut (lower arrow in b and b’) where they have. (d-e) Stills from 2-photon movies of ubi:shg-GFP; srpHemoGAL4 UAS-GFP embryos. Shg-GFP outlines all cells and hemocyte-expressed GFP is seen in cytoplasm. (d) Dorsal view of early st 11 single plane movie (Supplementary Information, Movie S1) showing the movement of hemocytes through surrounding cells into the tail (green arrow). (e) Dorsal view of a multi-plane movie (Supplementary Information, Movie S2) illustrating chain migration (green arrowhead) around the hindgut. Scale bars, (a-c) 25, (d) 10, or (e) 20 ⌈m. Anterior to left in all panels.

FIGURE 2. Identification of the gene, rhoL, which is expressed and required in hemocytes to permit migration into the tail

(a-d) In situ hybridizations with rhoL probe on wild type embryos show (a-c) hemocyte expression st 5-11 and (d) mesodermal expression st 12. Confocal images of fixed (e-g) st 12 and (h-l) st 14 embryos stained with anti-Cadherin (red) and anti-hemocyte expressed β-Gal (green) antibodies. In wild type embryos, hemocytes migrate (e) into the tail and (h) along the posterior vnc (indicated by white dashed oval). (f, i) In rhoL^XA12 embryos, hemocytes do not. (g, j) In rhoL^XA12 embryos expressing rhoL via the hemocyte driver 8-163GAL4, hemocyte migration (g) into the tail and (j) along the posterior vnc is restored. (k) Wild type and (l) rhoL^XA12 mutant hemocytes migrate normally along the dorsal vessel. (m) Quantitation of hemocyte migration into tail and along dorsal vessel (** P value: 3×10⁻²⁰, ns, not significant: P value: 0.3). Histograms throughout indicate mean ± s.e.m.. The number of embryos examined is recorded in this and all subsequent figures within the histogram. Error bars indicate s.e. throughout. (a-j) Scale bars in these and all other whole embryo pictures indicate 50 ⌈m. All embryos are oriented anterior to the left and dorsal up unless otherwise noted. (k-l) Scale bar, 20 ⌈m.

FIGURE 3. RhoL does not affect hemocyte actin structures or guidance, but is required to penetrate a Cadherin-containing barrier for migration into the tail

2-photon images of (a, c) wild type and (b, d) rhoL^XA12 embryos expressing Moesin-GFP in hemocytes under the control of the 8-163GAL4 driver. (a, b) Stills from 2-photon movies (Supplementary Information, Movie S4, Movie S5). (a) Wild type hemocytes move into the tail; but (b) rhoL^XA12 hemocytes move up to the tail (arrow in 0′) and produce protrusions towards it (arrow in 60′) but fail to enter (compare * in a and b). The elapsed number of minutes is indicated. (c, d) Single plane Z-sections show that RhoL is not required for the normal formation of lamellipodia or filopodia. (e) Matrix metalloproteases are not required for hemocyte movement into the tail (P values: left = 0.4, right = 0.5). (e, h) Quantitation of the number of hemocytes that move into the tail in the genotypes indicated. (f) Fewer hemocytes move into the tail in embryos carrying the rhoL^10-161 hypomorphic allele. (g) This defect is rescued by the addition of the shg^P34-1 mutant that decreases the expression of DE-Cadherin. (f-g) The tail is indicated by the white dashed oval. (h) P values: ** = 6×10⁻⁵, ns = 0.9. Scale bars, (a, b) 15 or (c, d) 5 ⌈m.

FIGURE 4. The Rap1 GEF Dizzy and the α-Integrin Inflated are required for tail invasion

Confocal images of fixed early st 12 embryos stained with anti-Cadherin (red) and (a, c) anti-hemocyte-expressed β-Gal or (b) CD2 antibodies (green) showing that (a, b, d) the Rap1 GEF Dizzy and (a, c, d) the Integrin α-subunit Inflated are required for movement into the tail, but not for movement along the dorsal vessel (Supplementary Information, Fig. S4). (d) Quantitation of the number of hemocytes that move into the tail in the indicated genotypes. The deficiency utilized is Df(2L)BSC185 which removes dizzyΔ1 completely. P values: ** left = 5×10⁻⁵, right = 8×10-¹⁷, both ns = 0.6.

FIGURE 5. RhoL is required for Rap1 to induce adhesion and relocalize from an intracellular concentration to the cell surface in hemocytes

(a, e) Wild type hemocytes located in lateral st 14 embryos do not form large clusters. (b, e) The expression of Rap1^V12, a DA form, induces hemocyte adhesion and clustering which can be alleviated by the presence of (c, e) the rhoL^10-161 hypomorphic allele. (d) The expression of RhoL by itself can lead to clustering. Quantitation of the fraction of hemocyte surfaces in contact with one another showed this was 42±3% in hemocytes overexpressing Rap^DA and 34±3% with RhoL (n .>17 hemocytes in 8 embryos each). (e) Quantitation of the largest number of hemocytes found in a cluster in embryos of indicated genotypes. P values: ** left = 4×10⁻¹⁰, right = 2×10⁻⁸, ns= 0.6. (f-g”) Confocal images from fixed embryos focusing on hemocytes at the junction with the tail or (h-i”) on a single hemocyte. (f-f’, h-h”) Rap1-GFP; srpHemoGAL4 UAS:CD2 and (g-g’, i-i”) Rap1-GFP; rhoL^10-161-GAL4 UAS:lacZ embryos stained with antibodies against (f-f’, h-h”) the membrane marker CD2 or (g-g”, i-i”) cytoplasmic β-Gal to visualize hemocytes (red) and anti-GFP (green) to visualize Rap1. (f-g) Insets indicate % of hemocytes displaying a Rap1 concentration in each genotype (arrow), showing that mutating rhoL increases Rap1 concentrations three fold. (f-i”) Column heads indicate panels show Rap1-GFP staining (f, g, h, i), hemocyte marker staining (h”, i”) or a merge of the two (f’, g’, h’, i’). (h) In wild type hemocytes Rap1 is found at the leading edge (arrow), and in (i) rhoL^10-161 mutants Rap1 is localized to an intracellular concentration that excludes cytoplasm (arrow). Scale bars, (a-d) 10, (f-g’) 20 or (h-i”) 5 ⌈m. (j) Model: To allow invasive migration and breach the DE-Cadherin barrier between epithelial cells, hemocytes require RhoL. The GEF Dizzy converts Rap1 to a GTP-bound form. RhoL causes Rap1-GTP to move from an intracellular concentration to the cell surface, where Rap1-GTP triggers increased Integrin affinity, permitting crossing of the DE-Cadherin barrier.