Uncoupling integrin adhesion and signaling: the betaPS cytoplasmic domain is sufficient to regulate gene expression in the Drosophila embryo

Abstract

Integrin cell surface receptors are ideally suited to coordinate cellular differentiation and tissue assembly during embryogenesis, as they can mediate both signaling and adhesion. We show that integrins regulate gene expression in the intact developing embryo by identifying two genes that require integrin function for their normal expression in Drosophila midgut endodermal cells. We determined the relative roles of integrin adhesion versus signaling in the regulation of these integrin target genes. We find that integrin-mediated adhesion is not required between the endodermal cells and the surrounding visceral mesoderm for integrin target gene expression. In addition, a chimeric protein that lacks integrin-adhesive function, but maintains the ability to signal, can substitute for the endogenous integrin and regulate integrin target genes. This chimera consists of an oligomeric extracellular domain fused to the integrin betaPS subunit cytoplasmic domain; a control monomeric extracellular domain fusion does not alter integrin target gene expression. Therefore, oligomerization of the 47-amino-acid betaPS intracellular domain is sufficient to initiate a signaling pathway that regulates gene expression in the developing embryo.

Figure 1

Loss of PS1 integrin results in changes in gene expression in the gut epithelia.(A) Schematic drawing of a portion of the midgut, showing the large endodermal cells outlined in blue, which express the PS1 integrin, surrounded by a thin layer of visceral muscles (pink), which express the PS2 integrin. (B–G) Midguts from wild-type and integrin mutant embryos that were dissected and stained for β-galactosidase produced by enhancer traps (258 and A3-2-66) or a gene construct (Mt). Relative to wild type (B–D), the absence of the PS1 integrin leads to an increase in 258 expression in the midgut (E), a decrease in Mt expression (F), and no change in A3-2-66 expression (G). Because the expression of these markers changes during early larval development, the embryos were carefully staged to avoid differences as a result of developmental arrest.

Figure 2

Possible models for integrin regulation of gene expression. Each section of the diagram shows two blue endodermal cells expressing the α_PS1β_PS integrin (blue and gray) attaching via the extracellular matrix (purple) to two pink visceral mesodermal cells that express the α_PS2β_PS integrin (pink and gray). In the first two models, the PS1 integrin holds the endoderm in close proximity to the visceral mesoderm so that either (1) secreted signals from the visceral mesoderm are received by a receptor on the endodermal cell surface (in green), or (2) an ECM component signals to a nonintegrin receptor (in purple). In the third model, PS1 integrin adhesion to the ECM also sends signals. The outcome of the signal is depicted as the repression of 258 expression (light blue nucleus). In the PS1 mutant embryos (bottom), all three types of signal can be disrupted by the loss of PS1 integrin function, resulting in the increased expression of 258 (dark blue nucleus).

Figure 3

Loss of integrin-mediated adhesion does not hinder the induction of endodermal Labial expression by Dpp secreted from the visceral mesoderm. (A) In wild-type embryos at stage 13, Labial expression (shown in black) is induced in those endodermal cells (arrowhead) in direct contact with visceral mesodermal cells (arrow) that express Decapentaplegic (not shown) under the control of the transcription factor Ultrabithorax (shown in brown). (B) This induction of Labial expression still occurs in the absence of PS integrin-mediated adhesion (lacking the β_PS subunit). Examination of older embryos (stage 16) shows that Labial expression is still maintained in the absence of PS integrins (D), as it is in the wild type (C).

Figure 4

Integrin-mediated adhesion between the endodermal and the visceral mesodermal cells is not required to regulate gene expression in the endoderm. (A–C) Dissected guts stained for actin with phalloidin conjugated to rhodamine to show the visceral mesoderm surrounding the gut. The continuous layer of visceral muscle seen in the wild type (A) is moderately disrupted in embryos that lack the PS1 integrin (B), and severely disrupted in embryos that lack the PS2 integrin (C). In D, the predicted result of the disruption of PS2 integrin-mediated adhesion of the visceral muscle to the endoderm is shown for each of the three models. If the signal is sent from the visceral mesoderm (model 1), then signaling will be lost in the PS2 mutant, whereas if the ECM provides the signal (models 2 and 3) then the signaling will be maintained. The latter is true as the absence of PS2 integrin does not change the expression pattern of any of the markers of (cf. E–G with Fig. 1A–C).

Figure 5

Dimerization of the β_PS cytoplasmic tail is sufficient to send signals that regulate gene expression. Antibody staining of the chimeras shows that both wild-type and dominant forms are expressed at similar levels in the embryonic midgut endoderm (end; A,B) with the GAL4 line 48Y. The increase in 258 expression that occurs in the absence of PS1 (F vs. C) is suppressed by the dominant chimera Torso^D/β_cyt (L), but not by the wild-type chimera Torso^WT/β_cyt (I), nor a control chimera Torso^D/punt_cyt (P). The region of the midgut that requires PS1 activity for the repression of 258 is marked with a black line in each panel. Similarly, the expression of the Mt transgene (D) requires PS1 integrin function (G), which can be functionally replaced with the dominant chimera Torso^D/β_cyt (M), but not by the wild-type chimera Torso^WT/β_cyt (J), nor Torso^D/punt_cyt (Q). (Right) Diagrams of the postulated effects of the different genotypes on 258 and Mt expression are shown. (K,O,R) Torso domains are green: (K,O) the β_cyt is gray; (R) the punt_cyt is pink. Although Torso^D/β_cyt sends similar signals to the endogenous PS1 integrin (E,O), neither the Torso^D/punt_cyt signal (red arrow, R) nor the nonoligomeric Torso^WT/β_cyt (K) alters 258 expression (left side of nucleus) or Mt expression (right side of nucleus).

Figure 6

Specific α subunits are not required for integrin signaling in the midgut to regulate 258 gene expression. As shown before, low levels of 258 expression in the anterior midgut require PS1 integrin expression (A,B). Replacement of the endogenous α_PS1 subunit with an α_PS1 subunit provided by GAL4 driven expression produces the wild-type 258 expression pattern (C). Expression of chimeric α subunits containing the extracellular domain of α_PS1 and the cytoplasmic domain of α_PS2 (D), or the extracellular domain of α_PS2 and the cytoplasmic domain of α_PS1 (E), and expression of the entire α_PS2 subunit (F), are all able to produce functional heterodimers that signal to repress 258 expression. The region of the midgut that requires the PS1 integrin for repression of 258 is indicated by the black lines.