Cross-Scale Integrin Regulation Organizes ECM and Tissue Topology

Abstract

The diverse morphologies of animal tissues are underlain by different configurations of adherent cells and extracellular matrix (ECM). Here, we elucidate a cross-scale mechanism for tissue assembly and ECM remodeling involving Cadherin 2, the ECM protein Fibronectin, and its receptor Integrin α5. Fluorescence cross-correlation spectroscopy within the zebrafish paraxial mesoderm mesenchyme reveals a physical association between Integrin α5 on adjacent cell membranes. This Integrin-Integrin complex correlates with conformationally inactive Integrin. Cadherin 2 stabilizes both the Integrin association and inactive Integrin conformation. Thus, Integrin repression within the adherent mesenchymal interior of the tissue biases Fibronectin fibrillogenesis to the tissue surface lacking cell-cell adhesions. Along nascent somite boundaries, Cadherin 2 levels decrease, becoming anti-correlated with levels of Integrin α5. Simultaneously, Integrin α5 clusters and adopts the active conformation and then commences ECM assembly. This cross-scale regulation of Integrin activation organizes a stereotypic pattern of ECM necessary for vertebrate body elongation and segmentation.

Figure 1. Regulation of FN matrix fibrillogenesis within the paraxial mesoderm

(A) A schematic showing the somites (green) and mesenchymal presomitic mesoderm (blue). The yellow box indicates the regions shown in B–E. (B–E) show a 3D reconstruction of the FN matrix in the paraxial mesoderm (see Movie S1). (A) A dorsal view of the tissue with anterior at the top. Positions of sections of the mature (C) and nascent (D) borders and presomitic mesoderm are indicated. Asterisk in C indicates somite border matrix contiguous with the dorsal surface of the tissue. Arrowhead in D indicates de novo matrix assembly within the nascent boundary. For the Fibronectin IHC analysis z-stacks were acquired and analyzed for 13 embryos from 3 independent stainings. In panels C–E, dorsal is up. (F) A schematic of cell transplantation from a labeled donor blastula into host blastula. The tbx6^−/− background is used since they make somite boundaries. Embryos develop until the 8–16 somite stage and clones are examined for FN matrix. (G) Isolated paraxial mesoderm clones lacking itgα5, due to itgα5 morpholino injection, are coated with FN by Itgα5 expressed on adjacent host paraxial mesoderm cells. Scale bars are 40 microns.

Figure 2. Quantification of Itgα 5 dynamics in the presomitic mesoderm

(A) A schematic of FCS data collection for fluorescently tagged proteins in the presomitic mesoderm. For FCS, the confocal volume (drawn approximately to scale in blue in A and B) was statically positioned in a cell membrane oriented orthogonal to the optical axis. (B) Confocal image of a zebrafish embryo expressing membrane RFP (mem-RFP). Scale bar is 10 μm. (C) Autocorrelation curve of cytosolic GFP (cyt-GFP), fit to 3D diffusion (Equation 1 supplemental methods). Inset, histogram of measured number of Itgα5-GFP molecules. (D) Representative set of 10 autocorrelation curves (gray) of Itgα5-GFP with the averaged curve used in fitting (green). Inset, the intensity fluctuations and binned intensities from this measurement. (E) Auto-correlation measurements of mem-RFP, Itgα5-RFP, and Cdh2-RFP. Fits to 2D diffusion and fluorophore fluctuation (solid line, Equation 3 supplemental methods) and to 2D diffusion only (dotted line, Equation 2). To facilitate comparison, diffusion components are normalized to 1. (F) Autocorrelation curves of mem-GFP-RFP illustrating the fast decay component is fluorophore specific, but the slow decay, i.e. diffusion, is identical in both autocorrelation curves as well the crosscorrelation curve. (G) Table of measured diffusion times and calculated diffusion coefficients ±SD.

Figure 3. Quantification of protein association in the presomitic mesoderm

(A) a schematic of cell transplantation and FCCS data collection. For FCCS, the confocal volume (drawn approximately to scale in blue in A and B) was positioned at a membrane interface between RFP and GFP expressing cells. (B) Confocal image of a mosaic embryo comprised of cells expressing mem-RFP and mem-GFP. Scale bar is 10μm. (C) Auto- and crosscorrelation measurements of Itgα5-RFP and Itgα5-GFP in a mosaic interface show low crosscorrelation. (D) Plots of crosscorrelation from individual measurements and mean values. (E) Table of crosscorrelation (Fcross) magnitude ±SD. Significance was examined using both t-test (two tailed, unequal variance) and Mann- Whitney U. There were no statistically significant differences in mem-GFP/mem-RFP compared to Itgα5-GFP/mem-RFP and Itgα5-GFP/Itgα5-RFP compared to Itgα5-GFP/Itgα5^FYLDD-RFP. Notably, either mem-GFP/mem-RFP or Itgα5-GFP/mem-RFP compared to either Itgα5-GFP/Itgα5-RFP or Itgα5-GFP/Itgα5^FYLDD-RFP differed with p<0.002. The Itgα5-RFP/Cdh2-GFP “trans” Fcross differs from the negative control (p<0.05) but not from the Itgα5-RFP/Cdh2-GFP “cis” Fcross. The Itgα5-GFP/Itgα5-RFP Fcross in the absence of Cdh2 is significantly lower (p<0.05) than the Itgα5-GFP/Itgα5-RFP Fcross in the presence of Cdh2. See Figure S1 for apparent Kd calculations.

Figure 4. Itgα 5 adopts the active, extended conformation independently of ligand binding

(A) A schematic of the bent and extended Itgα5β1. The SNAKA51 antibody recognizes an epitope on the extracellular domain of Itgα5 that is exposed when the Itgα5β1 adopts the extended, active conformation (see also Figure S2). The blue star represents the five amino acid substitutions in Itgα5^FYIDD that eliminate ligand binding. The magenta star represents the two amino acid substitutions in Itgα5^GAAKR that promote adoption of the extending active conformation. (B–E) Confocal images of MZ itgα5^−/− mutant embryos expressing Hs Itgα5β1-YFP (B) immunostained with the conformation-specific anti-Hs Itgα5 antibody SNAKA51 (C) and anti-FN antisera (D). Arrows in B indicate somite borders and the asterisks denote the lateral surface of the tissue. 6 experiments were performed with n=55 embryos analyzed. (E) To normalize the SNAKA51 signal to the level of YFP, a ratiometric topological heat map was generated. Warmer colors indicate higher relative levels of extended, active Itgα5β1. The same pattern of Itgα5 activation is observed in the absence of BiFC when using Hs Itgα5-GFP for ratio imaging (not shown). (F–I) show confocal images of a parallel analysis of MZ itgα5^−/− mutant embryos expressing Hs Itgα5^FYIDD ligand binding deficient Integrin. Arrowheads in (F) indicate ephemeral Itgα5^FYIDDβ1 clustering. 6 experiments were performed with n=52 embryos analyzed. In all panels, anterior is up and lateral is right. Scale bars are 20 μm.

Figure 5. Itgα5 conformation regulates Itgα5 clustering and FN matrix assembly in the presomitic mesoderm

Itgα5^GAAKR contains two amino acid substitutions that bias towards the extended, active conformation. (A) Hs Itgα5^GAAKRβ1-YFP clusters along somite boundaries (arrows) in MZ itgα5^−/− embryos but also inappropriately clusters in the presomitic mesoderm (asterisks). This inappropriate clustering colocalizes with Hs Itgα5^GAAKRβ1-YFP adoption of the active conformation (B and D) and FN aggregation (C). SNAKA51 and FN IHC were examined in 51 Hs Itgα5^GAAKRβ1-YFP expressing MZ itgα5^−/− embryos (5 experiments). (E–J) Hs Itgα5^GAAKRβ1-YFP clustering and FN-GFP aggregation in the presomitic mesoderm is unstable. Time-lapses of three Tg(tbx6:fn1a-GFP) embryos co-injected with the itgα5 morpholino and itgα5^GAAKR-RFP mRNA were acquired and analyzed. Three time points t=1 (E and F), t=15 (G and H) and t=30 (I and J) show the correlated changes in Itgα5^GAAKR-RFP clustering FN-GFP aggregation in the anterior presomitic mesoderm. Scale bars are 20 μm. The images are from a 94 minute time-lapse, with a z-stack spanning 14 μm acquired every 4 minutes (see Movie S2).

Figure 6. Cdh2 represses Itgα5 activation

(A) cdh2^−/− embryos produce significantly more ectopic small aggregates of FN matrix (red) in the mesenchyme of both the anterior (p<0.005, t-test) and (B) the posterior paraxial mesoderm (p<0.02, t-test). (C) tbx6^−/−;cdh2^−/− embryos produce significantly more FN fibrils (green) in the anterior paraxial mesoderm mesenchyme than tbx6^−/− controls (p<0.01, t-test). Mean values ± SEM are shown. (D) Ectopic FN assembly, Itgα5 clustering and adoption of the active Itgα5 conformation is observed in the PSM mesenchyme of cdh2 mutants, n=38 embryos. (E) The first and last timepoints of a 90 minute timelapse (1 frame every 3 min) of Cdh2-GFP and Itgα5-CFP localization during somite morphogenesis (see also Movie S3). Cdh2-GFP levels decrease at somite boundaries (asterisks) while Itgα5-CFP levels increase (arrows) due to clustering. Cdh2-GFP is also diminished along the surface of the paraxial mesoderm (arrowheads), n=4 timelapses. (F) Cdh2-GFP levels anti-correlate with site of FN assembly along somite boundaries and the tissue surface, n=8 embryos. Scale bars =40 μm.

Figure 7. FN matrix fibrillogenesis and remodeling at the somite boundary

(A) A schematic indicating the regions shown in subsequent figure panels. (B–G) show digital transverse sections of a 93 minute time-lapse of a nascent somite border in an Itgα5-RFP; Tg(tbx6:fn1a-GFP) embryo with 11 μm z-stacks of 1 μm intervals acquired every 3 minutes. Early: time-point 5 (12 minutes into the movie), late: time-point 31 (90 minutes into the movie). Arrow in B indicates Itgα5-RFP clustering prior to the appearance of detectable levels of FN matrix. See also Movie S4. Eleven time-lapses of itgα5-RFP; Tg(tbx6:fn1a-GFP) embryos were acquired and analyzed. (H–N) We acquired seven time-lapses of Tg(tbx6:fn1a-GFP) embryos. Shown are images from a 3D reconstruction of a 195 minute time-lapse with 40 μm z-stacks taken every 6.5 minutes. (H and J) timepoint 1. (I and K) timepoint 16, 97 minutes into the movie. Asterisks indicate boundaries. J and K are parasagittal sections while all other panels are dorsal views. Anterior is left in all panels. See also Movie S5. (L–N) Motion of the FN matrix was examined using particle tracking. Individual tracks were color coded to indicate whether they exhibited a net displacement along the dorsal-ventral axis (L), medial-lateral axis (M) or anterior-posterior axis (N). The ventral motion displays a segmental pattern corresponding to the somite boundaries (asterisks). Scale bars are 40 μm.

Figure 8. From collective Integrin repression to Integrin activation and Fibronectin matrix fibrillogenesis

(A–D) represent successive time points during somite border formation. Top images show tissue level changes in Fibronectin matrix while lower images show the underlying molecular processes at the cell membrane. (A) Within the unsegmented PSM, Integrin α5β1 is in the bent conformation and repression is maintained by physical interaction between Integrins on adjacent cells. Cdh2 promotes the association of Itgα5 on adjacent cell membranes and stabilizes the inactive Itgα5 conformation. Fibronectin dimers remain in soluble form in the extracellular space. This collective repression prevents FN fibrillogenesis internally within the tissue biasing fibrillogenesis to the tissue surface where collective repression is absent. (B) Cytoplasmic signals downstream of EphA4/Ephrinb2a, (light blue arrows) activate the Integrin heterodimer at the nascent somite border by inducing the extended conformation as well as Integrin clustering. Cdh2 levels diminish at the nascent boundary. (C) Activated Integrins bind soluble Fibronectin and assemble the dimers into an insoluble matrix. The Fibronectin matrix stabilizes Integrin clustering and induces Integrin signaling via Focal Adhesion Kinase (red). (D) Fibronectin matrix from the dorsal surface of the paraxial mesoderm involutes (yellow arrow) along the nascent border where it is integrated with the newly synthesized matrix to form a contiguous ECM between the tissue surface and the somite border.