Expression of 70 kD FN blocks FN fibrillogenesis but does not perturb expression and secretion of endogenous FN. (A-F) Extended focus confocal micrographs (thickness ~2 μm) of en face BCRs immunostained for FN or C-Cadherin. (G) Western blot of total embryo lysates and extracted blastocoel fluid probed with pAb 32F-J directed against endogenous FN (FL FN), and the anti-Myc mAb 9E10 that recognizes both the 70 kD and 40 kD myc-tagged FN constructs (FN-Myc). (H) Western blot of DOC extracted embryos probed for FN. DOC insoluble FN can be detected at tailbud [T] but not gastrula [G] stages. (I) Western blot of DOC extracted control and 70 kD FN gastrula stage embryos confirmed that FN is DOC soluble under both conditions. (UN: uninjected; con: control construct injected embryos; 70 kD: 70 kD FN-Myc injected embryos)

Blastopore closure and epiboly but not axial extension are defective in embryos expressing 70 kD FN. (A-B) Time-matched frames from movies of blastopore closure in representative sibling embryos expressing control (Con) or 70 kD FN (70 kD) constructs. Yellow arrowheads mark the approximate diameters of the blastopores. (C) A representative 70 kD FN gastrula with delayed blastopore closure initiates a putative second blastopore lip (red arrowhead). (D–F) Tailbud stage embryos; (D) uninjected (UN), (E) injected with 70 kD FN into both blastomeres at 2-cell stage, or (F) into the marginal region of two presumptive dorsal blastomeres at the 4-cell stage (DMZ: dorsal marginal zone). (G) Quantification of 70 kD FN tailbud embryo lengths normalized to the mean length of mock injected (dextran only) tailbud embryos from each batch. The horizontal lines represent the mean tailbud lengths. Numbers of all tailbud embryos analyzed are listed above each group. (H) Quantification of the mean width of the axial marker Chordin from in situ hybridizations. Error bars are ±SD (n= 9–10). (I–L) In situ hybridizations after completion of gastrulation (stg. 13) showing localization of Chordin mRNAs. Axial elongation in embryos expressing (L) 70 kD FN in the DMZ are comparable to (I) uninjected embryos. Broad expression of 70 kD FN results in two phenotypes: (J) embryos that fail to complete blastopore closure show a wide pattern of Chordin expression, and (K) embryos that complete blastopore closure express Chordin in a similar pattern to uninjected controls.

70 kD FN blocks epiboly, radial intercalation and randomizes mitotic spindle orientation. (A–E) Vitelline membranes were removed at (A,B) stage 10.5. After 5 hours at 14° C, uninjected gastrulae (C) were normal but 70 kD FN embryos (D) were mushroom shaped. The animal caps of 70 kD FN embryos were bumpy and wrinkled (E, red arrow) indicating that radial intercalation in the BCR was perturbed. (F-G) Confocal micrographs of midline sagitally bisected embryos immunostained for C-Cadherin (blue). Autofluorescence of the yolky endodermal cells appears green. White arrows indicate the BCR (bc, blastocoel; a, archenteron; m, margins of mesendoderm). (H–J) Confocal micrographs of sagitally bisected BCRs immunostained for C-Cadherin. Red lines indicate relative thickness of the BCRs. (K) Quantification of the average number of cells in the BCR (layer index) from 5 experiments (n=2–7 embryos per group). Error bars are ± SEM; results from Student’s t-tests are indicated. (L–N) Sagitally bisected BCRs immunostained for α-tubulin to visualize mitotic spindles. Yellow arrows: “in-plane” spindles (within 30° of the horizontal plane or single puncta in the center of a cell); yellow arrowhead: “out-of-plane” spindles (within 30° of the vertical plane) (Marsden and DeSimone, 2001). (O) Quantification of spindle orientation from a representative experiment. Red triangle represents increasing expression of 70 kD FN (n= 35–160, per group).

Overview of FN fibril functions at gastrulation. (A) Cartoon of 70 kD FN-induced BCR thickening. Direction of normal movements indicated by black arrows; BCR thickness indicated by blue brackets; red crosses represent FN fibrils; red dots represent non-fibrillar FN at cell surfaces. Embryos are oriented animal pole up and vegetal pole down. (B) FN fibrils on normal BCRs are more dense prior to contact with migrating mesendoderm and are more sparsely distributed as the mesendoderm migrates over the BCR (Davidson et al., 2004). Dense fibrils correlate with increased CCBD availability and can inhibit migration by promoting too much cell adhesion. Graph represents the decreasing rate of mesendoderm migration plated on a glass substrate coated with increasing FN density as described in methods. For each density of FN, 36–95 cells were examined from 3–8 experiments. Error bars are ±SEM. (C) Convergent extension in the DMZ requires FN expression, but not assembly into a fibrillar matrix.

Fibronectin constructs and strategy for inhibition of FN fibril assembly. (A) Schematic of Xenopus laevis FN and the corresponding control and 70 kD FN constructs used in this study. Location of epitope for mAb 4H2 in Type III₁₀ is indicated. (B) Representation of presumed mechanism of FN fibril inhibition by 70 kD FN based on the current literature. 70 kD FN binds to the N-terminal matrix assembly domain of endogenously expressed FN, thus blocking FN-FN interactions and fibrillogenesis but not the initial binding of endogenous dimeric FN to integrins at the cell surface. Additional FN dimers are accumulated into the BCR matrix as fibrillogenesis and gastrulation proceed in normal embryos but not in 70 kD FN embryos. This is possible even though the numbers of integrins engaged with FN under both conditions are equivalent.

Levels of total endogenous FN associated with BCR cell surfaces in the presence or absence of 70 kD FN as development proceeds. (A) Live, isolated BCRs were incubated with an anti-FN mAb 4H2 that recognizes the central cell binding domain (CCBD) where FN binds integrin α5β1. BCRs were washed, extracted and run on a non-reduced Western blot and then probed for anti-mouse IgG in order to detect the 4H2 antibody. BCRs incubated with and antibody directed against a cytoplasmic protein (anti-FAK mAb 2A7) was used as a negative control. At early stages (stg. 10–10.5) when fibril assembly begins, 4H2 binding to uninjected (UN) and 70 kD FN (70 kD) caps is comparable indicating that dimeric FN binding to integrins at BCR cell surfaces is equivalent. By late gastrula stages (stg. 11.5), 4H2 binding to 70 kD FN caps was ~30% that of UN control caps. In the 70 kD FN embryos, additional accumulation of FN is blocked, presumably due to limited availability of unoccupied integrins at the cell surface and the failure to form FN multimers. (B) Quantification of 4H2 binding to BCRs. Values (relative pixel densities of scanned gels) were normalized to control levels of FN, (N=4). Error bars are ±SEM

Fibrillar FN is not required for convergent extension in explants or whole embryos. (A–B) Keller sandwich explants made from control or 70 kD FN embryos from 6 experiments (n=2–5 per group) and immunostained for FN. Bright-field images at left and corresponding immunofluorescence at right. (C-D) High magnification, extended focus views of FN immunostaining from Keller explants, at the boundary (yellow dashes) of the notochord (n) and presomitic mesoderm (p). (E and G) 3-D rendered images of FN immunostaining from confocal z-series (~100 μm depth) obtained from the representative Keller sandwiches shown in (C) and (D). (F & H) Single confocal slice from within the through-focus region indicated by the red boxes in (E) and (G), immunostained for C-Cad (green) and FN (red). Movies of the z-series-through-focus and 3D rendering are available online (supplemental Movies 1 and 2). (I–K) Cross section of late stg. 12 gastrulae through the neural plate region (J) of uninjected (I) and 70 kD FN (K) embryos immunostained for C-Cad (green) and FN (red). Diagram at left in (J) after (Nieuwkoop and Faber, 1994).

Decreased adhesion and accelerated mesendodermal mantle closure accompanies loss of FN Fibrils. (A) Cartoon depiction of the area (black box) imaged in (B) and (C). The dotted ellipses trace the perimeter of the embryo equator (green) and mesendoderm margin (red). When viewed en face from the direction of the green arrow, two circles are observed. The ratio of the area bounded by the red circle to the green circle was calculated to represent extent of mesendodermal mantle closure in (E). (B, C) Extended focus confocal z-series of sagitally bisected gastrulae immunostained for C-Cad, through 70–100μm. Embryos are oriented with the animal pole up with partial views of the blastocoel (bc) at upper right. Red arrowheads in (C) indicate gaps between the migrating mesendoderm and the BCR. (D) Sagitally bisected 70 kD FN embryo following fusion of the mesendoderm margins. Green asterisk indicates space between the BCR and the detached mesendoderm (me). (E) Quantification of mesendodermal mantle closure between stg. 10.5 and stg. 11.5 from two independent experiments as illustrated in (A). Ratio of 1 means the mesendodermal mantle is at its maximal open position at the level of the equator and a ratio of 0 means that the mesendoderm margins have fused at the animal pole (i.e. closed position). Significance by t-test as indicated. Error bars plot the standard deviations. (Exp 1: n=9–10 and Exp 2: n=4–8 per group).