FN Matrix Is Required for Morphogenesis, but Not for Mesodermal Patterning in Whole Embryos
(A–C) (A) FN matrix assembly, as detected by immunofluorescence, is extensive in stage 12 embryos injected with control mAb 4H2. (B and C) Patterning of mesodermal ([B], Xnot; [C], Xbra) tissues is unaffected by mAb 4H2 injection, as detected by in situ hybridization.
(D–F) (D) Embryos injected with the FN function-blocking mAb 4B12 do not assemble an FN matrix, and the domains of expression of (E) Xnot and (F) Xbra are shorter and wider than in control embryos.
(G–I) (G) En face views of control cells along the inner surface of the blastocoel roof expressing the HA construct (green) support FN matrix assembly (red). (H and I) Patterning of mesodermal ([H], Xnot; [I], Xbra) tissues is normal in embryos expressing HA.
(J–L) (J) Embryos expressing the HAβ1 dominant-negative integrin construct (green) do not assemble an FN matrix (red). (K and L) Patterning of mesodermal ([K], Xnot; [L], Xbra) tissues is unaffected by HAβ1. However, the domains of expression of these marker genes are shorter and wider than in HA-expressing embryos ([H], Xnot; [I], Xbra).
The embryos in (G) and (J) were injected with HA or HAβ1 in one blastomere at the 2 cell stage. Embryos in (H), (I), (K), and (L) received transcript injections at two sites in each of the dorsal blastomeres at the 4 cell stage. The embryos shown in (H), (I), (K), and (L) are slightly older than those depicted in (B), (C), (E), and (F). Embryos are arranged with the blastopore down; anterior is up.
FN Matrix Is Required for Axial Extension
(A–O) (A) Keller sandwiches made from embryos expressing HA begin extending at (A) 4 hr and are fully extended by (B) 18 hr. Explants made from HAβ1-expressing embryos undergo little extension at (E) 4 hr and remain essentially unchanged by (F) 18 hr. Explants are arranged in this figure with the mesoderm to the left and neural and ectodermal tissue to the right. Expression of the HA construct has no effect on the patterning of (C) notochord or (D) somite tissue in 24 hr Keller sandwiches. Similarly, while morphogenesis of sandwiches expressing HAβ1 is disrupted, the patterning of (G) notochord and (H) somite tissues is unaffected. Notochord is revealed with the mAb Tor70, and somite is revealed with mAb 12-101. Keller sandwiches assemble FN fibrils during extension, and assembly is monitored by immunostaining and confocal microscopy. (I) Two hours after explants were assembled, FN fibrils can be observed in sandwiches, and extensive matrix is present by (J) 4 hr. (K) A cartoon of a Keller sandwich showing ectoderm (blue), neural tissue (green), mesoderm (red), and endoderm (pale yellow). The boxes indicate the areas represented in (L)–(O). In sandwiches expressing HA, FN underlies the area of neural tissue undergoing convergent extension (L) and defines boundaries surrounding notochord and somites (M). In sandwiches expressing HAβ1, FN fibril assembly is inhibited in both (N) neural and (O) mesodermal tissues.
Integrin Dominant Negative Disrupts Bipolar Cell Intercalations
(A–D) Confocal images of dextran-labeled deep cells were collected from (A and C) mesoderm and (B and D) neural tissues of Keller sandwich explants undergoing extension. Cells take on normal bipolar morphologies in both (A) mesoderm and (B) neural tissues of explants expressing HA. In explants made from embryos expressing HAβ1, there are no bipolar cells in either (C) mesoderm or (D) neural tissues. The red arrows indicate the anterior-posterior axis; anterior is toward the top of the figure in panels (A)–(D).
(E and F) In 18 hr explants that express HA, the notochord cells have converged toward the midline ([E], nuclei indicated by yellow arrowheads), producing a narrow structure ([E], red arrow). In HAβ1-expressing explants of a similar age, the notochord is broader ([F], red arrow), and the location of the notochord cell nuclei ([F], yellow arrowheads) indicates that cells have not converged toward the midline. Grayscale images (E) and (F) have been inverted to clarify cell boundaries.
FN Matrix Is Required for Elongate Cell Morphologies in Explants Undergoing Convergence and Extension Movements
(A–E) Keller sandwiches made by combining DMZs expressing HA and HAβ1. Elongate cell phenotypes ([A], dextran-labeled cells) are observed in the half of the explant that assembles FN ([B], FN revealed with 32F polyclonal Ab). In the half of the explant that does not assemble an FN matrix, cells take on a disorganized rounded phenotype, but a boundary between notochord and somitic mesoderm is still evident ([A], red arrowheads). (A) and (B) were obtained from 10 hr explants and represent a saggital section through an area similar to that indicated by the red box in Figure 2K. (C–E) Keller sandwiches were also made from a combination of DMZs expressing HA and HAβ1 colabeled with green and red dextrans, respectively. The HA-expressing mesoderm cells take on normal bipolar phenotypes ([C], grayscale image of green channel shown in [D]), unless surrounded by HAβ1-expressing cells ([C], red arrowheads). HAβ1-expressing cells have a rounded appearance ([D], red dextran; [E] is grayscale image of red channel shown in [D]), unless surrounded by HA-expressing cells ([E], red arrowheads).
Integrin Ligation Regulates Cadherin Adhesion and Is Independent of Receptor Levels at Cell Surface or FAK Phosphorylation.
(A) Animal cap blastomeres expressing HA adhere strongly to the FC-cadherin fusion protein (HA, 86 ± 12%), while activin decreases blastomere adhesion (HA + ACTIVIN, 57 ± 3.7%). Coincubation of activin-induced animal cap cells with 15 μg/ml of the GST-9.11 fusion protein encompassing the CCBD of FN restores adhesion to cadherin (HA + ACTIVIN + 9.11, 84 ± 4.5%). Cells expressing HAβ1 show reduced adhesion to the cadherin substrate (HAβ1, 65 ± 2.5%). This reduced adhesion is independent of activin induction (HAβ1 + ACTIVIN, 66 ± 7.5) and cannot be rescued with the FN fusion protein (HAβ1 + ACTIVIN + 9.11, 62 ± 4.9%).
(B and C) (B) Immunoprecipitation of cadherin and integrin receptors from surface-biotinylated DMZ cells reveals that surface expression of cadherins is unchanged between uninjected (CONT), HA-injected (HA), and HAβ1-injected (HAβ1) embryos. Similarly, surface levels of β1 integrins remain unchanged under the same conditions. Cadherins and integrins were immunoprecipitated with a polyclonal Ab (Xcad) and mAb 8C8, respectively, and were detected with avidin HRP. Integrin profiles reveal the presence of β1 and associated α subunits. (C) Immunoprecipitation of FAK from HA- or HAβ1-expressing embryos does not reveal differences in either the total amount of FAK or the level of phosphorylated FAK.
Cell Sorting of Dissociated DMZ Cells Is Altered by Expression of the Integrin Dominant-Negative Construct
Aggregates were made from HA- and/or HAβ1-injected DMZ-dissociated deep cell layers and were allowed to sort for 3 hr at room temperature.
(A) When HA-expressing green and red dextran-labeled cells are combined, they aggregate but do not sort.
(B) Red and green dextran-labeled cells expressing HAβ1 also aggregate but do not sort.
(C) Sorting also does not occur when green and red cells expressing HA are treated with the C-cadherin-activating antibody AA5.
(D and E) When HA-expressing cells (red) are combined with HAβ1 cells (green), cell sorting is evident after (D) 1 hr, and, by (E) 3 hr, the two cell populations are segregated; aggregates of red cells are shed at the periphery (data not shown). FN matrix can be detected in aggregates after 3 hr of association with cells expressing HA ([E], inset).
(F) Activation of C-cadherin by mAb AA5 inhibits the exclusion of HA (red) cells from the mixed aggregates. Three individual assays were performed on three separate spawnings for a total of nine aggregates of each type; representative samples are shown.
Integrin Ligation Promotes Cadherin-Dependent Reintegration of DMZ Cells
Red dextran-labeled stage 10 DMZ cells were dissociated and seeded back onto green dextran-labeled stage 10 intact DMZs. Vertical confocal sections were taken to observe the reintegration of the cells into the DMZ.
(A–D) Reintegration of DMZ cells requires integrin ligation. (A) Dissociated cells do not invade after 10 min. (B) Cells preincubated with CCBD of FN (GST-9.11) reintegrate rapidly.
(C) Reintegration of cells promoted by GST-9.11 binding is inhibited in the presence of an antibody that blocks integrin adhesion to GST-9.11 (4B12).
(D) A nonblocking anti-FN antibody (4H2) has no effect on integration behaviors.
(E–H) Integrin-mediated reintegration requires cadherins. (E) Dissociated DMZ cells do not integrate into the DMZ, (F) while those incubated with GST-9.11 reintegrate. (G) A C-cadherin function-blocking mAb (6B6) inhibits this reintegration. (H) Similarly, a mAb (AA5) that activates C-cadherin inhibits integration. (I–L) (I) Cells expressing the HA construct do not reintegrate into the DMZ, (J) while incubation with GST-9.11 promotes rapid integration. (K and L) This activity is inhibited by both mAb (K) 6B6 and (L) AA5.
(M–P) (M) HAβ1-expressing cells do not reintegrate, (N) even in the presence of GST-9.11. Nor do they integrate with the (O) function blocking C-cadherin antibody 6B6 or with the (P) adhesion-stimulating antibody AA5. Each of these experiments were repeated four times, and each condition was studied by analyzing the seeding of 10–12 cells per experiment. Individual cell behaviors were consistent from experiment to experiment (>95% repeatability; variability due to inaccurate placement of individual cells at explant periphery). Representative examples are shown.
Figure S1. Assays for Cell Sorting and Mobility
For cell-sorting assays, HA1-expressing embryos were coinjected with green dextran, and HA-expressing embryos were injected with red
dextran at the 1 cell stage. Stage 11 DMZs were excised and dissociated in Ca2/Mg2-free media. Dissociated cells were then combined
and allowed to aggregate under conditions described in Figure 6. For invasion assays, DMZs from embryos labeled with red dextran were
excised, dissociated, treated as described in Figure 7, and seeded onto excised green dextran-labeled DMZs.
Figure S2. Anti-FN Antibodies Inhibit Convergent
Extension in Open Face Explants
(A–F) (A, C, and E) Bright-field views of 16 hr
open-faced explants. (B, D, and F) The same
explants at higher magnification under epifluorescence
to reveal FN fibrils. Incubation of
open-faced explants in a mAb that blocks
FN fibril assembly gives variable results with
regard to both the (A and C) extent of extension
and the appearance of (B and D) FN fibrils.
A paucity of fibrils (B) is associated with
a lack of extension in these explants (A). Intermediate
extension is noted in some explants
incubated with 1F7 (C), and in these cases,
this correlates well with the appearance of
some fibrils (D). In all explants incubated with
control mAb 4H2, extension was robust and,
correspondingly, an extensive fibrillar network
was detected in the extending tissue
(F). Antibody treatments were as indicated in
each panel (4H2, control anti-FN mAb; 1F7,
FN-blocking mAb; Rb anti-FN, rabbit anti-
Xenopus FN polyclonal #32).