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Figure 1.
Fibronectin (FN) Localization in the Early Gastrula
(A) FN mucus on the blastocoel roof (arrow) and floor (arrowhead).
(B) FN on the blastocoel roof (arrow) and between migrating mesodermal cells (arrowhead).
(Reproduced, with permission, from R. Winklbauer et al., 1998, Dev. Dyn. 212, 335�345.)
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Fig. 1. Early gastrula, sagittal section. Animal pole (AP), vegetal pole
(VP), future dorsal (D) and ventral (V) sides indicated. Prospective
mesoderm dotted; arrow indicates direction of movement. Inner surface of
blastocoel roof (BCR) covered by FN fibril network, apical layer (AL) of
BCR peeled off the inner layer (IL) at the animal pole. BCR directly above
dorsal mesoderm is prospective neuroectoderm. Below the blastocoel
(BC), the blastocoel floor (BCF) is formed by the vegetal cell mass (VCM;
prospective endoderm). BP, blastopore, IBPL, inner blastopore lip.
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Fig. 2. FN localization in blastula stage embryos. aâc: Stained
sections, stage 6. a,b: Blastocoel and surface of blastomeres with
FN-containing mucus (arrowheads). Animal pole to the top. c: FN
between two vegetal cells (arrow). d: FN content (stage 101â2) in extract
from whole embryos (e) and in blastocoel mucus (m). -, empty lane.
Non-reducing conditions. Dimeric FN at 400 kD, monomeric FN at 250 kD.
Material corresponding to one embryo was loaded on each lane. aâc,
same magnification, bar 5 50 μm.
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Fig. 3. FN fibril formation on the BCR. Whole-mount of BCR from
stage 10 embryo. Bar 5 50 μm.
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Fig. 4. Kinetics of FN fibril formation. a: Percentage of cells with FN
fibrils as a function of developmental time. At the stages indicated, the
number of cells with FN fibrils was determined by examining about 500
cells for each BCR. Each circle represents a single BCR. Dashed line
indicates percentage of BCR cells adhering to FN; data from Winklbauer
(1988). b: Percentage of cells initiating fibril formation, as a function of the
overall progression of fibril formation. For the BCRs of a, the percentage
of cells carrying one to four fibrils was determined and plotted as a
function of the percentage of cells displaying any amount of fibrillar FN.
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Fig. 5. FN localization in the early gastrula. aâd: Dorsal side, stage
101â2, sagittal section. a: FN-containing mucus on the blastocoel floor
(arrowhead), FN fibril matrix on the BCR (large arrow). Bar 5 50 μm. b:
FN matrix (large arrow) between BCR (above) and migrating mesoderm
(below). FN staining at gaps between apical cells of the BCR (small
arrow), and around mesoderm cells (small double arrow). c: Lower end of
FN matrix (large arrow) at inner blastopore lip. Neuroectodermal BCR to
the left of the FN matrix, involuted mesoderm to the right. d: Cell surface
staining in vegetal prospective endoderm. bâd, same magnification, bar5
50 μm. eâh, FN-stained whole-mounts, stage 101 gastrula. e: Border
(arrowheads) between BCR and blastocoel floor (BCF). f: Inner side of
apical BCR layer (top) peeled before fixation from complementary surface
of inner BCR layer (bottom). FN deposits in gaps between apical cells
(Fig. 5b for sectional view) indicated by small arrows. g: Substrate side of
migrating mesoderm, BCR removed before fixation. h: Fractured surface
of vegetal prospective endoderm. eâh, same magnification, bar 5 50 μm.
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Fig. 6. FN fibril formation on explants. a: Stage 91â2 BCR explant
cultured for 3 hr with its inner surface exposed to the medium. b: Same as
in a, but with a coverslip at a close distance above explant. c: Stage 81â2
BCR cultured for 5 hr with its inner surface apposed to a thin layer of
agarose. d: Same as c, but fixed 1 hr before gastrulation. e,f: Apical layer
of stage 91â2 BCR peeled off, and newly exposed surfaces of inner BCR
layer (e) or apical layer (f) apposed to agarose for 3 hr. g: Cut surface of
mesoderm explant isolated at stage 10 and fixed after 2 hr on agarose. h:
Cut surface of vegetal cell mass (isolated at stage 10) after 2 hr on
agarose. Same magnification, bar 5 50 μm.
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Fig.7. Assembly of exogenous FN into fibrils. aâd: Stage 91â2 BCRs
cultured for 2 hr in 4 μg/ml of bovine plasma FN (a), 15 μg/ml (b), 60 μg/ml
(c), and 250 μg/ml (d) of FN. e: Stage 8 BCR cultured for 2 hr in 250 μg/ml
of FN. f: Stage 10 vegetal cell mass, 2 hr in 250 μg/ml of FN. g,h: Stage
81â2 embryos injected with 200 nl of 1 mg/ml bovine plasma FN and fixed at
stages 91â2 (g) and 101â2 (h). All stained with antibody to bovine FN. Same
magnification, bar 5 50 μm.
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Fig. 8. Cell adhesion and fibril formation. a,b: Stage 91â2 BCR
incubated for 3 hr on agarose in 160 μm/ml of Fab fragment of antibody to
XB/U- and EP/C-cadherin (a) or control Fab fragment from preimmune
serum (b). c,d: Stage 91â2 BCR, used to condition substrate for 3 hr in the
presence of cadherin antibody (c) or control antibody (d). e,f: Substrate
conditioned for 2 hr by small stage 91â2 BCR explants. gâi: Small stage 91â2
BCR pieces covered by large stage 91â2 BCR explant during conditioning.
Fibrils form under covering BCR (g), not under small test explant when its
apical layer is present (h), under test explant with apical layer removed (i).
Same magnification, bar 5 25 μm.
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