Cell-cell contact landscapes in Xenopus gastrula tissues

Abstract

Molecular and structural facets of cell-cell adhesion have been extensively studied in monolayered epithelia. Here, we perform a comprehensive analysis of cell-cell contacts in a series of multilayered tissues in the Xenopus gastrula model. We show that intercellular contact distances range from 10 to 1,000 nm. The contact width frequencies define tissue-specific contact spectra, and knockdown of adhesion factors modifies these spectra. This allows us to reconstruct the emergence of contact types from complex interactions of the factors. We find that the membrane proteoglycan Syndecan-4 plays a dominant role in all contacts, including narrow C-cadherin-mediated junctions. Glypican-4, hyaluronic acid, paraxial protocadherin, and fibronectin also control contact widths, and unexpectedly, C-cadherin functions in wide contacts. Using lanthanum staining, we identified three morphologically distinct forms of glycocalyx in contacts of the Xenopus gastrula, which are linked to the adhesion factors examined and mediate cell-cell attachment. Our study delineates a systematic approach to examine the varied contributions of adhesion factors individually or in combinations to nondiscrete and seemingly amorphous intercellular contacts.

Keywords: Xenopus; cadherin; cell adhesion; glycocalyx; syndecan.

Fig. 1.

Cell–cell contacts in the X. laevis gastrula. (A) Midsagittal semithin section of stage 11 Xenopus gastrula outlining dorsal tissues: Ectoderm (Ecto; green), chordamesoderm (CMe; yellow), prechordal mesoderm (PCMe; orange), leading-edge mesendoderm (LEMe; red), and endoderm (Endo; blue). Arrows: D, dorsal; V, vegetal. Epithelial layer, bottle cells (purple), and ventral mesoderm (VeMe; magenta) were not analyzed. (B–F) TEM images. (B) Ectoderm with cell highlighted in green. (C) Pair contact between interstitial gaps (i) with narrow (yellow arrowheads) and wide regions (purple arrowheads). (D) Narrow <50-nm contact with sections filled with dense material (white arrowheads) and membrane pits and membrane-adjacent elongate vesicles (red arrowheads). (E) Wide contact, width determined by measuring intercellular distance in 100-nm intervals (red dotted lines). (F) Contact angle 2θ between cells at the ends of contact (i). (G) Mean relative abundance of narrow (<50 nm) and wide (>50 nm) contacts and gaps. (H) Contact angles between cells as in F. (I and J) Total length of adhesive contacts and gaps as a function of cell circumference. Error bars represent SD of the mean. For P values refer to SI Appendix, Tables S2 and S3. n = 24 embryos for all tissues. P > 0.05 n.s., P < 0.05*, P < 0.01**, P < 0.001***, P < 0.0001****. (Scale bars: black, 100 μm in A; 10 μm in B; and white, 1 μm.)

Fig. 2.

Tissue-specific contact spectra in the X. laevis gastrula. Contact widths were binned at 50-nm intervals, and their respective abundance was determined for (A) ectoderm, (B) chordamesoderm, (C) prechordal mesoderm, (D) leading-edge mesendoderm, and (E) endoderm. Recurrent peaks are highlighted by shaded regions. n = 24 embryos for all tissues.

Fig. 3.

Knockdown of adhesion factors disrupts cell contact patterns. (A–J) Semithin sections of single and double morphant Xenopus gastrulae. Ecto, ectoderm, Endo, endoderm. Dotted outlines demarcate leading-edge mesendoderm (red), prechordal mesoderm (orange), and chordamesoderm (yellow). White arrowheads indicate tip of archenteron invagination. Invagination is almost completely inhibited in treated embryos except in C, where it is increased. Dorsal is to the Left, animal to the Top. (Scale bars, 100 μm.)

Fig. 4.

Knockdown of C-cadherin and Syndecan-4. (A–D and F–I) TEM images of wild-type, C-cad, and Syn-4 morphant ectoderm and prechordal mesoderm. Single cells are highlighted in green or orange. (B′–D′ and G′–I′) Difference spectra for ectoderm and prechordal mesoderm. The Left-most, white column in each graph denotes interstitial gaps. Colored columns indicate changes in abundance, which are statistically significant (P < 0.05) and represent ≥50% changes in morphants compared with wild type (blue, decrease; red, increase). Gray shades highlight positions of recurrent peaks. (E and J) Abundance of narrow and wide contacts and of gaps. Error bars represent SD of the mean. For P values refer to Datasets S2 and S3. (Scale bars: black, 10 μm; white, 15 μm.) Wild type: n = 24 embryos; C-cad-MO: n = 14; Syn-4-MO: n = 10; and C-cad-MO+Syn-4-MO: n = 8.

Fig. 5.

Knockdown of FN. (A–D and F–I) TEM images of wild-type and FN, C-cad/FN, and FN/Syn-4 morphant ectoderm and prechordal mesoderm, with single cells highlighted. (B′–D′ and G′–I′) Difference spectra for ectoderm and prechordal mesoderm. Colored columns show changes in abundance as in Fig. 4. Positions of recurrent peaks are shaded in gray. (E and J) Summary of the abundance of narrow and wide contacts and of gaps. Error bars represent SD of the mean. For P values refer to Datasets S2 and S3. (Scale bars: black, 10 μm; white, 15 μm.) Wild type: n = 24 embryos; FN-MO: n = 12; C-cad-MO+FN-MO: n = 15; and FN-MO+Syn-4-MO: n = 9.

Fig. 6.

Knockdown of HA synthases. (A–D and F–H) TEM images of wild-type and Has-1, Has-2, and C-cad/Has-1 morphant ectoderm and prechordal mesoderm; single cells are highlighted. (B′–D′ and G′–H′) Difference spectra for ectoderm and prechordal mesoderm. Colored columns show changes in abundance as in Fig. 4. (E and I) Abundance of narrow and wide contacts and of gaps. Error bars represent SD of the mean. For specific P values refer to Datasets S2 and S3. (Scale bars: black, 10 μm; white, 15 μm.) Wild type: n = 24 embryos; Has-1-MO: n = 10; Has-2-MO: n = 11; and C-cad-MO+Has-1-MO: n = 9.

Fig. 7.

La³⁺ staining of interstitial gaps. (A) Low magnification view of La³⁺-stained stretches in contacts and interstitium. Interstitial gap (i) surfaces (B) displaying little to no lanthanum staining, (C) contiguous, dense plaques; (D–E′) a bush-like glycocalyx; or (F–G) a fine fibrillar glycocalyx based on dense plaques or a zone of dense lumps (black arrowheads). (E and E′) A bush-like glycocalyx is embedded in a faintly stained material (pink arrowheads). (H) Gaps are often filled with fine, wavy fibrils (white arrowhead). (I) Aggregates of straight and wavy fibrils attached to dense plaques on the cell surface or (J) unattached in the interstitium, together with dense lumps.

Fig. 8.

La³⁺ staining of cell–cell contacts. (A) Narrow contact 15 to 30 nm wide. (B) La³⁺-stained contact increasing from 30 to 100 nm from Left to Right, ending at interstitial gap. (C) A 30- to 70-nm-wide contact with alternating La³⁺-stained and unstained stretches. (D) Homogeneously stained contact, width increasing from 40 to 120 nm, ending in unstained wide contact (Right). Patches of cytoplasmic staining are visible near the contact. (E) La³⁺-stained bush-like glycocalyx with thick fibrils in 125- to 250-nm-wide contact ending on the Right in unstained 125-nm contact. (F) Wide contact gradually opening into an interstitial gap. Bush-like glycocalyx with thick fibrils extending from dense plaques, often in alternating positions on opposite membranes (Inset). (G) A 350-nm-wide contact with fine fibrils emanating from dense plaques. (H) Micrometer-wide contact with fibril aggregates and dense plaques extending between opposing cell surfaces. Interstitial gaps, i. Black arrowheads indicate La³⁺-stained stretches; white arrowheads, unstained stretches; and orange arrowheads, cytoplasmic La³⁺ staining near contacts.

Fig. 9.

Modulation of glycocalyx structure by adhesion factors. TEM images of La³⁺-stained structures in morphants. (A and A′) C-cad and (B and B′) FN morphant prechordal mesoderm with plaques (black arrowheads), thin fibrils (white arrowheads), and (A′ and B′) fibril aggregates containing dense lumps in interstitium (i). (C and C′) Has-1 morphant prechordal mesoderm with plaque layer in 120-nm contact (white arrow) and in gap (i), and (C′) with 40-nm plaque layers on either side of 200-nm wide contact. (D) Has-2 morphant ectoderm, glycocalyx-filled contact from 15 to 130 nm wide. (D′) Gap with plaque layer in Has-1-MO mesoderm. (E and E′) Syn-4 morphant mesoderm. (E) A 200- to 400-nm contact with small, sparse lumps (black arrowheads) and a fibril (white arrowheads). (E′) Fibril aggregates without lumps.

Fig. 10.

Model of glycocalyx-mediated contacts in the Xenopus gastrula. Reconstruction of contact types in ectoderm (A) and prechordal mesoderm (B), based on difference spectra and La³⁺ staining. Solid and dashed lines indicate regions of width spectrum that are strongly or weakly reduced by single adhesion factor knockdown. Gray inhibitory arrows indicate supposed indirect inhibition of contacts. Gray box, reconstructed contact types; asterisks, results from double knockdown were used in deductions. (C) La³⁺ staining reveals different glycocalyx structures in the Xenopus gastrula. Only La³⁺-binding components of surface coats are visualized (dark red); others like FN or yet unidentified factors (pink) must be assumed to contribute.