Chemokine Signaling Controls Endodermal Migration During Zebrafish Gastrulation

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Science  03 Oct 2008:
Vol. 322, Issue 5898, pp. 89-92
DOI: 10.1126/science.1160038


Directed cell movements during gastrulation establish the germ layers of the vertebrate embryo and coordinate their contributions to different tissues and organs. Anterior migration of the mesoderm and endoderm has largely been interpreted to result from epiboly and convergent-extension movements that drive body elongation. We show that the chemokine Cxcl12b and its receptor Cxcr4a restrict anterior migration of the endoderm during zebrafish gastrulation, thereby coordinating its movements with those of the mesoderm. Depletion of either gene product causes disruption of integrin-dependent cell adhesion, resulting in separation of the endoderm from the mesoderm; the endoderm then migrates farther anteriorly than it normally would, resulting in bilateral duplication of endodermal organs. This process may have relevance to human gastrointestinal bifurcations and other organ defects.

Acrucial feature of vertebrate embryogenesis is the coordinated morphogenesis of germ layers (endoderm, mesoderm, and ectoderm) during gastrulation (1). Interactions between the endoderm and mesoderm specify organ locations and symmetries (2). Defects in the endoderm alter the morphogenesis of mesodermal organs (e.g., heart, kidneys, and blood), whereas mesodermal defects disrupt the locations of the liver and pancreas (25). Morphogenesis is regulated by Wnt (6) and Nodal signaling (7) when cells are intermingled in a bipotential “mesendoderm” (8). However, relatively little is known about germ layer–specific pathways that establish organ rudiments. In zebrafish, mesendodermal organ progenitors involute at the gastrula margin (blastopore) and move anteriorly toward the animal pole (future head) while converging toward the midline (convergent extension).

The chemokine receptor CXCR4 controls directional migration in many contexts and is expressed in the endoderm. It is up-regulated by the endodermal determinants Mixer and Sox17β (913) and is required for gastrointestinal vascularization (14). Of the two closely related zebrafish Cxcr4s, Cxcr4b regulates the migration of many cell types (12, 1518), but no roles have been reported for Cxcr4a during embryogenesis.

Zebrafish embryos deficient in Cxcr4a or Cxcl12b, generated by injection with antisense morpholino oligonucleotides (MO), appeared morphologically normal (Fig. 1, A to C, and fig. S1). However, analysis of Tg(gutGFP)s854 transgenic embryos in which the entire gut fluoresces [(19); GFP, green fluorescent protein] revealed duplications of endodermal organs at 56 hours post-fertilization (hpf) (Fig. 1, D to F, and fig. S2), including the pancreas (normally on the right; fig. S3, A to F) and liver (normally on the left; fig. S3, A to C and G to I), a phenotype we call “viscera bifida” (cxcl12bMO, 78%, n = 23; cxcr4aMO, 68%, n = 37). At 26 hpf, the intestine was also split bilaterally (Fig. 1, G to I; 12bMO, 78%, n = 18; 4aMO, 76%, n = 34), as revealed by foxa2 expression, whereas the floor plate of the neural tube was unaffected (Fig. 1, H and I). No defects were detected in the mesoderm or ectoderm (fig. S3, J to O, and fig. S4).

Fig. 1.

cxcl12b and cxcr4a are required for endodermal morphogenesis. (A to C) Bright-field images, live embryos, 30 hpf; (A) control, (B) cxcl12b morphant, (C) cxcr4a morphant. (D to L) Dorsal views, anterior to the top. [(D) to (F)] Tg(gutGFP)s854, 56 hpf, reveals duplicated liver (L) and pancreas (P) (asterisks) in confocal projections of cxcl12b (E) and cxcr4a (F) morphants. g, gut; hd, hepatic duct; pd, pancreatic duct. [(G) to (I)] foxa2, 26 hpf; the intestinal rod (ir) bifurcates, but not the floor plate (asterisks); pe, pharyngeal endoderm. [(J) to (L)] Double in situ hybridizations for sox32 (endoderm, blue) and ntl (mesoderm, red), 6 hpf; sox32+ cells, but not ntl+ or sox32+ forerunner cells (FRCs, arrowheads), move anteriorly (toward the animal pole and away from the margin). (M to O) foxa2, 8 hpf; lateral view. Morphant endoderm moves anteriorly (arrowheads indicate margin). (P) Quantitation of displacement of foxa2+ cells at dorsal (D), lateral (L, 90° from D), and ventral (V, 180° from D) positions, 8 hpf. Controls, red bars; cxcl12b morphants, blue bars; cxcr4a morphants, gold bars.

During gastrulation, cxcl12b is expressed in mesoderm (Fig. 2, A, C, and E, andfig. S5, A and B), whereas cxcr4a is expressed in endoderm (Fig. 2, B, D, and F, and fig. S5, E and F), and both require Nodal signaling (fig. S5, C, D, G, and H), suggesting that chemokine signaling regulates endoderm-mesoderm interactions. At the onset of gastrulation (6 hpf), sox32+ endodermal cells appeared normal in number but were displaced slightly anteriorly in cxc12b (83%, n = 82) and cxcr4a (74%, n = 62) morphants (Fig. 1, J to L). Displacement became more pronounced by 8 hpf, as revealed by foxa2 expression (Fig. 1, M to O; 12bMO, 88%, n = 34; 4aMO, 85%, n = 55). In controls, the trailing edge of foxa2+ cells was 50 to 100 μm from the margin, whereas in morphants this gap was larger by a factor of >3 (178 to 275 μm dorsally, 170 to 310 μm laterally, 186 to 340 μm ventrally) (Fig. 1P). The leading edge was also displaced up to 100 μm, particularly ventrally. Displacement was not due to precocious endodermal internalization (fig. S6), which suggests a later requirement for chemokine signaling in restricting endodermal movements anteriorly, toward the animal pole. We refer to this as endodermal tethering.

Fig. 2.

Mesoderm expresses cxcl12b and endoderm requires cxcr4a cell-autonomously. (A to F) Whole-mount in situ hybridizations, dorsal right [except (C) and (D), transverse sections of gastrulae]. (A) cxcl12b in mesoderm, 8 hpf; arrow indicates margin. (B) cxcr4a in endoderm. (C) Double in situ hybridizations for cxcl12b (blue) and tbx16 (red), 8 hpf, confirms coexpression in region indicated by arrows, but not in endoderm (arrowhead). (D) Endodermal cxcr4a expression. (E) cxcl12b in lateral mesoderm (arrow), 10 hpf. (F) cxcr4a in endoderm and prechordal plate (PCP, arrow), 10 hpf. (G to L) cxcr4a morphant (green; fluorescein) and control cells (red; rhodamine) grafted into unlabeled hosts, lateral views at 4.3 to 4.5 hpf [(G), (I), (K)] and 8 to 8.5 hpf [(H), (J), (L)]. (M) Average leading and trailing positions of control (red bar) and cxcr4a morphant (green bar) transplanted cells (n = 7).

Despite displacement of the endoderm, the mesoderm was unaffected, as assayed by no tail (ntl) at 6 hpf (Fig. 1, J to L), tbx16 in paraxial mesoderm at 8 hpf (fig. S4, A to C), and hand2 in lateral plate mesoderm at 11 hpf (LPM; fig. S4, D to F). Our results reveal an early distinction between endodermal and mesodermal cell behaviors before they separate from the mesendoderm, thereby implicating the Cxcl12b-Cxcr4a system as among the earliest known signals in endodermal morphogenesis.

If Cxcl12b in mesoderm binds Cxcr4a in endodermal cells to restrict (tether) their movements, Cxcr4a should be required cell-autonomously in the endoderm. To test this, we transplanted endoderm-targeted cxcr4a morphant cells into wild-type hosts (Fig. 2, G to L). As an internal control, these cells were cotransplanted with wild-type endoderm into the same locations (Fig. 2, G, I, and K) in unlabeled hosts at 4 hpf, and cell distributions were compared 4 hours later (Fig. 2, H, J, and L). cxcr4a morphant endodermal cells moved, on average, 200 μm farther anteriorly than did controls (Fig. 2M), demonstrating a cell-autonomous requirement.

How does Cxcl2b-Cxcr4a signaling regulate endoderm migration? Because cxcl12b is maternally deposited and localized to the mesoderm, from which endodermal cells separate during gastrulation, it seems unlikely to act as a chemoattractant here. However, increasing evidence suggests that chemokine signaling modulates extracellular matrix (ECM) proteins [e.g., fibronectin (FN), laminin] and their receptors (integrins), which are required for gastrulation (2024). FN in ECM binds secreted CXCL12 and presents it to CXCR4, causing its redistribution to leading edges of migrating cells (25). Of several fnsin zebrafish, fn1 is expressed by mesoderm during gastrulation (26). Thus, FN in the mesodermal ECM might bind and present Cxcl12b to Cxcr4a-expressing endoderm, sensitizing it to chemokines. CXCR4 activation by CXCL12 also enhances integrin-dependent adhesion of renal carcinoma and small-cell lung cancer cells to FN (22, 27). Thus, we considered both FNs and integrins as potential downstream effectors during endoderm migration.

If this is correct, interfering with FN-integrin interactions should also disrupt endoderm migration. To test this, we treated gastrulating zebrafish embryos with RGD peptides (containing the tripeptide motif Arg-Gly-Asp), which bind integrins and block signaling (28). This caused anterior displacement of the endoderm at 8 hpf, similar to cxcl12b and cxcr4a morphants (Fig. 3, A and B; 40%, n = 52), and delayed convergence of endoderm toward the midline (fig. S7, A to F; 55%, n = 20). In contrast, convergence of LPM, which is required for gut morphogenesis (5), was unaffected (fig. S7, G and H), as in morphants (fig. S4, D to F). RGD peptide treatments of transgenic Tg(gutGFP)s854 embryos caused viscera bifida (Fig. 3, C and D; 42%, n = 65) or situs inversus (fig. S7, I and J; 14%, n = 64), even when applied at mid- to late gastrula stages (viscera bifida, 32%, n = 22; situs inversus, 18%, n = 22), indicating that integrin-dependent interactions are essential throughout gastrulation.

Fig. 3.

FN-integrin–mediated cell adhesion restricts anterior migration of endoderm. (A and B) Whole-mount in situ hybridizations for foxa2, 8 hpf; lateral view, dorsal (right). foxa2+ cells move anteriorly in RGD-treated embryos. (C and D) Tg(gutGFP)s854, 56 hpf; liver (L) and pancreas (P) duplications (asterisks) in RGD-treated embryos. (E) Reduced adhesion of cxcr4a morphant endoderm to a FN1-coated surface, and rescue by itgb1b overexpression (P = 0.003 and 0.0003, respectively). (F to K) foxa2, 30 hpf; dorsal views, anterior to the top, showing expression in pharyngeal endoderm (pe), floor plate (asterisk), and intestinal rod (ir, arrowhead), which bifurcates in morphants (white arrows). Injection of 50 pg (I), 75 pg (J), and 100 pg (K) of itgb1b mRNA rescues the intestine partially [arrowheads, (I) and (J)] or completely [arrowhead, (K)].

These results argue against the presentation of FN-bound Cxcl12b to Cxcr4a and instead suggest that chemokines control ECM-integrin–dependent adhesive interactions of the endoderm. To test this, we conducted in vitro cell adhesion assays to determine the ability of cxcr4a morphant endodermal cells to adhere to FN-coated surfaces. Relative to controls, the number of morphant cells that remained attached was reduced by one-third; adhesion was rescued by coinjection of integrin beta 1b (itgb1b) mRNA (Fig. 3E), which confirmed that chemokine signaling directly regulates adhesion of endoderm to FN.

Molecular interactions between CXCL12 and CXCR4 up-regulate levels of integrin α and β mRNAs in renal carcinoma cells to enhance their adhesion to FN (22). Thus, Cxcl12b-Cxcr4a signaling in the endoderm may similarly regulate integrin levels. Of the integrin βs expressed in zebrafish, itgb1b is expressed maternally and ubiquitously during gastrulation (29). Quantitative real-time polymerase chain reaction (qPCR) revealed a reduction in itgb1b mRNA levels in whole embryos and in cxcr4aMO endoderm (fig. S7K); this finding suggests that the link between chemokines and integrins is, at least in part, a transcriptional one.

If endodermal defects in cxcl12b-cxcr4a morphants reflect disruption of ECM-integrin signaling, injecting itgb1b mRNA into morphants should rescue these defects. itgb1b mRNA injected into cxcl12b and cxcr4a morphants rescued intestinal bifurcations in a dose-dependent manner (Fig. 3, F to K), either unilaterally (50 to 75 pg; Fig. 3, I and J) or completely (100 pg; Fig. 3K and table S1). Taken together, our results suggest that Cxcl12b-Cxcr4a interactions promote integrin-mediated adhesion to tether the endoderm to the mesoderm during gastrulation.

Cell adhesion is a key regulator of gastrulation movements. E-cadherin mediates epiboly and anterior migration of prechordal mesoderm (30), integrin-αB allows mesendodermal cells to crawl on FN along the blastocoel roof (31), and fn1 controls migration of myocardial progenitors toward the midline (26, 32)—a process that also requires another chemokine, apelin (33, 34). However, gut defects have generally been interpreted as secondary to defects in mesoderm migration. In contrast, our studies reveal an earlier requirement for ECM-integrin interactions directly in endoderm migration.

We have shown that endoderm migration toward the anterior is genetically separable from other gastrulation movements (35). We propose that chemokine-dependent expression of integrin tethers the endoderm to the mesoderm, and that loss of this tether releases the endoderm to move anteriorly (Fig. 4); a secondary result is viscera bifida, because endodermal cells on either side [presumably containing organ progenitors (8)] have farther to converge dorsally and do not reach the midline in time to fuse. Viscera bifida–like syndromes in humans, including intestinal cysts and ectopic pancreatic or liver tissue, are relatively common and are not associated with spina bifida (ectoderm) (36), and defects in the CXCL12-CXCR4 signaling pathway may be an underlying cause.

Fig. 4.

A chemokine-mediated tether model for endodermal morphogenesis. Diagrams of a wild-type (WT) gastrula at 6, 8, and 10 hpf, and cxcl12b/cxcr4a morphants or RGD-treated (MO/RGD) embryos, lateral view, animal pole (top), dorsal (right). Endoderm, blue; mesoderm, red. Arrows: epiboly (1), involution (2), convergent extension (3), and anterior migration (4). (A) At onset of gastrulation, cxcl12b+ mesoderm tethers cxcr4a+ endoderm, which coordinates mesendoderm migration. (B and C) By the end of gastrulation, midline convergence clears ventral cells. (D) In morphants or RGD-treated embryos, endoderm released from this tether migrates anteriorly. (E and F) Displaced endoderm in anterior and ventral positions does not reach the midline [ectopic ventral blue dots in (F)], leading to organ duplication. Enlarged cells at center depict a molecular model in which Cxcl12b from mesoderm signals through Cxcr4a in endoderm to up-regulate itgb1b expression, which binds FN1 in the mesodermal extracellular matrix.

For zebrafish endodermal cells, regulation of migration by controlling adhesion reconciles the recent observation that involuted endodermal cells initially move via a “random walk” rather than the directed migration displayed by the mesoderm (37). Classically, chemokines are cytokines that induce chemotaxis in responding cells; CXCL12-CXCR4 interactions control homing of hematopoietic stem cells to the bone marrow, as well as migration of germ cells, neuronal progenitors, and several metastatic cancers (1518, 27). In some of these cases, however, there is evidence for a system more like the tether described here, where receptor-expressing cells are confined to a territory defined by ligand-expressing cells. Thus, chemokine-dependent changes in adhesion to the ECM may influence cell migration rates and directionality in many developmental and disease contexts.

Supporting Online Material

Materials and Methods

Figs. S1 to S7

Table S1


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