EphrinB1/EphB3b Coordinate Bidirectional Epithelial-Mesenchymal Interactions Controlling Liver Morphogenesis and Laterality

Abstract

Positioning organs in the body often requires the movement of multiple tissues, yet the molecular and cellular mechanisms coordinating such movements are largely unknown. Here, we show that bidirectional signaling between EphrinB1 and EphB3b coordinates the movements of the hepatic endoderm and adjacent lateral plate mesoderm (LPM), resulting in asymmetric positioning of the zebrafish liver. EphrinB1 in hepatoblasts regulates directional migration and mediates interactions with the LPM, where EphB3b controls polarity and movement of the LPM. EphB3b in the LPM concomitantly repels hepatoblasts to move leftward into the liver bud. Cellular protrusions controlled by Eph/Ephrin signaling mediate hepatoblast motility and long-distance cell-cell contacts with the LPM beyond immediate tissue interfaces. Mechanistically, intracellular EphrinB1 domains mediate EphB3b-independent hepatoblast extension formation, while EpB3b interactions cause their destabilization. We propose that bidirectional short- and long-distance cell interactions between epithelial and mesenchyme-like tissues coordinate liver bud formation and laterality via cell repulsion.

Keywords: EphB3; EphrinB1; bidirectional; lateral plate mesoderm; liver; migration; morphogenesis; protrusion; repulsion; zebrafish.

Graphical abstract

Figure 1

Hepatoblast Polarization Coincides with Liver Budding (A–D) Stages of liver budding: Schematic (A) and confocal projections of corresponding stages with Tg(XlEef1a1:GFP)^s854 marking the endoderm and Prox1 hepatoblasts; ventral views (B–D). (E and F) EphrinB1 staining highlights cell shapes at the start of budding (E) and when a bud is apparent (F). Morphometric measurements were performed on serial coronal sections of the bud (E and F); elongated hepatoblasts (L/W ≥ 2) are shown in green. (G–I) Quantification of hepatoblast shape in control embryos at 26 and 32 hpf: (G) proportion of elongated cells per bud; SEs are shown, (H) L/W distribution for one representative bud; (I) orientation of elongated hepatoblasts with respect to the anteroposterior axis. (J–L) Time lapse of Tg(sox17:GFP)-positive foregut starting around 25 hpf (J) shows distinct hepatoblast movements during liver budding (K) and onset of outgrowth (L); dorsal views. TagBFP-nls (gray) marks nuclei for tracking of liver (yellow), gut (magenta), and pancreas progenitors (cyan). (M and N) Hepatoblasts from different anteroposterior positions migrate with distinct orientation. (M) Rose plots show the distribution of angular displacement with respect to the embryonic midline for 28 min intervals (blue sectors) and the angle of mean displacement per cell for the entire period (red arrow). (N) Line plots representing directionality of displacement over time show individual angular cell displacement for various liver (red hues) and gut progenitors (blue hues). Scale bars represent 40 μm. ^∗∗∗p < 0.001. See also Figure S1; Movies S1, S2, and S4.

Figure 2

Hepatoblasts Form Filopodia- and Lamellipodia-like Protrusions during Liver Budding (A–B′) Mosaic UAS:lyn-citrine or ubi:lyn-tdTomato expression shows EphrinB1⁺ hepatoblasts form lamellipodia (arrows in A′) and filopodia-like extensions (B and B′). Extensions (white arrowheads) connect the LPM (yellow arrowhead) and hepatoblasts (red arrowhead) over several cell diameters (B and B′). Dashed lines outline the LPM (white) and endoderm (green). (C and C′) Utrophin-GFP highlights actin in the cortical network and protrusions of hepatoblasts (arrowheads). Dashed lines delineate endoderm (green) and LPM (white). (D) Quantification of hepatoblast protrusions shows an increase of lamellipodia and decrease of filopodia-like extensions during budding. (E) Time lapse of migrating hepatoblasts during liver budding and early outgrowth; dorsal views. Membrane labeling with UAS:lyn-Citrine shows filopodia in the direction of outgrowth (white arrows) and toward the LPM (yellow arrows) and lamellipodia (red arrowheads); stills of Movie S3. Scale bars represent 30 μm. ^∗∗p < 0.01, ^∗∗∗p < 0.001). See also Movie S3.

Figure 3

Compromised Protrusion Formation Correlates with Liver Budding Defects (A–F) Latrunculin B (0.1 μg/ml) treatment during liver budding (26–32 hpf) leads to significantly less hepatoblast protrusions (A–C; DMSO clones, n = 7; Lat B clones, n = 9, N = 1), and a significantly longer Prox1 domain (bracket) (D–F; DMSO, n = 23; Lat B, n = 13, N = 2). (G–L) Hepatoblasts expressing Cdc42^T17N-GFP during liver budding (26–32 hpf) form significantly less protrusions compared to controls and Cdc42-GFP (G–I; control clones, n = 10; Cdc42-GFP clones, n = 16; Cdc42^T17N-GFP clones, n = 18, N = 1) and a significantly longer EphrinB1 domain (bracket) (J–L; control, n = 7; Cdc42-GFP, n = 18; Cdc42^T17N-GFP, n = 16, N = 2). N indicates the number of experiments. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

Figure 4

Complementary EphrinB1 and EphB3b Expression Controls Hepatoblast Positioning and Cell-Shape Changes in Liver Bud Formation (A–F‴) At 22 hpf, EphrinB1 and EphB3b expression largely overlaps in future hepatoblasts and the LPM (A–A‴). Complementary expression of both factors coincides with the start of liver budding: EphrinB1 in hepatoblasts and EphB3b in the gut and LPM (B–B‴). At 32 hpf, hepatoblasts are located more posteriorly and medially compared with controls in MO-ephrinb1 (C–D‴), in MO-ephb3b (E–E‴), and upon conditional UAS:ephrinb1^EC expression (F–F‴). (A–F′) ventral views of confocal projections, anterior to the top; (A″–F″) transverse sections of the foregut, as indicated by the dashed line in (A′–F′), and matching schematics (A‴–F‴); yellow arrowheads specify the midline and white brackets the length of the Prox1 domain. (G–I) Cell shapes were determined with EphrinB1-staining at 32 hpf (see Figure 1F). Quantification of hepatoblast shape in control, MO-ephrinb1, and MO-ephb3b embryos: (G) L/W distribution for one representative bud; (H) proportion of elongated cells per bud; SEs are shown; and (I) orientation of elongated hepatoblasts with respect to the anteroposterior axis. ^∗p < 0.05. See also Figure S2.

Figure 5

EphB3b Controls LPM Polarity and Asymmetric Movement (A–E′) EphrinB1 and EphB3b regulate LPM polarity in the foregut region. α-ZO-1 staining in MO-ephb3b embryos reveals that junctions in the LPM form similar to controls at 22 hpf (A–B′); while at 32 hpf, ZO-1 is mislocalized (arrowheads) in MO-ephrinb1 and MO-ephb3b embryos (C–E′). Yellow arrowheads specify the midline and reveal impaired gut looping in MO-ephrinb1 and MO-ephb3b (C–E). (F–G′) casanova mutants exhibit ZO-1 localization defects in the LPM (arrowheads) at 32 hpf. (A–G′) Transverse sections at liver level, left side to the right; dashed lines delineate the endoderm (green) and LPM (yellow); (A′–G′) are magnifications of the areas indicated by a box in (A–G).

Figure 6

Opposing EphrinB1 and EphB3b Functions Control Formation of Hepatoblast Protrusions (A–G) EphrinB1 and EphB3b regulate hepatoblast protrusion number and morphology. Sparse labeling reveals fewer and shorter filopodia in MO-ephrinb1 hepatoblasts than in controls (A and B). Conversely, MO-ephb3b hepatoblasts show more complex, branched extensions (C). In contrast to EphrinB1 and EphrinB1^6F (D and E), EphrinB1^ΔV fails to rescue extension formation in MO-ephrinb1 (F). Arrows indicate representative protrusions (A–F). Quantification of hepatoblast protrusion types in various conditions; comparative p values are shown in Table S1 (G). (H) Hepatoblast lamellipodia orientation is randomized in MO-ephb3b. (A–C) lyn-Citrine and lyn-Tomato outline hepatoblasts, (D–F) EphrinB1 staining highlights overexpression of different forms of EphrinB1, (A–F) ventral projections, anterior to the top. SEs are shown. Scale bars represent 10 μm. See also Figure S3; Table S1.

Figure 7

Asymmetric EphB3b Can Exert Repulsive Activity during Liver Budding (A–C) EphB3b expression is higher on the right than the left LPM at 24 hpf (A and A′). Quantification of EphB3b expression measuring overall fluorescent intensity (B) and intensity profile (C). Fluorescent intensity profiles show high EphB3b at cell membranes (red arrowheads) of the right but not left LPM; numbered arrows in (A) indicate the position of profiles. The right LPM-hepatoblast interface (profile 3) shows highest EphB3b expression. High DAPI levels indicate nuclei position (blue arrowheads). (D–F) Ectopic EphB3b^ΔICD expression alters hepatoblast position. Compared with mosaic lyn-Tomato (D), mosaic EphB3b^ΔICD expression on the left LPM (E) or left LPM and hepatoblasts (F) at 26 hpf causes positioning of Prox1⁺ hepatoblasts away from the clone at 32 hpf. EphrinB1 is absent from membranes in 3–7 hepatoblasts next to EphB3b^ΔICD clones (white arrowheads), without altering Prox1 expression (E′ and F′). (E′) Inset shows an EphrinB1 signal in EphB3b^ΔICD protrusion, indicating direct cell interaction. (A, A′ and D–F′) Transverse sections at liver level, left side to the right; yellow arrowhead specifies the midline; lines delineate the left and right LPM (white), gut (green), hepatoblasts (red). ^∗∗p < 0.01. See also Figures S4 and S5.