Examination of Tg(hand2:EGFP) Embryos Reveals Novel Cell Rearrangements during LPM Migration

(A–E) Time course analysis of Tg(hand2:EGFP) expression in the LPM during gut looping. Ten Tg(hand2:EGFP) embryos were fixed every hour between 24 and 30 hpf, and stained for GFP (green) and phalloidin (red).

(F) Diagram of LPM migration. The gut is in yellow, the left LPM is in red, the right LPM is in blue, and the Tg(hand2:EGFP)-expressing cells are in green. The dark red line represents the apical side of the LPM epithelium. Arrows indicate the direction of migration of the Tg(hand2:EGFP)-expressing cells.

(G–K) Perturbing spaw function randomized the laterality of the rearrangements of the Tg(hand2:EGFP)-expressing cells. Compared to uninjected controls (G; n = 12), of 53 embryos injected with 10 ng of spaw MO, 30% showed the wild-type pattern (H); 25% showed the reverse pattern (I); 28% had both the left and right LPM migrate on top of the gut while the Tg(hand2:EGFP)-expressing cells on both sides remained ventral (J); and 17% had both the left and right LPM migrate ventrally and the Tg(hand2:EGFP)-expressing cells on both sides “rolled” dorsally (K).

All images are transverse sections, dorsal to the top. Dashed lines outline the LPM. L, left; NC, notochord; R, right; So, somite. The scale bar represents 10 μm.

Bmp Signaling Regulates hand2 Expression in the LPM

(A and B) Within the gut-looping region of wild-type embryos, bmp2b (A) is expressed in the LPM, whereas chordin (B) is expressed in the ventral portion of the somites. Dashed lines mark the somite/LPM boundary.

(C) Wild-type embryo stained for GFP (green) and phosphorylated Smad1/5/8 (red).

(D) The same embryo as shown in (C) but with red channel only. Dashed lines outline the LPM.

(E) Fluorescence intensity of the phosphorylated Smad staining in the LPM cells (mean ± SEM). Forty Tg(hand2:EGFP)-expressing cells and 40 Tg(hand2:EGFP)-nonexpressing cells from seven wild-type embryos were analyzed. Asterisks indicate statistical significance: ^***p < 0.001; ^****p < 0.0001.

(F–H) Wild-type and Tg(hsp70l:bmp2b)^f13 embryos were heat shocked at the 21-somite stage, and stained for GFP (green) and phalloidin (red) at 30 hpf. White dashed lines outline the LPM. Yellow dashed lines mark the midline. Tg(hand2:EGFP) was expressed mostly in the ventral half of the LPM in wild-types (F), but was expressed throughout the LPM in the gut-looping region in Tg(hsp70l:bmp2b)^f13 embryos (G and H). Eight of 32 Tg(hsp70l:bmp2b)^f13 embryos examined exhibited no gut looping (G); the remaining 24 exhibited leftward gut looping (H).

(I–K) Compared to wild-type control (I), hand2 expression in the LPM (asterisks) was decreased (K) or absent (J) in Tg(hsp70l:dnBmpr-GFP)^w30 embryos after heat shock.

(L–N) Expression of wnt2bb in the LPM was present in wild-type and Tg(hsp70l:dnBmpr-GFP)^w30 embryos after heat shock. Dashed lines outline the notochord and LPM.

(O–Q) Phalloidin staining of wild-type control (O) and Tg(hsp70l:dnBmpr-GFP)^w30 (P and Q) embryos. When heat shock was applied at the 21-somite stage, 9 of 34 embryos exhibited no gut looping (P); the remaining 25 exhibited leftward gut looping (Q). White dashed lines outline the LPM. Yellow dashed lines mark the midline.

All images, except (I)–(K), are transverse sections, dorsal to the top. (I)–(K) are dorsal views, anterior to the top. L, left; NC, notochord; R, right. The scale bars represent 40 μm. See also Figure S1.

Hand2 Is Required for Gut Looping and LPM Migration

(A) foxa3 expression in wild-type and han^c99 mutants. Upper panel: the gut has looped to the left by 35 hpf in wild-types (22/22), but stays in the midline in han^c99 mutants (17/18). Dashed lines mark the midline. Lower panel: in wild-type by 48 hpf, the liver and pancreas are positioned on the left and right side of the gut, respectively (35/35). In han^c99 mutants, the liver (arrows) and/or pancreas (asterisks) is duplicated (12/12).

(B) At the 20-somite stage (19 hpf), spaw is expressed in the left LPM (arrows) in wild-types (70/70), but is absent from the LPM in most han^c99 mutants, or is bilateral in a few han^c99 mutants.

(C–G) Time course analysis shows that the Tg(hand2:EGFP)-expressing cells in han^c99 mutants fail to undergo asymmetric rearrangements. Ten han^c99 mutants were analyzed at each time point. Dashed lines outline the LPM.

(H) Diagram of LPM migration in han^c99 mutants. The gut is in yellow, the left LPM is in red, the right LPM is in blue, and the Tg(hand2:EGFP)-expressing cells are in green. The dark red line represents the apical side of the LPM epithelium. Arrows indicate the direction of migration of the Tg(hand2:EGFP)-expressing cells.

(A and B) Dorsal views, anterior to the top.

(C–H) Transverse sections, dorsal to the top.

L, left; Li, liver; NC, notochord; Pa, pancreas; R, right; So, somite. The scale bars represent 50 μm. See also Figure S2.

han^c99 Mutants Exhibit Prolonged Deposition of Laminin at the LPM/Gut Boundary

(A–F) Time course analysis of Tg(hand2:EGFP) expression and laminin deposition during LPM migration. Ten wild-type (A–C) and eight han^c99 mutants (D–F) were fixed at the stages indicated and stained for laminin (red) and GFP (green). Dashed lines outline the notochord.

(A–C) In wild-type, laminin becomes diminished along the migratory path of Tg(hand2:EGFP)-expressing cells (arrows).

(D–F) In han^c99 mutants, laminin deposition persists along the entire LPM/gut boundary (arrows).

(G) The LPM in lamβ1^s804 heterozygotes undergoes asymmetric migration (28/30).

(H–J) Out of 29 lamβ1^s804 mutants examined, 6 showed incomplete asymmetric LPM migration (H), 18 had both the left and right LPM remain on top of the gut (I), and 5 had the LPM located lateral to the gut (J). Arrows point to the aberrant intrusion of LPM cells into the gut. Dashed lines outline the LPM.

(K) Percentages (mean ± SEM) of han^c99 mutants (n = 21) from han^c99+/- incrosses and han^c99 single mutants (n = 56) from han^c99+/-;lamβ1^s804+/- incrosses showing gut-looping phenotypes according to foxa3 expression. Three independent crosses were examined.

All images are transverse sections, dorsal to the top. L, left; NC, notochord; R, right. The scale bars represent 40 μm. See also Figure S3.

Asymmetric LPM Migration Requires the Diminishment of Laminin by MMPs

(A and B) MMP inhibition blocked the asymmetric LPM migration. Tg(hand2:EGFP) embryos were treated with DMSO (A) or 10 μM GM6001 (B) from 24 hpf, and stained for GFP (green) and phalloidin (blue) at 30 hpf. Dashed lines outline the LPM.

(C and C′) Laminin (red) deposition was diminished near the Tg(hand2:EGFP)-expressing cells in the DMSO-treated controls (C, arrows), but persisted along the entire LPM/gut boundary in GM6001-treated embryos (C′, arrows).

(D) Proportions (mean ± SEM) of wild-type and han^c99+/- embryos showing gut-looping phenotypes according to foxa3 expression. Three independent experiments were conducted. Two hundred wild-type and 200 genotyped han^c99+/- embryos treated with DMSO or 5 μM GM6001 were examined. Asterisks indicate statistical significance: ^*p < 0.05; ^**p < 0.01.

(A–C′) Transverse sections, dorsal to the top. L, left; R, right. The scale bar represents 40 μm. See also Figure S2.

Loss of Hand2 Function Reduces MMP Activity along the LPM/Gut Boundary

(A) Diagram showing the microinjection of the DQ-collagen IV substrate into the embryo. Lateral view, anterior to the left. The blue dot marks the position where gut looping occurs.

(B) Tg(hand2:EGFP) embryos were injected with DQ-collagen IV at 26 hpf, fixed at 27 hpf, and stained for GFP (blue) and phalloidin (red). The fluorescein signal caused by cleavage of the DQ-collagen substrate is shown in green.

(C) In embryos preinjected with GM6001, almost no fluorescein signal could be detected.

(D and E) Wild-type (D) and han^c99-/- (E) embryos were coinjected with DQ-collagen and Alex549-conjugated collagen at 27 hpf and fixed at 28 hpf. The spots where the fluorescence intensity was measured are marked by pink and orange bars.

(F) MMP activity represented as normalized fluorescence intensity (mean ± SEM). Six wild-type and six han^c99 mutants were examined at each time point. Asterisks indicate statistical significance: ^*p < 0.05.

(B–E) Transverse sections, dorsal to the top. L, left; NC, notochord; NT, neural tube; R, right; So, somite. The scale bars represent 40 μm.

Expression of mmp Genes and Their Inhibitors Is Altered in hand2 Mutants

(A) Comparison of mmp and timp expression in wild-type and han^s40-/- samples by quantitative real-time PCR (two independent experiments, three replicates of wild-type and han^s40-/- samples each time). The results are represented as relative expression levels that are normalized to β-actin (mean ± SEM).

(B) Proportion (mean ± SEM) of wild-type embryos (n = 29), wild-type embryos injected with 0.5 ng of mmp14a MO (n = 44), han^c99 heterozygotes (n = 55), and han^c99 heterozygotes injected with 0.5 ng of mmp14a MO (n = 70) showing gut-looping phenotypes according to foxa3 expression. Three independent experiments were conducted.

(A and B) Asterisks indicate statistical significance: *p < 0.05; **p < 0.01.

(C) Diagram of Hand2 regulating asymmetric LPM migration during zebrafish gut-looping morphogenesis. See also Figure S4.

Bmp signaling regulates hand2 expression in the lateral plate mesoderm (LPM)
(A-B) Transverse section of Tg(hand2:EGFP) embryo stained with hand2 in situ probe (red), anti-GFP antibody (green), and Topro to label the nuclei (blue). Both hand2 mRNA and Tg(hand2:EGFP) are expressed in the ventral half of the LPM, and the most medial LPM cells appear to downregulate hand2 expression.
(C-E) Expression of hand2 in wild-type (C), Tg(hsp70l:bmp2b)^f13 (D), and Tg(hsp70l:dnBmpr- GFP)^w30 (E) embryos that were heat-shocked at the 5-somite stage (11.66 hpf) and fixed at the 15-somite stage (16.5 hpf). Bmp2b overexpression leads to an expansion of hand2 expression, while blocking Bmp signaling slightly reduces it by the 15-somite stage. (F-G) Tg(hand2:EGFP) expression in wild-type (F) and Tg(hsp70l:bmp2b)^f13 (G) embryos that were heat-shocked at the 5-somite stage and fixed at 30 hpf. Heat-shocked Tg(hsp70l:bmp2b)^f13 embryos expressed Tg(hand2:EGFP) throughout the LPM, and the epithelial polarity of the LPM cells seemed to be affected. White dashed lines outline the LPM and yellow dashed lines mark the midline.
(H-I) Inhibiting Bmp signaling by heat-shocking Tg(hsp70l:dnBmpr-GFP)^w30 embryos at the 5- somite stage abolished hand2 expression in the LPM (asterisk) by 30 hpf. (J-K) Tg(hsp70l:dnBmpr-GFP)^w30 embryos expressed the LPM marker wnt2bb after heat-shock at the 5-somite stage. Dashed lines mark the notochord and LPM. (L-M) As revealed by phalloidin labeling, Tg(hsp70l:dnBmpr-GFP)^w30 embryos failed to undergo leftward gut looping after heatshock at the 5-somite stage. White dashed lines outline the LPM and yellow dashed lines mark the midline.
(C-E, H-I) Dorsal views, anterior to the top. (A-B, F-G, J-M) Transverse sections, dorsal to the top. L, left; NC, notochord; R, right. Scale bars, 40 μm.

The LPM in the gut-looping region of han^c99 mutants does not exhibit impaired epithelial polarity or altered Fibronectin deposition
(A-B) In wild-type and han^c99-/- embryos, ZO-1 (red) is localized to the apical side of the LPM epithelium. Dashed lines outline the LPM.
(C-D) Expression of fibronectin1/fn1 gene within the gut-looping region in wild-type and han^{c99- /-} embryos. Dashed lines outline the notochord (NC) and the LPM.
(E-F) Deposition of Fn protein along the LPM/gut boundary appeared to be comparable between wild-type (E) and han^c99-/- (F) embryos at 30 hpf.
(G-H) Deposition of Fn also appeared to be comparable between wild-type embryos treated with DMSO (G) and embryos treated with 10 μM of the MMP inhibitor GM6001 (H) between 24 and 30 hpf. All images show transverse sections, dorsal to the top. In (E-H), asterisks mark Fn deposition along the LPM/gut boundary. L, left; R, right. Scale bars, 40 μm.

Expression levels of laminin and integrin genes are not dramatically altered in han^s40 mutants compared to wild-type.
(A) Strategy of FACS sorting and quantitative real-time PCR. Tg(hand2:EGFP) wild-type or han^s40-/- embryos were raised until 28 hpf. The trunk region posterior to the optic vesicles and anterior to the yolk extension (between the two green lines) was dissected out. Within this region, Tg(hand2:EGFP) is mainly expressed in the fin bud (arrowheads) and the LPM (asterisk).
(B) Comparison of laminin (lam) and integrin (itg) expression in wild-type and han^s40-/- samples by quantitative real-time PCR (two independent experiments, three replicates of wild-type and han^s40-/- samples each time). The results are represented as relative expression levels that are normalized to β-actin (mean±SEM). None of the genes examined showed more than a 2-fold difference in their expression levels between wild-type and han^s40 mutants.

Expression of mmp14a, timp2a, and timp2b in wild-type and han^c99-/- embryos at 28 hpf For each gene, 10 wild-type and 10 han^c99-/- embryos were examined. (A-F) are dorsal views, anterior to the top. Squares mark the gut-looping region. (A′-F′) are transverse sections, dorsal to the top. Dashed lines outline the notochord and the LPM. L, left; NC, notochord; R, right. Scale bars, 40 μm.

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