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doi: 10.1242/10.1242/dev.00400

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The role of the zebrafish nodal-related genes squint and cyclops in patterning of mesendoderm

Scott T. Dougan^1,*, Rachel M. Warga², Donald A. Kane², Alexander F. Schier³ and William S. Talbot^1,

¹ Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305, USA
² Department of Biology, University of Rochester, Rochester, NY 14627, USA
³ Developmental Genetics Program, Skirball Institute of Biomolecular Medicine, Department of Cell Biology, New York University School of Medicine, New York, NY 10016, USA
^* Present address: Department of Cellular Biology, University of Georgia, 724 Biological Sciences Building, Athens, GA 30602, USA

View larger version (26K): [in a new window]   Fig. 1. Two models for the action of Nodal signals in patterning the mesendoderm along the dorsoventral axis in zebrafish embryos. In each case, the gradient of shading at the margin represents the putative distribution of Nodal signals, with the darkest shade indicating the highest concentration. The fate maps shown in the second column are loosely based on those of Kimmel et al. (Kimmel et al., 1990). Mesendoderm is shaded red, green or blue, depending on the position along the dorsoventral axis, and regions generating both mesoderm and endoderm shaded darker than regions producing mesoderm alone. (A) High levels of Nodal signals specify dorsal mesendodermal fates, intermediate levels specify lateral mesendodermal fates and low levels determine ventral mesendoderm. In this model, Nodal signals act in a dorsal-to-ventral gradient to pattern the mesendoderm. The gradient shown here is only one of many such gradients that could be drawn consistent with the evidence. However, all versions of the gradient model predict that reductions in the level of Nodal function would result in the transformation of dorsal marginal cells to more ventrolateral fates (illustrated in the right-hand panels in A). (B) Nodal activity is uniformly distributed along the dorsoventral axis, but a gradient of Nodal signals along the animal-vegetal axis patterns the germ layers. Independent dorsalizing factors pattern the mesendoderm along the dorsoventral axis (represented by the red arrow). This model predicts that endodermal cells (darker colors near the margin) are transformed to more animal fates as levels of Nodal signals are reduced (right-hand panels in B). Dorsalizing factors remain to establish dorsoventral pattern. Ne, neuroectoderm; D, dorsal mesendoderm; L, lateral mesendoderm; V, ventral mesendoderm, Y, yolk.

View larger version (79K): [in a new window]   Fig. 4. Time course of dorsal mesendoderm development in sqt-/-; cyc+/+, sqt-/-; cyc+/- and sqt-/-; cyc-/- mutants. gsc expression in wild-type (A), sqt-/-; cyc+/+ (B), sqt-/-; cyc+/- (C) and sqt-/-; cyc-/- (D) embryos. For each stage, wild-type, sqt-/-; cyc+/+, sqt-/-; cyc+/- are siblings; the sqt-/-; cyc-/-, mutants were derived from a separate cross that was processed in parallel. The images for panels C6 and D6 come from a separate cross of sqt+/-; cyc+/- adults. gsc expression initiates in sqt-/-; cyc+/+, sqt-/-; cyc+/- and sqt-/-; cyc-/- embryos (A1,B1,C1,D1), but is rapidly lost (B2,C2,D2). In sqt-/-; cyc+/+ embryos, gsc is expressed only in a few cells at 5 h, but steadily increases throughout gastrulation such that it is often indistinguishable from wild-type at bud stage (B2-B6). Dorsal views are shown, except for animal views at 5 and 10 h. The genotypes of all embryos shown were determined by PCR after photography, except for panels C6 and D6, which were determined by morphology. In the sqt+/-; cyc+/+ intercross depicted in A6, B6, only 3/51 embryos displayed reduced gsc expression; the rest of the embryos, including the remaining sqt mutants, have a wild-type pattern as shown. Notably, we found no reduction of gsc expression in cycm294 homozygotes at 8 h (data not shown). This contrasts with earlier work on cycb16 (Thisse et al., 1994), which is now known to be a deficiency that removes other genes in the cyclops region of linkage group 12 (Talbot et al., 1998).

View larger version (88K): [in a new window]   Fig. 2. Analysis of genetic interaction between sqt and cyc. Images of 28 hour embryos from a sqt+/-; cyc+/- intercross (A-O) or cross of sqt+/-; cyc+/- to sqt+/-; cyc+/+ parents (P-U). Phenotypes were scored at 6 h, 1 d and 5 d; an embryo representative of each phenotypic class is shown. (A-C) sqt-/-; cyc-/- embryos lack head and trunk mesoderm and endoderm derivatives, and display severe cyclopia (C). Tail mesoderm still forms in these embryos, as indicated by the presence of tail somites (A). (D-F) Trunk somites, heart and blood form in sqt-/-; cyc+/- embryos (D). These embryos have strong midline defects, including a reduced notochord and missing floor plate (E) as well as cyclopia (F), which is indicative of defects in prechordal plate mesoderm. These defects are typically more severe than those observed in sibling sqt-/-; cyc+/+ embryos (G-I), which are often indistinguishable from wild type (M-O), and can survive to adulthood. Some sqt-/-; cyc+/+ embryos display mild cyclopia (I), but have normal notochords and floor plates. (J-L) The defects in sqt+/+; cyc-/- embryos include a curved body axis (J), missing floor plate (K) and cyclopia (L); these embryos have apparently normal trunk somites and notochord (K). In other clutches, the majority of sqt-/-; cyc+/- embryos have truncated notochords and fused somites (R), unlike typical sqt-/-; cyc+/+ siblings (Q). Kupffer's vesicle is apparent in the tailbuds of 12-14 h wild-type embryos (S, arrow), but is reduced or absent in sqt-/-; cyc+/+ and sqt+/+; cyc-/- embryos (T,U, arrows). Anterior is towards the left in A-R, except for head views in C,F,I,L,O. Posterior is towards the left in S-U. No, notochord; Som, somites. The genotypes of all embryos shown were determined by PCR after photography.

View larger version (51K): [in a new window]   Fig. 9. ß-catenin regulates sqt and cyc, and requires sqt to induce gsc. Wild-type embryos were injected with ß-catenin (A,C,E) or lacZ (B,D,F) mRNA, and processed for in situ hybridization. Overexpression of ß-catenin induces cyc expression at 6 h (A), but not at 4 h (E, sphere) or 5 h (C, 40% epiboly). (G-J) Analysis of gsc expression at 5 hours in embryos from a sqt+/- intercross injected with ß-catenin or lacZ mRNA. ß-catenin mRNA (G), but not lacZ mRNA (H), can induce ectopic gsc expression in wild-type embryos. ß-catenin mRNA does not induce normal levels of gsc expression in sqt-/- mutants (I), but ectopic patches of weak gsc are often observed in these embryos, indicating that ß-catenin has some activity in sqt mutants (arrows, I). ß-catenin induces sqt expression at 3.3 h (K), but not at 5 h (M, 40% epiboly). Animal views; dorsal towards right when apparent.

View larger version (94K): [in a new window]   Fig. 3. Analysis of crossregulation between squint and cyclops. Time course of sqt expression in wild-type (A-E), sqt mutant (J) and cyc mutant embryos (F-I). cyc expression in wild-type (K-O), sqt-/-; cyc+/+ (P-S) and sqt-/-; cyc+/- (T-V) embryos. Developmental stages are indicated at the bottom, except for E and J, which are 5 h embryos as noted in the panels. Although the absence of sqt transcripts in sqt mutants is consistent with an autoregulatory role for sqt, it is also possible that the 1.9 kb insertion in the sqtcz35 allele affects the stability of the message. Animal pole views are shown for all embryos prior to 6 h, and dorsal views are shown for embryos at 6 and 8 h, except for the lateral images in D,I. The genotypes of all embryos shown were determined by PCR after photography, except for E and J, for which the genotypes were inferred from the phenotypic ratio evident in progeny from a sqt/+ intercross (10 mutants/33 progeny).

View larger version (48K): [in a new window]   Fig. 5. Endoderm development in sqt-/-; cyc+/+ and sqt-/-; cyc+/- mutants. Expression of axial/foxa2 (A-F) or sox17 (G-L) in wild-type (A,D,G,J), sqt-/-; cyc+/+ (B,E,H,K) and sqt-/-; cyc+/- (C,F,I,L) embryos at 6 h (A-C,G-I; animal pole views) and 8 h (D-F,J-L; dorsal views). The reduction of endodermal expression of axial/foxa2 and sox17 in sqt-/-; cyc+/+ and sqt-/-; cyc+/- embryos is particularly apparent on the dorsal margin (E,F,K,L). Arrows in J-L indicate the position of dorsal forerunner cells. (M) The total number of endodermal precursors expressing axial/foxa2 at 8 h in wild-type embryos (n=3), sqt-/-; cyc+/+ embryos (n=4) and sqt-/-; cyc+/- embryos (n=5). (N) Reduction of axial/foxa2 expressing endodermal cells at different positions along the dorsoventral axis (dorsal midline set at zero degrees, ventral midline at 180 degrees). Height of each bar indicates the number of marginal cell tiers along the animal-vegetal axis expressing axial/foxa2. No cells at the dorsal midline expressed axial/foxa2 in sqt-/-; cyc+/- embryos. The genotype is indicated by color, as shown in M. The genotypes of all embryos shown were determined by PCR after photography.

View larger version (79K): [in a new window]   Fig. 6. Analysis of markers for ventrolateral mesoderm and neurectoderm in sqt-/-; cyc+/+ and sqt-/-; cyc+/- embryos. The genes, genotypes and stages analyzed are shown. Fusion of paraxial and adaxial myod expression domains is apparent in sqt-/-; cyc+/- embryos (C,F). In wild-type, sqt-/-; cyc+/+ and sqt-/-; cyc+/- embryos, spt (G-L), tbx6 (M-O) and vox (P-R) are each strongly expressed around the ventrolateral margin. tbx6 and vox are normally excluded from the dorsal margin, and this region of exclusion is expanded in sqt-/-; cyc+/- embryos (O,R). spt expression in the prechordal plate (J, arrow), is reduced in sqt-/-; cyc+/+ (arrow) and absent in sqt-/-; cyc+/- embryos, while marginal expression is excluded from a larger dorsal sector in sqt-/-; cyc+/- embryos (I,L) than in sqt-/-; cyc+/+ or wild-type embryos. ntl is also reduced dorsally in sqt-/-; cyc+/+ (T) and absent dorsally in sqt-/-; cyc+/- (U) embryos. (V-X) Expression of neural marker cyp26. Lateral views, dorsal towards the right. Arrow indicates marginal domain of cyp26 expression, arrowheads indicate vegetal extent of neural domain. The neural expression is shifted slightly towards the margin in sqt-/-; cyc+/+ embryos (W), and more dramatically in sqt-/-; cyc+/- embryos (X). In X, the dorsal marginal domain of cyp26 expression is located at a more animal position than the ventrolateral marginal domain, apparently because of abnormal morphogenetic movements of dorsal cells in sqt-/-; cyc+/- embryos. Dorsal views with anterior towards the left (A-F), animal views with dorsal to the right (G-I,P-R,S-U), dorsal views with animal pole upwards (J-O) and lateral views with animal pole upwards (V-X). The genotypes of all embryos shown were determined by PCR after photography.

View larger version (38K): [in a new window]   Fig. 7. Fate map of wild-type, sqt-/-; cyc+/+, sqt-/-; cyc+/- and sqt-/-; cyc-/- embryos. Positions of clones along the animal-vegetal and dorsoventral axes are depicted on a graphic representation of an embryo, in which each line represents a different row of cells. The `0' line represents cells in contact with the YSL. In the dorsal region, clones generating neural fates arose at least eight cell rows animal to the margin in wild-type (A, top panel). By contrast, sqt-/-; cyc+/+ (B) and sqt-/-; cyc+/- (C) embryos, clones at the dorsal margin produced spinal cord, hindbrain and midbrain. (D) As previously reported, dorsal marginal cells in sqt-/-; cyc-/- adopt neural fates. Two ventrolateral clones adopted tail muscle fates. The genotypes of all embryos shown were determined by PCR after photography.

View larger version (90K): [in a new window]   Fig. 8. Aberrant morphogenesis of dorsal clones in sqt-/-; cyc+/- embryos. Morphogenesis and resulting fates of dorsal marginal clones (A,B) and ventrolateral marginal clones (C,D) in wild-type (A,C), and in sqt-/-; cyc+/- embryos (B,D) were examined during the time periods indicated. Tracings of the behavior of individual cells within each clone are shown (A'-D'). Dorsal marginal cells migrate towards the vegetal pole with the movements of epiboly, and begin to involute at the onset of gastrulation at 50% epiboly (A). Some cells do not involute and contribute to the tail (asterisk in A'). In sqt-/-; cyc+/- embryos (B), dorsal marginal cells fail to involute (B'). Whereas the dorsal marginal cells became hatching gland, pharyngeal endoderm and endothelium near the eye in wild type (A'', blue circle), these cells became floorplate in sqt-/-; cyc+/- embryos (B'', blue circles). Arrows in A'' and B'' mark labeled notochord cells. In many sqt-/-; cyc+/- embryos, a region devoid of cells is created at the dorsal midline, possibly generated by the abnormal movements of dorsal marginal cells. By contrast, morphogenetic movements of ventrolateral marginal cells in wild-type (C,C') and sqt-/-; cyc+/- embryos (D,D') are indistinguishable, each undergoing the normal movements of epiboly, involution and convergence (Warga and Kimmel, 1990). Labeled cells formed heart endothelium (C'',D''; yellow circles), pronephric duct (C'',D''; white circles), fin bud (C'',D''; green circles), and muscle (C'',D''; black circles) in both wild-type and mutant clones. Other labeled cells (red) are in the EVL. The genotypes of all embryos shown were determined by PCR after photography.

View larger version (46K): [in a new window]   Fig. 10. ß-catenin protein is localized in the nucleus at the correct time and place to endogenously regulate squint expression. ß-catenin protein was detected by immunohistochemistry in whole-mount assays, after which embryos were sectioned. Arrows in B-D indicate boundary between blastomeres and the yolk syncytial layer (YSL). (A) ß-catenin in the nucleus of a marginal blastomere at 2.25 h (128-cell stage), the earliest stage detected. (B,C) Nuclear ß-catenin is observed in dorsal blastomeres in embryos at 3 h (1000-cell stage), both in the enveloping layer (B,C), and in the deep layer (B). (D) ß-catenin accumulates in nuclei of the dorsal YSL at 3.3 h (high stage), but it is also seen in dorsal blastomeres. ß-catenin was associated with membranes of blastomeres at all stages examined, consistent with its role in cell adhesion. Dorsal is towards the right.