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Hairy/E(spl)-related (Her) genes are central components of the segmentation oscillator and display redundancy with the Delta/Notch signaling pathway in the formation of anterior segmental boundaries in the zebrafish

Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA and Department of Organismal Biology and Anatomy, University of Chicago, Chicago, IL 60637, USA

View larger version (95K): [in a new window]   Fig. 1. Developmental expression of her7 and comparison with other segmentation and cyclic genes. Embryos at epiboly stages are shown in whole mount from a dorsovegetal view (A,B). A lateral view of the tail tip of a 25-somite embryo is shown in F. Comparison by two-color in situ hybridization of segmental and cyclic genes in the presomitic mesoderm in embryos at 14 hpf (10 somites) is shown in dorsal view after flat mounting with anterior upwards (C-E,G-J). In all panels, her7 has been developed in blue/black. (C-E) Domains of cyclic gene expression at stages III, I+ and II, according to the nomenclature of Jiang et al. (Jiang et al., 2000); these stages are repeated in G-J. The co-expression of myod (G), her1 (H), dlc (I) and dld (J) are shown in red. (K) Diagram of the relative positions of the domains of cyclic gene expression (her7, black; her1, green; dlc, red; dld, blue) to each other within a combined domain moving anteriorly (arrow) in the PSM. (L) The gene expression cycle experienced multiple times by individual cells in the PSM, inferred from the spatial relationships (K).

View larger version (84K): [in a new window]   Fig. 2. Properties of her7-targeted morpholinos. The efficacy and specificity of the morpholinos targeted to her7 in preventing translation (B-I) and in stabilizing target mRNAs (J-L) are shown. The structure of the 5'UTR of the her7 mRNA is shown in A, illustrating the binding sites of the morpholinos her7m1 (spanning the ATG) and her7m2 (more 5'). The uppercase C indicates a consistent sequence difference in TLF, *AB and GH (recently derived) strains from the database sequence. The relationship of the 5'UTR to the entire her7 transcript, as well as to control constructs using the gfp-coding region is shown below in block form. GFP fluorescence is shown in living embryos between 6 and 10 somite stages in B,D,F,H, and the presence of injected gfp mRNA is shown in whole mount at the same stage (C,E,G,I). (B,C) Injected with gfp mRNA; (D,E) co-injected with gfp mRNA and 7 ng/µl her7m2; (F,G) her7 5'UTR-gfp and control morpholino; and (H,I) her7 5'UTR-gfp and 7 ng/µl her7m2. The distribution of endogenous and exogenous her7 mRNA at bud stage (10 hpf) is shown in uninjected whole mounted embryos (J), after injection of her7 mRNA (K), and co-injection of her7 mRNA and 5 ng/µl her7m1 (L).

View larger version (144K): [in a new window]   Fig. 3. Effect of reduction of her7 function on segmentation. The myotome boundaries of the trunk marked by twitchin expression are shown in 26 hpf embryos in lateral view, anterior towards the left and dorsal upwards (A-D). The expression of segmental genes in the presomitic mesoderm and trunk somites of embryos at 14 hpf (10 somites) are shown in dorsal view after flat mounting with anterior upwards (F-U). Arrows and arrowheads indicate localized defects and brackets indicate the extent of larger regions of abnormalities. The effect of increasing her7m2 dose on myotome boundaries at 26 hpf is shown in A-D. (A) Uninjected control. Embryos injected with 1 ng/µl (B), 3 ng/µl (C) and 5 ng/µl (D) her7m2. (E) Histogram showing the restricted distribution of the most anterior segmental defect, assayed at 26 hpf. The effect of increasing her7m1 dose on the expression of myod is shown in F-I. (F) Uninjected control, (G) injected with 3 ng/µl, (H) 4 ng/µl and (I) 5 ng/µl her7m1. The disruption of segment organization and polarity by her7m2 injection is shown in J-U. The expression of dld (J,K), notch5 (L,M), paraxial protocadherin (N,O), mespa (P,Q), mespb (R,S) and notch1a (T,U) is shown for uninjected controls (J,L,N,P,R,T) and after injection with 7 ng/µl her7m2 (K,M,O,Q,S,U, indicated with +). Scale bar: in A, 250 µm for A-D.

View larger version (77K): [in a new window]   Fig. 4. Expression of cyclic genes in response to reduction of function of her7. The expression of cyclic genes in the presomitic mesoderm of zebrafish at 14 hpf (10 somites) is shown in dorsal view after flat mounting (A-D,G-J,M-P). Expression of dlc (A-D), her1 (G-J) and her7 (M-P). (A,G,M) Two uninjected control embryos with different stage cyclic gene expression; (B-D,H-J,N-P) the classes of increasing severity of cyclic expression domain disruption after injection of 5 ng/µl her7m1. The effect of increasing her7m2 dose on cyclic domain organization is shown in histograms E,K,Q, assayed at 14 hpf (10 somites) for dlc, her1 and her7, respectively. The developmental progression of the disrupted cyclic expression domain phenotype after injection of 7 ng/µl her7m2 is shown in histograms F,L,R for dlc, her1 and her7, respectively.

View larger version (113K): [in a new window]   Fig. 5. Expression of her7 in Delta/Notch segmentation mutants. Embryos at bud stage (A-D) and the ten somite stage (E-I) have been dissected from the yolk and flat mounted with anterior upwards after in situ hybridization with her7 riboprobe. Scale bar: 250 µm. Expression patterns of her7 are shown in wild type (A,E), and in the bea (B,F), aei (C,G), des (D,H) and wit (I) mutant backgrounds. Embryos with cyclic expression domains at stage III and II are shown in E.

View larger version (129K): [in a new window]   Fig. 6. Analysis of the interaction of her7 with the aei (after eight), des (deadly seven), bea (beamter) and wit (white tail) mutations in the Delta/Notch signaling pathway. The myotome boundaries of the anterior trunk marked by twitchin expression are shown in 26 hpf embryos in lateral view, anterior towards the left and dorsal upwards (A-F). The expression of cyclic genes is shown in the presomitic mesoderm of embryos at 14 hpf (10 somites) in a dorsal view after flat mounting with anterior up (K-Y). (A) aei, (B) des and (C) bea uninjected; (D) aei, (E) des and (F) bea injected with 7 ng/µl her7m2. Arrows in A-F indicate the position of the anterior limit of segmental defects in each embryo. Scale bar in A: 50 µm for A-F. Histograms comparing the effect of her7m2 injection on the anterior limit of segmental defects at 26 hpf in aei (H), des (I), and bea (J) genetic backgrounds. In K-Y, a pair of embryos are shown, with uninjected on the left and injected (7 ng/µl her7m2) on the right (indicated with +). An average of 15 embryos injected with either her7m1 or her7m2 was analyzed per treatment. K-O, P-T and U-Y show the expression of dlc, her1 and her7, respectively, and the columns show the wild type (K,P,U), bea (L,Q,V), aei (M,R,W), des (N,S,X) and wit (O,T,Y) genetic backgrounds.

View larger version (91K): [in a new window]   Fig. 7. Effect of reduction of her1 function on segmentation and cyclic gene expression. The myotome boundaries of the trunk marked by twitchin expression are shown in 26 hpf embryos in lateral view, anterior towards the left and dorsal upwards (B,C). The expression of segmental and cyclic genes in the presomitic mesoderm and trunk somites of embryos at 14 hpf (10 somites) are shown in dorsal view after flat mounting with anterior up (D-K). Arrows and arrowheads indicate localized defects and brackets indicate the extent of larger regions of abnormalities. Scale bar in B: 250 µm for A,B. Embryos prior to somitic furrow formation at 10 hpf (bud stage) are shown in lateral view (L,M) and in dorsal view after flat mounting (N). Arrows indicate the anterior-most expression of genes in the PSM, arrowheads show the location of the yolk plug (vegetal). Scale bar in N: 100 µm. (A) Diagram of the position of the her1m1 morpholino with respect to the translation start site (+1, ATG) of the her1 mRNA. (B) Uninjected wild-type control. (C) Wild type injected with 5 ng/µl her1m1. Expression of myod (D,E), dlc (F,G), her1 (H,I) and her7 (J,K) in uninjected embryos (D,F,H,J) and in embryos injected with 5 ng/µl her1m1 (E,G,I,K). Expression of mespa (L) and her1 (M) and co-expression of mespa (blue) and her1 (red) prior to somite formation (N).

View larger version (81K): [in a new window]   Fig. 8. Effect of reduction of her1 and her7 function on somite morphogenesis. The appearance of the paraxial mesoderm in live embryos is shown in lateral view at 11.5 hpf (four somites; A,B) and 14 hpf (10 somites; C,D) in uninjected wild type embryos (A,C) and in wild type injected with 2.5 ng/µl of both her1m1 and her7m2 (B,D). Arrowheads indicate the position of normal somitic furrows in A,C, and delayed partial furrows in D. The position of nuclei demarcating epithelial boundaries in the anterior trunk paraxial mesoderm is shown in dorsal view of flat mounted 14 hpf (10 somite) embryos stained with 1 µM Hoechst 34222 (E,F). (E) Wild type uninjected control, (F) wild type injected with 2.5 ng/µl of both her1m1 and her7m2. Arrowheads indicate the position of the first nine intersomitic furrows in E and five abnormally spaced and oriented furrows in F, which is a montage of two focal planes. Scale bar in E: 50 µm for E,F.

View larger version (109K): [in a new window]   Fig. 9. Analysis of the interaction between her7 and her1 on segmentation and cyclic gene expression in wild-type and beamter embryos. The myotome boundaries of the trunk, marked by twitchin expression, and the sclerotome, marked by twist in the tail, are shown in 26 hpf embryos in lateral view, anterior towards the left and dorsal upwards (A-D, W,X and E,F, respectively). The expression of segmental and cyclic genes in the presomitic mesoderm and trunk somites are shown in dorsal view after flat mounting with anterior upwards for embryos at 14 hpf (10 somites) (G-P,U,V) and at three somites (T). Expression of dlc is shown in whole-mount embryos from 80% epiboly to bud stage in a dorsovegetal oblique view (Q-S). Arrows indicate localized defects and brackets indicate the extent of larger regions of abnormalities. Scale bar in A: 250 µm for A-D,W,X. (A) Uninjected wild-type control. (B) Wild-type injected with 2.5 ng/µl of both her1m1 and her7m2, (C) 5 ng/µl her1m1 or (D) 5 ng/µl her7m2. Scale bar in E: 50 µm. In each panel (G-V), a pair of embryos is shown, with uninjected on the left and injected with 2.5 ng/µl of both her1m1 and her7m2 on the right, indicated with + (or top and bottom respectively in Q-S). Expression of myod (G), notch5 (H), fgf8 (I), lfng (J) notch1a (K), papc (L), mespa (M), mespb (N), her1 (O), her7 (P), dlc (Q-U) and dld (V). In Q-S, arrowheads indicate regions of the PSM that exhibit clear stripe and interstripe (i.e. segmental) patterns in control, but not in injected embryos, and the diffuse nature of the anterior-most expression domain in injected embryos is highlighted with asterisks in R,S. (W) Uninjected bea control and (X) bea injected with 2.5 ng/µl of both her1m1 and her7m2. (Y) Histogram comparing the position of the anterior limit of segmental defects in 26 hpf bea controls, and wild type and bea mutants injected with 2.5 ng/µl of both her1m1 and her7m2.

View larger version (34K): [in a new window]   Fig. 10. Model of molecular interactions in HER-linked Delta/Notch oscillator: cell-autonomous and non-autonomous, or phase-adjusting aspects. Proposed molecular interactions of the core segmentation oscillator are represented in diagrammatic form inside an abstracted PSM cell. Normal type and capital letters indicate proteins and lowercase italics indicate genes and RNA. Black lettering and arrows indicate high levels or activity, whereas gray represent low levels or activity. Open ‘B’ arrowhead represents the basal level of stimulus to the dlc gene throughout the PSM, revealed in the absence of oscillator activity. The Notch receptor is ubiquitously expressed in the PSM. This core oscillator mechanism is hypothesized to run throughout the PSM, but may be modified by other factors at different locations in the PSM, for example, DeltaD in the tailbud, FGF signaling in the posterior PSM and the fss gene in the anterior PSM. See text for details. (A-D) The cell-autonomous oscillator: (A) basal dlc expression (asterisk indicates role of DeltaD potentially limited to the tailbud), (B) translation of dlc mRNA to DeltaC protein and activation of Notch signaling feedback to amplify dlc levels, (C) activation of her1 and her7 expression, (D) translation of her1 and her7 mRNA to Her1 and Her7 proteins that repress expression of her1, her7 and dlc. Cycle returns to A and repeats. (E) Intercellular or phase-adjusting function: cell on right in stage C (from above) activates Notch signaling in a neighboring cell (on left) that was previously phase-delayed relative to the right hand cell and phase-advances it from stage A into B by providing exogenous DeltaC.