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First published online March 15, 2004
doi: 10.1242/10.1242/dev.01031

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Two zebrafish Notch-dependent hairy/Enhancer-of-split-related genes, her6 and her4, are required to maintain the coordination of cyclic gene expression in the presomitic mesoderm

Andrea Pasini^1,*,, Yun-Jin Jiang^2, and David G. Wilkinson^1,

¹ Division of Developmental Neurobiology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 7AA, UK
² Vertebrate Development Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, UK

View larger version (57K): [in a new window]   Fig. 1. (A-C) her6 is weakly expressed by cells in the blastoderm margin (arrows) throughout epiboly. (A) Dorsal view of a 70% epiboly embryo. (B,C) Lateral (B) and vegetal (C) pole view of a 90% epiboly embryo. In A and B, anterior is towards the top; in B, dorsal is rightwards. In A and B, the arrowhead indicates her6 expression in the anterior epiblast. (D-F) After the end of epiboly, her6 is expressed in the tailbud until about the five-somite stage (arrows in D,E), in the posterior compartment of all somites and in two stripes in the anterior PSM. (D) Vegetal pole view of a two-somite stage embryo, dorsal is rightwards. (E,F) Dorsal views of a four-somite stage (E) and a 10-somite stage (F) embryos. Arrowheads indicate her6 expression in the margins of the neural plate (E) and in the posterior lateral mesoderm (F). (G,H) her6 expression identifies the posterior compartment of the forming somite (S0) and of the first prospective somite (S-1). her6 expression (dark blue staining in G and H) overlaps with the expression of the prospective posterior compartment marker, myod (red staining in G), and intercalates with the expression of the prospective anterior compartment marker, papc (red staining in H).

View larger version (113K): [in a new window]   Fig. 2. The expression of her6 within the paraxial mesoderm is under the control of the Notch pathway. Two-cell stage embryos were injected in one cell with a dominant-blocking (A) or a constitutively active (B) form of the Notch effector Suppressor-of-Hairless [Su(H)] and analysed at 6- to 10-somite stage. In both A and B, the injected side is on the right, the control non-injected side is on the left. Anterior is towards the top. her6 is ectopically expressed throughout the PSM in embryos injected with the active form Su(H)Ank (B) and downregulated in both the anterior PSM and the segmented paraxial mesoderm in embryos expressing the dominant blocking Su(H)DBM (A).

View larger version (69K): [in a new window]   Fig. 3. Alterations of Her6 and Her4 expression disrupt paraxial mesoderm segmentation and AP somitic polarity without affecting the specification of A or P somitic compartments. Dorsal view of whole-mounted (A-C,H-J) or flat-mounted embryos at 7-15 ss. Anterior is towards the top. Two-cell stage embryos were injected in one cell only. In all cases, the injected side is on the right, the control non-injected side on the left. Red (D,E,G,K,L,N) or brown (O) staining marks the tracer co-injected with the various mRNAs. (A-C,H-J) Embryos injected with wt-her6 or her6VP16 mRNAs, have abnormal somites with irregular boundaries. Living embryos injected with wt-her6 (A) or her6VP16 (H) and lacZ mRNA as a lineage tracer. (B,I) The same embryos as in A and H, respectively, after fixation and X-gal staining. (C,J) Higher magnification views of the anterior somites of living embryos injected with wt-her6 and her6VP16, respectively. (D,K,O) In embryos injected with wt-her6, her6VP16 and wt-her4, respectively, the segmental expression of myod in the paraxial mesoderm is irregular. (E,F,L,M,P) Expression of A (lfng, papc) and P (ephrinB2) somite compartment markers is disrupted in embryos with altered expression and/or function of Her6 and Her4. Identical abnormalities are observed for lfng in wt-her6-injected embryos (E) and for ephrinB2 in her6VP16-injected embryos (L). Markers are irregularly expressed throughout the segmented paraxial mesoderm. The segmental expression pattern of papc, which is normally restricted to the prospective A compartment of the forming and newly formed somites, is disorganised in embryos injected with wt-her6 (F), her6-VP16 (M) or wt-her4 (P). (G,N) Alterations of Her6 expression or function do not affect the determination of the A somitic compartment. Injection of wt-her6 (G) or her6-VP16 (N) both disrupt the expression pattern of the A compartment specification gene, mespb, without leading to its up- or downregulation compared with the control noninjected side.

View larger version (52K): [in a new window]   Fig. 4. Alterations of Her6 and Her4 expression and/or activity disrupt the coordinate cyclic expression of deltaC and her1. Flat-mounted embryos at 10-12 ss. Two-cell stage embryos were injected in one cell only. In all cases, the injected side is on the right. Red (A,C,D,F,G) or brown (H,J,K) staining labels the injected side. The pattern of sharp deltaC stripes is disrupted in the anterior PSM of embryos injected with wt-her6 (A), her6-VP16 (D,E) or wt-her4 (H,I). In the anterior PSM of her6-VP16- (E') and wt-her4- (I') injected embryos, a random mixture of cells expressing deltaC at different levels is found. (E',I') Higher magnifications of the boxed regions in E and I, respectively. The sharp stripes of her1 expression are replaced by a random mixture of her1-positive and her1-negative cells throughout the PSM of embryos injected with wt-her6 (B-C''), her6-VP16 (F-G'') or wt-her4 (J-K''). (C',C'',G',G'',K',K'') Magnified views of the boxed areas in C, G and K, respectively, to allow a better comparison between injected and noninjected side of the embryos.

View larger version (76K): [in a new window]   Fig. 5. (A) her4MO (1 µM) is sufficient to suppress Her4 synthesis in a radioactive in vitro translation assay without blocking her6 mRNA translation (left panel). her6MO (1 µM) reduces Her6 synthesis without affecting Her4 (right panel). (B-G') Somitogenesis is disrupted in Her6, Her4- and Her6+Her4-depleted embryos. Forty-eight hour embryos stained with anti-myosin heavy chain antibody. Anterior is towards the left. (B-G) Low-magnification images to compare the general morphology of control and experimental embryos. Details of somite and intersomitic boundary morphology are shown in the corresponding high-magnification images. Black arrows indicate complete intersomitic boundaries, white arrows indicate irregular and/or defective intersomitic boundaries, white arrowheads mark myosin fibrils crossing intersomitic boundaries. (B) Control embryo. (B') High magnification of the boxed area in B. (C-C''') In 3 ng her6MO-injected embryos, somitogenesis is initiated normally but somites and intersomitic boundaries become progressively more irregular. (C',C'',C''') High magnification of the anterior, intermediate and posterior boxed areas in C, respectively. (E) her4MO-injected (3 ng) embryos develop normally and only the tail tip is affected. (D,F) The effect of her6MO and her4MO on somitogenesis is dose dependent. her6MO- (D) and her4MO- (F) (6 ng) injected embryos have a bent body axis and the onset of somitic abnormalities is shifted rostrally. D',F' are magnified views of the boxed areas in D and F. In D', somites as anterior as number 16 have ill-defined boundaries. In F', the last normal intersomitic boundary occurs at somite 25. (G,G') Somitogenesis defects occur earlier in embryos co-injected with 3 ng each her6MO and her4MO than in embryos injected with 6 ng of either her6MO or her4MO. (G') High magnification of the boxed area in G: intersomitic boundaries are ill-defined (white arrows) and irregularly spaced (black ruler at the bottom of the picture). (G'') A high-magnification view of the boxed area in (G'): large myosin fibrils span several somites. The expression pattern of her6 in the PSM is not dependent on the function of the Her6 protein. 10 ss control (H) or her6MO-injected (I) embryos probed for the expression of her6. The levels of her6 transcript are higher in the somites, anterior PSM, notochord/ventral neural tube and ectoderm of her6MO-injected embryos (I) than in the corresponding tissues of control embryos (H). The segmental expression of her6 in the somites and the anterior PSM is not affected. (H',H'',I',I') sections of the embryos in H and I cut along the broken lines marked by the corresponding small letters. The her6 signal is stronger in the anterior PSM of her6MO-injected embryos than in control ones, but no accumulation of her6 transcript is detected in the intermediate/posterior PSM.

View larger version (75K): [in a new window]   Fig. 6. Control (A,C,E,G,I,K) or her6MO-injected (B,D,F,H,J,L) embryos probed for the expression of the Notch pathway genes notch1a/des (A-D), lfng (E,F), notch6 (G,H), notch5 (I,J) and notch1b (K,L) at 14- to 18 ss. Staining was developed for exactly the same amount of time on control MO- and her6MO-injected embryos, to allow for a precise comparison of the gene expression levels. Levels of notch1a/des expression are high in the posterior PSM, decrease towards the anterior PSM and become very low in the somites (A,C). The transition between PSM and somites is defined by a sharp drop in notch1a/des expression. In 6 ng her6MO-injected embryos (B,D), this downregulation is impaired and expression of notch1a/des persists in the newly formed somites. No abnormalities are observed in the expression pattern of notch1b, notch5, notch6 (H,J,L). Expression of lfng is upregulated, but not disrupted, in the neural tube and newly formed somites (F).

View larger version (76K): [in a new window]   Fig. 7. Control (A-L) or morpholino-injected (M-) embryos hybridised with probes against deltaC (A-K,M-T',Y-ß) or her1 (I-L; U-X; ,) at different time points. (A-L) In control embryos, stripes of deltaC and her1 are maintained throughout somitogenesis. (D',D'') Higher magnifications of the boxed areas in the anterior and posterior PSM, respectively, of the embryo in D. (I') High magnification of the boxed area in I. (M-X) In embryos injected with 3 ng her6MO+3 ng her4MO, the periodicity of deltaC and her1 expression breaks down between 8 and 10 ss and is lost by 14 ss. deltaC stripes become blurred between 8 and 10 ss (M-O). Between 10 and 14 ss, the anterior stripes fuse (P,Q), lose their homogeneous character (compare R', a magnification of the boxed area in R, with D') and are replaced by an irregular domain expressing deltaC at different levels (S,S',T,T'). (S',T') Higher magnifications of the boxed areas in the anterior PSM of S and T. (S'') Higher magnification of the posterior boxed area in S, showing that an irregular deltaC pattern is also found in the posterior PSM (compare S'' with D''). her1 stripes become blurred around 10 ss (compare V,V' with I,I') and by 14 ss a mixture of cells expressing her1 at different levels is found throughout the PSM (W-X). (Y-) In embryos injected with 6 ng her6MO the periodicity of deltaC expression is maintained at 14 ss (Y), breaks down around 20 ss (Z) and is lost by 26 ss (). (,) disruption of her1 starts around 18 ss and is complete by 26 ss. (ß) In an embryo injected with 3 ng her6MO, only weak deltaC abnormalities are visible at 26 ss.