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Two linked hairy/Enhancer of split-related zebrafish genes, her1 and her7, function together to refine alternating somite boundaries

Clarissa A. Henry^1,*, Michael K. Urban^1,*, Kariena K. Dill¹, John P. Merlie¹, Michelle F. Page¹, Charles B. Kimmel² and Sharon L. Amacher^1,

¹ Department of Molecular and Cell Biology, University of California, Berkeley CA 94720-3200, USA
² Institute of Neuroscience, University of Oregon, Eugene, OR 97403-1254, USA
^* These authors contributed equally
The authors dedicate this paper to the memory of Loan Thanh Nguyen, our labmate and friend

View larger version (61K): [in a new window]   Fig. 1. her1 and her7 are included in the b567 deficiency and b567 mutant embryos have somitic defects. (A,B) Dorsal view of embryos hybridized with an in situ hybridization screen cocktail including pax2, forkhead6, floating head, valentino and her1. (A) Wild-type embryo; (B) b567 mutant embryo lacking her1 expression (arrows). (C,D) Although somite formation is disrupted in b567 mutant embryos (D) as compared to wild-type embryos (C), overall embryo morphology is normal at this stage. (E) Map of the b567 deficiency. A total of 25 markers, including SSLPs (z-markers), ESTs (prefixed by fa, fb and fc), and cloned genes (italicized), were PCR-amplified from DNA prepared from b567+ and b567– embryos. Markers that failed to amplify from b567– DNA samples are indicated in red. Map positions are indicated in cM (indicating relative position on the integrated map [ZMAP] that includes all 6 independent mapping panels) and in cR (indicating position on the T51 mapping panel, on which 18 of the tested 25 markers have been mapped). Approximate centromere position is indicated by a thick black box. The solid red line indicates the extent of the deficiency and the dashed red line indicates the possible location of the telomeric breakpoint. The b567 deficiency deletes approximately 92-174 cR (15-22 cM) of LG 5.

View larger version (149K): [in a new window]   Fig. 2. AP patterning of somites is disrupted in her1+7 MO-injected and b567 mutant embryos. WT denotes wild-type embryos, and MO denotes her1+7 MO-injected embryos. All panels are dorsal views with anterior towards the top. Developmental stages are indicated at the bottom right. The segmental expression of papc and myoD is disrupted throughout somitogenesis in MO-injected and b567 mutant embryos (A-H). The Notch ligands deltaD and deltaC are expressed throughout the presumptive somite rather than being restricted to the anterior or posterior half, respectively (I-L). Expression of a Notch receptor, notch5, is also disrupted (M,N). In addition, the expression of mespA is downregulated and not segmental in b567 mutant embryos (O,P). Both ephrin B2 (Q,R) and fak (S,T) are expressed throughout the paraxial mesoderm instead of in posterior half-somites.

View larger version (109K): [in a new window]   Fig. 3. Somites in b567 mutant embryos are enlarged in the AP dimension. Whereas live 24 hpf wild-type embryos have reiterated somites over regular intervals (A, arrows), b567 mutant embryos (B) have enlarged somites (arrows) with weak boundaries in between (arrowhead). (C-E) F59 staining highlights the large, irregular boundaries in both b567 mutant and her1+7 MO-injected embryos. Again, arrows denote strong boundaries and arrowheads denote weak boundaries. Molecular evidence of large somites is seen by expression of a titin homolog (F,G).

View larger version (73K): [in a new window]   Fig. 4. Somites in her1+7 MO-injected embryos are enlarged in the AP dimension. All panels are confocal micrographs with black and white inversed, side views of ß-catenin staining in 17- to 18-somite embryos. A'-F', are the same confocal micrographs as A-F, without any pseudocoloring. (A,B) Wild-type embryo. (A) Anterior somites are chevron-shaped and posterior somites are more block shaped. Red-brown and blue colors denote alternating somites. The anteriormost somite shown in (A) is somite 11, panels C and E are at approximately the same AP position. (B) A higher magnification view of A showing the epithelial, cuboidal border cells (yellow and pink) that flank the intersomitic boundary. (C-F) her1+7 MO-injected embryos. Somites in these embryos are larger in the AP dimension. It is important to note that the stronger somite boundaries that are denoted by pseudocoloring are defined as such by their 3-dimensional structure: these boundaries occupy at least 90% of the dorsal-ventral dimension and 30% of the mediolateral dimension (as determined by viewing all of the focal planes, not just the one shown). (D,F) Some weak attempts at boundary formation in between stronger boundaries (arrowheads) are seen in these confocal sections. The weak boundaries do not meet our stronger boundary criteria.

View larger version (113K): [in a new window]   Fig. 5. her1 and her7 are partially redundant. (A-C) Confocal micrographs, side views of embryos stained for ß-catenin to outline cell boundaries. A', B' and C' are non-colorized versions of the confocal micrographs. (A) The first 2-3 somites in her1 MO-injected embryos (15 somite stage) are disrupted. The arrowhead denotes an attempt at a somite boundary that does not extend fully in either the mediolateral or dorsoventral dimensions. All other boundaries appear normal. (B) While anterior somites are normal in her7 MO-injected embryos (15 somite stage), posterior somites are enlarged. The first somite shown is somite 9. (C) Interior boundaries partially recover over time in her7 MO-injected embryos (22 somite stage). Strong boundaries are highlighted in blue. Weak boundaries are shown in red. (D-I) deltaD expression correlates with abnormal somite morphology in her7 MO-injected embryos. Early deltaD expression (2 somite stage) is unaffected in her7 MO-injected embryos compared to control embryos (D,E), but later (10 somite stage), deltaD expression is disrupted (F,I) similar to her1+7 MO-injected embryos (G). Interestingly, presomitic mesoderm expression of deltaD is partially disrupted in her1 MO-injected embryos (H), but somite morphology is normal posterior to somite 3 (A).

View larger version (26K): [in a new window]   Fig. 6. Alternate somite boundaries are strengthened in her7 MO-injected embryos. Embryos were examined approximately every 30-40 minutes. The somites that had formed at the beginning of the experiment (A,B,C) are shown in black. Anterior is towards the top and left in A-C, and at the left in D,E. Development at the first time point is shown in red, the second in green, the third in pink, and the fourth in purple. In wild-type embryos (A), a strong somite boundary forms at every time point. Weak boundaries are sometimes seen as somites are forming, but were not observed in this particular embryo. In her7 MO-injected embryos (B,C), somite boundary formation is altered in 3 ways. First, weak boundaries persist much longer than in wild-type embryos and are therefore more frequently observed (dashed lines). Secondly, weak and disorganized attempts form in a segmental fashion but only alternate boundaries are strengthened (solid lines). Finally, large somites sometimes subdivide into 2 normal somites after the formation of many more posterior somites. (D,E) A cartoon summary of somite formation in wild-type (D) and her7 MO-injected embryos (E). In wild-type embryos, somites form in an anterior- to-posterior fashion approximately every 30 minutes. In her7 MO-injected embryos, weak boundaries (dashed lines) form in a segmental fashion. However, only some weak boundaries are strengthened (solid lines).