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her1 and the notch pathway function within the oscillator mechanism that regulates zebrafish somitogenesis

Scott A. Holley^*,, Dörthe Jülich, Gerd-Jörg Rauch, Robert Geisler and Christiane Nüsslein-Volhard

Max Planck-Institut für Entwicklungsbiologie, Tübingen, Germany
Present address: Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, USA

View larger version (58K): [in a new window]   Fig. 1. des is notch1. Morphological phenotypes of (A) wild-type and (B) desP37A embryos at about the 15-somite stage. des embryos form the first seven to nine somites but not the posterior somites. Anterior is leftwards. (C) des was mapped to linkage group 21 between z20701 and z7925, while notch1 was mapped between z27387 and z7925 (z15810). Genetic distance from the top of linkage group 21 (left) is given in cM. (D) Four independent alleles of des were sequenced. desAXO1B has a 7 bp insertion (5'-TGTGCAG-3') between bases 2738 and 2739, creating a frame-shift and premature stop, seven codons to the C terminus. desH35B has a T to A transition at base 4552, converting a Cys to a stop. desP37A has a T to A transition at base 186, creating a Leu to Gln substitution within the hydrophobic domain of the signal peptide (SP). desM145B has a G to A transition at base 6683, causing a Val to Met substitution. There are no obvious differences between these alleles in the severity of the somite phenotype. Nucleotide and amino acid sequences refer to the published wild-type sequences (Bierkamp and Campos-Ortega, 1993). TM, transmembrane domain.

View larger version (64K): [in a new window]   Fig. 2. Comparison of the oscillating pattern of deltaC expression with the non-oscillating expression of deltaD and notch1. (A) In wild-type embryos, notch1 is always expressed uniformly throughout the PSM, indicating that its expression does not oscillate. myoD expression is shown in red. (B-I) Wild-type embryos were staged and analyzed as previously described (Holley et al., 2000). (B-E) Representative early and late 12-somite stage embryos stained for deltaC (C) (B,C) or deltaD (D) (D,E) in blue and counterstained for myoD in red. In each panel, anterior is upwards. (F-I) Graphs depicting the distances that separate anterior of deltaC stripes 13 and 14 at the early (F) and late (G) 12 somite stage. The distances separating deltaD stripes 13 and 14 at the early and late 12 somite stages are depicted in H and I, respectively. Note that in H, no data points exist because the more posterior deltaD stripe has not formed yet. Measurements were made in pixels and later converted to number of cells (8 pixels/cell). Mean values are represented by broken lines. The mean values for F and G were compared using a two-sample t-test. The difference between the means in F and G is 25.25 pixels (3.1 cells) with a 95% C.I. from 18.7 to 31.7 pixels indicating that the differences between the data in F and G are significant. This indicates that, as for her1, the distance between consecutive deltaC stripes decreases as the somite cycle progresses.

View larger version (109K): [in a new window]   Fig. 3. Injection of morpholinos specific to notch1, deltaD, her1 or deltaC perturbs somite formation. Embryos injected with (A) notch1mo (four experiments; n=201; 97% affected) or (B) deltaDmo (four experiments; n=127; 99% affected) form the anterior seven to nine somites but fail to make regular posterior segments. (C,D) Dorsal views of her1mo1 (4 experiments n=200; 91% affected) or deltaCmo1 (6 experiments; n=545; 78% affected) injected embryos, respectively. In contrast to aei/deltaD and des/notch1, her1 and deltaC are necessary for the formation of both the anterior and posterior somites. Arrowheads in D indicate the misplaced somite borders. (E-P) The expression patterns of her1 and deltaC seen in wild-type, bea and morpholino-injected embryos. These embryos are between the 8 and 12 somite stages. Anterior is upwards. (E,K) Wild-type expression patterns of her1 and deltaC, respectively. Injection of Notch1mo causes defects in her1 expression (F) (two experiments; n=133; 100% affected) and deltaC expression (L) (two experiments; n=38; 100% affected) identical to that observed in des embryos. Injection of deltaDmo recapitulates the pattern of gene expression that is observed in aei/deltaD embryos (not shown). Injection of deltaCmo1 disrupts her1 expression (G) (four experiments; n=109; 98% affected) and deltaC expression (M) (6 experiments; n=168; 100% affected). Injection of a second deltaC morpholino, deltaCmo2, that does not overlap the sequence of deltaCmo1, produces the same defect in the expression of both her1 (H) (four experiments; n=145; 100% affected) and deltaC (N) (four experiments; n=187; 100% affected). Conversely, a control morpholino identical to deltaCmo1, except for four nucleotide substitutions, deltaCmoC, has no effect on the expression of either her1 (I) (three experiments; n=62; 0% affected) or deltaC (O) (3 experiments; n=56; 0% affected). (J,P) Expression of her1 and deltaC, respectively, in beaM98B embryos. beaM98B embryos were collected from a mating of homozygous beaM98B adults. deltaD expression is the same in all of the mutants and knockdown embryos, with the exception of 15% of fss embryos, as previously noted (not shown) (Holley et al., 2000). For F-H and L-N, percentages are in reference to n, the number of pre-sorted morphologically affected embryos examined. The specificity of the individual morpholinos is illustrated by the fact that: (1) both the deltaDmo and notch1mo phenocopy their known mutant phenotypes; and (2) deltaCmo1 and deltaCmo2 produce the identical phenotype, while deltaCmoC produces no phenotype.

View larger version (133K): [in a new window]   Fig. 4. Loss of her1 function eliminates all evidence of the oscillations in gene expression. These embryos are between the 8 and 12 somite stages. In all panels, anterior is upwards. (A) The wild-type her1 expression pattern is observed in all embryos injected with a control morpholino, her1moC, that is identical to her1mo1, except for four nucleotide substitutions (four experiments; n=182; 0% affected). (B,B’) injection of her1mo1 into wild-type embryos leads to a de-repression of her1 expression (three experiments; n=76; 100% affected). (B’’,B’’’) injection of a second her1 morpholino, her1mo2, which does not overlap the sequence of her1mo1, produces the identical defect in her1 expression (three experiments; n=128; 100% affected). Notice that there is no heterogeneity in the levels of expression between neighboring cells. (C) Wild-type expression pattern of deltaC is seen in embryos injected with her1moC (three experiments; n=162; 0% affected). (D,D’) in embryos injected with her1mo1, deltaC expression is reduced throughout the posterior and intermediate PSM (three experiments; n=77; 100% affected). In the anterior PSM, deltaC is expressed in a smooth domain that undergoes a refinement in the anteriormost PSM. This refinement appears to originate from the anterior and creates stripes of deltaC expression that can be later seen in the somitic mesoderm. (D’’,D’’’) injection of her1mo2 produces the identical defect (three experiments; n=122; 99% affected). (E-E’’’) The refinement of deltaC expression is lost in her1mo1;aei/deltaDAR33 embryos. Additionally, the stripes of deltaC expression in the somitic mesoderm are lost.

View larger version (103K): [in a new window]   Fig. 5. The epistatic relationship between her1 and the notch pathway changes along the anteroposterior axis of the PSM. Anterior is upwards. (A-G) her1 expression is in blue and that of myoD is in red. (A) In wild-type embryos, stripes of her1 expression are seen throughout the PSM. This embryo is a sibling of the mutant aei/deltaDAR33 embryo shown in B. In aei/deltaD embryos, no stripes of her1 expression are observed, and her1 is always exclusively expressed in a ‘salt and pepper’ pattern in the anterior PSM (between the broken lines). (C) her1 stripes form in fssAE114 embryos, but expression in the anterior PSM is always lost. (D) A fssAE114;aei/deltaDAR33 double mutant sibling of the embryo shown in C. In fss;aei/deltaD double mutant embryos, no stripes of her1 expression are formed (as in aei/deltaD embryos) and there is no expression of her1 in the anterior PSM (as in fss embryos). (E) Injection of deltaCmo1 into fssAE114 embryos produces a pattern of her1 expression similar to that observed in fss;des/notch1 and fss;aei/deltaD double mutant embryos: no stripes of expression are formed and no expression is seen in the anterior PSM (four experiments; n=123; 99% affected). (F) fssAE114;beaM98B double mutant embryos also lack both stripes of her1 expression and expression in the anterior PSM. These embryos were derived from a cross between double homozygous parents. (G) Injection of her1mo1 into fssAE114 embryos produced a her1 expression pattern identical to that of her1mo embryos (three experiments; n=158; 99% affected). A parallel analysis of deltaC expression yielded similar results (not shown). (H-N) All panels show deltaC expression. (H) deltaC expression in wild-type embryos. (I) her1mo1 embryos (four experiments 72% of 97 embryos) exhibit a refining stripe within the deltaC expression domain in the anteriormost PSM, arrow. In her1mo1;fssAE114 embryos (J) (two experiments 0% of 184 embryos), her1mo1;aei/deltaDAR33 embryos (K) (four experiments 0.5% of 202 embryos), her1mo1;notch1mo1 embryos (L) (three experiments 0.7% of 153 embryos), her1mo1; deltaCmo1 (M) (three experiments 0.6% of 169 embryos) and her1mo1; beaM98B (N) (two experiments 1% of 86 embryos), this refining stripe is lost. Sometimes in these double mutant embryos the pattern (stripe) of repression is converted into a ‘salt and pepper’ pattern, arrowheads in K-M.

View larger version (29K): [in a new window]   Fig. 6. A summary of the genetic analysis of the functions of her1 and the notch pathway during somitogenesis. (A) Anterior is upwards. (I) the notch pathway and bea are required to generate the oscillating expression of deltaC and her1 in the posterior and intermediate PSM. her1 probably functions within the oscillator and feeds back on the notch pathway to create the oscillating pattern of both deltaC and her1. (II) fss functions downstream of the notch pathway but upstream of her1 in the anterior PSM. (III) Slightly later, the notch pathway, bea and fss function in the anteriormost PSM/somitic mesoderm. (B) A model in which the notch pathwayher1notch pathway circuit creates the oscillations in gene expression. While the actual oscillator is probably more complicated, this model reflects the present data. Two states are represented: one in which her1 transcription is on (left) and one in which her1 transcription is off (right). Activation of Notch via interaction with Delta expressed on the surface of neighboring cells causes the activation of her1 transcription (left). The subsequent increase in Her1 would then act in a negative feedback loop to repress its own transcription (right). In time, the amount of Her1 would drop below a threshold and would allow her1 transcription to be activated again (left). Non-autonomous effects of the oscillations may be mediated by DeltaD, DeltaC or perhaps an unidentified ligand. The data addressing this aspect of the oscillations are not easily interpreted (represented by the ‘?’ and the broken arrow).