DEV020891 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Expression of en regulators is affected in wol embryos. Embryos derived from wild-type or wol¹ germline clone females were incubated with probes against the regulators of odd-numbered en stripes, and are shown in lateral views with ventral down. In embryos just prior to gastrulation, the paired (prd) pattern consists of seven strong primary stripes and seven weaker secondary stripes (A). In wol mutant embryos, primary stripes four and five (corresponding to en stripes seven and nine) are weaker than the other primary stripes (B). It is possible that the failure to reach high levels of Prd in primary stripes four and five is the reason why en stripes seven and nine are not activated in wol embryos. Cellularizing wild-type (wt) embryos contain seven runt (run) stripes (C), whereas expression of stripes five and six are reduced in wol mutants (D). In cellularized wild-type embryos, seven odd skipped (odd) stripes are present (E). Only six odd stripes are detected in wol embryos (F). Embryos at the beginning of germband elongation normally contain 14 regularly spaced sloppy paired 1 (slp1) stripes (G). In wol embryos, the spacing is irregular (H).

• Supplemental Figure S2 -

  Fig. S2. Defects in posterior gene expression in wol embryos is not caused by a failure in the terminal or maternal posterior system. Gene expression was compared between wild-type and wol¹ germline clone embryos. The tailless (tll) gene is expressed at the posterior pole and as a stripe close to the anterior pole in response to activation of the terminal Torso receptor tyrosine kinase. tll expression is not affected in wol embryos (B) as compared with wild type (A). Posterior gap gene expression is also controlled by Hunchback (Hb), which is absent from the posterior of wild-type embryos owing to translational repression by Nanos (Nos). The nos expression patterns in wild-type and wol mutants are indistinguishable (C,D). Hb protein is absent from the posterior of both wild-type (E) and wol (F) embryos, although the amounts of Hb protein may be somewhat reduced in wol mutants.

• Supplemental Figure S3 -

  Fig. S3. Gastrulation defects in wol embryos. Wild-type embryos and embryos derived from wol¹ germline clones were incubated with a rho probe and are shown from a ventral view. In cellularizing embryos, two lateral bands are present in both wild-type (A) and wol mutant (B) embryos. In germband elongated wild-type embryos (C), the two bands have merged at the ventral midline due to mesoderm invagination. In wol embryos (D), mesoderm invagination is incomplete and the two rho bands fail to fuse. Mesoderm invagination is known to depend on the snail and twist genes. Consistent with the normal early rho pattern, expression of snail and twist is uncompromised in wol embryos (not shown), suggesting that wol is needed downstream of mesoderm specification for proper cell movements to occur.

• Supplemental Figure S4 -

  Fig. S4. Drosophila ALG5, but not wol mutant Drosophila ALG5, can complement a growth defect of an alg5 mutant S. cerevisiae strain. Combination of the Δalg5 mutation with the wbp1-2 allele, encoding a mutant form of an oligosaccharyl transferase subunit, results in a synthetic growth defect. YG355 (Δalg5 wbp1-2) cells were transformed with yeast expression vector containing Drosophila ALG5 cDNAs and grown on selective medium lacking uracil. Cells were collected in their logarithmic phase, six times diluted 1/6 starting with OD₅₄₆ of 25×10⁵ cells/ml and spotted on YPD plates, followed by incubation at 23°C, 30°C, 32°C or 35°C for 96 hours. The growth defect of the Δalg5 wbp1-2 strain is most pronounced at higher temperatures, and could be rescued by yeast WBP1 (pWBP1), yeast ALG5 (pScALG5), and fly ALG5 (pDmALG5) cDNAs, partially by fly ALG5 with the wol² mutation, and not at all by ALG5 with the wol¹ mutation or the vector (mock). This indicates that wol¹ is a stronger allele than wol². Consistent with this, one of the transgenic ALG5 lines could rescue the lethality and fertility of both wol alleles, whereas another transgenic line could rescue wol² but not wol¹ due to position effects.