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In vivo mapping of the functional regions of the DEAD-box helicase Vasa.

Dehghani M, Lasko P - Biol Open (2015)

Bottom Line: We identified novel domains in the N- and C-terminal regions of the protein that are essential for localization, transposon repression, posterior patterning, and pole cell specification.One such functional region, the most C-terminal seven amino acids, is specific to Vas orthologues and is thus critical to distinguishing Vas from other closely related DEAD-box helicases.Surprisingly, we also found that many eGFP-Vas proteins carrying mutations that would be expected to abrogate DEAD-box helicase function localized to the nuage and posterior pole, and retained the capacity to support oogenesis, although they did not function in embryonic patterning, pole cell specification, grk activation, or transposon repression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, McGill University, 3649 Promenade Sir William Osler, Montréal, QC H3G 0B1, Canada.

No MeSH data available.


Dorsal-ventral patterning in eggs produced by vasPH165 females carrying different egfp-vas constructs.(A) Dorsal appendage formation. The green bars indicate the percentage of the embryos with two separate or partly fused dorsal appendages. The beige and yellow bars represent the portion of the embryos with fused or no dorsal appendages, respectively. Data from vasPH165 controls are presented at the far right. Error bars indicate SEM, n (number of females tested) >50 for all genotypes. Asterisks indicate a significant difference compared to vas+ (p<0.05). (B) Grk expression. Red bars indicate the percentage of stage 8 egg chambers positively stained for localized Grk. Error bars represent SEM from three different replicates. n (number of stage 8 egg chambers in each replicate) >50 for all genotypes.
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f03: Dorsal-ventral patterning in eggs produced by vasPH165 females carrying different egfp-vas constructs.(A) Dorsal appendage formation. The green bars indicate the percentage of the embryos with two separate or partly fused dorsal appendages. The beige and yellow bars represent the portion of the embryos with fused or no dorsal appendages, respectively. Data from vasPH165 controls are presented at the far right. Error bars indicate SEM, n (number of females tested) >50 for all genotypes. Asterisks indicate a significant difference compared to vas+ (p<0.05). (B) Grk expression. Red bars indicate the percentage of stage 8 egg chambers positively stained for localized Grk. Error bars represent SEM from three different replicates. n (number of stage 8 egg chambers in each replicate) >50 for all genotypes.

Mentions: Females for vas function fail to translate grk mRNA at wild-type levels (Styhler et al., 1998; Tomancak et al., 1998), and the consequent reduction of secreted Grk ligand available to adjacent follicle cells results in ventralization of their patterning, which can be observed through effects on the structure and position of the dorsal appendages of the eggshell. As patterning becomes more ventralized, the dorsal appendages move closer together, then fuse, and then become absent altogether. We compared the percentages of eggs produced from each line expressing a different eGFP-Vas mutant in a vasPH165 mutant background that had two dorsal appendages (separate or partly fused), a single fully fused appendage, or no appendages, as an index of Gurken (Grk) expression (Fig. 3A). Using this assay, we found that most N-terminal deletions of eGFP-Vas had only modest, but nevertheless statistically significant, effects on dorsal appendage formation (Fig. 3A; supplementary material Table S1). Larger N-terminal deletions had somewhat larger effects with greater statistical significance, and fewer than 20% of eggs produced from females expressing only eGFP-Vas deleted for amino acids 3-200 had two dorsal appendages (Fig. 3A; supplementary material Table S1). In contrast to what we observed for oogenesis, all mutations in the conserved helicase domains severely compromised dorsal appendage formation (less than 10% of embryos with two dorsal appendages; Fig. 3A), and again D554A had the most extreme effect (no embryos with two appendages). The three C-terminal mutations we examined behaved divergently in this assay; deletion of amino acids 636-646 or a point mutation in a conserved residue within this segment (R644A) had only modest but statistically significant effects, while deletion of the seven most C-terminal amino acids (655-661) produced nearly as severe a phenotype as some of the mutations in conserved DEAD-box domains (Fig. 3A; supplementary material Table S1).


In vivo mapping of the functional regions of the DEAD-box helicase Vasa.

Dehghani M, Lasko P - Biol Open (2015)

Dorsal-ventral patterning in eggs produced by vasPH165 females carrying different egfp-vas constructs.(A) Dorsal appendage formation. The green bars indicate the percentage of the embryos with two separate or partly fused dorsal appendages. The beige and yellow bars represent the portion of the embryos with fused or no dorsal appendages, respectively. Data from vasPH165 controls are presented at the far right. Error bars indicate SEM, n (number of females tested) >50 for all genotypes. Asterisks indicate a significant difference compared to vas+ (p<0.05). (B) Grk expression. Red bars indicate the percentage of stage 8 egg chambers positively stained for localized Grk. Error bars represent SEM from three different replicates. n (number of stage 8 egg chambers in each replicate) >50 for all genotypes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400588&req=5

f03: Dorsal-ventral patterning in eggs produced by vasPH165 females carrying different egfp-vas constructs.(A) Dorsal appendage formation. The green bars indicate the percentage of the embryos with two separate or partly fused dorsal appendages. The beige and yellow bars represent the portion of the embryos with fused or no dorsal appendages, respectively. Data from vasPH165 controls are presented at the far right. Error bars indicate SEM, n (number of females tested) >50 for all genotypes. Asterisks indicate a significant difference compared to vas+ (p<0.05). (B) Grk expression. Red bars indicate the percentage of stage 8 egg chambers positively stained for localized Grk. Error bars represent SEM from three different replicates. n (number of stage 8 egg chambers in each replicate) >50 for all genotypes.
Mentions: Females for vas function fail to translate grk mRNA at wild-type levels (Styhler et al., 1998; Tomancak et al., 1998), and the consequent reduction of secreted Grk ligand available to adjacent follicle cells results in ventralization of their patterning, which can be observed through effects on the structure and position of the dorsal appendages of the eggshell. As patterning becomes more ventralized, the dorsal appendages move closer together, then fuse, and then become absent altogether. We compared the percentages of eggs produced from each line expressing a different eGFP-Vas mutant in a vasPH165 mutant background that had two dorsal appendages (separate or partly fused), a single fully fused appendage, or no appendages, as an index of Gurken (Grk) expression (Fig. 3A). Using this assay, we found that most N-terminal deletions of eGFP-Vas had only modest, but nevertheless statistically significant, effects on dorsal appendage formation (Fig. 3A; supplementary material Table S1). Larger N-terminal deletions had somewhat larger effects with greater statistical significance, and fewer than 20% of eggs produced from females expressing only eGFP-Vas deleted for amino acids 3-200 had two dorsal appendages (Fig. 3A; supplementary material Table S1). In contrast to what we observed for oogenesis, all mutations in the conserved helicase domains severely compromised dorsal appendage formation (less than 10% of embryos with two dorsal appendages; Fig. 3A), and again D554A had the most extreme effect (no embryos with two appendages). The three C-terminal mutations we examined behaved divergently in this assay; deletion of amino acids 636-646 or a point mutation in a conserved residue within this segment (R644A) had only modest but statistically significant effects, while deletion of the seven most C-terminal amino acids (655-661) produced nearly as severe a phenotype as some of the mutations in conserved DEAD-box domains (Fig. 3A; supplementary material Table S1).

Bottom Line: We identified novel domains in the N- and C-terminal regions of the protein that are essential for localization, transposon repression, posterior patterning, and pole cell specification.One such functional region, the most C-terminal seven amino acids, is specific to Vas orthologues and is thus critical to distinguishing Vas from other closely related DEAD-box helicases.Surprisingly, we also found that many eGFP-Vas proteins carrying mutations that would be expected to abrogate DEAD-box helicase function localized to the nuage and posterior pole, and retained the capacity to support oogenesis, although they did not function in embryonic patterning, pole cell specification, grk activation, or transposon repression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, McGill University, 3649 Promenade Sir William Osler, Montréal, QC H3G 0B1, Canada.

No MeSH data available.