ALG-1, a microRNA argonaute, promotes vulva induction in C. elegans

Abstract

Signaling by the LET-60 Ras GTPase/ MPK-1 Extracellular Regulated Kinase pathway specifies the vulva cell fate in C. elegans . The let-7 miRNA family negatively regulates LET-60 Ras but other miRNAs can also modulate vulva induction. To determine the impact of globally reducing miRNA function on LET-60 Ras-mediated vulva induction we analyzed the effect of loss of the ALG-1 miRNA regulator on vulva development . Contrary to our expectations, we find that ALG-1 promotes vulva induction independently of LET-60 Ras. We found that the reduced vulva cell fate induction of alg-1 deletion mutants could be due to delayed development of the vulva, or a requirement to maintain the competence of the uninduced precursor cells.

Figure 1. alg-1 deletion alleles cause a partial vulvaless phenotype

( A ) Schematic of vulva induction. Six vulva precursor cells (VPCs) P3.p-P8.p are competent to be induced to adopt vulval cell fates. However, only the VPCs closest to the anchor cell (AC) are induced to adopt 1° and 2° vulval fates which undergo three rounds of cell division to generate 22 vulval cells. The remaining VPCs adopt the 3° non-vulval fate and divide once with the exception of P3.p, which 50% of the time loses competence and adopts the fused (F) fate without dividing. Created using Biorender ( B ) Differential interference contrast images of normal (top) and partially induced (bottom) vulvas of L4 stage alg-1 ( tm369 ) deletion mutant strain. In the top image (anterior is left, ventral is down) yellow asterisks mark the visible nuclei of P5.p and P7.p descendants. White arrow marks the uninduced posterior daughter of P4.p. In the bottom image (anterior is right) yellow asterisks mark the visible nuclei of the descendants of P5.p and a yellow arrow marks the P7.p cell that failed to divide and appears to have adopted a fused fate. White arrow marks the uninduced anterior daughter of P8.p. ( C ) Bar graph/scatter dot plot depicting vulva induction in various alg-1 and let-60 mutants. Each dot represents the VPC induction score for one animal and the bar represents the average VPC induction score for the strain. A score of 3 is wild type (WT), <3 is vulvaless, and >3 is multivulva. Since the VPCs are still competent to be induced after the first division, half inductions are common when signaling is compromised. A one-way ANOVA with multiple comparisons was used to determine the significance between average VPC inductions scores ** P<0.01 , *** P<0.001 and **** P<0.0001. A Fisher's exact test was used to determine the significance of the vulvaless phenotypes ## P<0.01 , ### P<0.001 and #### P<0.0001. ns, not significant. ( D ) Bar graph depicting the number of animals with normal alae or defective alae that were either faint or incomplete in wild type, alg-1 ( gk214 ) and alg-1 ( tm492 ). A Fisher's exact test was used to determine the difference in normal versus defective (faint + incomplete) alae between wild type and the alg-1 mutants. **** P<0.0001. ( E ) Bar graph depicting the % Fused fate adopted by each VPC in the alg-1 mutants compared to that expected for wild type. Between 30-34 animals were scored for the vulval phenotypes depicted in panels C and E.