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. 2015 Aug;200(4):1201-18.
doi: 10.1534/genetics.115.179184. Epub 2015 Jun 15.

Robust Distal Tip Cell Pathfinding in the Face of Temperature Stress Is Ensured by Two Conserved microRNAS in Caenorhabditis elegans

Samantha L Burke  1 , Molly Hammell  2 , Victor Ambros  3
Affiliations

Robust Distal Tip Cell Pathfinding in the Face of Temperature Stress Is Ensured by Two Conserved microRNAS in Caenorhabditis elegans

Samantha L Burke et al. Genetics. 2015 Aug.

Abstract

Biological robustness, the ability of an organism to maintain a steady-state output as genetic or environmental inputs change, is critical for proper development. MicroRNAs have been implicated in biological robustness mechanisms through their post-transcriptional regulation of genes and gene networks. Previous research has illustrated examples of microRNAs promoting robustness as part of feedback loops and genetic switches and by buffering noisy gene expression resulting from environmental and/or internal changes. Here we show that the evolutionarily conserved microRNAs mir-34 and mir-83 (homolog of mammalian mir-29) contribute to the robust migration pattern of the distal tip cells in Caenorhabditis elegans by specifically protecting against stress from temperature changes. Furthermore, our results indicate that mir-34 and mir-83 may modulate the integrin signaling involved in distal tip cell migration by potentially targeting the GTPase cdc-42 and the beta-integrin pat-3. Our findings suggest a role for mir-34 and mir-83 in integrin-controlled cell migrations that may be conserved through higher organisms. They also provide yet another example of microRNA-based developmental robustness in response to a specific environmental stress, rapid temperature fluctuations.

Keywords: distal tip cell migrations; mir-29; mir-34; mir-83; robustness.

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Figures

Figure 1
Figure 1
mir-83(n4638); mir-34(gk437) mutants have gonad migration defects. (A) The C. elegans gonad consists of two U-shaped gonad arms. The shape of each arm is created by the migration path of the DTC. The two DTCs are initially located ventrally near the midbody. Each DTC migrates away from the midbody before turning dorsally, migrating to the dorsal body wall muscle, and then migrating back toward the midbody. The path of each DTC can be inferred by the location of the respective gonad arm. Body wall muscle in red, germline in gray, DTCs in black, uterus designated with “U.” (B) mir-83(n4638); mir-34(gk437) mutants have a gonad migration defect. The improper location of the mature gonad arms implies that DTCs did not migrate along the normal path. In the arm pictured (bottom), the DTC (black arrow) migrated too far, passing the vulva (white asterisk), and is displaced ventrally, as compared to N2 (top). Penetrance quantified in Figure 2. (C) Improper DTC migration causes a rearrangement of internal organs, due to space constraints. In N2 worms, the intestine is positioned laterally with respect to the dorsal/ventral axis (indicated by a white, dashed line), and the proximal and distal segments of the gonad are positioned ventrally and dorsally, respectively, on the other side from the intestine. In the mir-83(n4638); mir-34(gk437) worm shown here, the displaced gonad caused a displacement of the intestine. Black arrowheads point to the adult lateral alae, which are positioned on the left and right sides of the animal.
Figure 2
Figure 2
The gonad migration defect is significantly enhanced in mir-83(n4638); mir-34(gk437) double mutants. Gonad arm morphology in young adult hermaphrodites was used to score for defects in DTC migrations during larval development at (A) 20° or (B) under an oscillating temperature regimen (15° for 15 min, 25° for 15 min, repeated from plating eggs until young adulthood). (C) Integrated transgenes expressing either mir-34, maIs387 or mir-83, maIs391, under their natural promoters rescue single mutants. Both VT3289 and VT3294 maIs387; maIs391; mir-83(n4638); mir-34(gk437) were produced by crossing VT3106 maIs387; mir-34(gk437) to VT3110 maIs391; mir-83(n4638), shown in D. As expected, the newly isolated mir-83(n4638); mir-34(gk437) double mutant, VT3289, displays the migration defect while the double mutant carrying both rescue transgenes, VT3294, appears normal. Significance asterisks compare to N2. Penetrance in VT2595 is not significantly different from penetrance in VT3289. Animals were cycled as described in B. ***P-value ≤ 0.005, *0.01 < P ≤ 0.05, not significant (n.s.) if P > 0.05.
Figure 3
Figure 3
Other miRNAs implicated in gonad migration function in separate pathways. (A) Previously implicated miRNA mutants were tested for enhancement of gonad migration defects by temperature oscillations. Worms were either maintained at a steady 20° throughout development or were subjected to oscillating temperature (15° for 15 min, 25° for 15 min) throughout development (“cycled”). (B) The mir-259(n4106) mutation was crossed into the mir-83(n4638); mir-34(gk437) strain to assess potential genetic interactions. Worms were subjected to an oscillating temperature regimen (15° for 15 min, 25° for 15 min, repeated until young adulthood). The difference in migration defective (mig) phenotype penetrance between mir-83(n4638); mir-34(gk437) and mir-83(n4638); mir-259(n4106); mir-34(gk437) is not significant. ***P-value ≤ 0.005. (C) Phenotype penetrance for the mir-259(n4106) mutation in combination with either the mir-34(gk437) mutation or the mir-83(n4638) mutation was compared to that in the mir-83(n4638); mir-34(gk437) double mutant. Worms were raised at 20° or cycled during the temperature-sensitive period discussed in Figure 4 [20° for 16 hr (15° for 15 min, 25° for 15 min, repeated three additional times), 20° until young adulthood]. ***P-value ≤ 0.005.
Figure 4
Figure 4
Temperature oscillations within a limited 2-hr window cause the migration defective phenotype enhancement in mir-83(n4638); mir-34(gk437) mutants. The temperature was oscillated every 15 min for a total of 2 hr (15° for 15 min, 25° for 15 min, repeated four times). (A) Temperature oscillations occurred prior to the birth of the DTCs (12 to 14 hr after plating starved L1’s on HB101-seeded NGM plates). (B) Temperature oscillations occurred over an interval (16 to 18 hr after plating starved L1’s on HB101) corresponding to the time of DTC birth. (C) Temperature oscillations occurred after the birth of the DTCs (20 to 22 hr after plating starved L1’s on HB101). ***P-value ≤ 0.005, **0.005 < P ≤ 0.01, significance asterisks compare N2 to mir-83(n4638); mir-34(gk437) mutants.
Figure 5
Figure 5
The mir-83(n4638); mir-34(gk437) migration defect is suppressed in cdc-42 and pat-3 heterozygotes. The penetrance of the migration defective phenotype is significantly reduced in mir-83(n4638); mir-34(gk437) mutants when (A) carrying one copy of cdc-42, (B) raised on cdc-42 RNAi food as compared to empty vector control, (C) carrying one copy of pat-3, or (D) raised on pat-3 RNAi food as compared to empty vector control. Arrested L1’s were plated on HB101-seeded NGM plates to restart development. Once plated, temperature was held at 20° for 16 hr, then cycled as follows: 15° for 15 min, 25° for 15 min, four times, then held at 20° until young adulthood. ***P-value ≤ 0.005, **0.005 < P ≤ 0.01, *0.01 < P ≤ 0.05, P > 0.05 not significant (n.s.).
Figure 6
Figure 6
mir-83(n4638); mir-34(gk437) mutants produce normal sized self-broods and reduced cross-broods. (A) The total number of living offspring was counted to determine brood size for N2 and mir-83(n4638); mir-34(gk437) worms raised at 20° throughout development or subjected to temperature cycles (15° for 15 min, 25° for 15 min, repeated four times) from 16 to 18 hr of development after plating starved L1’s on HB101. There is no statistically significant difference between any of the groups. (B) Individual 4-day-old adult hermaphrodites that had exhausted their self-progeny were crossed to four 1-day-old N2 males and number of cross-brood progeny was counted. The average cross-brood size was reduced for mir-83(n4638); mir-34(gk437) hermaphrodites whether they were raised at 20° or subjected to temperature cycles (15° for 15 min, 25° for 15 min, repeated four times) from 16 to 18 hr of development. Averages were compared using an unpaired t-test, performed by PRISM. ***P-value ≤ 0.005. Each group was compared to the three others. The absence of significance asterisks denotes a lack of statistical significance.
Figure 7
Figure 7
mir-83(n4638); mir-34(gk437) mutants have a decreased lifespan compared to wild type. Wild-type (N2) and mir-83(n4638); mir-34(gk437) embryos were harvested by hypochlorite treatment and synchronized L1 larvae were obtained by hatching overnight in M9. Larvae were plated on HB101-seeded NGM plates and raised (A) continuously at 20° or (B) subjected to temperature cycles (15° for 15 min, 25° for 15 min, repeated four times) from 16 to 18 hr of development after plating starved L1’s on HB101. For each longevity assay shown in panels A and B, 100 animals per replicate, three replicates per condition, were plated as L4’s (day 0) and tracked until their death. Alive or dead was determined by prodding worms on their nose and looking for a reaction. Daily averages were compared using a two sample t-test for means. ***P-value ≤ 0.005, **0.005 < P ≤ 0.01, *0.01 < P ≤ 0.05.
Figure 8
Figure 8
A model proposing robustness functions for mir-34 and mir-83 through dampening noisy cdc-42 and pat-3 expression in the face of temperature changes. (A) Environmental stresses, such as changing temperatures, may lead to fluctuations in the expression levels of various transcripts, which could challenge cellular proteomic homeostasis. Repression of protein production by miRNAs can provide a means of stabilizing protein output from fluctuating target transcripts. (B) In the case of mir-34 and mir-83, the fidelity of DTC migration is proposed to be maintained in part by inhibiting noisy cdc-42 and pat-3 expression incited by unstable environmental temperature.
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