A new deletion allele of sma-4

Figure.

1A. Location of the jj278 deletion. 1B. Results of body size measurement. jj278 worms are small. e729 is the canonical allele of sma-4 and was used as a control. Body size measurements were conducted as previously described (TIAN et al. 2013). Hermaphrodite worms were imaged at the L4.1 stage based on vulva development (MOK et al. 2105), Body lengths were measured from the images using the Segmented Line tool of Fiji. Statistical analysis was carried out using Student’s t test. The mean body length of N2 worms is normalized to 100%. Error bars represent 95% confidence intervals for the normalized body length.­ The numbers inside each bar represent the numbers of animals measured for the specific genotype. *** P<0.0001.

Description

sma-4 encodes the co-Smad of the BMP pathway in C. elegans, which is also known as the Sma/Mab pathway (Savage et al. 1996). Null mutations in core components of this pathway, including the BMP ligand DBL-1, the receptors SMA-6 and DAF-4, and the R-Smads SMA-2 and SMA-3, all result in a small body size phenotype without significantly compromising viability (Gumienny and Savage-Dunn 2013). However, we found that even after multiple rounds of out-crossing, two deletion alleles of sma-4, ok3140 and tm4731, caused late larval lethality and embryonic lethality, respectively. This observation suggests that either SMA-4 has DBL-1/BMP-independent functions that are required for viability, or ok3140 and tm4731 have closely linked lethal mutations. To distinguish between these two possibilities, we generated a deletion allele of sma-4 using CRISPR/Cas9-mediated non-homologous end joining. This allele, jj278, contains a 3,556bp deletion (position: Chromosome III: 5,816,203….5,819,759) that deletes almost the entire coding region of sma-4 (Figure 1A) and represents a true molecular null. jj278 animals are viable and fertile, but are smaller than wild-type animals (Figure 1B), like loss-of-function mutants in other core BMP pathway members (Savage et al. 1996; Krishna et al. 1999). This result suggests that ok3140 and tm4731 likely have closely linked lethal mutations not caused by their respective sma-4 deletions.

Reagents

Plasmids and oligos used to generate jj278:

All sgRNA plasmids were using the pRB1017 backbone (ARRIBERE et al. 2014).

pJKL1171 (sma-4 N-sgRNA #1):

JKL-1692 (F): TCTTGGTCGAATAATGTTTCATCC

JKL-1693 (R): AAACGGATGAAACATTATTCGAC

pJKL1172(sma-4 N-sgRNA #2):

JKL-1694 (F): TCTTGACGGCTGAGATGTCATACC

JKL-1695 (R): AAACGGTATGACATCTCAGCCGT

pANM1 (sma-4 C-sgRNA #1):

ANM-12 (F): TCTTGCACTGTCAGGCATTATCGC

ANM-13 (R): AAACGCGATAATGCCTGACAGTGC

pANM2 (sma-4 C-sgRNA #2):

ANM-10 (F): TCTTGCAGCGATAATGCCTGACAG

ANM-11 (R): AAACCTGTCAGGCATTATCGCTGC

Oligos used to genotype jj278:

Expected sizes: Wild-type: 4.097kb + 295bp; jj278: 541bp

JKL-1104: CATGAATATGAGAAACTGCTGG

ANM-14: CCGTTGTCACCTCGAATCAC

ANM-15: GCTCTGCTCCATACAAAGGATC

Strain: LW5558: sma-4(jj278) III. Will be sent to the CGC.