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Saul-Wilson Syndrome (SWS) is an ultra-rare, autosomal dominant skeletal dysplasia syndrome discovered in 1990; only 16 patients have been identified to date (Saul and Wilson 1990; Ferreira et al. 2018, OMIM#: 618150). The disease is characterized by short stature, various craniofacial abnormalities, shortened fingers and toes, and speech and physical developmental delay (Ferreira 2020). SWS is caused by a missense mutation in the COG4 gene, resulting in a G516R residue change. Other pathogenic mutations have been observed in this gene and all are clustered at the C-terminal end of the protein (R724W, R729W, R729A, E764A). These are associated with Congenital Disorder of Glycosylation type 2j (CDGIIj). This is a recessive disease characterized by mild psychomotor delay, mild dysmorphic features, epilepsy, and defective sialylation (Reynders et al. 2009). Besides the mild developmental delay, this disease seems to share virtually no phenotypic similarity with SWS.

We had previously identified the causative mutation in emb-14(g43ts) as an Ala1425Thr missense mutation in the C. elegans fasn-1 gene on LGI (Jaramillo-Lambert et al. 2015). At that time, it was suggested that the emb-14 gene be renamed fasn-1. FASN-1 is a fatty acid synthase orthologous to human FASN (Lee et al. 2010). The g43ts allele is a tight, highly penetrant temperature-sensitive allele. There are presumably two other alleles, g14ts and fr8. We characterized the osmotic sensitivity of g14ts embryos, as well as the brood size and embryonic viability of the g14ts allele. We also demonstrated that g14ts failed to complement the tight temperature-sensitive allele, g43ts. We also determined the sequence alteration of g14ts to be a Gly1830Arg missense mutation upon sequencing PCR fragments that covered the entire fasn-1 gene (Fig. 1A). fr8 was previously identified as an M241I missense mutation in the fasn-1 gene. Interestingly, 40% of fasn-1(fr8) embryos fail to hatch (Lee et al. 2010). The brood size of the g14ts and g43ts strains were variable but statistically different than N2, the wildtype strain (Fig. 1B). Brood size was determined by moving mothers each day to fresh plates and counting the number of hatched larvae and unhatched dead embryos on each plate 24 hrs after moving the mothers. Daily transfers were performed until the mothers stopped laying fertilized embryos. Brood size was defined as the sum of hatched larvae and unhatched dead embryos. Embryonic viability (% hatching) at 25°C was quite variable for g14ts (Fig 1C). However, g43ts embryos failed to hatch at 25°C, while the wildtype embryos hatched >98% (Fig. 1C). To confirm that g14ts was an allele of fasn-1, we did a complementation assay with g43ts (Fig. 1D). We used males marked with a pharyngeal GFP marker (mIs10) to confirm that cross progeny were being analyzed. Half of the F1 hermaphrodites should be g14ts/g43ts while half should be just g43ts/+. All F1 animals were then shifted to 25°C as L4 larvae, and the embryonic viability of each mother was quantified (Fig. 1E). Embryonic viability was bimodal, likely reflecting that half the F1 failed to complement at 25°C, and the other half were g43ts/+ mothers with high levels of embryonic hatching. To quantify the defective eggshells of the fasn-1 mutants, N2, g14ts, and g43ts were subjected to DAPI staining. Young gravid hermaphrodites were washed in M9 buffer three times and dissected with 23 G x 3/4” needles. Different stage mitotic embryos were transferred to a homemade hanging drop chamber made by molten Vaseline (Edgar and Goldstein 2010). The chamber was filled with Blastomere Culture Medium (BCM) with 5 μg/ml DAPI. The images were captured by high-resolution confocal microscopy with a Nikon 60 X 1.2 NA water objective with 1 μm z-step size. Images were generated by custom Fiji Image J>Stacks>Z project (Linkert et al. 2010, Schindelin et al. 2012). As expected, N2 embryos were not permeable to DAPI (other than the first polar body; Fig. 1F) while fasn-1(g43ts) embryos were highly permeable. fasn-1(g14ts) embryos were variably osmotically sensitive with only 14.4% showing DAPI permeability (Fig. 1G, H). fr8 was also reported to be permeable to the lipophilic dye Nile red (Lee et al. 2010). Of the three existing alleles, g43ts is the tightest and most penetrant. Suppressor screens with this allele would be quite straight forward.

Presentation of poster 1457B at TAGC 2020 Online. Files include a PDF of the poster (Sloan_TAGC_Online_1457B.PDF), a ~10-minute audio walkthrough of the poster material (Sloan_TAGC_Online_1457B_Audio.mp3), and a transcript of the audio walkthrough (Sloan_TAGC_Online_1457B_Transcript of Audio.PDF).

SPE-11 is a sperm protein required to initiate embryonic development. Mutations of the spe-11 gene result in a strict paternal-effect lethal phenotype. Wild-type oocytes fertilized by sperm from homozygous spe-11 males leads to abnormal development, but the reciprocal interaction, spe-11 mutant oocytes fertilized by wild-type male sperm, results in normal development (Hill et al. 1989). Sperm from homozygous spe-11 males are morphologically normal and do not display motility defects (L’Hernault et al. 1988). However, the absence of functional SPE-11 results in embryonic failure at early stages. Embryonic defects include failure to complete oocyte meiosis (an event that takes place upon fertilization), formation of a weak eggshell, and improper orientation of the first mitotic spindle (Hill et al. 1989). In addition, Spe-11 embryos fail to complete cytokinesis during the first division resulting in multinucleated one-cell embryos that eventually die (Hill et al. 1989). The embryonic lethality phenotype conferred by spe-11 mutant sperm is rescued when SPE-11 is ectopically expressed in oocytes demonstrating that SPE-11 functions during early embryogenesis and is not required during sperm development (Browning and Strome 1996). Furthermore, SPE-11 expression in oocytes did not disrupt oocyte activation because these oocytes were still capable of fertilization and did not precociously form an eggshell (i.e. prior to fertilization, Browning and Strome 1996). SPE-11 is a 299 amino acid protein with no significant similarity to other proteins. SPE-11 is first detected in spermatocytes in the gonad of males as foci that eventually unite to form a ring around the condensed DNA of mature sperm (Browning and Strome 1996). It is predicted to be a very highly hydrophilic protein with five potential nuclear localization sequences (NLS) (Browning and Strome 1996). SPE-11 has no obvious protein domains (Browning and Strome 1996). To define functional units of the SPE-11 protein, we generated a series of GFP::SPE-11 mutant constructs along with a GFP::SPE-11 rescuing construct (Figure 1A) and created transgenic worm lines in the null spe-11(ok2143) background (deletion of exons two through six out of seven total exons) through microparticle bombardment. To determine functionality of each SPE-11 mutant construct we assayed the transgenic lines for SPE-11 localization in late meiotic prophase and in post-meiotic sperm and for rescue of the embryonic lethality phenotype. Similar to previous reports of SPE-11 localization, the wild-type GFP-SPE-11 construct forms foci around diplotene nuclei that eventually coalesce into a perinuclear ring in spermatids (Browning and Strome 1996) and rescues embryonic lethality (Avg. number of viable progeny=175.3, Figure 1B and 1C). We started our mutant analysis by creating a single integrated, transgenic line carrying a mutant version of the first putative NLS (GFP::SPE-11NLS1). GFP::SPE-11NLS1 both rescues the embryonic lethality of the spe-11(ok2143) mutant and localizes correctly in the male germ line (Figure 1B and 1C). We have also generated a single integrated line with the fifth putative NLS mutated (GFP::SPE-11NLS5). This line localizes correctly during the diplotene substage of meiotic prophase, however it fails to encircle the nuclei of post-meiotic sperm and fails to rescue the embryonic lethality of the spe-11(ok2143) mutant (Figure 1B and 1C). This failed rescue along with the localization defect suggests that the amino acids mutated in GFP::SPE-11NLS5 are important for SPE-11 function and localization. We also generated a SPE-11 construct in which three basic amino acids in the last ten amino acids of the C-terminus are changed to Ala (GFP::SPE-11AAAmut). Note that the GFP::SPE-11NLS5 mutant has two additional basic residues changed to Ala (see Reagents section). Interestingly, the GFP::SPE-11AAAmut mutant had a wild-type localization pattern, yet it completely fails to rescue the embryonic lethality of the spe-11(ok2143) mutant (Avg. number of viable progeny=4.6, Figure 1B and 1C). The majority of existing spe-11 mutant alleles truncate the SPE-11 protein suggesting that the C-terminus is very important for function. To test the importance of the C-terminus on SPE-11 function we generated an additional transgenic line, one in which only the last five amino acids are removed (GFP::SPE-11∆295-299). This line has a significantly lower number of viable progeny than the GFP::SPE-11 rescuing construct (p

The 26S proteasome is one of the major proteolytic machineries in cells and is composed of nearly 33 different subunits (Budenholzer et al., 2017; Chen et al., 2008; Papaevgeniou and Chondrogianni, 2014). The subunits are arranged as one or two 19S regulatory particle(s) capping a cylindrical catalytic 20S core particle (Budenholzer et al., 2017). Each subunit is highly conserved from yeast to mammals, and proper function of the proteasome is crucial for survival of organisms (Papaevgeniou and Chondrogianni, 2014). Spatiotemporal expression of specific subunits and their roles in regulating the proteasome activity is still not clearly understood. RPN-12 is a subunit of the 19S regulatory particle (RP) of the 26S proteasome in Caenorhabditis elegans (Boehringer et al., 2012; Takahashi et al., 2002). In C. elegans, loss or down regulation of a single proteasome subunit causes embryonic lethality, with the exception of three of the 19S RP subunits: RPN-10, RPN-12, and DSS-1 (Keith et al., 2016; Pispa et al., 2008; Shimada et al., 2006; Takahashi et al., 2002). While the roles of RPN-10 and DSS-1 were previously studied, more detailed investigation into the function of RPN-12 has not been performed. A heterozygous mutant containing a 952bp deletion of the coding region of rpn-12 was generated [rpn-12(av93)] using CRISPR/Cas9 genome editing technology. The heterozygous rpn-12 null mutant was then homozygosed and determined to be homozygous viable with apparent fertility issues. Only 36% of the rpn-12(av93) hermaphrodites produced progeny due to sperm production defects (this phenotype will be published elsewhere). Since efficient proteasome activity is important for the longevity of most organisms, we wanted to investigate whether rpn-12(av93) mutants may have an altered lifespan compared to wild type animals (Saez and Vilchez, 2014; Vilchez et al., 2012). To determine whether the lifespan of rpn-12(av93) mutant animals was affected, lifespan assays were conducted at 20°C and 25°C. The mean lifespan of rpn-12(av93) hermaphrodites (15.94 days) was not significantly different from wild type hermaphrodites (15.54 days) at 20°C. Interestingly, at 25°C the mean lifespan of rpn-12(av93) hermaphrodites (15.59 days) was increased compared to wild type animals (12.80 days). However, the maximum lifespan of both rpn-12(av93) and wild type hermaphrodites was not significantly different at 25°C. The lifespan analysis at 20°C was performed three times (cumulative wild type n=240 and mutant n=192) and at 25°C was performed twice (cumulative wild type n=185 and mutant n=135). Our data suggests that RPN-12 is not essential for viability and lifespan of C. elegans under normal conditions (20ºC), but that absence of RPN-12 can result in a significant increase in mean lifespan under heat stress (25ºC).