Doitsidou, Maria et al. (2019) MicroPubl Biol

Hobert, Oliver, Doitsidou, Maria

[MicroPubl Biol, 2019]

We screened for mutants that affect expression of dopaminergic neuron identity, using a transcriptional reporter for expression of the dopamine transporter dat-1. We previously published and characterized a number of mutants that affect dat-1 expression in different neuron types (Doitsidou et al., 2008). Four alleles that we did not publish in our original screening paper are described here. While wild-type animals only display a single dat-1::gfp(+) neuron pair in the midbody region, the PDE neuron pair from the postdeirid lineage, all 4 mutant alleles display ectopic dat-1::gfp expression along the anterior/posterior axis of the animal (Fig.1A,B). Postdeirid lineage duplication defects were previously described in animals lacking the bHLH transcription factor lin-22/Hairy (Wrischnik and Kenyon, 1997). We find that the canonical lin-22 allele, n372, indeed displays dat-1::gfp expression defects similar to those observed in our mutants (Fig.1B). We sequenced the lin-22 locus in all of our four, independently isolated alleles. Two of them are premature stop codons, one is a missense mutation affecting a conserved leucine residue and all display a similar penetrance of defects (Fig.1B,C). The fourth and weakest allele, ot269, displayed no sequence alteration in the lin-22 coding sequence or in exon/intron boundaries. ot269 failed to complement ot267, ot268, ot287 and the canonical lin-22 allele n372. Furthermore, the ot269 phenotype was rescued by injection of the fosmid WRM0627dG07, which contains lin-22 and one additional complete gene. We found that ot269 harbors a single nucleotide change in the upstream intergenic region of lin-22, almost 5kb away from the start of the gene (sequence change shown in Fig.1B). Subsequent work has shown that this mutation affects a binding site for a GATA transcription factor (Katsanos et al. 2017).

Godini, Rasoul et al. MicroPubl Biol

Pocock, Roger, Godini, Rasoul, Langebeck-Jensen, Kasper

[MicroPubl Biol, 2020]

The BAG neurons control multiple aspects of Caenorhabditis elegans behavior, such as sensing environmental gases (oxygen and carbon dioxide), regulation of systemic fat levels and egg laying (Brandt et al. 2012; Guillermin et al. 2011; Juozaityte et al. 2017; Zimmer et al. 2009). To identify factors that control BAG specification, we performed a forward genetic mutagenesis screen using the Pgcy-33::gfp reporter, which is exclusively expressed in the BAG neurons. We isolated a new allele (rp4) that exhibits a loss of Pgcy-33::gfp expression. Using the one-step whole-genome sequencing and SNP mapping strategy (Doitsidou et al. 2010) we mapped the genetic lesion to the egl-46 gene, encoding a zinc finger transcription factor homologous to mammalian INSM1/2, which we had previously shown to be important for BAG specification (Rojo Romanos et al. 2015). The new lesion we identified egl-46(rp4) (TGC>TAC) causes a single amino acid change in a highly conserved cysteine residue (C185Y) that lies in the first zinc finger domain of EGL-46, which would be predicted to affect DNA binding. Analysis of Pgcy-33::gfp expression in the rp4 allele reveals that it exhibits the same phenotype as the previously published rp13 deletion allele, which is an out-of-frame deletion that removes the zinc finger domains (Rojo Romanos et al. 2015). Therefore, rp4 acts as a strong loss-of-function/null allele and may be of use to those researchers interested in elucidating additional functions of EGL-46.

Minevich, Gregory et al. MicroPubl Biol

Bernstein, Alex, Mei, Kevin, Minevich, Gregory, Poole, Richard J, Hobert, Oliver

[MicroPubl Biol, 2019]

We are interested in isolating mutants that affect lineage specification in the nervous system of the nematode C. elegans.The harsh touch sensory neuron PVD, generated by the postdeirid lineage, can been labeled with two transgenically expressed fluorophores,ser-2::gfp (otIs138 transgene)(Tsalik et al., 2003) and dat-1:rfp (vsIs33transgene)(Chase et al., 2004). Using a otIs138; vsIs33 double transgenic strain, we screened for EMS-induced mutations in which both markers fail to be expressed in PVD and isolated two strains in which the majority of animals fail to display reporter expression in PVD (ot642mutant strain: 56% animals showed no marker expression in PVD expression; ot575 mutant strain: 71% of animals; n=54). ot575and ot642fail to complement each other. Animals that carry these alleles are viable, fertile and display no obvious morphological abnormalities. Both strains were subjected to Illumina whole genome re-sequencing, one in combination with Hawaiian SNP mapping (ot642)(Doitsidou et al., 2010) the other in combination with variant discovery mapping (ot575)(Minevich et al., 2012). These two orthogonal mapping approaches revealed that both mutant strains carry the exact same alteration: a loss of 405,058 bp from the extreme left end of the X chromosome (Figure 1). ot575and ot642were isolated from separate rounds of screens so these mutants were not progeny of the same initial parent. Each of these strains were isolated with the otIs138[ser2prom3::gfp]transgene that is also located on chromosome X so it is possible that this transgene somehow contributes to chromosome instability in some way or form. We confirmed the deletion with PCR primers located within this deletion, which yielded a PCR product from wild-type animals, but not mutant animals. Since the size of the deletions classifies these alleles as deficiencies, we renamed these alleles otDf575and otDf642. The deletion in otDf575 and otDf642eliminates 50 protein-coding genes (one of them, R04A9.1, which carries the deletion breakpoint, is cut in half), at least 20 pseudogenes and a number of regulatory RNAs, ranging from several 21U RNAs to three miRNA encoding genes, mir-258.1, mir-258.2and mir-800. The protein coding genes lost in these strains include a number of different functional categories, including a number of gene regulatory factors: two transcription factors (elk-2and ceh-93, a homeobox gene), two SET-domain containing chromatin factors (set-28,set-33), an argonaute protein (nrde-3) and the polycomb-group gene, sor-3. We find that the sor-3 locus alone is responsible for the PVD mutant phenotype of otDf575animals, because otDf575fails to complement sor-3(bp185) (5/21 ot575/bp185transheterozygous animals show PVD loss) and sor-3(bp185) homozygotes show the same PVD mutant phenotype as otDf575and otDf642mutant animals (68% penetrant; n=22). sor-3codes for a protein containing an MBT repeat domain that displays methylated histone binding activity and exists in the PcG proteins SCM and Sfmbt in other organisms (Yang et al., 2007). Previous work showed that loss of sor-3leads to expression of ectopic dopaminergic and serotonergic male ray neuron fates (Yang et al., 2007). Furthermore, in sor-3mutants, the Hox genes egl-5and lin-39are ectopically expressed outside their usual domain (Yang et al., 2007). Ectopic expression of the mab-5 gene has previously been shown to eliminate the production of the entire postdeirid lineage (that produces the PVD neuron)(Salser and Kenyon, 1996). We hypothesize that sor-3 mutants affect PVD neuron differentiation via ectopic expression of endogenous mab-3.