Predicted glycosyltransferases promote development and prevent spurious cell clumping in the choanoflagellate S. rosetta

Abstract

In a previous study we established forward genetics in the choanoflagellate Salpingoeca rosetta and found that a C-type lectin gene is required for rosette development (Levin et al., 2014). Here we report on critical improvements to genetic screens in S. rosetta while also investigating the genetic basis for rosette defect mutants in which single cells fail to develop into orderly rosettes and instead aggregate promiscuously into amorphous clumps of cells. Two of the mutants, Jumble and Couscous, mapped to lesions in genes encoding two different predicted glycosyltransferases and displayed aberrant glycosylation patterns in the basal extracellular matrix (ECM). In animals, glycosyltransferases sculpt the polysaccharide-rich ECM, regulate integrin and cadherin activity, and, when disrupted, contribute to tumorigenesis. The finding that predicted glycosyltransferases promote proper rosette development and prevent cell aggregation in S. rosetta suggests a pre-metazoan role for glycosyltransferases in regulating development and preventing abnormal tumor-like multicellularity.

Light-regulated collective contractility in a multicellular choanoflagellate

“…S8). Importantly, we found no evidence for the intercellular bridges, shared ECM, or filopodial contacts that mediate multicellularity in other choanoflagellate species (6,7,10,36).…”

Genome editing enables reverse genetics of multicellular development in the choanoflagellateSalpingoeca rosetta

“…We next sought to use genome editing as a general tool for reverse genetics in choanoflagellates. To this end, we targeted rosetteless (rtls), one of only three genes known to be required for rosette development in S. rosetta (Levin et al, 2014;Wetzel et al, 2018). A prior forward genetic screen linked the first rosette defect mutant to an allele, rtls tl1 , in which a T to C transition in the 5'-splice site of intron 7 (S. rosetta supercontig 8: 427,804 nt; Fairclough et al, 155 2013) was associated with the disruption of rtls expression and rosette development ( Fig.…”

“…A forward genetic screen was established to hunt for mutants that were unable to develop into rosettes and resulted in the identification of genes required for rosette development (Levin 65 et al, 2014;Wetzel et al, 2018). The first of these (Levin et al, 2014), rosetteless encodes a Ctype lectin protein that localizes to the interior of rosettes ( Fig.…”

“…However, the screen also 70 underscored the necessity for targeted genetics in S. rosetta. Because of inefficient mutagenesis in S. rosetta, forward genetics has been laborious: out of 37,269 clones screened, only 16 rosette-defect mutants were isolated and only three of these have been mapped to genes (Levin et al, 2014;Wetzel et al, 2018). Establishing genome editing would accelerate direct testing of gene candidates identified through forward genetic screens, differential gene 75 expression, and/or genomic comparisons.…”

“…given organism , the delivery of the Cas9 endonuclease bound to a programmable guide RNA (gRNA) can direct DNA cleavage at a target site to introduce mutations from co-delivered DNA templates or from untemplated repair errors that cause insertions or deletions (Rouet et al, 1994;Choulika et al, 1995;Bibikova et al, 2001;. While the delivery of macromolecules into choanoflagellate cells 85 has been a longstanding barrier for establishing reverse genetic tools, we recently established a robust method to transfect S. rosetta with DNA plasmids for expressing transgenes , which allowed us to perform genetic complementation (Wetzel et al, 2018). Despite having established a method for gene delivery in S. rosetta, the lack of knowledge about DNA repair mechanisms in choanoflagellates and low-transfection efficiency (~1%) presented 90 challenges for establishing genome editing, particularly without a proven selectable marker to enrich for editing events.…”