CEPsh glia development is not required for general AWC identity or AWC asymmetry.

The Caenorhabditis elegans VAB-3/Pax6 homeodomain protein was previously shown to play a role in both the development of cephalic sheath (CEPsh) glia and asymmetric differentiation of AWC olfactory neuron subtypes AWC ON /AWC OFF . Here we show that vab-3 is not required for the specification of general AWC identity. We also show that some vab-3 mutant alleles with defective CEPsh glia development displayed wild-type AWC asymmetry. These results suggest that vab-3 has separable roles in CEPsh glia development and AWC asymmetry. Together, our results suggest that general AWC identity and AWC asymmetry are not dependent on the development of CEPsh glia. Copyright:

( A ) Representative images of wild type and vab-3(ns157) mutants expressing integrated transgenes of the CEPsh identity marker hlh-17p::GFP and general AWC identity marker odr-1p::DsRed or odr-1p::TagRFP in the adult stage. Arrows indicate the cell body of AWC and CEPsh glia; asterisks indicate the AWB cell body. Scale bar, 10 μm. Anterior to the left and ventral to the bottom. ( B ) Representative images of wild type and vab-3(e648) mutants expressing integrated transgenes of the AWC ON marker str-2p::GFP and general AWC identity marker odr-1p::TagRFP in the adult stage. Arrows indicate the cell body of AWC; asterisks indicate the AWB cell body. Scale bar, 10 μm. Anterior to the left and ventral to the bottom. ( C ) Expression of integrated transgenes of the CEPsh identity marker hlh-17p::GFP (i), general AWC identity and AWC axon outgrowth/morphology/guidance marker odr-1p::TagRFP (ii, iii), and the AWC ON asymmetry marker str-2p::GFP or str-2p::TagRFP (iv) in wild type and vab-3 mutants. Animals were scored in adults. n, total number of animals scored. ( D ) Genomic structure and position of the vab-3 locus in chromosome X. vab-3 has three alternatively spliced isoforms, vab-3a-c (wormbase.org, release WS248).

Description

The C. elegans nerve ring, considered to be the brain of the worm, is enveloped by sheet-like processes of four CEPsh glia (Figure 1A) (White et al., 1986). CEPsh glia was shown to control axon guidance and branching of sensory neurons, including AWC olfactory neurons, in the nerve ring (Yoshimura et al., 2008). It was also shown that normal axon guidance and AWC axon contact are required for asymmetric differentiation of the AWC neuron pair (Figure 1B) (Troemel et al., 1999). These findings prompted us to determine whether the development of CEPsh glia regulates two subsequent steps of AWC development, general AWC identity specification and AWC asymmetry. To address these questions, we studied candidate genes previously implicated in both CEPsh glia development and AWC development. mls-2 , encoding an HMX/NKX MLS-2 transcription factor, and vab-3 , encoding a homolog of homeodomain protein Pax6, have been shown to regulate the development of CEPsh glia (Figure 1A and 1C) (Yoshimura et al., 2008). We and others previously showed that mls-2 plays a role in general AWC identity and AWC asymmetry (Kim et al., 2010; Hsieh et al., 2021). Since AWC asymmetry defects observed in mls-2(lf) mutants result from indirect defects in general AWC identity and direct defects in AWC asymmetry, it would not be straightforward to determine whether CEPsh glia development regulates AWC asymmetry using mls-2 mutants. vab-3 , like mls-2 , is also required for AWC asymmetry (Figure 1B and 1C) (Troemel et al., 1999). But it was unknown whether vab-3 regulates general AWC identity.

In addition to CEPsh glia development and AWC asymmetry, a wide array of vab-3 mutations have been isolated to implicate its roles in other different aspects of development, including neuronal fate specification, anterior epidermal fate specification, head morphogenesis, axon guidance in the anterior nervous system, gonadal distal cell migration, blast cell fate specification in the rectal epidermis, and male tail morphogenesis (Lewis and Hodgkin, 1977; Hodgkin, 1983; Chamberlin and Sternberg, 1995; Chisholm and Horvitz, 1995; Zhang and Emmons, 1995; Nishiwaki, 1999; Troemel et al., 1999; Zallen et al., 1999; Cinar and Chisholm, 2004; Yoshimura et al., 2008; Doitsidou et al., 2010; Brandt et al., 2019). Here, four vab-3 alleles were analyzed for CEPsh glia and AWC development phenotypes (Figure 1D). vab-3(e648) and vab-3(ju468) alleles are nonsense mutations leading to truncations of the homeodomain (Chisholm and Horvitz, 1995; Cinar and Chisholm, 2004). vab-3(ns157) and vab-3(e1796) alleles have missense mutations in the paired domain and homeodomain, respectively (Zhang and Emmons, 1995; Yoshimura et al., 2008). All vab-3 mutant alleles examined showed various defects in the expression of the CEPsh identity marker hlh-17p::GFP (Figure 1Ci), as shown previously (Yoshimura et al., 2008). In particular, 85-86% of vab-3(ju468) and vab-3(e1796) mutants lost the expression of hlh-17p::GFP in all four CEPsh glia cells (Figure 1Ci).

To determine whether vab-3 plays a role in general AWC identity, an integrated odr-1p::TagRFP marker was crossed into these four vab-3 mutant alleles. All these vab-3 mutant alleles displayed completely wild-type expression of the general AWC identity marker odr-1p::TagRFP in both AWC neurons (Figure 1B and 1Cii). These results suggest that vab-3 , unlike mls-2 , is not required for general AWC identity. These results also suggest that the development of CEPsh glia is not required for general AWC identity.

The AWC axon outgrowth, morphology, and guidance were also examined using the odr-1p::TagRFP marker in the vab-3 mutants. More than 90% of vab-3(e648) and vab-3(ju468) mutants, while ~20% of vab-3(e1796) and vab-3(ns157) mutants, exhibited defects in AWC axon development (Figure 1B and 1Ciii), similar to previous results from vab-3(ns157) mutants (Yoshimura et al., 2008). To determine whether AWC axon defects correlate with AWC asymmetry defects in vab-3 mutants and whether CEPsh glia is required for AWC asymmetry, the expression of an integrated AWC ON asymmetry marker str-2p::GFP or str-2p::TagRFP was examined in these vab-3 mutants. Similar to previous results from vab-3(e648) mutants (Troemel et al., 1999; Su et al., 2006), the AWC ON marker was not expressed in either AWC cell in the majority of vab-3(e648) and vab-3(ju468) mutants (Figure 1B and 1Civ). By contrast, the expression of the AWC ON marker was normal in vab-3(e1796) and vab-3(ns157) mutants, (Figure 1Civ). Our results showed that the penetrance of AWC asymmetry defects correlates with that of AWC axon defects. While vab-3(ju468) and vab-3(e1796) mutants showed a similar degree of CEPsh glia identity defects (Figure 1Ci), they displayed substantially different penetrance of defective AWC axon and AWC asymmetry phenotypes (Figure 1Ciii and 1Civ). These results suggest that vab-3 has independent functions in the regulation of CEPsh glia development and AWC asymmetry; that vab-3 regulates AWC asymmetry via controlling AWC axon guidance; and that the homeodomain is essential for VAB-3 function in AWC asymmetry. However, AWC phenotypes of vab-3(e648) , which only affects the long isoform vab-3a but not short isoforms vab-3b or vab-3c (Figure 1D), suggest that the homeodomain-only isoforms ( vab-3b and vab-3c ) are not sufficient for normal AWC development. Together, our results support the notion that CEPsh glia is not required for general AWC identity or AWC asymmetry.

Strain	Genotype	Source
CB648	vab-3(e648) X	(Hodgkin, 1983)
CZ3391	vab-3(ju468) X	(Cinar and Chisholm, 2004)
CB3304	vab-3(e1796) X	(Zhang and Emmons, 1995)
OS1912	nsIs105 [hlh-17p::GFP] I ; vab-3(ns157) X	(Yoshimura et al., 2008)
OS1914	nsIs105 [hlh-17p::GFP] I	(Yoshimura et al., 2008)
IX641	nsIs105 [hlh-17p::GFP] I ; oyIs44 [ odr-1p::DsRed; lin-15(+) ] V (Lanjuin et al., 2003)	This study
IX6360	nsIs105 [hlh-17p::GFP] I ; vyIs56 [ odr-1p::TagRFP ] III (Alqadah et al., 2016); vab-3(ns157) X	This study
IX2242	kyIs140 [str-2p::GFP] I ; vyIs56 [ odr-1p::TagRFP ] III (Alqadah et al., 2016)	This study
IX5979	kyIs140 [str-2p::GFP] I ; vyIs56 [ odr-1p::TagRFP ] III; vab-3(e648) X	This study
IX2626	nsIs105 [hlh-17p::GFP] I; vab-3(e648) X	This study
IX6361	nsIs105 [hlh-17p::GFP] I ; vyIs96 [ str-2p::TagRFP ] V; vab-3(ju468) X	This study
IX5672	nsIs105 [hlh-17p::GFP] I ; vyIs96 [ str-2p::TagRFP ] V; vab-3(e1796) X	This study
IX5989	nsIs105 [hlh-17p::GFP] I ; vyIs96 [ str-2p::TagRFP ] V; vab-3(ns157) X	This study
IX5960	kyIs140 [str-2p::GFP] I ; vyIs56 [ odr-1p::TagRFP ] III; vab-3(ju468) X	This study
IX6357	kyIs140 [str-2p::GFP] I ; vyIs56 [ odr-1p::TagRFP ] III; vab-3(e1796) X	This study
IX6358	kyIs140 [str-2p::GFP] I ; vyIs56 [ odr-1p::TagRFP ] III; vab-3(ns157) X	This study