Comparing expression patterns of CRISPR/Cas9-engineered reporter alleles with fosmid-based reporter transgenes.

Comparing expression patterns of CRISPR/Cas9-engineered reporter alleles with fosmid-based reporter transgenes.</p

<i>tab-1</i> regulates the differentiation of various neurons in the ABala lineage.

Fig 7A: In tab-1(ok2198) mutants, expression of both nlp-42(syb3238) and NeuroPAL reporters in AIN is lost. tab-1(ok2198) mutants also showed defects in unc-17(otIs576) reporter expression in AIN and AVD. No loss of reporter expression was observed in ttx-3(ot22) mutants. Representative images of wild type and mutant worms are shown with 10 μm scale bars. Fig 7B: tab-1 is expressed in various neurons derived from the ABala lineage (adapted from Ma et al., 2021). In tab-1(ok2198) mutants, defects in NeuroPAL reporter expression, including ultrapanneuronal (UPN) reporter expression, are seen in neurons which express tab-1 embryonically. Representative images of wild type and mutant worms are shown with 10 μm scale bars. In all panels, neurons of interest are outlined in solid white when expressing wildtype reporter colors, and dashed white when one or all colors are lost. P-values were calculated by Fisher’s exact test.</p

<i>ceh-14</i> affect differentiation of several neuron classes, in combination with different homeobox genes.

Fig 3A:ceh-14(ot900) mutant animals show a loss of neuropeptide-encoding gene expression in PVW, including a promoter fusion reporter transgene for flp-22 (ynIs50) and a flp-27 CRISPR reporter (syb4413), while expression of the neuropeptide CRISPR reporters for flp-21 (syb3212) and nlp-13 (syb3411) is unaffected. Neuron of interest is outlined in solid white when expressing wildtype reporter colors, and dashed white when one or all colors are lost. Representative images of wild type and mutant worms are shown with 10 μm scale bar. Graphs compare expression in wild type and mutant worms with the number of animals examined listed at the bottom of the bar. P-values were calculated by Fisher’s exact test. Fig 3B: ceh-14(ot900) mutant animals show a loss of AIM marker expression, including an eat-4 CRISPR reporter (syb4257) and NeuroPAL (otIs669) in AIM. Additionally, unc-86(ot1158) as well as mls-2(cc615) mutant animals show expression defects of NeuroPAL (otIs669) in AIM. Neuron of interest is outlined in solid white when expressing wildtype reporter colors, and dashed white when one or all colors are lost. Representative images of wild type and mutant worms are shown with 10 μm scale bars. Graphs compare expression in wild type and mutant worms with the number of animals examined listed at the bottom of the bar. P-values were calculated by Fisher’s exact test. Fig 3C: ceh-14(ot900) and unc-86(ot1158) mutant animals show a loss of neuropeptide-encoding gene expression in AIM using the CRISPR/Cas9-engineered reporter alleles nlp-51(syb2805) and nlp-73(syb4406). mls-2(cc615) mutant animals diminish, but do not extinguish expression in of nlp-51(syb2805) and nlp-73(syb4406) in AIM. Expression in RIP is unaffected by ceh-14(ot900), unc-86(ot1158), and mls-2(cc615). Neurons are outlined in solid black and a dashed black line represents loss of expression. Asterisks indicate ectopic expression of unidentified neurons. Graphs compare expression in wild type and mutant worms (on vs off or on vs dim) with the number of animals examined listed at the bottom of the bar. P-values were calculated by Fisher’s exact test.</p

A regulatory map of transcription factors with a role in terminal neuron differentiation.

The basis for this data is taken mainly from [35] and supplemented with data from this paper, as well as others, including [6, 7, 17, 50, 59, 62, 79]. Criteria to be included in this list is that the transcription factor is expressed throughout embryonic and postembryonic development of the respective neuron type (i.e. is likely involved not only in initiation, but also maintenance of terminal differentiation programs) and the existence of mutant data that support a role in controlling marker genes. The homeobox part of this table is graphically presented in Fig 12. (XLSX)</p

Derepression of dopaminergic terminal feature and dopaminergic regulatory signature in <i>unc-86</i> mutants.

Fig 12A:unc-86(ot1158) and unc-86(n846) mutant animals ectopically express markers of ADE identity, including NeuroPAL (otIs669), reporter transgenes for genes involved in dopamine synthesis, including bas-1 (otIs226), cat-1 (otIs224), cat-2 (nIs118), cat-4(otIs225) and dat-1(vtIs1), and a flp-33 CRISPR reporter (syb3195). Representative images of wild type and mutant worms are shown with 10 μm scale bars. Graphs compare expression on the left side (ADEL) and the right side (ADER) in wild type and mutant worms with the number of animals examined listed at the bottom of the bar. Fig 12B: unc-86(ot1158) mutant animals show a derepression of CRISPR/Cas9-engineered reporter alleles of ceh-43(syb5073) and ast-1(vlc19) in cells of the anterior deirid lineage. Representative images of wild type and mutant worms are shown with 10 μm scale bars (non-neuronal expression depicted with an asterisk). Graphs compare expression on the left side (ADEL and AIZL) and the right side (ADER and AIZR) in wild type and mutant worms with the number of animals examined listed at the bottom of the bar. Fig 12C, FigD: ast-1 and ceh-43 are epistatic to unc-86. The derepression of expression of the cat-2 reporter transgene (otIs199) in unc-86(ot1158) mutant is suppressed in an ast-1(ot417) or an ast-1(ot406) mutant background. Note that both alleles are hypomorphic alleles (null alleles are lethal) [13,78]. Neurons of interest are outlined in solid white when expressing WT reporter colors, and dashed white when one or all colors are lost. Representative images of wild type and mutant worms are shown with 10 μm scale bars. Graphs compare expression on the left side (ADEL) and the right side (ADER) in wild type and mutant worms with the number of animals examined listed at the bottom of the bar. In all panels, neurons of interest are outlined in solid white when expressing wildtype reporter colors, and dashed white when one or all colors are lost and P-values were calculated by Fisher’s exact test.</p

Regulation of neuron identity across the <i>C</i>. <i>elegans</i> nervous system by homeodomain transcription factors.

Functional analysis of homeobox gene family, overlayed onto the homeobox expression matrix from Fig 1B. Red boxes indicate that a homeodomain transcription factor is expressed in and likely acts as a terminal selector for a given neuron type (based on extent of functional marker analysis). Orange boxes indicate that a homeodomain transcription factor has a more restricted function in a given neuron class. Gray boxes indicate that a homeodomain transcription factor is expressed in that given neuron type, but not necessarily functionally analyzed and white boxes indicate that a homeodomain transcription factor is not expressed in that given neuron type. Panneuronal and non-neuronal homeoboxes were excluded from this representation because they do not contribute to unique neuron type codes. Neuron types along the x axis are clustered by transcriptomic similarity using the Jaccard index (see methods) and homeobox genes along the y axis are clustered similarly by their similar expression profiles in shared neuron types. See S4 Table for tabular list of genes and cells on which this matrix is based.</p

Numerical representation of homeobox expression data.

This data uses the expression data from S1 and S2 Tables. (EPS)</p

Graphical representation of the homeobox mutant alleles that we generated by CRISPR/Cas9 genome engineering.

Deletions were generated by CRISPR/Cas9 genome engineering using an oligo-repair template. Identical deletions introduced into different reporter strain backgrounds get separate allele names. For lin-11, the deletions ot1025 and ot1026 are the same, but ot1241 is different by 4 nucleotides, even though the same oligo-mediated repair template was used. (EPS)</p

<i>unc-39</i> controls differentiation of the AIA interneuron class.

unc-39R203Q mutant animals (either canonical e257 allele or CRISPR/Cas9 genome engineered ot1173 allele with identical nucleotide change) were analyzed. Fig 2A: unc-39 affects the cholinergic identity of the AIA interneuron class (unc-17 reporter allele syb4491 and a cho-1 promoter fragment which is part of the otIs653 array), and other AIA terminal identity markers: reporter alleles dmsr-2(syb4514), ins-1(syb5452) and flp-19(syb3278), and a mgl-1 promoter fragment otIs327. We did not quantify changes in AIA in the NeuroPAL color code, because it is variable in wild type. Representative images of wild type and mutant worms are shown with 10 μm scale bars. Graphs compare expression in wild type and mutant worms with the number of neurons examined listed at the bottom of the bar. P-values were calculated by Fisher’s exact test. Fig 2B: unc-39 affects the expression of the tagged eya-1 locus (nIs352 transgene) in AIA.</p

Ring interneuron (RIC, RIH, RIR) differentiation defects in homeobox gene mutants.

Fig 9A:unc-62(e644) mutant animals show a loss of RIC marker expression, including an eat-4 CRISPR reporter (syb4257) and NeuroPAL (otIs669). Representative images of wild type and mutant worms are shown with 10 μm scale bars. Graphs compare expression in wild type and mutant worms with the number of animals examined listed at the bottom of the bar. P-values were calculated by Fisher’s exact test. Fig 9B: unc-86(ot1158) mutant animals show loss of RIH and RIR marker expression of an extrachromosomal cho-1prom3 reporter (otEx4530) [105]. Representative images of wild type and mutant worms are shown with 10 μm scale bars. Graphs compare expression in wild type and mutant worms with the number of animals examined listed at the bottom of the bar. In all panels, neurons of interest are outlined in solid white when expressing wildtype reporter colors, and dashed white when one or all colors are lost. P-values were calculated by Fisher’s exact test.</p