Studies of Caenorhabditis elegans neuronal cell fate

The specification and development of nervous system diversity is a driving question in the field of Neurobiology. The overarching goals of the projects described in this thesis are to describe tools to aid in the description of nervous system development and to show the use of the described tools to study nervous system development in the nematode Caenorhabditis elegans. The first chapter of this thesis describes a complete map of the male C. elegans nervous system using a tool developed in the lab to uniquely label all neurons in the C. elegans nervous system, NeuroPAL. The second chapter of this thesis largely focuses on a well-studied homeobox gene, unc-86, and its role in fate transformations in dopaminergic and GABAergic neuron types. These two seemingly disparate projects are united in their effort to investigate nervous system development and neuronal fate determination. NeuroPAL is a multicolor transgene that uniquely labels all neurons of the C. elegans hermaphrodite nervous system and here I show it can be used to disambiguate all 93 neurons of the male-specific nervous system. I demonstrate the wide utility of NeuroPAL to visualize and characterize numerous features of the male-specific nervous system, including mapping the expression of gfp-tagged reporter genes and neuron fate analysis. NeuroPAL can be used in combination with any gfp-tagged reporters to unambiguously map the expression of any gene of interest in the male, or hermaphrodite, nervous system. Furthermore, NeuroPAL is used in mutants of several developmental patterning genes to confirm previously described defects in neuronal identity acquisition. Additionally, I show that NeuroPAL can be used to uncover novel neuronal fate losses and identity transformations in these mutants because of the unique labeling of every neuron. Lastly, we show that even though the male-specific neurons are generated throughout all four larval stages, the neurons only terminally differentiate in the fourth and final larval stage, termed ‘just-in-time’ differentiation. In the second part of this thesis, I describe a few examples of mutant analysis of homeobox gene family members and describe their function in the C. elegans nervous system. I focus largely on a couple potential examples of homeotic fate transformations in mutants of the POU homeobox gene, unc-86. In unc-86 mutants, I describe the ectopic expression of multiple GABAergic terminal identity features in one cell in the head of C. elegans. I raise the hypothesis that this cell may be a transformation of a non-GABAergic ring interneuron, RIH, into that of its GABAergic sister cell, AVL, in unc-86 mutants. While ectopic dopaminergic neurons were previously described in unc-86 mutants, I expand the study to show the ectopic expression of all dopaminergic synthesis and packaging genes. I show support that all non-dopaminergic anterior deirid neurons, ADA, AIZ, FLP, and RMG, lose the expression of some of their wild type terminal fate genes and transform to a fate like that of their dopaminergic sister cell, ADE, as assessed by NeuroPAL expression. Taken together, these studies describe tools and methods for studying nervous system development as well as describe many examples of cell fate transformations

Widespread employment of conserved C. elegans homeobox genes in neuronal identity specification.

Homeobox genes are prominent regulators of neuronal identity, but the extent to which their function has been probed in animal nervous systems remains limited. In the nematode Caenorhabditis elegans, each individual neuron class is defined by the expression of unique combinations of homeobox genes, prompting the question of whether each neuron class indeed requires a homeobox gene for its proper identity specification. We present here progress in addressing this question by extending previous mutant analysis of homeobox gene family members and describing multiple examples of homeobox gene function in different parts of the C. elegans nervous system. To probe homeobox function, we make use of a number of reporter gene tools, including a novel multicolor reporter transgene, NeuroPAL, which permits simultaneous monitoring of the execution of multiple differentiation programs throughout the entire nervous system. Using these tools, we add to the previous characterization of homeobox gene function by identifying neuronal differentiation defects for 14 homeobox genes in 24 distinct neuron classes that are mostly unrelated by location, function and lineage history. 12 of these 24 neuron classes had no homeobox gene function ascribed to them before, while in the other 12 neuron classes, we extend the combinatorial code of transcription factors required for specifying terminal differentiation programs. Furthermore, we demonstrate that in a particular lineage, homeotic identity transformations occur upon loss of a homeobox gene and we show that these transformations are the result of changes in homeobox codes. Combining the present with past analyses, 113 of the 118 neuron classes of C. elegans are now known to require a homeobox gene for proper execution of terminal differentiation programs. Such broad deployment indicates that homeobox function in neuronal identity specification may be an ancestral feature of animal nervous systems

<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

Numerical representation of homeobox expression data.

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

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

Homeodomain regulatory map, organized by expressing neuron.

This is an updated version of a table from [6]. (XLSX)</p

List of strains used in this paper.

(DOCX)</p

The Eyeless/Pax6 ortholog <i>vab-3</i> controls the identity of neurons in the anterior ganglion.

Fig 5A: In a vab-3(ot1243) mutant allele, many neurons in the anterior ganglion lose their NeuroPAL coloring (from otIs669) and expression of the eat-4 reporter allele (syb4257). Notably, there are much less blue neurons (URY/URA/URB but URX seem present), and the bright green OLQ and turquoise OLL are never seen. 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 (for n = 10 WT worms / 7 vab-3 mutant) examined listed at the bottom of the bar. P-values were calculated by Fisher’s exact test. Similar results were observed with a larger deletion allele, ot1269 (S7 Fig). Fig 5B: In vab-3 mutant worms (ot1239, ot1238, all carrying the same lesion, introduced into respective reporter background; S7 Fig), the OLL and URYmarkers nlp-66(syb4403), eat-4prom8 (otIs521) are affected. Markers are more frequently lost in the ventral URY than the dorsal URY. vab-3 mutants also ectopically express nlp-66 in hypodermal cells. 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. 5C: In vab-3 mutant worms (ot1240, ot1269; S7 Fig), URA and URB identities are affected as seen with the markers sri-1 (otIs879) (URB, OLL) and a promoter fragment of unc-17/VAchT (prom9; otEx7705) [105] expressed in IL2/URA/URB. 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 (sri-1) or neurons (unc-17prom9) examined listed at the bottom of the bar. vab-3 does not affect expression of an unc-17 reporter allele in the IL2 neurons, and we can therefore infer that the remaining positive cells labeled with unc-17prom9, are the IL2 neurons and the lost expression is in URA/URB. P-values were calculated by Fisher’s exact test. Fig 5D: Markers of OLQ neuron identity (ocr-4 kyEx581, ttll-9 otIs850 and des-2 otEx7697) are fully lost in the vab-3(ot1237) mutant animals. 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.</p

Expression pattern of <i>ast-1(vlc19)</i> reporter allele.

ast-1 CRISPR/Cas9-engineered reporter allele, vlc19, [79] is expressed in the following head neuron classes: ADE, AIN, AIZ, ASG, AVG, CEPD, CEPV, I4, I5, M3, M5, RIV, RMD, RMDD, RMDV, SMBD, SMBV, SMDD, and SMDV. Expression in the midbody, ventral nerve cord, and tail was not examined. (EPS)</p