Title: Genetic Basis of Neuronal Subtype Differentiation in Caenorhabditis elegans
Description: A central question of developmental neurobiology is how the extraordinary variety of cell types in the nervous system is generated. A large body of evidence suggests that transcription factors acting as terminal selectors control cell fate determination by directly activating cell type-specific gene regulatory programs during neurogenesis. Neurons within the same class often further differentiate into subtypes that have distinct cellular morphology, axon projections, synaptic connections, and neuronal functions. The molecular mechanism that controls the subtype diversification of neurons sharing the same general fate is poorly understood, and only a few studies have addressed this question, notably the motor neuron subtype specification in developing vertebrate spinal cord and the segment-specific neuronal subtype specification of the peptidergic neurons in Drosophila embryonic ventral nerve cord. In this dissertation, I investigate the genetic basis of neuronal subtype specification using the Touch Receptor Neurons (TRNs) of Caenorhabditis elegans.

The six TRNs are mechanosensory neurons that can be divided into four subtypes, which are located at various positions along the anterior-posterior (A-P) axis. All six neurons share the same TRN fate by expressing the POU-domain transcription factor UNC-86 and the LIM domain transcription factor MEC-3, the terminal selectors that activate a battery of genes (referred as TRN terminal differentiation genes) required for TRN functions. TRNs also have well-defined morphologies and synaptic connections, and therefore serve as a great model to study neuronal differentiation and subtype diversification at a single-cell resolution. This study primarily focuses on the two embryonically derived TRN subtypes, the anterior ALM and the posterior PLM neurons; each contains a pair of bilaterally symmetric cells. Both ALM and PLM neurons have a long anteriorly-directed neurite that branches at the distal end; the PLM, but not the ALM, neurons are bipolar, having also a posteriorly-directed neurite. ALM neurons form excitatory gap junctions with interneurons that control backward movement and inhibitory chemical synapses with interneurons that control forward movement, whereas PLM neurons do the reverse.

Therefore, the clear differences between ALM and PLM neurons offer the opportunity to identify the mechanisms controlling subtype specification. Using the TRN subtypes along the A-P axis, I first found that the evolutionarily conserved Hox genes regulate TRN differentiation by both promoting the convergence of ALM and PLM neurons to the common TRN fate (Chapter II) and inducing posterior subtype differentiation that distinguishes PLM from the ALM neurons (Chapter III). First, distinct Hox proteins CEH-13/lab/Hox1 and EGL-5/Abd-B/Hox9-13, acting in ALM and PLM neurons respectively, promote the expression of the common TRN fate by facilitating the transcriptional activation of TRN terminal selector gene mec-3 by UNC-86. Hox proteins regulate mec-3 expression through a binary mechanism, and mutations in ceh-13 and egl-5 resulted in an “all or none” phenotype: ~35% of cells lost the TRN cell fate completely, whereas the rest ~65% of cells express the TRN markers at the wild-type level. Therefore, Hox proteins contribute to cell fate decisions during terminal neuronal differentiation by acting as reinforcing transcription factors to increase the probability of successful transcriptional activation.

Second, Hox genes also control TRN subtype diversification through a “posterior induction” mechanism. The posterior Hox gene egl-5 induces morphological and transcriptional specification in the posterior PLM neurons, which distinguish them from the ALM. This subtype diversification requires EGL-5-induced repression of TALE cofactors, which antagonize EGL-5 functions, and the activation of rfip-1, a component of recycling endosomes, which mediates Hox activities by promoting subtype-specific neurite outgrowth. Thus, these results suggest that neuronal subtype diversification along the A-P axis is mainly driven by the posterior Hox genes, which induces the divergence of posterior subtypes away from the common state of the neuron type. I have also performed an RNAi screen to identify novel regulators of the TRN fate and identified the LIM domain-binding protein LDB-1 and the Zinc finger homeodomain transcription factor ZAG-1 as part of the regulatory network that determines TRN fate (Chapter IV).

LDB-1 binds to and stabilizes MEC-3 and is also required for the activation of TRN terminal differentiation genes by MEC-3. ZAG-1 promotes TRN fate by preventing the expression other transcription factors EGL-44 and EGL-46, which inhibits the expression of TRN fate by competing for the cis-regulatory elements normally bound by the TRN fate selectors UNC-86/MEC-3. The mutual inhibition between ZAG-1 and EGL-44 establishes a bistable switch that regulates cell fate choice between TRNs and FLP neurons. I also investigated the genetic basis of neuronal morphogenesis using TRNs. By conducting a forward genetic screen searching for mutants with TRN neurite outgrowth defects, I identified a series of genes required for axonal outgrowth and guidance in TRNs. Following a few genes identified from the screen, genetic studies have revealed two novel mechanisms for neuritogenesis. First, Dishevelled protein DSH-1 attenuates the strength of Wnt signaling to allow the PLM posterior neurite to grow against the gradient of repulsive Wnt proteins, which are enriched at the posterior side of PLM cell body and normally repel the axons toward the anterior (Chapter V).

Second, guanine nucleotide exchange factor UNC-73 and TIAM-1 promotes anteriorly and posteriorly directed neurite outgrowth, respectively; and outgrowth in different directions can suppress each other by competing for the limited neurite extension capacity (Chapter VI). As side projects, I performed mRNA expression profiling using isolated and separated populations of in vitro cultured ALM and PLM neurons and identified hundreds of genes differentially expressed between the two subtypes (Appendix I). I have also studied subtype differentiation of the VC motor neurons in the ventral nerve cord of C. elegans and discovered a mechanism by which histone modification patterns the expression of subtype-specific genes during terminal neuronal differentiation (Appendix II). In summary, my doctoral research established a framework for the study of neuronal subtype specification using the C. elegans TRNs and uncovered the genetic mechanisms for a variety of aspects of terminal neuronal differentiation.

By investigating the generation of neuron type and subtype diversity in a well-defined model organism, my study provides novel insights for understanding the development of the nervous system.