Our lab is interested in understanding the molecular basis of neuronal development, regeneration, and degeneration using a variety of genetics, genomics, and cell biology methods. To circumvent the extreme complexity of mammalian nervous systems, we are using the nematode Caenorhabditis elegans as a model system to address many fundamental questions in neuroscience. Compared to the human brain that contains ~100 billion neurons, the C. elegans nervous system only contains 302 neurons, of which the anatomy and neuronal connection were mapped. This relative simplicity and the ease of genetic manipulation allow us to make discoveries about the organizing principles of neuronal development. Given the orthology between C. elegans and human genes, our findings also provide insight into the molecular mechanisms underlying nerve cell development in humans.
Within developmental neurobiology, we are mainly working on cell fate specification and neurite development. In addition, we are also using C. elegans to study the molecular mechanisms of axonal regeneration and to model neurodegenerative diseases. Finally, we are also interested in understanding the evolution of neuron types by studying the genomic basis of the neurodevelopment patterns in C. elegans and related species.
Currently, we are pursuing the following directions.
1. Identifying cell fate regulators in C. elegans nervous system
C. elegans nervous system in adult hermaphrodites contains 302 neurons, which can be categorized into 118 morphologically distinct classes. Cell fate regulators of ~70% of those neuron types were identified (Hobert, 2016, WIREs Dev Biol), making it one of the best understood nerve systems in terms of differentiation. We are in the process of identifying the cell fate determinants for the rest ~30% neuron types using a combination of forward genetic screen, RNAi screen, and one-hybrid DNA/protein interaction screen. The goal is to have a complete map of the factors that regulate the fate specification of every type of neuron in an animal. This systematic approach would provide important insights into our understanding of the organization of a nervous system.
(Image from the Hobert lab)
As an example, we worked on identifying the regulators of the touch receptor neuron (TRN) fate through an RNAi screen and discovered a three-factor (UNC-86, MEC-3, and ZAG-1) combinatorial code that is only expressed in the six TRNs and regulates its fate specification (Zheng et al., 2018, Development). The three transcription factors appear to have distinct functions in controlling the process of fate specification. Importantly, the mutual inhibition between ZAG-1 and EGL-44/EGL-46 regulates the binary cell fate choice between TRN and FLP neurons through a bistable regulatory loop. We are currently working on identifying the factors that regulate the cell fate choice between TRN and PVD.
For another example, we investigated the role of Hox genes in terminal neuronal differentiation. Hox genes encode conserved homeodomain transcription factors that control the body plan along the anterior-posterior (A-P) axis in embryos. In addition to their functions in embryogenesis, our studies using the TRNs found that Hox genes also regulate terminal differentiation of neurons by 1) acting as "transcriptional guarantors" to ensure activation of terminal selectors and robust fate acquisition (Zheng et al., 2015, Cell Rep.) and 2) by acting as "subtype inducers" to induce diversification among the neurons that share the same general cell fate (Zheng et al., 2015, Neuron). Those studies provided important examples for understanding the role of Hox genes in terminal neuronal differentiation.
More recently, we mapped the expression of the six Hox genes in the entire nervous system of C. elegans and explored their functions in the differentiation of other neurons. We found Hox expression in 97 (32%) of the 302 neurons in adult hermaphrodites and our analysis of the fate markers for these neurons revealed that Hox genes regulate the differentiation of 29 (25%) of the 118 classes of C. elegans neurons. Hox genes not only regulate the specification of terminal neuronal fates through multiple mechanisms but also control subtype diversification along the A-P axis. The widespread involvement of Hox genes in neuronal differentiation indicates their roles in establishing complex nervous systems (Zheng et al., 2022, PLoS Genetics).
Going forward, we are interested in understanding the cell fate regulators that control the fate decision between two sister cells. For instance, the mechanisms that control the fate choice between PLM and its sister ALN neurons. We are searching for such regulators through forward genetic screens. Recent single-cell transcriptomic data also provided candidates for direct tests.
2. Identifying novel regulators of microtubule (MT) stability during neurite growth and axonal regeneration.
Previous studies using the Touch Receptor Neurons (TRNs) in C. elegans have identified several important molecular mechanisms of neurite guidance and extensions, such as Dishevelled attenuating Wnt repelling activity (Zheng et al., 2015, PNAS) and GEFs controlling the directional specificity of neurite growth (Zheng et al., 2016, PNAS).
More recently, we found that mutations in tubulin genes dramatically affect neurite growth by altering MT stabilities (Zheng et al., 2017, MBoC). In general, three types of mutations (loss-of-function, antimorphic, and neomorphic gain-of-function) were found to have distinct effects on MT stability and neurite growth. Structure-function analysis revealed a close relationship between the location of the mutation on the tubulin structure and their effects. Antimorphic mutations map to the GTP binding site and intradimer and interdimer interfaces and cause severe neurite growth defects, while neomorphic mutations map to the exterior surface and cause ectopic neurite growth. These studies also have clinical relevance because mutations in human tubulins cause a wide range of neurodevelopmental disorders. Using the TRNs, we are able to model those human mutations and study their impact at cellular levels.
The neomorphic tubulin mutations are particularly interesting because they increase MT stability and cause ectopic neurite growth. We have used these mutants as a sensitized background to discover novel regulators of MT structure and stability. Through these suppressor screens, more tubulin mutations were discovered and their effects on MT dynamics were studied (Lee et al., 2021, MBoC). Interestingly, we have also discovered complex genetic interactions among the tubulin missense mutations, including epistatic, synthetic, and balancing interactions.
C. elegans, like other metazoans, have multiple tubulin isotypes and each of the tubulins undergoes a wide range of post-translational modifications (PTMs), which prompted the "tubulin code" hypothesis (reviewed in Lu and Zheng et al., 2022, Front Cell Dev Biol). To understand this code, we are currently working on how the tubulin PTMs affect neurite growth in C. elegans through CRISPR-mediated genome engineering.
Moreover, the regulation of MTs is also critical for axonal regeneration. Since the TRNs in C. elegans is an important model for axonal regeneration upon laser axotomy, we are also studying the regenerative function of those MT regulators identified in our screen in an effort to discover molecular targets for regeneration.
3. Modeling microbe-host interaction in neurodegenerative disease in C. elegans
We are interested in using the simple nematode C. elegans to model neurodegenerative diseases, like Alzheimer's disease and Parkinson's disease. The transparency of the animal allows direct visualization of live neurons; the ease to perform genetic manipulation in a controlled experiment allows modeling of the disease condition and identification of genetic components contribute to the disease; the small size and short life cycle of the animal allow large-scale screens to discover compounds that can prevent or slow down neurodegeneration.
In recent work, we screened the E. coli genome to identify genes that contribute to neurodegeneration in a Parkinson's disease model, where the human alpha-synuclein is expressed in C. elegans neurons. This screen yielded 38 pro-neurodegenerative genes. In follow-up studies, we focused on the role of the bacteria amyloid fibril curli, made of the CsgA and CsgB proteins. We found that the bacteria-derived curli could enter host neurons to promote alpha-synuclein aggregation through a cross-seeding mechanism. Removing curli from the bacteria reduced alpha-synuclein aggregation, restored mitochondrial healthy, and rescued neurodegeneration (Wang et al., 2021, PNAS). These studies suggest that targeting curli release from the microbes may serve as a novel therapeutic intervention for neurodegenerative diseases. Interestingly, through literature review, we found that at least 50% of the neuroprotective compounds identified in animal models have anti-biofilm activities, which may be mediated by their suppression of curli production (Wang and Zheng, 2022, Front Pharmacol).
We are currently investigating other pro-neurodegenerative microbial factors identified from the screen.
4. Comparative genomics to study the evolutionary origin of neuronal diversity
As Theodosius Dobzhansky said, "Nothing in biology makes sense except in the light of evolution." One fascinating question about the development of the nervous system is how the extraordinary diversity of neuron types is evolved. We try to address this question by understanding the genetic mechanisms underlying the generation of new neuron types and the loss of old neuron types. We are conducting comparative genomics and comparative developmental neurobiology studies to understand how the differences in genomic sequence among nematodes contribute to the difference in their nervous systems.
Our recent work combined refined phylostratification and single-cell transcriptomic data to analyze the evolutionary age (or the transcriptomic age index; TAI) of different cell types throughout the development of C. elegans and showcased how the transcriptome age at the single-cell level could provide insight into the cellular basis of developmental innovation and help understand the functional diversity and evolutionary origin of cell types (Ma and Zheng, 2023, PNAS). We identified a period of the lowest TAI during mid-embryogenesis, which corresponds to the phylotypic stage, supporting the developmental hourglass model. The analysis revealed an older transcriptome age of germline precursors compared to somatic tissues in early embryos and that the variation in transcriptome ages among the cell and tissue types grew bigger at late embryonic and larval stages as cells differentiate. Among the 128 neuron types in the C. elegans nervous system, a group of chemosensory neurons and their downstream interneurons expressed very young transcriptomes and may contribute to adaptation in recent evolution. Finally, the variation in TAI among the neuron types, as well as the age of their fate regulators, led us to hypothesize the evolutionary history of some neuron types.
Moreover, in the process of analyzing the inter- and intra-specific variation of the genomic sequences, we also made discoveries about the genetic diversity of C. elegans. For example, we found that F-box and csGPCR genes, especially the Srw family csGPCRs, showed much more intraspecific diversity than other gene families among the C. elegans wild isolates. These natural variants showed signs of positive selection and may contribute to the adaptation of the wild isolates (Ma et al., 2021, Genome Biol Evol).
Currently, we are comparing C. elegans with other Caenorhabditis species and P. pacificus for the difference in neuron counts and neuronal fate specification programs. We are also interested in intraspecies variation in nervous systems and are investigating this question using a collection of C. elegans wild isolates that are extracted from different geographic locations globally. By employing a series of bioinformatics methods, we are searching for natural variations in the genome of C. elegans that lead to variations in the pattern of neuronal development and then use experimental methods to verify whether strains that carry those genetic variations would indeed develop a nervous system that deviates from that of the classical C. elegans strains. We aim at understanding to what degree the neuronal development program is invariant within the same species.