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1. Molecular analyses of zebrafish V0v spinal interneurons and identification of transcriptional regulators downstream of Evx1 and Evx2 in these cells

   https://doi.org/10.1186/s13064-023-00176-w  

   England, Samantha J. ; Rusnock, Amber K. ; Mujcic, Amra ; Kowalchuk, Angelica ; de Jager, Sarah ; Hilinski, William C. ; Juárez-Morales, José L. ; Smith, Matthew E. ; Grieb, Ginny ; Banerjee, Santanu ; et al ( November 2023 , Neural Development)

   V0v spinal interneurons are highly conserved, glutamatergic, commissural neurons that function in locomotor circuits. We have previously shown that Evx1 and Evx2 are required to specify the neurotransmitter phenotype of these cells. However, we still know very little about the gene regulatory networks that act downstream of these transcription factors in V0v cells.

   To identify candidate members of V0v gene regulatory networks, we FAC-sorted wild-type andevx1;evx2double mutant zebrafish V0v spinal interneurons and expression-profiled them using microarrays and single cell RNA-seq. We also used in situ hybridization to compare expression of a subset of candidate genes inevx1;evx2double mutants and wild-type siblings.

   Our data reveal two molecularly distinct subtypes of zebrafish V0v spinal interneurons at 48 h and suggest that, by this stage of development,evx1;evx2double mutant cells transfate into either inhibitory spinal interneurons, or motoneurons. Our results also identify 25 transcriptional regulator genes that require Evx1/2 for their expression in V0v interneurons, plus a further 11 transcriptional regulator genes that are repressed in V0v interneurons by Evx1/2. Two of the latter genes arehmx2andhmx3a. Intriguingly, we show that Hmx2/3a, repress dI2 interneuron expression ofskor1aandnefma, two genes that require Evx1/2 for their expression in V0v interneurons. This suggests that Evx1/2 might regulateskor1aandnefmaexpression in V0v interneurons by repressing Hmx2/3a expression.

   This study identifies two molecularly distinct subsets of zebrafish V0v spinal interneurons, as well as multiple transcriptional regulators that are strong candidates for acting downstream of Evx1/2 to specify the essential functional characteristics of these cells. Our data further suggest that in the absence of both Evx1 and Evx2, V0v spinal interneurons initially change their neurotransmitter phenotypes from excitatory to inhibitory and then, later, start to express markers of distinct types of inhibitory spinal interneurons, or motoneurons. Taken together, our findings significantly increase our knowledge of V0v and spinal development and move us closer towards the essential goal of identifying the complete gene regulatory networks that specify this crucial cell type.

    

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2. Coordinated control of neuronal differentiation and wiring by sustained transcription factors

   https://doi.org/10.1126/science.add1884  

   Özel, Mehmet Neset ; Gibbs, Claudia Skok ; Holguera, Isabel ; Soliman, Mennah ; Bonneau, Richard ; Desplan, Claude ( December 2022 , Science)

   INTRODUCTION Neurons are by far the most diverse of all cell types in animals, to the extent that “cell types” in mammalian brains are still mostly heterogeneous groups, and there is no consensus definition of the term. The Drosophila optic lobes, with approximately 200 well-defined cell types, provides a tractable system with which to address the genetic basis of neuronal type diversity. We previously characterized the distinct developmental gene expression program of each of these types using single-cell RNA sequencing (scRNA-seq), with one-to-one correspondence to the known morphological types. RATIONALE The identity of fly neurons is determined by temporal and spatial patterning mechanisms in stem cell progenitors, but it remained unclear how these cell fate decisions are implemented and maintained in postmitotic neurons. It was proposed in Caenorhabditis elegans that unique combinations of terminal selector transcription factors (TFs) that are continuously expressed in each neuron control nearly all of its type-specific gene expression. This model implies that it should be possible to engineer predictable and complete switches of identity between different neurons just by modifying these sustained TFs. We aimed to test this prediction in the Drosophila visual system. RESULTS Here, we used our developmental scRNA-seq atlases to identify the potential terminal selector genes in all optic lobe neurons. We found unique combinations of, on average, 10 differentially expressed and stably maintained (across all stages of development) TFs in each neuron. Through genetic gain- and loss-of-function experiments in postmitotic neurons, we showed that modifications of these selector codes are sufficient to induce predictable switches of identity between various cell types. Combinations of terminal selectors jointly control both developmental (e.g., morphology) and functional (e.g., neurotransmitters and their receptors) features of neurons. The closely related Transmedullary 1 (Tm1), Tm2, Tm4, and Tm6 neurons (see the figure) share a similar code of terminal selectors, but can be distinguished from each other by three TFs that are continuously and specifically expressed in one of these cell types: Drgx in Tm1, Pdm3 in Tm2, and SoxN in Tm6. We showed that the removal of each of these selectors in these cell types reprograms them to the default Tm4 fate. We validated these conversions using both morphological features and molecular markers. In addition, we performed scRNA-seq to show that ectopic expression of pdm3 in Tm4 and Tm6 neurons converts them to neurons with transcriptomes that are nearly indistinguishable from that of wild-type Tm2 neurons. We also show that Drgx expression in Tm1 neurons is regulated by Klumpfuss, a TF expressed in stem cells that instructs this fate in progenitors, establishing a link between the regulatory programs that specify neuronal fates and those that implement them. We identified an intronic enhancer in the Drgx locus whose chromatin is specifically accessible in Tm1 neurons and in which Klu motifs are enriched. Genomic deletion of this region knocked down Drgx expression specifically in Tm1 neurons, leaving it intact in the other cell types that normally express it. We further validated this concept by demonstrating that ectopic expression of Vsx (visual system homeobox) genes in Mi15 neurons not only converts them morphologically to Dm2 neurons, but also leads to the loss of their aminergic identity. Our results suggest that selector combinations can be further sculpted by receptor tyrosine kinase signaling after neurogenesis, providing a potential mechanism for postmitotic plasticity of neuronal fates. Finally, we combined our transcriptomic datasets with previously generated chromatin accessibility datasets to understand the mechanisms that control brain wiring downstream of terminal selectors. We built predictive computational models of gene regulatory networks using the Inferelator framework. Experimental validations of these networks revealed how selectors interact with ecdysone-responsive TFs to activate a large and specific repertoire of cell surface proteins and other effectors in each neuron at the onset of synapse formation. We showed that these network models can be used to identify downstream effectors that mediate specific cellular decisions during circuit formation. For instance, reduced levels of cut expression in Tm2 neurons, because of its negative regulation by pdm3 , controls the synaptic layer targeting of their axons. Knockdown of cut in Tm1 neurons is sufficient to redirect their axons to the Tm2 layer in the lobula neuropil without affecting other morphological features. CONCLUSION Our results support a model in which neuronal type identity is primarily determined by a relatively simple code of continuously expressed terminal selector TFs in each cell type throughout development. Our results provide a unified framework of how specific fates are initiated and maintained in postmitotic neurons and open new avenues to understanding synaptic specificity through gene regulatory networks. The conservation of this regulatory logic in both C. elegans and Drosophila makes it likely that the terminal selector concept will also be useful in understanding and manipulating the neuronal diversity of mammalian brains. Terminal selectors enable predictive cell fate reprogramming. Tm1, Tm2, Tm4, and Tm6 neurons of the Drosophila visual system share a core set of TFs continuously expressed by each cell type (simplified). The default Tm4 fate is overridden by the expression of a single additional terminal selector to generate Tm1 ( Drgx ), Tm2 ( pdm3 ), or Tm6 ( SoxN ) fates. 

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3. Transcriptomic changes in single yeast cells under various stress conditions

   https://doi.org/10.1186/s12864-023-09184-w  

   Su, Yangqi ; Xu, Chen ; Shea, Jonathan ; DeStephanis, Darla ; Su, Zhengchang ( February 2023 , BMC Genomics)

   The stress response ofSaccharomyces cerevisiaehas been extensively studied in the past decade. However, with the advent of recent technology in single-cell transcriptome profiling, there is a new opportunity to expand and further understanding of the yeast stress response with greater resolution on a system level. To understand transcriptomic changes in baker’s yeastS. cerevisiaecells under stress conditions, we sequenced 117 yeast cells under three stress treatments (hypotonic condition, glucose starvation and amino acid starvation) using a full-length single-cell RNA-Seq method.

   We found that though single cells from the same treatment showed varying degrees of uniformity, technical noise and batch effects can confound results significantly. However, upon careful selection of samples to reduce technical artifacts and account for batch-effects, we were able to capture distinct transcriptomic signatures for different stress conditions as well as putative regulatory relationships between transcription factors and target genes.

   Our results show that a full-length single-cell based transcriptomic analysis of the yeast may help paint a clearer picture of how the model organism responds to stress than do bulk cell population-based methods.

    

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4. Transcriptome profiling of the Caenorhabditis elegans intestine reveals that ELT-2 negatively and positively regulates intestinal gene expression within the context of a gene regulatory network

   https://doi.org/10.1093/genetics/iyad088  

   Williams, Robert T. P. ; King, David C. ; Mastroianni, Izabella R. ; Hill, Jessica L. ; Apenes, Nicolai W. ; Ramirez, Gabriela ; Miner, E. Catherine ; Moore, Andrew ; Coleman, Karissa ; Nishimura, Erin Osborne ; et al ( May 2023 , GENETICS)

   ELT-2 is the major transcription factor (TF) required for Caenorhabditis elegans intestinal development. ELT-2 expression initiates in embryos to promote development and then persists after hatching through the larval and adult stages. Though the sites of ELT-2 binding are characterized and the transcriptional changes that result from ELT-2 depletion are known, an intestine-specific transcriptome profile spanning developmental time has been missing. We generated this dataset by performing Fluorescence Activated Cell Sorting on intestine cells at distinct developmental stages. We analyzed this dataset in conjunction with previously conducted ELT-2 studies to evaluate the role of ELT-2 in directing the intestinal gene regulatory network through development. We found that only 33% of intestine-enriched genes in the embryo were direct targets of ELT-2 but that number increased to 75% by the L3 stage. This suggests additional TFs promote intestinal transcription especially in the embryo. Furthermore, only half of ELT-2's direct target genes were dependent on ELT-2 for their proper expression levels, and an equal proportion of those responded to elt-2 depletion with over-expression as with under-expression. That is, ELT-2 can either activate or repress direct target genes. Additionally, we observed that ELT-2 repressed its own promoter, implicating new models for its autoregulation. Together, our results illustrate that ELT-2 impacts roughly 20–50% of intestine-specific genes, that ELT-2 both positively and negatively controls its direct targets, and that the current model of the intestinal regulatory network is incomplete as the factors responsible for directing the expression of many intestinal genes remain unknown.

    

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5. Regulation of regeneration in Arabidopsis thaliana

   https://doi.org/10.1007/s42994-023-00121-9  

   Islam, Md Khairul ; Mummadi, Sai Teja ; Liu, Sanzhen ; Wei, Hairong ( November 2023 , aBIOTECH)

   We employed several algorithms with high efficacy to analyze the public transcriptomic data, aiming to identify key transcription factors (TFs) that regulate regeneration inArabidopsis thaliana. Initially, we utilized CollaborativeNet, also known as TF-Cluster, to construct a collaborative network of all TFs, which was subsequently decomposed into many subnetworks using the Triple-Link and Compound Spring Embedder (CoSE) algorithms. Functional analysis of these subnetworks led to the identification of nine subnetworks closely associated with regeneration. We further applied principal component analysis and gene ontology (GO) enrichment analysis to reduce the subnetworks from nine to three, namely subnetworks 1, 12, and 17. Searching for TF-binding sites in the promoters of the co-expressed and co-regulated (CCGs) genes of all TFs in these three subnetworks and Triple-Gene Mutual Interaction analysis of TFs in these three subnetworks with the CCGs involved in regeneration enabled us to rank the TFs in each subnetwork. Finally, six potential candidate TFs—WOX9A, LEC2, PGA37, WIP5, PEI1, and AIL1 from subnetwork 1—were identified, and their roles in somatic embryogenesis (GO:0010262) and regeneration (GO:0031099) were discussed, so were the TFs in Subnetwork 12 and 17 associated with regeneration. The TFs identified were also assessed using the CIS-BP database and Expression Atlas. Our analyses suggest some novel TFs that may have regulatory roles in regeneration and embryogenesis and provide valuable data and insights into the regulatory mechanisms related to regeneration. The tools and the procedures used here are instrumental for analyzing high-throughput transcriptomic data and advancing our understanding of the regulation of various biological processes of interest.

    

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