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1. Comparative transcriptome analyses of the Drosophila pupal eye

   https://doi.org/10.1093/g3journal/jkaa003  

   DeAngelis, Miles W ; Coolon, Joseph D ; Johnson, Ruth I ( January 2021 , G3 Genes|Genomes|Genetics)

   Lott, S (Ed.)

   Abstract Tissue function is dependent on correct cellular organization and behavior. As a result, the identification and study of genes that contribute to tissue morphogenesis is of paramount importance to the fields of cell and developmental biology. Many of the genes required for tissue patterning and organization are highly conserved between phyla. This has led to the emergence of several model organisms and developmental systems that are used to study tissue morphogenesis. One such model is the Drosophila melanogaster pupal eye that has a highly stereotyped arrangement of cells. In addition, the pupal eye is postmitotic that allows for the study of tissue morphogenesis independent from any effects of proliferation. While the changes in cell morphology and organization that occur throughout pupal eye development are well documented, less is known about the corresponding transcriptional changes that choreograph these processes. To identify these transcriptional changes, we dissected wild-type Canton S pupal eyes and performed RNA-sequencing. Our analyses identified differential expression of many loci that are documented regulators of pupal eye morphogenesis and contribute to multiple biological processes including signaling, axon projection, adhesion, and cell survival. We also identified differential expression of genes not previously implicated in pupal eye morphogenesis such as components of the Toll pathway, several non-classical cadherins, and components of the muscle sarcomere, which could suggest these loci function as novel patterning factors. 

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2. Gene age shapes the transcriptional landscape of sexual morphogenesis in mushroom-forming fungi (Agaricomycetes)

   https://doi.org/10.7554/eLife.71348  

   Merényi, Zsolt ; Virágh, Máté ; Gluck-Thaler, Emile ; Slot, Jason C ; Kiss, Brigitta ; Varga, Torda ; Geösel, András ; Hegedüs, Botond ; Bálint, Balázs ; Nagy, László G ( February 2022 , eLife)

   Multicellularity has been one of the most important innovations in the history of life. The role of gene regulatory changes in driving transitions to multicellularity is being increasingly recognized; however, factors influencing gene expression patterns are poorly known in many clades. Here, we compared the developmental transcriptomes of complex multicellular fruiting bodies of eight Agaricomycetes and Cryptococcus neoformans , a closely related human pathogen with a simple morphology. In-depth analysis in Pleurotus ostreatus revealed that allele-specific expression, natural antisense transcripts, and developmental gene expression, but not RNA editing or a ‘developmental hourglass,’ act in concert to shape its transcriptome during fruiting body development. We found that transcriptional patterns of genes strongly depend on their evolutionary ages. Young genes showed more developmental and allele-specific expression variation, possibly because of weaker evolutionary constraint, suggestive of nonadaptive expression variance in fruiting bodies. These results prompted us to define a set of conserved genes specifically regulated only during complex morphogenesis by excluding young genes and accounting for deeply conserved ones shared with species showing simple sexual development. Analysis of the resulting gene set revealed evolutionary and functional associations with complex multicellularity, which allowed us to speculate they are involved in complex multicellular morphogenesis of mushroom fruiting bodies. 

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3. Mutation of self-binding sites in the promoter of the MrpC transcriptional regulator leads to asynchronous Myxococcus xanthus development

   https://doi.org/10.3389/fmicb.2023.1293966  

   McLaughlin, Maeve ; Higgs, Penelope I. ( November 2023 , Frontiers in Microbiology)

   MrpC, a member of the CRP/Fnr transcription factor superfamily, is necessary to induce and control the multicellular developmental program of the bacterium,Myxococcus xanthus. During development, certain cells in the population first swarm into haystack-shaped aggregates and then differentiate into environmentally resistant spores to form mature fruiting bodies (a specialized biofilm).mrpCtranscriptional regulation is controlled by negative autoregulation (NAR).

   Wild type and mutantmrpCpromoter regions were fused to a fluorescent reporter to examine effects onmrpCexpression in the population and in single cellsin situ. Phenotypic consequences of the mutantmrpCpromoter were assayed by deep convolution neural network analysis of developmental movies, sporulation efficiency assays, and anti-MrpC immunoblot. In situ analysis of single cell MrpC levels in distinct populations were assayed with an MrpC-mNeonGreen reporter.

   Disruption of MrpC binding sites within themrpCpromoter region led to increased and broadened distribution ofmrpCexpression levels between individual cells in the population. Expression ofmrpCfrom the mutant promoter led to a striking phenotype in which cells lose synchronized transition from aggregation to sporulation. Instead, some cells abruptly exit aggregation centers and remain locked in a cohesive swarming state we termed developmental swarms, while the remaining cells transition to spores inside residual fruiting bodies.In situexamination of a fluorescent reporter for MrpC levels in developmental subpopulations demonstrated cells locked in the developmental swarms contained MrpC levels that do not reach the levels observed in fruiting bodies.

   Increased cell-to-cell variation inmrpCexpression upon disruption of MrpC binding sites within its promoter is consistent with NAR motifs functioning to reducing noise. Noise reduction may be key to synchronized transition of cells in the aggregation state to the sporulation state. We hypothesize a novel subpopulation of cells trapped as developmental swarms arise from intermediate levels of MrpC that are sufficient to promote aggregation but insufficient to trigger sporulation. Failure to transition to higher levels of MrpC necessary to induce sporulation may indicate cells in developmental swarms lack an additional positive feedback signal required to boost MrpC levels.

    

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4. 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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5. Identifying the Gene Regulatory Network of the Starvation-Induced Transcriptional Activator Nla28

   https://doi.org/10.1128/jb.00265-22  

   Ma, Muqing ; Garza, Anthony G. ; Lemon, David J. ; Caro, Eduardo A. ; Ritchie, Linnea ; Ryan, Charles ; Spearing, Victoria M. ; Murphy, Kimberly A. ; Welch, Roy D. ( December 2022 , Journal of Bacteriology)

   O'Toole, George (Ed.)

   ABSTRACT Myxococcus xanthus copes with starvation by producing fruiting bodies filled with dormant and stress-resistant spores. Here, we aimed to better define the gene regulatory network associated with Nla28, a transcriptional activator/enhancer binding protein (EBP) and a key regulator of the early starvation response. Previous work showed that Nla28 directly regulates EBP genes that are important for fruiting body development. However, the Nla28 regulatory network is likely to be much larger because hundreds of starvation-induced genes are downregulated in a nla28 mutant strain. To identify candidates for direct Nla28-mediated transcription, we analyzed the downregulated genes using a bioinformatics approach. Nine potential Nla28 target promoters (29 genes) were discovered. The results of in vitro promoter binding assays, coupled with in vitro and in vivo mutational analyses, suggested that the nine promoters along with three previously identified EBP gene promoters were indeed in vivo targets of Nla28. These results also suggested that Nla28 used tandem, imperfect repeats of an 8-bp sequence for promoter binding. Interestingly, eight of the new Nla28 target promoters were predicted to be intragenic. Based on mutational analyses, the newly identified Nla28 target loci contained at least one gene that was important for starvation-induced development. Most of these loci contained genes predicted to be involved in metabolic or defense-related functions. Using the consensus Nla28 binding sequence, bioinformatics, and expression profiling, 58 additional promoters and 102 genes were tagged as potential Nla28 targets. Among these putative Nla28 targets, functions, such as regulatory, metabolic, and cell envelope biogenesis, were assigned to many genes. IMPORTANCE In bacteria, starvation leads to profound changes in behavior and physiology. Some of these changes have economic and health implications because the starvation response has been linked to the formation of biofilms, virulence, and antibiotic resistance. To better understand how starvation contributes to changes in bacterial physiology and resistance, we identified the putative starvation-induced gene regulatory network associated with Nla28, a transcriptional activator from the bacterium Myxoccocus xanthus . We determined the mechanism by which starvation-responsive genes were activated by Nla28 and showed that several of the genes were important for the formation of a highly resistant cell type. 

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