In-depth transcriptomic analysis of Anopheles gambiae hemocytes uncovers novel genes and the oenocytoid developmental lineage.

BACKGROUND: Hemocytes are immune cells that patrol the mosquito hemocoel and mediate critical cellular defense responses against pathogens. However, despite their importance, a comprehensive transcriptome of these cells was lacking because they constitute a very small fraction of the total cells in the insect, limiting the study of hemocyte differentiation and immune function. RESULTS: In this study, an in-depth hemocyte transcriptome was built by extensive bulk RNA sequencing and assembly of hemocyte RNAs from adult A. gambiae female mosquitoes, based on approximately 2.4 billion short Illumina and about 9.4 million long PacBio high-quality reads that mapped to the A. gambiae PEST genome (P4.14 version). A total of 34,939 transcripts were annotated including 4,020 transcripts from novel genes and 20,008 novel isoforms that result from extensive differential splicing of transcripts from previously annotated genes. Most hemocyte transcripts identified (89.8%) are protein-coding while 10.2% are non-coding RNAs. The number of transcripts identified in the novel hemocyte transcriptome is twice the number in the current annotation of the A. gambiae genome (P4.14 version). Furthermore, we were able to refine the analysis of a previously published single-cell transcriptome (scRNAseq) data set by using the novel hemocyte transcriptome as a reference to re-define the hemocyte clusters and determine the path of hemocyte differentiation. Unsupervised pseudo-temporal ordering using the Tools for Single Cell Analysis software uncovered a novel putative prohemocyte precursor cell type that gives rise to prohemocytes. Pseudo-temporal ordering with the Monocle 3 software, which analyses changes in gene expression during dynamic biological processes, determined that oenocytoids derive from prohemocytes, a cell population that also gives rise to the granulocyte lineage. CONCLUSION: A high number of mRNA splice variants are expressed in hemocytes, and they may account for the plasticity required to mount efficient responses to many different pathogens. This study highlights the importance of a comprehensive set of reference transcripts to perform robust single-cell transcriptomic data analysis of cells present in low abundance. The detailed annotation of the hemocyte transcriptome will uncover new facets of hemocyte development and function in adult dipterans and is a valuable community resource for future studies on mosquito cellular immunity.

Sleep need-dependent changes in functional connectivity facilitate transmission of homeostatic sleep drive.

How the homeostatic drive for sleep accumulates over time and is released remains poorly understood. In Drosophila, we previously identified the R5 ellipsoid body (EB) neurons as putative sleep drive neurons1 and recently described a mechanism by which astrocytes signal to these cells to convey sleep need.2 Here, we examine the mechanisms acting downstream of the R5 neurons to promote sleep. EM connectome data demonstrate that R5 neurons project to EPG neurons.3 Broad thermogenetic activation of EPG neurons promotes sleep, whereas inhibiting these cells reduces homeostatic sleep rebound. Perforated patch-clamp recordings reveal that EPG neurons exhibit elevated spontaneous firing following sleep deprivation, which likely depends on an increase in extrinsic excitatory inputs. Our data suggest that cholinergic R5 neurons participate in the homeostatic regulation of sleep, and epistasis experiments indicate that the R5 neurons act upstream of EPG neurons to promote sleep. Finally, we show that the physical and functional connectivity between the R5 and EPG neurons increases with greater sleep need. Importantly, dual patch-clamp recordings demonstrate that activating R5 neurons induces cholinergic-dependent excitatory postsynaptic responses in EPG neurons. Moreover, sleep loss triggers an increase in the amplitude of these responses, as well as in the proportion of EPG neurons that respond. Together, our data support a model whereby sleep drive strengthens the functional connectivity between R5 and EPG neurons, triggering sleep when a sufficient number of EPG neurons are activated. This process could enable the proper timing of the accumulation and release of sleep drive.

Astroglial Calcium Signaling Encodes Sleep Need in Drosophila.

Sleep is under homeostatic control, whereby increasing wakefulness generates sleep need and triggers sleep drive. However, the molecular and cellular pathways by which sleep need is encoded are poorly understood. In addition, the mechanisms underlying both how and when sleep need is transformed to sleep drive are unknown. Here, using ex vivo and in vivo imaging, we show in Drosophila that astroglial Ca2+ signaling increases with sleep need. We demonstrate that this signaling is dependent on a specific L-type Ca2+ channel and is necessary for homeostatic sleep rebound. Thermogenetically increasing Ca2+ in astrocytes induces persistent sleep behavior, and we exploit this phenotype to conduct a genetic screen for genes required for the homeostatic regulation of sleep. From this large-scale screen, we identify TyrRII, a monoaminergic receptor required in astrocytes for sleep homeostasis. TyrRII levels rise following sleep deprivation in a Ca2+-dependent manner, promoting further increases in astrocytic Ca2+ and resulting in a positive-feedback loop. Moreover, our findings suggest that astrocytes then transmit this sleep need to a sleep drive circuit by upregulating and releasing the interleukin-1 analog Spätzle, which then acts on Toll receptors on R5 neurons. These findings define astroglial Ca2+ signaling mechanisms encoding sleep need and reveal dynamic properties of the sleep homeostatic control system.

Morning and Evening Circadian Pacemakers Independently Drive Premotor Centers via a Specific Dopamine Relay.

Many animals exhibit morning and evening peaks of locomotor behavior. In Drosophila, two corresponding circadian neural oscillators-M (morning) cells and E (evening) cells-exhibit a corresponding morning or evening neural activity peak. Yet we know little of the neural circuitry by which distinct circadian oscillators produce specific outputs to precisely control behavioral episodes. Here, we show that ring neurons of the ellipsoid body (EB-RNs) display spontaneous morning and evening neural activity peaks in vivo: these peaks coincide with the bouts of locomotor activity and result from independent activation by M and E pacemakers. Further, M and E cells regulate EB-RNs via identified PPM3 dopaminergic neurons, which project to the EB and are normally co-active with EB-RNs. These in vivo findings establish the fundamental elements of a circadian neuronal output pathway: distinct circadian oscillators independently drive a common pre-motor center through the agency of specific dopaminergic interneurons.

A Genetic Toolkit for Dissecting Dopamine Circuit Function in Drosophila.

The neuromodulator dopamine (DA) plays a key role in motor control, motivated behaviors, and higher-order cognitive processes. Dissecting how these DA neural networks tune the activity of local neural circuits to regulate behavior requires tools for manipulating small groups of DA neurons. To address this need, we assembled a genetic toolkit that allows for an exquisite level of control over the DA neural network in Drosophila. To further refine targeting of specific DA neurons, we also created reagents that allow for the conversion of any existing GAL4 line into Split GAL4 or GAL80 lines. We demonstrated how this toolkit can be used with recently developed computational methods to rapidly generate additional reagents for manipulating small subsets or individual DA neurons. Finally, we used the toolkit to reveal a dynamic interaction between a small subset of DA neurons and rearing conditions in a social space behavioral assay.

Genome-wide discovery of active regulatory elements and transcription factor footprints in Caenorhabditis elegans using DNase-seq.

Deep sequencing of size-selected DNase I-treated chromatin (DNase-seq) allows high-resolution measurement of chromatin accessibility to DNase I cleavage, permitting identification of de novo active cis-regulatory modules (CRMs) and individual transcription factor (TF) binding sites. We adapted DNase-seq to nuclei isolated from C. elegans embryos and L1 arrest larvae to generate high-resolution maps of TF binding. Over half of embryonic DNase I hypersensitive sites (DHSs) were annotated as noncoding, with 24% in intergenic, 12% in promoters, and 28% in introns, with similar statistics observed in L1 arrest larvae. Noncoding DHSs are highly conserved and enriched in marks of enhancer activity and transcription. We validated noncoding DHSs against known enhancers from myo-2, myo-3, hlh-1, elt-2, and lin-26/lir-1 and recapitulated 15 of 17 known enhancers. We then mined DNase-seq data to identify putative active CRMs and TF footprints. Using DNase-seq data improved predictions of tissue-specific expression compared with motifs alone. In a pilot functional test, 10 of 15 DHSs from pha-4, icl-1, and ceh-13 drove reporter gene expression in transgenic C. elegans Overall, we provide experimental annotation of 26,644 putative CRMs in the embryo containing 55,890 TF footprints, as well as 15,841 putative CRMs in the L1 arrest larvae containing 32,685 TF footprints.

Molecular dissection of cis-regulatory modules at the Drosophila bithorax complex reveals critical transcription factor signature motifs.

At the Drosophila melanogaster bithorax complex (BX-C) over 330kb of intergenic DNA is responsible for directing the transcription of just three homeotic (Hox) genes during embryonic development. A number of distinct enhancer cis-regulatory modules (CRMs) are responsible for controlling the specific expression patterns of the Hox genes in the BX-C. While it has proven possible to identify orthologs of known BX-C CRMs in different Drosophila species using overall sequence conservation, this approach has not proven sufficiently effective for identifying novel CRMs or defining the key functional sequences within enhancer CRMs. Here we demonstrate that the specific spatial clustering of transcription factor (TF) binding sites is important for BX-C enhancer activity. A bioinformatic search for combinations of putative TF binding sites in the BX-C suggests that simple clustering of binding sites is frequently not indicative of enhancer activity. However, through molecular dissection and evolutionary comparison across the Drosophila genus we discovered that specific TF binding site clustering patterns are an important feature of three known BX-C enhancers. Sub-regions of the defined IAB5 and IAB7b enhancers were both found to contain an evolutionarily conserved signature motif of clustered TF binding sites which is critical for the functional activity of the enhancers. Together, these results indicate that the spatial organization of specific activator and repressor binding sites within BX-C enhancers is of greater importance than overall sequence conservation and is indicative of enhancer functional activity.

Disruption of the abdominal-B promoter tethering element results in a loss of long-range enhancer-directed Hox gene expression in Drosophila.

There are many examples within gene complexes of transcriptional enhancers interacting with only a subset of target promoters. A number of molecular mechanisms including promoter competition, insulators and chromatin looping are thought to play a role in regulating these interactions. At the Drosophila bithorax complex (BX-C), the IAB5 enhancer specifically drives gene expression only from the Abdominal-B (Abd-B) promoter, even though the enhancer and promoter are 55 kb apart and are separated by at least three insulators. In previous studies, we discovered that a 255 bp cis-regulatory module, the promoter tethering element (PTE), located 5' of the Abd-B transcriptional start site is able to tether IAB5 to the Abd-B promoter in transgenic embryo assays. In this study we examine the functional role of the PTE at the endogenous BX-C using transposon-mediated mutagenesis. Disruption of the PTE by P element insertion results in a loss of enhancer-directed Abd-B expression during embryonic development and a homeotic transformation of abdominal segments. A partial deletion of the PTE and neighboring upstream genomic sequences by imprecise excision of the P element also results in a similar loss of Abd-B expression in embryos. These results demonstrate that the PTE is an essential component of the regulatory network at the BX-C and is required in vivo to mediate specific long-range enhancer-promoter interactions.

Dissecting the regulatory switches of development: lessons from enhancer evolution in Drosophila.

Cis-regulatory modules are non-protein-coding regions of DNA essential for the control of gene expression. One class of regulatory modules is embryonic enhancers, which drive gene expression during development as a result of transcription factor protein binding at the enhancer sequences. Recent comparative studies have begun to investigate the evolution of the sequence architecture within enhancers. These analyses are illuminating the way that developmental biologists think about enhancers by revealing their molecular mechanism of function.

Functional evolution of cis-regulatory modules at a homeotic gene in Drosophila.

It is a long-held belief in evolutionary biology that the rate of molecular evolution for a given DNA sequence is inversely related to the level of functional constraint. This belief holds true for the protein-coding homeotic (Hox) genes originally discovered in Drosophila melanogaster. Expression of the Hox genes in Drosophila embryos is essential for body patterning and is controlled by an extensive array of cis-regulatory modules (CRMs). How the regulatory modules functionally evolve in different species is not clear. A comparison of the CRMs for the Abdominal-B gene from different Drosophila species reveals relatively low levels of overall sequence conservation. However, embryonic enhancer CRMs from other Drosophila species direct transgenic reporter gene expression in the same spatial and temporal patterns during development as their D. melanogaster orthologs. Bioinformatic analysis reveals the presence of short conserved sequences within defined CRMs, representing gap and pair-rule transcription factor binding sites. One predicted binding site for the gap transcription factor KRUPPEL in the IAB5 CRM was found to be altered in Superabdominal (Sab) mutations. In Sab mutant flies, the third abdominal segment is transformed into a copy of the fifth abdominal segment. A model for KRUPPEL-mediated repression at this binding site is presented. These findings challenge our current understanding of the relationship between sequence evolution at the molecular level and functional activity of a CRM. While the overall sequence conservation at Drosophila CRMs is not distinctive from neighboring genomic regions, functionally critical transcription factor binding sites within embryonic enhancer CRMs are highly conserved. These results have implications for understanding mechanisms of gene expression during embryonic development, enhancer function, and the molecular evolution of eukaryotic regulatory modules.

Non-genic transcription at the Drosophila bithorax complex functional activity of the dark matter of the genome.

Drosophila melanogaster is a powerful model system for the study of gene regulation due to its short generation time, high fertility and the availability of various genetic tools to manipulate the genome. Investigation into the regulation of homeotic genes and their role in embryonic patterning during development was pioneered in Drosophila. Recently, the molecular mechanisms responsible for regulating gene expression in the bithorax complex have been the focus of active study. Many of these studies have pointed to the importance of cis-regulatory modules, genetic sequences that direct the temporal and spatial patterns of gene expression over large genomic distances. Additional components of the regulatory code have emerged beyond the primary DNA sequence. In particular, non-genic transcription is an important mechanism for controlling gene expression either through direct transcriptional mechanisms that mediate dynamic epigenetic control of the chromatin environment or through functional activity of the RNA products.

Between transcription and translation: Re-defining RNA and regulation.

The diverse functional roles for RNA molecules in cells of the developing embryo have been an area of intense study in the last few years. Progress reported at the 49(th) Annual Drosophila Research Conference in San Diego, California highlighted many of the varied mechanistic activities for RNAs. In particular, talks at the 'RNA Biology' platform session provided a great deal of insight into the function of RNA transcripts and their associated protein complexes. The topics covered included: (1) a large-scale screen examining the localization of mRNAs during embryonic development, (2) mechanisms of mRNA transport in different cell types, (3) localization-dependent repression of mRNA translation and (4) the activity of the RNAi machinery in insulator-mediated chromatin structures. Our journey through the modern RNA world clearly indicates that we should be considering a much more expansive role for RNAs in molecular biology.

The abdominal-B promoter tethering element mediates promoter-enhancer specificity at the Drosophila bithorax complex.

At the Drosophila bithorax complex many distinct classes of cis-regulatory modules work collectively during development to control gene expression. Abdominal-B (Abd-B) is one of three homeotic genes in the BX-C and is expressed in specific presumptive abdominal segments in the embryo. The transcription of Abd-B is tightly controlled by an array of cis-regulatory modules that direct its expression over extended genomic distances. These regulatory modules include promoters, insulators, silencers, enhancers, promoter targeting sequences and the recently identified promoter tethering element (PTE). To activate gene expression at the endogenous complex, enhancers located >50 kb away must bypass intervening insulators to interact with the Abd-B promoter. The molecular mechanisms that allow enhancers to bypass insulators are not currently well understood. In this short article, we report on a novel mechanism for insulator bypass involving the PTE. In addition, we use bioinformatic analysis across twelve Drosophila genomes to identify putative cis-regulatory sequences that may be capable of facilitating specific promoter-enhancer interactions at the bithorax complex and propose a model for their molecular function during development.