The cranial sensory ganglia represent populations of neurons with distinct functions, or sensory modalities. The production of individual ganglia from distinct neurogenic placodes with different developmental pathways provides a powerful model to investigate the acquisition of specific sensory modalities. To date there is a limited range of gene markers available to examine the molecular pathways underlying this process.

During the splicing reaction, the 5' intron end is joined to the branchpoint nucleotide, selecting the next exon to incorporate into the mature RNA and forming an intron lariat, which is excised. Despite a critical role in gene splicing, the locations and features of human splicing branchpoints are largely unknown. We use exoribonuclease digestion and targeted RNA-sequencing to enrich for sequences that traverse the lariat junction and, by split and inverted alignment, reveal the branchpoint. We identify 59,359 high-confidence human branchpoints in >10,000 genes, providing a first map of splicing branchpoints in the human genome. Branchpoints are predominantly adenosine, highly conserved, and closely distributed to the 3' splice site. Analysis of human branchpoints reveals numerous novel features, including distinct features of branchpoints for alternatively spliced exons and a family of conserved sequence motifs overlapping branchpoints we term B-boxes, which exhibit maximal nucleotide diversity while maintaining interactions with the keto-rich U2 snRNA. Different B-box motifs exhibit divergent usage in vertebrate lineages and associate with other splicing elements and distinct intron-exon architectures, suggesting integration within a broader regulatory splicing code. Lastly, although branchpoints are refractory to common mutational processes and genetic variation, mutations occurring at branchpoint nucleotides are enriched for disease associations.

Microglia form the immune system of the brain. Previous studies in cell cultures and animal models suggest altered activation states and cellular senescence in the aged brain. Instead, we analyzed 3 transcriptome data sets from the postmortem frontal cortex of 381 control individuals to show that microglia gene markers assemble into a transcriptional module in a gene coexpression network. These markers predominantly represented M1 and M1/M2b activation phenotypes. Expression of genes in this module generally declines over the adult life span. This decrease was more pronounced in microglia surface receptors for microglia and/or neuron crosstalk than in markers for activation state phenotypes. In addition to these receptors for exogenous signals, microglia are controlled by brain-expressed regulatory factors. We identified a subnetwork of transcription factors, including RUNX1, IRF8, PU.1, and TAL1, which are master regulators (MRs) for the age-dependent microglia module. The causal contributions of these MRs on the microglia module were verified using publicly available ChIP-Seq data. Interactions of these key MRs were preserved in a protein-protein interaction network. Importantly, these MRs appear to be essential for regulating microglia homeostasis in the adult human frontal cortex in addition to their crucial roles in hematopoiesis and myeloid cell-fate decisions during embryogenesis.

Autism spectrum disorder (ASD) is a complex neurodevelopmental disease whose underpinning molecular mechanisms and neural substrates are subject to intense scrutiny. Interestingly, the cerebellum has emerged as one of the key brain regions affected in ASD. However, the genetic and molecular mechanisms that link the cerebellum to ASD, particularly during development, remain poorly understood. To gain insight into the genetic and molecular mechanisms that might link the cerebellum to ASD, we analysed the transcriptome dynamics of a developing cell population highly enriched for Purkinje cells of the mouse cerebellum across multiple timepoints. We identified a single cluster of genes whose expression is positively correlated with development and which is enriched for genes associated with ASD. This ASD-associated gene cluster was specific to developing Purkinje cells and not detected in the mouse neocortex during the same developmental period, in which we identified a distinct temporally regulated ASD gene module. Furthermore, the composition of ASD risk genes within the two distinct clusters was significantly different in their association with intellectual disability (ID), consistent with the existence of genetically and spatiotemporally distinct endophenotypes of ASD. Together, our findings define a specific cluster of ASD genes that is enriched in developing PCs and predicts co-morbidity status.

Cichlid fishes are famous for large, diverse and replicated adaptive radiations in the Great Lakes of East Africa. To understand the molecular mechanisms underlying cichlid phenotypic diversity, we sequenced the genomes and transcriptomes of five lineages of African cichlids: the Nile tilapia (Oreochromis niloticus), an ancestral lineage with low diversity; and four members of the East African lineage: Neolamprologus brichardi/pulcher (older radiation, Lake Tanganyika), Metriaclima zebra (recent radiation, Lake Malawi), Pundamilia nyererei (very recent radiation, Lake Victoria), and Astatotilapia burtoni (riverine species around Lake Tanganyika). We found an excess of gene duplications in the East African lineage compared to tilapia and other teleosts, an abundance of non-coding element divergence, accelerated coding sequence evolution, expression divergence associated with transposable element insertions, and regulation by novel microRNAs. In addition, we analysed sequence data from sixty individuals representing six closely related species from Lake Victoria, and show genome-wide diversifying selection on coding and regulatory variants, some of which were recruited from ancient polymorphisms. We conclude that a number of molecular mechanisms shaped East African cichlid genomes, and that amassing of standing variation during periods of relaxed purifying selection may have been important in facilitating subsequent evolutionary diversification.

Low-complexity regions (LCRs) within proteins sequences are often considered to evolve neutrally even though recent studies reported evidence for selection acting on some of them. Because of their widespread distribution among eukaryotes genomes and the potential deleterious effect of expansion/contraction of some of them in humans, low-complexity sequences are of major interest and numerous studies have attempted to describe their dynamic between genomes as well as the factors correlated to their variation and to assess their selective value. However, due to the scarcity of individual genomes within a species, most of the analyses so far have been performed at the species level with the implicit assumption that the variation both in composition and size within species is too small relative to the between-species divergence to affect the conclusions of the analysis. Here we used the available genomes of 14 Plasmodium falciparum isolates to assess the relationship between low-complexity sequence variation and factors such as nucleotide polymorphism across strains, sequence composition, and protein expression. We report that more than half of the 7,711 low-complexity sequences found within aligned coding sequences are variable in size among strains. Across strains, we observed an increasing density of polymorphic sites toward the LCR boundaries. This observation strongly suggests the joint effects of lowered selective constraints on low-complexity sequences and a mutagenic effect of these simple sequences.

Karl Ernst Von Baer noted that species tend to show greater morphological divergence in later stages of development when compared to earlier stages. Darwin originally interpreted these observations via a selectionist framework, suggesting that divergence should be greatest during ontogenic stages in which organisms experienced varying 'conditions of existence' and opportunity for differential selection. Modern hypotheses have focused on the notion that genes and structures involved in early development will be under stronger purifying selection due to the deleterious pleiotropic effects of mutations propagating over the course of ontogeny, also known as the developmental constraint hypothesis.

Much of the morphological diversity in eukaryotes results from differential regulation of gene expression in which transcription factors (TFs) play a central role. The nematode Caenorhabditis elegans is an established model organism for the study of the roles of TFs in controlling the spatiotemporal pattern of gene expression. Using the fully sequenced genomes of three Caenorhabditid nematode species as well as genome information from additional more distantly related organisms (fruit fly, mouse, and human) we sought to identify orthologous TFs and characterized their patterns of evolution.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder, affecting 1% of the population over 65 years characterized clinically by both motor and non-motor symptoms accompanied by the preferential loss of dopamine neurons in the substantia nigra pars compacta. Here, we sequenced the exomes of 244 Parkinson's patients selected from the Oxford Parkinson's Disease Centre Discovery Cohort and, after quality control, 228 exomes were available for analyses. The PD patient exomes were compared to 884 control exomes selected from the UK10K datasets. No single non-synonymous (NS) single nucleotide variant (SNV) nor any gene carrying a higher burden of NS SNVs was significantly associated with PD status after multiple-testing correction. However, significant enrichments of genes whose proteins have roles in the extracellular matrix were amongst the top 300 genes with the most significantly associated NS SNVs, while regions associated with PD by a recent Genome Wide Association (GWA) study were enriched in genes containing PD-associated NS SNVs. By examining genes within GWA regions possessing rare PD-associated SNVs, we identified RAD51B. The protein-product of RAD51B interacts with that of its paralogue RAD51, which is associated with congenital mirror movements phenotypes, a phenotype also comorbid with PD.

East African lake cichlids are one of the most impressive examples of an adaptive radiation. Independently in Lake Victoria, Tanganyika, and Malawi, several hundreds of species arose within the last 10 million to 100,000 years. Whereas most analyses in cichlids focused on nucleotide substitutions across species to investigate the genetic bases of this explosive radiation, to date, no study has investigated the contribution of structural variants (SVs) in the evolution of adaptive traits across the three Great Lakes of East Africa.

Whilst much sequencing effort has focused on key mammalian model organisms such as mouse and human, little is known about the relationship between genome sequencing techniques for non-model mammals and genome assembly quality. This is especially relevant to non-model mammals, where the samples to be sequenced are often degraded and of low quality. A key aspect when planning a genome project is the choice of sequencing data to generate. This decision is driven by several factors, including the biological questions being asked, the quality of DNA available, and the availability of funds. Cutting-edge sequencing technologies now make it possible to achieve highly contiguous, chromosome-level genome assemblies, but rely on high-quality high molecular weight DNA. However, funding is often insufficient for many independent research groups to use these techniques. Here we use a range of different genomic technologies generated from a roadkill European polecat (Mustela putorius) to assess various assembly techniques on this low-quality sample. We evaluated different approaches for de novo assemblies and discuss their value in relation to biological analyses.

Seminal studies of vertebrate protein evolution speculated that gene regulatory changes can drive anatomical innovations. However, very little is known about gene regulatory network (GRN) evolution associated with phenotypic effect across ecologically diverse species. Here we use a novel approach for comparative GRN analysis in vertebrate species to study GRN evolution in representative species of the most striking examples of adaptive radiations, the East African cichlids. We previously demonstrated how the explosive phenotypic diversification of East African cichlids can be attributed to diverse molecular mechanisms, including accelerated regulatory sequence evolution and gene expression divergence.

The divergence of regulatory regions and gene regulatory network (GRN) rewiring is a key driver of cichlid phenotypic diversity. However, the contribution of miRNA-binding site turnover has yet to be linked to GRN evolution across cichlids. Here, we extend our previous studies by analyzing the selective constraints driving evolution of miRNA and transcription factor (TF)-binding sites of target genes, to infer instances of cichlid GRN rewiring associated with regulatory binding site turnover. Comparative analyses identified increased species-specific networks that are functionally associated to traits of cichlid phenotypic diversity. The evolutionary rewiring is associated with differential models of miRNA- and TF-binding site turnover, driven by a high proportion of fast-evolving polymorphic sites in adaptive trait genes compared with subsets of random genes. Positive selection acting upon discrete mutations in these regulatory regions is likely to be an important mechanism in rewiring GRNs in rapidly radiating cichlids. Regulatory variants of functionally associated miRNA- and TF-binding sites of visual opsin genes differentially segregate according to phylogeny and ecology of Lake Malawi species, identifying both rewired, for example, clade-specific and conserved network motifs of adaptive trait associated GRNs. Our approach revealed several novel candidate regulators, regulatory regions, and three-node motifs across cichlid genomes with previously reported associations to known adaptive evolutionary traits.

Long non-coding RNA (lncRNA) function is mediated by the process of transcription or through transcript-dependent associations with proteins or nucleic acids to control gene regulatory networks. Many lncRNAs are transcribed in the ventricular-subventricular zone (V-SVZ), a postnatal neural stem cell niche. lncRNAs in the V-SVZ are implicated in neurodevelopmental disorders, cancer, and brain disease, but their functions are poorly understood. V-SVZ neurogenesis capacity declines with age due to stem cell depletion and resistance to neural stem cell activation. Here we analyzed V-SVZ transcriptomics by pooling current single-cell RNA-seq data. They showed consistent lncRNA expression during stem cell activation, lineage progression, and aging. In conjunction with epigenetic and genetic data, we predicted V-SVZ lncRNAs that regulate stem cell activation and differentiation. Some of the lncRNAs validate known epigenetic mechanisms, but most remain uninvestigated. Our analysis points to several lncRNAs that likely participate in key aspects of V-SVZ stem cell activation and neurogenesis in health and disease.

If sequencing was possible only for genomes, and not for RNAs or proteins, then functional protein-coding exons would be recognizable by their unusual patterns of nucleotide composition, specifically a high GC content across the body of exons, and an unusual nucleotide content near their edges. RNAs and proteins can, of course, be sequenced but the extent of functionality of intergenic long noncoding RNAs (lncRNAs) remains under question owing to their low nucleotide conservation. Inspired by the nucleotide composition patterns of protein-coding exons, we sought evidence for functionality across lncRNA loci from diverse species. We found that such patterns across multiexonic lncRNA loci mirror those of proteincoding genes, although to a lesser degree: Specifically, compared with introns, lncRNA exons are GC rich. Additionally we report evidence for the action of purifying selection to preserve exonic splicing enhancers within human multiexonic lncRNAs and nucleotide composition in fruit fly lncRNAs. Our findings provide evidence for selection for more efficient rates of transcription and splicing within lncRNA loci. Despite only a minor proportion of their RNA bases being constrained, multiexonic intergenic lncRNAs appear to require accurate splicing of their exons to transact their function.

We describe the genome of the western painted turtle, Chrysemys picta bellii, one of the most widespread, abundant, and well-studied turtles. We place the genome into a comparative evolutionary context, and focus on genomic features associated with tooth loss, immune function, longevity, sex differentiation and determination, and the species' physiological capacities to withstand extreme anoxia and tissue freezing.

The tempo and mode of evolutionary change during speciation have remained contentious until recently. While much of the evidence claiming speciation is an abrupt and rapid process comes from fossil data, recent molecular phylogenetics show that the background of gradual evolution is often broken by accelerated rates of molecular evolution during speciation. However, what kinds of genes affect or are affected by speciation remains unexplored. Our analysis of 4843 protein-coding genes in five species of the Drosophila melanogaster subgroup shows that while ~70% of genes follow clock-like evolution, between 17-19.67% of loci show signatures of accelerated rates of evolution in recently formed species. These genes show 2-3-fold higher rates of substitution in recently diverged species compared to older species. This fraction of loci affects a diverse range of functions. Only a small proportion of reproductive genes experience speciation-related accelerated changes but many sex-and -reproduction related genes show an interesting pattern of persistent rapid evolution suggesting that sex-and-reproduction related genes are under constant selective pressures. The identification of loci associated with accelerated evolution allows us to address the mechanisms of rapid evolution and speciation, which in our study appears to be a combination of both selection and rapid demographical changes.

Paneth cells are key epithelial cells that provide an antimicrobial barrier and maintain integrity of the small-intestinal stem cell niche. Paneth cell abnormalities are unfortunately detrimental to gut health and are often associated with digestive pathologies such as Crohn's disease or infections. Similar alterations are observed in individuals with impaired autophagy, a process that recycles cellular components. The direct effect of autophagy impairment on Paneth cells has not been analysed. To investigate this, we generated a mouse model lacking Atg16l1 specifically in intestinal epithelial cells, making these cells impaired in autophagy. Using three-dimensional intestinal organoids enriched for Paneth cells, we compared the proteomic profiles of wild-type and autophagy-impaired organoids. We used an integrated computational approach combining protein-protein interaction networks, autophagy-targeted proteins and functional information to identify the mechanistic link between autophagy impairment and disrupted pathways. Of the 284 altered proteins, 198 (70%) were more abundant in autophagy-impaired organoids, suggesting reduced protein degradation. Interestingly, these differentially abundant proteins comprised 116 proteins (41%) that are predicted targets of the selective autophagy proteins p62, LC3 and ATG16L1. Our integrative analysis revealed autophagy-mediated mechanisms that degrade key proteins in Paneth cell functions, such as exocytosis, apoptosis and DNA damage repair. Transcriptomic profiling of additional organoids confirmed that 90% of the observed changes upon autophagy alteration have effects at the protein level, not on gene expression. We performed further validation experiments showing differential lysozyme secretion, confirming our computationally inferred downregulation of exocytosis. Our observations could explain how protein-level alterations affect Paneth cell homeostatic functions upon autophagy impairment.This article has an associated First Person interview with the joint first authors of the paper.

Spikelets are the fundamental building blocks of Poaceae inflorescences, and their development and branching patterns determine the various inflorescence architectures and grain yield of grasses. In wheat (Triticum aestivum), the central spikelets produce the most and largest grains, while spikelet size gradually decreases acropetally and basipetally, giving rise to the characteristic lanceolate shape of wheat spikes. The acropetal gradient corresponds with the developmental age of spikelets; however, the basal spikelets are developed first, and the cause of their small size and rudimentary development is unclear. Here, we adapted G&T-seq, a low-input transcriptomics approach, to characterize gene expression profiles within spatial sections of individual spikes before and after the establishment of the lanceolate shape. We observed larger differences in gene expression profiles between the apical, central, and basal sections of a single spike than between any section belonging to consecutive developmental time points. We found that SHORT VEGETATIVE PHASE MADS-box transcription factors, including VEGETATIVE TO REPRODUCTIVE TRANSITION 2 (VRT-A2), are expressed highest in the basal section of the wheat spike and display the opposite expression gradient to flowering E-class SEPALLATA1 genes. Based on multi-year field trials and transgenic lines, we show that higher expression of VRT-A2 in the basal sections of the spike is associated with increased numbers of rudimentary basal spikelets. Our results, supported by computational modeling, suggest that the delayed transition of basal spikelets from vegetative to floral developmental programs results in the lanceolate shape of wheat spikes. This study highlights the value of spatially resolved transcriptomics to gain insights into developmental genetics pathways of grass inflorescences.

Platelets and erythrocytes constitute over 95% of all hematopoietic stem cell output. However, the clonal dynamics of HSC contribution to these lineages remains largely unexplored.