Combinatorial Code Research Articles

The Bruce effect: Representational stability and memory formation in the accessory olfactory bulb of the female mouse.

SUMMARYIn the Bruce effect, a mated female mouse becomes resistant to the pregnancy-blocking effect of the stud. Various lines of evidence suggest that this form of behavioral imprinting results from reduced sensitivity of the female’s accessory olfactory bulb (AOB) to the stud’s chemosignals. However, the AOB’s combinatorial code implies that diminishing responses to one individual will distort representations of other stimuli. Here, we record extracellular responses of AOB neurons in mated and unmated female mice while presenting urine stimuli from the stud and from other sources. We find that, while initial sensory responses in the AOB (within a timescale required to guide social interactions) remain stable, responses to extended stimulation (as required for eliciting the pregnancy block) display selective attenuation of stud-responsive neurons. Such temporal disassociation could allow attenuation of slow-acting endocrine processes in a stimulus-specific manner without compromising ongoing representations that guide behavior.

Dachshund acts with Abdominal-B to trigger programmed cell death in the Drosophila central nervous system at the frontiers of Abd-B expression.

A striking feature of the nervous system pertains to the appearance of different neural cell subtypes at different axial levels. Studies in the Drosophila central nervous system reveal that one mechanism underlying such segmental differences pertains to the segment‐specific removal of cells by programmed cell death (PCD). One group of genes involved in segment‐specific PCD is the Hox homeotic genes. However, while segment‐specific PCD is highly precise, Hox gene expression is evident in gradients, raising the issue of how the Hox gene function is precisely gated to trigger PCD in specific segments at the outer limits of Hox expression. The Drosophila Va neurons are initially generated in all nerve cord segments but removed by PCD in posterior segments. Va PCD is triggered by the posteriorly expressed Hox gene Abdominal‐B (Abd‐B). However, Va PCD is highly reproducible despite exceedingly weak Abd‐B expression in the anterior frontiers of its expression. Here, we found that the transcriptional cofactor Dachshund supports Abd‐B‐mediated PCD in its anterior domain. In vivo bimolecular fluorescence complementation analysis lends support to the idea that the Dachshund/Abd‐B interplay may involve physical interactions. These findings provide an example of how combinatorial codes of transcription factors ensure precision in Hox‐mediated PCD in specific segments at the outer limits of Hox expression.

Cracking the encoding of human scent in the mosquito brain

In a recent study, Zhao et al. decipher how the olfactory system encodes human versus animal odors in the mosquito Aedes aegypti. By combining genome engineering, invivo calcium imaging, advanced chemistry, and behavioral analysis, the authors provide compelling evidence that the discriminatory coding of host odors is surprisingly simple - and bridges labeled line with combinatorial coding.

Using Single Cell Omics to Understand Posterior Neural Crest Differentiation in Zebrafish

Neural crest cells (NCCs) are a migratory, multipotent, and transient stem cell population essential to vertebrate development. Born along the neural tube, NCCs are regionally specified along the anterior‐posterior axis to contribute to a vast number of cell lineages; such as craniofacial cartilage, pigment cells, and large aspects of the peripheral nervous system. In our recently published single‐cell RNA‐sequencing (scRNA‐seq) analysis of posterior NCC lineages in zebrafish embryos, we discovered a combinatorial expression code of transcripts that encode for posterior Hox transcription factors, which was especially specific to NCC‐derived neural lineages. While Hox expression combinations had been characterized within pre‐migratory and recently delaminated cranial NCCs in prior studies, such a high‐resolution signature had not been described within the context of posterior NCC lineages during their differentiation. Here, we report a lifetime atlas of NCC and NCC‐derived cells along the posterior zebrafish body, greatly expanding our prior analyses. To achieve this, several published and publicly‐available zebrafish scRNA‐seq studies characterizing NCC and NCC‐derived cells were computationally merged with our prior data sets and integrated, encompassing: 24 hours post fertilization (hpf), 48‐50 hpf, 68‐70 hpf, 5 days post fertilization (dpf), juvenile, and adult tissues—producing a cohesive distribution of cell identities. Within the lifetime atlas, various Hox gene transcript signatures were observed where numerous discrete cell lineages formed over time and unique Hox expression codes resolved within these incipient lineages. The generation of a multi‐stage comprehensive transcriptional atlas from multiple scRNA‐seq studies will serve as a valuable tool for the NCC and zebrafish communities. Additionally, this atlas of cell lineages demonstrates the complexity and robustness of combinatorial Hox expression codes in posterior NCC throughout development and suggests possible functional roles therein.

Odor-Induced Vomiting Is Combinatorially Triggered by Palp Olfactory Receptor Neurons That Project to the Lobus Glomerulatus in Locust Brain.

Although vomiting is commonly recognized as a protective reaction in response to toxic stimuli, the elaborate sensory processes and necessary molecular components are not fully clear, which is due to a lack of appropriate experimental animal models. Vomiting reflex to volatile chemicals renders locust one candidate for vomiting model. Here, we identified a panel of chemical cues that evoked evident vomiting in locust nymphs and demonstrated the selected combinatorial coding strategy that palps but not antennae olfactory receptor neurons (ORNs) employed. Specifically, knocking down individual palp odorant receptors (ORs) such as OR17, OR21, and OR22 attenuated the vomiting intensity evoked by E-2-hexenal and hexanal, while suppression of OR12 and OR22 augmented vomiting to E-2-hexenal and 2-hexanone, respectively. Furthermore, dual-RNAi treatment against OR17 or OR21 together with OR22 resulted in a much lower response intensity than that of individual OR suppression. Furthermore, OR12 was revealed in palp sensilla basiconica (pb) subtype 3 to tune the neuronal decaying activity to E-2-hexenal. Finally, anterograde labeling indicated that palp ORNs primarily projected into the lobus glomerulatus (LG), and the projection neurons (PNs) in the LG further projected into the accessary calyx (ACA). Together, the establishment of an olfaction-inducible vomiting model in locusts deepens the understanding of olfactory coding logics and provides an opportunity to clarify the neural basis underlying animal vomiting.

Inferring mammalian tissue-specific regulatory conservation by predicting tissue-specific differences in open chromatin

BackgroundEvolutionary conservation is an invaluable tool for inferring functional significance in the genome, including regions that are crucial across many species and those that have undergone convergent evolution. Computational methods to test for sequence conservation are dominated by algorithms that examine the ability of one or more nucleotides to align across large evolutionary distances. While these nucleotide alignment-based approaches have proven powerful for protein-coding genes and some non-coding elements, they fail to capture conservation of many enhancers, distal regulatory elements that control spatial and temporal patterns of gene expression. The function of enhancers is governed by a complex, often tissue- and cell type-specific code that links combinations of transcription factor binding sites and other regulation-related sequence patterns to regulatory activity. Thus, function of orthologous enhancer regions can be conserved across large evolutionary distances, even when nucleotide turnover is high.ResultsWe present a new machine learning-based approach for evaluating enhancer conservation that leverages the combinatorial sequence code of enhancer activity rather than relying on the alignment of individual nucleotides. We first train a convolutional neural network model that can predict tissue-specific open chromatin, a proxy for enhancer activity, across mammals. Next, we apply that model to distinguish instances where the genome sequence would predict conserved function versus a loss of regulatory activity in that tissue. We present criteria for systematically evaluating model performance for this task and use them to demonstrate that our models accurately predict tissue-specific conservation and divergence in open chromatin between primate and rodent species, vastly out-performing leading nucleotide alignment-based approaches. We then apply our models to predict open chromatin at orthologs of brain and liver open chromatin regions across hundreds of mammals and find that brain enhancers associated with neuron activity have a stronger tendency than the general population to have predicted lineage-specific open chromatin.ConclusionThe framework presented here provides a mechanism to annotate tissue-specific regulatory function across hundreds of genomes and to study enhancer evolution using predicted regulatory differences rather than nucleotide-level conservation measurements.

Transcription Factors in the Regulation of Leydig Cell Gene Expression and Function.

Cell differentiation and acquisition of specialized functions are inherent steps in events that lead to normal tissue development and function. These processes require accurate temporal, tissue, and cell-specific activation or repression of gene transcription. This is achieved by complex interactions between transcription factors that form a unique combinatorial code in each specialized cell type and in response to different physiological signals. Transcription factors typically act by binding to short, nucleotide-specific DNA sequences located in the promoter region of target genes. In males, Leydig cells play a crucial role in sex differentiation, health, and reproductive function from embryonic life to adulthood. To better understand the molecular mechanisms regulating Leydig cell differentiation and function, several transcription factors important to Leydig cells have been identified, including some previously unknown to this specialized cell type. This mini review summarizes the current knowledge on transcription factors in fetal and adult Leydig cells, describing their roles and mechanisms of action.

Construction and Characterization of Optical Orthogonal Codes (n,w,1) for Fast Frequency Hopping-Optical Code Division Multiple Access

This paper investigates a new combinatorial construction approach based on cyclic difference packings for the construction of an optical orthogonal code that is suitable as a time spreading sequence for fast frequency hopping optical code division multiple access (OCDM) systems. The proposed construction provides a solution to the electrical decoding delay problem when compared to its optical counterpart, as well as other optimization issues. The criteria for optimizing the performance of such an OCDMA system are provided, namely: the flexibility and simplicity in constructing the optimal code for any length and weight, a reduction in the encoding/decoding complexity that complies with changes in the fast frequency hopping system, and the provision of system transfer transparency, a decoding rule that exploits the embedded asymmetric error correcting capability of the code. Our neighbor difference approach refers to the partition of different solutions into sub-classes, given in the correlation matrix form. It takes into account both direct and recursive combinatorial code construction methods, and the resulting characteristics are evaluated accordingly. A performance analysis based on the OCDMA channel is given, and a comparison with other difference family solutions is performed.

Simultaneous visualization of DNA loci in single cells by combinatorial multi-color iFISH

Single-molecule DNA fluorescence in situ hybridization (FISH) techniques enable studying the three-dimensional (3D) organization of the genome at the single cell level. However, there is a major unmet need for open access, high quality, curated and reproducible DNA FISH datasets. Here, we describe a dataset obtained by applying our recently developed iFISH method to simultaneously visualize 16 small (size range: 62–73 kilobases, kb) DNA loci evenly spaced on chromosome 2 in human cells, in a single round of hybridization. We show how combinatorial color coding can be used to precisely localize multiple loci in 3D within single cells, and how inter-locus distances scale inversely with chromosome contact frequencies determined by high-throughput chromosome conformation capture (Hi-C). We provide raw images and 3D coordinates for nearly 10,000 FISH dots. Our dataset provides a free resource that can facilitate studies of 3D genome organization in single cells and can be used to develop automatic FISH analysis algorithms.

Transcriptional Regulation and Implications for Controlling Hox Gene Expression.

Hox genes play key roles in axial patterning and regulating the regional identity of cells and tissues in a wide variety of animals from invertebrates to vertebrates. Nested domains of Hox expression generate a combinatorial code that provides a molecular framework for specifying the properties of tissues along the A–P axis. Hence, it is important to understand the regulatory mechanisms that coordinately control the precise patterns of the transcription of clustered Hox genes required for their roles in development. New insights are emerging about the dynamics and molecular mechanisms governing transcriptional regulation, and there is interest in understanding how these may play a role in contributing to the regulation of the expression of the clustered Hox genes. In this review, we summarize some of the recent findings, ideas and emerging mechanisms underlying the regulation of transcription in general and consider how they may be relevant to understanding the transcriptional regulation of Hox genes.

Parallel detection of SARS-CoV-2 epitopes reveals dynamic immunodominance profiles of CD8+ T memory cells in convalescent COVID-19 donors.

ObjectivesHigh‐magnitude CD8+ T cell responses are associated with mild COVID‐19 disease; however, the underlying characteristics that define CD8+ T cell‐mediated protection are not well understood. The antigenic breadth and the immunodominance hierarchies of epitope‐specific CD8+ T cells remain largely unexplored and are essential for the development of next‐generation broad‐protective vaccines. This study identified a broad spectrum of conserved SARS‐CoV‐2 CD8+ T cell epitopes and defined their respective immunodominance and phenotypic profiles following SARS‐CoV‐2 infection.MethodsCD8+ T cells from 51 convalescent COVID‐19 donors were analysed for their ability to recognise 133 predicted and previously described SARS‐CoV‐2‐derived peptides restricted by 11 common HLA class I allotypes using heterotetramer combinatorial coding, which combined with phenotypic markers allowed in‐depth ex vivo profiling of CD8+ T cell responses at quantitative and phenotypic levels.ResultsA comprehensive panel of 49 mostly conserved SARS‐CoV‐2‐specific CD8+ T cell epitopes, including five newly identified low‐magnitude epitopes, was established. We confirmed the immunodominance of HLA‐A*01:01/ORF1ab1637–1646 and B*07:02/N105–113 and identified B*35:01/N325–333 as a third epitope with immunodominant features. The magnitude of subdominant epitope responses, including A*03:01/N361–369 and A*02:01/S269–277, depended on the donors' HLA‐I context. All epitopes expressed prevalent memory phenotypes, with the highest memory frequencies in severe COVID‐19 donors.ConclusionSARS‐CoV‐2 infection induces a predominant CD8+ T memory response directed against a broad spectrum of conserved SARS‐CoV‐2 epitopes, which likely contributes to long‐term protection against severe disease. The observed immunodominance hierarchy emphasises the importance of T cell epitopes derived from nonspike proteins to the overall protective and cross‐reactive immune response, which could aid future vaccine strategies.

Universal Gröbner bases of toric ideals of combinatorial neural codes

In the 1970s, O'Keefe and Dostrovsky discovered that certain neurons, called place cells, in an animal's brain are tied to its location within its arena. A combinatorial neural code is a collection of $0/1$-vectors which encode the patterns of co-firing activity among the place cells. Gross, Obatake, and Youngs have recently used techniques from toric algebra to study when a neural code is $0$- $1$-, or $2$-inductively pierced: a property that allows one to reconstruct a Venn diagram-like planar figure that acts as a geometric schematic for the neural co-firing patterns. This article examines their work closely by focusing on a variety of classes of combinatorial neural codes. In particular, we identify universal Gr\"obner bases of the toric ideal for these codes.

Embedding dimension phenomena in intersection complete codes

Two tantalizing invariants of a combinatorial code $${\mathcal {C}}\subseteq 2^{[n]}$$ are $${{\,\mathrm{cdim}\,}}({\mathcal {C}})$$ and $${{\,\mathrm{odim}\,}}({\mathcal {C}})$$ , the smallest dimension in which $${\mathcal {C}}$$ can be realized by convex closed or open sets, respectively. Cruz, Giusti, Itskov, and Kronholm showed that for intersection complete codes $${\mathcal {C}}$$ with $$m+1$$ maximal codewords, $${{\,\mathrm{odim}\,}}({\mathcal {C}})$$ and $${{\,\mathrm{cdim}\,}}({\mathcal {C}})$$ are both bounded above by $$\max \{2,m\}$$ . Results of Lienkaemper, Shiu, and Woodstock imply that $${{\,\mathrm{odim}\,}}$$ and $${{\,\mathrm{cdim}\,}}$$ may differ, even for intersection complete codes. We add to the literature on open and closed embedding dimensions of intersection complete codes with the following results: A key tool in our work is the study of sunflowers: arrangements of convex open sets in which the sets simultaneously meet in a central region, and nowhere else. We use Tverberg’s theorem to study the structure of “k-flexible" sunflowers, and consequently obtain new lower bounds on $${{\,\mathrm{odim}\,}}({\mathcal {C}})$$ for intersection complete codes $${\mathcal {C}}$$ .

An explainable artificial intelligence approach for decoding the enhancer histone modifications code and identification of novel enhancers in Drosophila

BackgroundEnhancers are non-coding regions of the genome that control the activity of target genes. Recent efforts to identify active enhancers experimentally and in silico have proven effective. While these tools can predict the locations of enhancers with a high degree of accuracy, the mechanisms underpinning the activity of enhancers are often unclear.ResultsUsing machine learning (ML) and a rule-based explainable artificial intelligence (XAI) model, we demonstrate that we can predict the location of known enhancers in Drosophila with a high degree of accuracy. Most importantly, we use the rules of the XAI model to provide insight into the underlying combinatorial histone modifications code of enhancers. In addition, we identified a large set of putative enhancers that display the same epigenetic signature as enhancers identified experimentally. These putative enhancers are enriched in nascent transcription, divergent transcription and have 3D contacts with promoters of transcribed genes. However, they display only intermediary enrichment of mediator and cohesin complexes compared to previously characterised active enhancers. We also found that 10–15% of the predicted enhancers display similar characteristics to super enhancers observed in other species.ConclusionsHere, we applied an explainable AI model to predict enhancers with high accuracy. Most importantly, we identified that different combinations of epigenetic marks characterise different groups of enhancers. Finally, we discovered a large set of putative enhancers which display similar characteristics with previously characterised active enhancers.

The genetic informational network: how DNA conveys semantic information.

The question of whether "genetic information" is a merely causal factor in development or can be made sense of semantically, in a way analogous to a language or other type of representation, has generated a long debate in the philosophy of biology. It is intimately connected with another intense debate, concerning the limits of genetic determinism. In this paper I argue that widespread attempts to draw analogies between genetic information and information contained in books, blueprints or computer programs, are fundamentally inadequate. In development, gene exons are the central part of an intricate and densely ramified semantic Genetic Informational Network. DNA in the entire genome is in a state of continuous positive and negative feedback with itself and with its 'environment', and is 'read' and acted upon by the cell in various alternative and complementary ways. The linear combinatorial coding relation between codons and amino acids is but one aspect of semantic genetic information, which is, when considered in its entirety, a far wider and richer concept.

A harmonized atlas of mouse spinal cord cell types and their spatial organization

Single-cell RNA sequencing data can unveil the molecular diversity of cell types. Cell type atlases of the mouse spinal cord have been published in recent years but have not been integrated together. Here, we generate an atlas of spinal cell types based on single-cell transcriptomic data, unifying the available datasets into a common reference framework. We report a hierarchical structure of postnatal cell type relationships, with location providing the highest level of organization, then neurotransmitter status, family, and finally, dozens of refined populations. We validate a combinatorial marker code for each neuronal cell type and map their spatial distributions in the adult spinal cord. We also show complex lineage relationships among postnatal cell types. Additionally, we develop an open-source cell type classifier, SeqSeek, to facilitate the standardization of cell type identification. This work provides an integrated view of spinal cell types, their gene expression signatures, and their molecular organization.

Discrete cis-acting element regulates developmentally timed gene-lamina relocation and neural progenitor competence invivo.

SUMMARYThe nuclear lamina is typically associated with transcriptional silencing, and peripheral relocation of genes highly correlates with repression. However, the DNA sequences and proteins regulating gene-lamina interactions are largely unknown. Exploiting the developmentally timed hunchback gene movement to the lamina in Drosophila neuroblasts, we identified a 250 bp intronic element (IE) both necessary and sufficient for relocation. The IE can target a reporter transgene to the lamina and silence it. Endogenously, however, hunchback is already repressed prior to relocation. Instead, IE-mediated relocation confers a heritably silenced gene state refractory to activation in descendent neurons, which terminates neuroblast competence to specify early-born identity. Surprisingly, we found that the Polycomb group chromatin factors bind the IE and are required for lamina relocation, revealing a nuclear architectural role distinct from their well-known function in transcriptional repression. Together, our results uncover in vivo mechanisms underlying neuroblast competence and lamina association in heritable gene silencing.

Orthogonal neural codes for speech in the infant brain

Creating invariant representations from an everchanging speech signal is a major challenge for the human brain. Such an ability is particularly crucial for preverbal infants who must discover the phonological, lexical, and syntactic regularities of an extremely inconsistent signal in order to acquire language. Within the visual domain, an efficient neural solution to overcome variability consists in factorizing the input into a reduced set of orthogonal components. Here, we asked whether a similar decomposition strategy is used in early speech perception. Using a 256-channel electroencephalographic system, we recorded the neural responses of 3-mo-old infants to 120 natural consonant-vowel syllables with varying acoustic and phonetic profiles. Using multivariate pattern analyses, we show that syllables are factorized into distinct and orthogonal neural codes for consonants and vowels. Concerning consonants, we further demonstrate the existence of two stages of processing. A first phase is characterized by orthogonal and context-invariant neural codes for the dimensions of manner and place of articulation. Within the second stage, manner and place codes are integrated to recover the identity of the phoneme. We conclude that, despite the paucity of articulatory motor plans and speech production skills, pre-babbling infants are already equipped with a structured combinatorial code for speech analysis, which might account for the rapid pace of language acquisition during the first year.

A 4D single-cell protein atlas of transcription factors delineates spatiotemporal patterning during embryogenesis.

Complex biological processes such as embryogenesis require precise coordination of cell differentiation programs across both space and time. Using protein-fusion fluorescent reporters and four-dimensional live imaging, we present a protein expression atlas of transcription factors (TFs) mapped onto developmental cell lineages during Caenorhabditis elegans embryogenesis, at single-cell resolution. This atlas reveals a spatiotemporal combinatorial code of TF expression, and a cascade of lineage-specific, tissue-specific and time-specific TFs that specify developmental states. The atlas uncovers regulators of embryogenesis, including an unexpected role of a skin specifier in neurogenesis and the critical function of an uncharacterized TF in convergent muscle differentiation. At the systems level, the atlas provides an opportunity to model cell state-fate relationships, revealing a lineage-dependent state diversity within functionally related cells and a winding trajectory of developmental state progression. Collectively, this single-cell protein atlas represents a valuable resource for elucidating metazoan embryogenesis at the molecular and systems levels.

Gelsolin and dCryAB act downstream of muscle identity genes and contribute to preventing muscle splitting and branching in Drosophila

A combinatorial code of identity transcription factors (iTFs) specifies the diversity of muscle types in Drosophila. We previously showed that two iTFs, Lms and Ap, play critical role in the identity of a subset of larval body wall muscles, the lateral transverse (LT) muscles. Intriguingly, a small portion of ap and lms mutants displays an increased number of LT muscles, a phenotype that recalls pathological split muscle fibers in human. However, genes acting downstream of Ap and Lms to prevent these aberrant muscle feature are not known. Here, we applied a cell type specific translational profiling (TRAP) to identify gene expression signatures underlying identity of muscle subsets including the LT muscles. We found that Gelsolin (Gel) and dCryAB, both encoding actin-interacting proteins, displayed LT muscle prevailing expression positively regulated by, the LT iTFs. Loss of dCryAB function resulted in LTs with irregular shape and occasional branched ends also observed in ap and lms mutant contexts. In contrast, enlarged and then split LTs with a greater number of myonuclei formed in Gel mutants while Gel gain of function resulted in unfused myoblasts, collectively indicating that Gel regulates LTs size and prevents splitting by limiting myoblast fusion. Thus, dCryAB and Gel act downstream of Lms and Ap and contribute to preventing LT muscle branching and splitting. Our findings offer first clues to still unknown mechanisms of pathological muscle splitting commonly detected in human dystrophic muscles and causing muscle weakness.