Embryonic Mouse Brain Research Articles

Defining Candidate Imprinted loci in Bos taurus.

Using a whole-genome assembly of Bos taurus, I applied my bioinformatics strategy to locate candidate imprinting control regions (ICRs) genome-wide. In mammals, genomic imprinting plays essential roles in embryogenesis. In my strategy, peaks in plots mark the locations of known, inferred, and candidate ICRs. Genes in the vicinity of candidate ICRs correspond to potential imprinted genes. By displaying my datasets on the UCSC genome browser, one could view peak positions with respect to genomic landmarks. I give two examples of candidate ICRs in loci that influence spermatogenesis in bulls: CNNM1 and CNR1. I also give examples of candidate ICRs in loci that influence muscle development: SIX1 and BCL6. By examining the ENCODE data reported for mice, I deduced regulatory clues about cattle. I focused on DNase I hypersensitive sites (DHSs). Such sites reveal accessibility of chromatin to regulators of gene expression. For inspection, I chose DHSs in chromatin from mouse embryonic stem cells (ESCs) ES-E14, mesoderm, brain, heart, and skeletal muscle. The ENCODE data revealed that the SIX1 promoter was accessible to the transcription initiation apparatus in mouse ESCs, mesoderm, and skeletal muscles. The data also revealed accessibility of BCL6 locus to regulatory proteins in mouse ESCs and examined tissues.

Dysregulation of immune and metabolism pathways in maternal immune activation induces an increased risk of autism spectrum disorders

AimsMaternal immune activation (MIA) via infection during pregnancy is known to be an environmental risk factor for neurodevelopmental disorders and the development of autism spectrum disorders (ASD) in the offspring, but it still remains elusive that the molecular relevance between infection-induced abnormal neurodevelopmental events and an increased risk for ASD development. Main methodsFully considering the extremely high genetic heterogeneity of ASD and the universality of risk-gene with minimal effect-sizes, the gene and pathway-based association analysis was performed with the transcriptomic and DNA methylation landscapes of temporal human embryonic brain development and ASD, and the time-course transcriptional profiling of MIA. We conducted the transcriptional profiling of mouse abnormal neurodevelopment two days following induced MIA via LPS injection at E10.5. Key findingsA novel evidence was proved that illustrated altering four immune and metabolism-related risk pathways, including starch and sucrose metabolism, ribosome, protein processing in endoplasmic reticulum, and retrograde endocannabinoid signaling pathway, which were prominent involvement in the process of MIA regulating abnormal fetal brain development to induce an increased risk of ASD. Here, we have observed that almost all key genes within these risk pathways are significantly differentially expressed at embryonic days (E) 10.5–12.5, which is considered to be the optimal coincidence window of mouse embryonic brain development to study the intimate association between MIA and ASD using mouse animal models. SignificanceThere search establishes that MIA causes dysregulation of immune and metabolic pathways, which leads to abnormal embryonic neurodevelopment, thus promoting development of ASD symptoms in offspring.

Identification of novel microRNAs in the embryonic mouse brain using deep sequencing

Many advances in small RNA-seq technology and bioinformatics pipelines have been made recently, permitting the discovery of novel miRNAs in the embryonic day 15.5 (E15.5) mouse brain. We aimed to improve miRNA discovery in this tissue to expand our knowledge of the regulatory networks that underpin normal neurodevelopment, find new candidates for neurodevelopmental disorder aetiology, and deepen our understanding of non-coding RNA evolution. A high-quality small RNA-seq dataset of 458 M reads was generated. An unbiased miRNA discovery pipeline identified fifty putative novel miRNAs, six of which were selected for further validation. A combination of conservation analysis and target functional prediction was used to determine the authenticity of novel miRNA candidates. These findings demonstrate that miRNAs remain to be discovered, particularly if they have the features of other small RNA species.

Abstract 6018: Sequencing of 5-hydroxymethylcytosine to single base resolution

Abstract DNA methylation is an epigenetic regulator of gene expression with important functions in development and diseases such as cancer. The modified cytosines, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are identified using Illumina sequencing. Libraries are generated using either NEBNext® EM-seq™, an enzyme-based workflow, or by using sodium bisulfite conversion. These methods, however, cannot differentiate between 5mC and 5hmC. Distinguishing these modifications is important as there is increased interest in 5hmCs role in regulating gene expression. Methods currently exist to enable discrimination of 5mC and 5hmC, for example, oxBS-seq and TAB-seq. These methods still rely upon sodium bisulfite conversion and have reduced data quality due to fragmentation and loss of DNA. Here we describe an enzymatic method that enables specific detection of 5hmC. 5hmC is detected using two enzymatic steps. Firstly, 5hmCs are glucosylated, which protects them from subsequent deamination by APOBEC. In contrast, cytosines and 5mCs are deaminated resulting in their conversion to uracil and thymine, respectively. During Illumina sequencing deaminated cytosine and 5mC are read as thymine and the glucosylated 5hmC are read as cytosine. Subtractive analysis of 5hmC data from EM-seq data, which detects both 5mC and 5hmC, permits the identification of individual 5mC and 5hmC sites. 5hmC data were generated for inputs of 0.1 ng to 200 ng DNA isolated from E14 mouse embryonic stem cells and human brain. The 5hmC libraries had similar characteristics to EM-seq libraries, including expected insert sizes due to intact DNA molecules, low duplication rates and minimal GC bias. T4147 phage DNA was used as an internal control, as all cytosines are 5-hydroxymethylated, with 98-99% of cytosines identified as 5hmC. 5mC and 5hmC levels were also profiled during E14 cell differentiation for 10 days. Interestingly, 5hmC levels decreased whereas 5mC levels increased particularly during the first five days of differentiation. LC-MS/MS quantification of this same DNA mirrored the changes observed by sequencing. The ability to discriminate between 5mC and 5hmC will provide key insights into the role of these cytosine modifications in development and disease. Citation Format: Daniel J. Evanich, V. K. Chaithanya Ponnaluri, Vaishnavi Panchapakesa, Ariel Erijman, Matthew A. Campbell, Nan Dai, Bradley W. Langhorst, Romualdas Vaisvila, Louise Williams. Sequencing of 5-hydroxymethylcytosine to single base resolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6018.

Viral delivery of L1CAM promotes axonal extensions by embryonic cerebral grafts in mouse brain.

Cell replacement therapy is expected as a new and more radical treatment against brain damage. We previously reported that transplanted human cerebral organoids extend their axons along the corticospinal tract in rodent brains. The axons reached the spinal cord but were still sparse. Therefore, this study optimized the host brain environment by the adeno-associated virus (AAV)-mediated expression of axon guidance proteins in mouse brain. Among netrin-1, SEMA3, and L1CAM, only L1CAM significantly promoted the axonal extension of mouse embryonic brain tissue-derived grafts. L1CAM was also expressed by donor neurons, and this promotion was exerted in a haptotactic manner by their homophilic binding. Primary cortical neurons cocultured on L1CAM-expressing HEK-293 cells supported this mechanism. These results suggest that optimizing the host environment by the AAV-mediated expression of axon guidance molecules enhances the effect of cell replacement therapy.

Spatial epigenome–transcriptome co-profiling of mammalian tissues

Emerging spatial technologies, including spatial transcriptomics and spatial epigenomics, are becoming powerful tools for profiling of cellular states in the tissue context1–5. However, current methods capture only one layer of omics information at a time, precluding the possibility of examining the mechanistic relationship across the central dogma of molecular biology. Here, we present two technologies for spatially resolved, genome-wide, joint profiling of the epigenome and transcriptome by cosequencing chromatin accessibility and gene expression, or histone modifications (H3K27me3, H3K27ac or H3K4me3) and gene expression on the same tissue section at near-single-cell resolution. These were applied to embryonic and juvenile mouse brain, as well as adult human brain, to map how epigenetic mechanisms control transcriptional phenotype and cell dynamics in tissue. Although highly concordant tissue features were identified by either spatial epigenome or spatial transcriptome we also observed distinct patterns, suggesting their differential roles in defining cell states. Linking epigenome to transcriptome pixel by pixel allows the uncovering of new insights in spatial epigenetic priming, differentiation and gene regulation within the tissue architecture. These technologies are of great interest in life science and biomedical research.

Chitosan-alginate porous scaffold incorporated with hydroalcoholic Zingiber officinale Roscoe extract for neural tissue engineering

Context: Preparing a suitable substrate for the culture of neural stem cells and their proliferation in neural tissue engineering is of paramount importance. Aims: To evaluate the effect of the hydroalcoholic Zingiber officinale extract incorporated in the chitosan-alginate scaffold (Chi-Alg-Zo) on nerve tissue. Methods: The porous scaffolds developed in the present study were investigated in terms of their surface properties, chemical interaction, crystallinity, thermal stability, porosity percentage, pore sizes, degradability, and water absorption properties. To this end, the following tests were performed: Field emission scanning electron microscope (FE-SEM), Fourier-transform infrared spectroscopy (FTIR), X-ray powder diffraction (XRD), thermal gravimetric analysis (TGA), porosity based on liquid replacement, Image-J analysis, and water degradation and absorption test. Mouse neural stem/progenitor cells (NS/PCs) were harvested from the embryonic mouse brain. NSCs were seeded on scaffolds incorporated with hydroalcoholic Z. officinale extract. The MTT assay was done for the survival and the proliferation of neural stem/progenitor cells (NS/PCs) on scaffolds. Results: Results indicated the good capacity of Chi-Alg-Zo for proliferation and differentiation into glial (astrocytes and oligodendrocytes) lineages. A suitable surface, which was provided for cellular interaction, led to the advancement of cell survival, connectivity, proliferation, and separation of NSCs. Conclusions: The present study evaluated the separation of stem cells on the scaffold, finding that the expression of the glial fibrillary acidic protein (GFAP) and Oligo4 markers was higher in Chi-Alg scaffolds containing hydroalcoholic Z. officinale extract. Chi-Alg-Zo scaffolds could be suitable candidates for neural tissue engineering.

Disorder of Golgi Apparatus Precedes Anoxia-Induced Pathology of Mitochondria.

Mitochondrial malfunction and morphologic disorganization have been observed in brain cells as part of complex pathological changes. However, it is unclear what may be the role of mitochondria in the initiation of pathologic processes or if mitochondrial disorders are consequences of earlier events. We analyzed the morphologic reorganization of organelles in an embryonic mouse brain during acute anoxia using an immunohistochemical identification of the disordered mitochondria, followed by electron microscopic three-dimensional (3D) reconstruction. We found swelling of the mitochondrial matrix after 3 h anoxia and probable dissociation of mitochondrial stomatin-like protein 2 (SLP2)-containing complexes after 4.5 h anoxia in the neocortex, hippocampus, and lateral ganglionic eminence. Surprisingly, deformation of the Golgi apparatus (GA) was detected already after 1 h of anoxia, when the mitochondria and other organelles still had a normal ultrastructure. The disordered GA showed concentrical swirling of the cisternae and formed spherical onion-like structures with the trans-cisterna in the center of the sphere. Such disturbance of the Golgi architecture likely interferes with its function for post-translational protein modification and secretory trafficking. Thus, the GA in embryonic mouse brain cells may be more vulnerable to anoxic conditions than the other organelles, including mitochondria.

Differential modularity of the mammalian Engrailed 1 enhancer network directs sweat gland development.

Enhancers are context-specific regulators of expression that drive biological complexity and variation through the redeployment of conserved genes. An example of this is the enhancer-mediated control of Engrailed 1 (EN1), a pleiotropic gene whose expression is required for the formation of mammalian eccrine sweat glands. We previously identified the En1 candidate enhancer (ECE) 18 cis-regulatory element that has been highly and repeatedly derived on the human lineage to potentiate ectodermal EN1 and induce our species' uniquely high eccrine gland density. Intriguingly, ECE18 quantitative activity is negligible outside of primates and ECE18 is not required for En1 regulation and eccrine gland formation in mice, raising the possibility that distinct enhancers have evolved to modulate the same trait. Here we report the identification of the ECE20 enhancer and show it has conserved functionality in mouse and human developing skin ectoderm. Unlike ECE18, knock-out of ECE20 in mice reduces ectodermal En1 and eccrine gland number. Notably, we find ECE20, but not ECE18, is also required for En1 expression in the embryonic mouse brain, demonstrating that ECE20 is a pleiotropic En1 enhancer. Finally, that ECE18 deletion does not potentiate the eccrine phenotype of ECE20 knock-out mice supports the secondary incorporation of ECE18 into the regulation of this trait in primates. Our findings reveal that the mammalian En1 regulatory machinery diversified to incorporate both shared and lineage-restricted enhancers to regulate the same phenotype, and also have implications for understanding the forces that shape the robustness and evolvability of developmental traits.

SMPD4 regulates mitotic nuclear envelope dynamics and its loss causes microcephaly and diabetes.

Biallelic loss-of-function variants in SMPD4 cause a rare and severe neurodevelopmental disorder with progressive congenital microcephaly and early death. SMPD4 encodes a sphingomyelinase that hydrolyses sphingomyelin into ceramide at neutral pH and can thereby affect membrane lipid homeostasis. SMPD4 localizes to the membranes of the endoplasmic reticulum and nuclear envelope and interacts with nuclear pore complexes (NPC). We refine the clinical phenotype of loss-of-function SMPD4 variants by describing five individuals from three unrelated families with longitudinal data due to prolonged survival. All individuals surviving beyond infancy developed insulin-dependent diabetes, besides presenting with a severe neurodevelopmental disorder and microcephaly, making diabetes one of the most frequent age-dependent non-cerebral abnormalities. We studied the function of SMPD4 at the cellular and organ levels. Knock-down of SMPD4 in human neural stem cells causes reduced proliferation rates and prolonged mitosis. Moreover, SMPD4 depletion results in abnormal nuclear envelope breakdown and reassembly during mitosis and decreased post-mitotic NPC insertion. Fibroblasts from affected individuals show deficient SMPD4-specific neutral sphingomyelinase activity, without changing (sub)cellular lipidome fractions, which suggests a local function of SMPD4 on the nuclear envelope. In embryonic mouse brain, knockdown of Smpd4 impairs cortical progenitor proliferation and induces premature differentiation by altering the balance between neurogenic and proliferative progenitor cell divisions. We hypothesize that, in individuals with SMPD4-related disease, nuclear envelope bending, which is needed to insert NPCs in the nuclear envelope, is impaired in the absence of SMPD4 and interferes with cerebral corticogenesis and survival of pancreatic beta cells.

CD206+ macrophages transventricularly infiltrate the early embryonic cerebral wall to differentiate into microglia.

The relationships between tissue-resident microglia and early macrophages, especially their lineage segregation outside the yolk sac, have been recently explored, providing a model in which a conversion from macrophages seeds microglia during brain development. However, spatiotemporal evidence to support such microglial seeding in situ and to explain how it occurs has not been obtained. By cell tracking via slice culture, intravital imaging, and Flash tag-mediated or genetic labeling, we find that intraventricular CD206+ macrophages, which are abundantly observed along the inner surface of the mouse cerebral wall, frequently enter the pallium at embryonic day 12. Immunofluorescence of the tracked cells show that postinfiltrative macrophages in the pallium acquire microglial properties while losing the CD206+ macrophage phenotype. We also find that intraventricular macrophages are supplied transepithelially from the roof plate. This study demonstrates that the "roof plate→ventricle→pallium" route is an essential path for microglial colonization into the embryonic mouse brain.

Microglia isolation from aging mice for cell culture: A beginner's guide.

Microglia, the innate immune cell of the central nervous system, play significant roles in brain development, maintenance, homeostasis, and neuroinflammation. Although numerous methods have been developed to isolate microglia from embryonic or postnatal mouse brains, still major difficulties exist in isolating microglia from adult mice, often resulting in low yield and risk of cellular activation. Therefore, there is a need for a more efficient method to isolate pure and high-yield microglia from adult mice to study various neurodegenerative diseases. The aim of this study was to develop a fully functional protocol for the isolation of microglia by comparing different protocols. We investigated the efficacy of three protocols in terms of cell yield, purity, cellular activation, cellular aging, and migration properties and proposed the modified protocol (PROTOCOL 1), which provides an optimal yield of functional microglial cells with a minimum of material and equipment and allows young researchers with little experience to isolate microglia and helps them to delve deeper into the world of neuroscience.

A region-dependent allele-biased expression of Dopa decarboxylase in mouse brain.

Genomic imprinting is an epigenetic event in which genes are expressed only from either the paternal or maternal allele. Dopa decarboxylase (Ddc), is an imprinted gene that encodes an enzyme which catalyzes the conversion of L-dopa to dopamine. Although Ddc has been reported to be paternally expressed in embryonic and neonatal hearts, its expression pattern in the brain has been controversial. To visualize Ddc-expressing neurons, we established a knock-in mouse carrying a humanized Kusabira orange 1 (hKO1) reporter cassette at the Ddc locus (Ddc-hKO1). The expression of Ddc-hKO1 was detected in all known Ddc-positive cells in the brains of embryonic, neonatal, adult, and aged mice. We further developed an efficient purification method for Ddc-hKO1-positive neurons using a cell sorter. RNA sequencing analysis confirmed the enrichment of dopaminergic, serotonergic and cholinergic neurons in Ddc-hKO1-positive cell population recovered using this method. A detailed analysis of Ddc-hKO1 paternally and maternally derived heterozygous mice combined with immunostaining revealed that Ddc was preferentially expressed from the maternal allele in ventral tegmented area (VTA), substantia nigra pars compacta (SNc), and retrorubral field (RRF); while it was expressed from both alleles in dorsal raphe nucleus (DR). These results indicate that Ddc exhibit an allele-specific expression pattern in different brain regions, presumably reflecting the diverse regulatory mechanisms of imprinting.

Decreasing Wapl dosage partially corrects embryonic growth and brain transcriptome phenotypes in Nipbl+/- embryos.

Cohesin rings interact with DNA and modulate the expression of thousands of genes. NIPBL loads cohesin onto chromosomes, and WAPL takes it off. Haploinsufficiency for NIPBL causes a developmental disorder, Cornelia de Lange syndrome (CdLS), that is modeled by Nipbl+/- mice. Mutations in WAPL have not been shown to cause disease or gene expression changes in mammals. Here, we show dysregulation of >1000 genes in WaplΔ/+ embryonic mouse brain. The patterns of dysregulation are highly similar in Wapl and Nipbl heterozygotes, suggesting that Wapl mutations may also cause human disease. Since WAPL and NIPBL have opposite effects on cohesin's association with DNA, we asked whether decreasing Wapl dosage could correct phenotypes seen in Nipbl+/- mice. Gene expression and embryonic growth are partially corrected, but perinatal lethality is not. Our data are consistent with the view that cohesin dynamics play a key role in regulating gene expression.

CUT & RUN to Profile Chromatin-Bound Proteins in Primary Mouse Neural Progenitor Cells.

Cleavage under targets and release using nuclease (CUT & RUN) is an innovative method to profile histone modifications and chromatin-bound proteins genome-wide. CUT & RUN offers two distinct advantages of requiring much fewer cells and providing strong signal-to-noise ratios in deep-sequencing data. Here, we describe a workflow starting from dissociation and sorting of mouse embryonic brains, CUT & RUN, and DNA library preparation to deep sequencing. With our workflow, researchers can obtain high-quality sequencing data to profile histones and chromatin-associated proteins by using as few as 100,000 neural progenitor cells (NPCs).

Dynamic changes in spatiotemporal transcriptome reveal maternal immune dysregulation of autism spectrum disorder

Maternal immune activation (MIA) during pregnancy is known to be an environmental risk factor for neurodevelopment and autism spectrum disorder (ASD). However, it is unclear at which fetal brain developmental windows and regions MIA induces ASD-related neurodevelopmental transcriptional abnormalities. The non-chasm differentially expressed genes (DEGs) involved in MIA inducing ASD during fetal brain developmental windows were identified by performing the differential expression analysis and comparing the common DEGs among MIA at four different gestational development windows, ASD with multiple brain regions from human patients and mouse models, and human and mouse embryonic brain developmental trajectory. The gene set and functional enrichment analyses were performing to identify MIA dysregulated ASD-related the fetal neurodevelopmental windows and brain regions and function annotations. Additionally, the networks were constructed using Cytoscape for visualization. MIA at E12.5 and E14.5 increased the risk of distinct brain regions for ASD. MIA-driven transcriptional alterations of non-chasm DEGs, during the coincidence brain developmental windows between human and mice, involving ASD-relevant synaptic components, as well as immune- and metabolism-related functions and pathways. Furthermore, a great number of non-chasm brain development-, immune-, and metabolism-related DEGs were overlapped in at least two existing ASD-associated databases, suggesting that the others could be considered as the candidate targets to construct the model mice for explaining the pathological changes of ASD when environmental factors (MIA) and gene mutation effects co-occur. Overall, our search supported that transcriptome-based MIA dysregulated the brain development-, immune-, and metabolism-related non-chasm DEGs at specific embryonic brain developmental window and region, leading to abnormal embryonic neurodevelopment, to induce the increasing risk of ASD.

Spatially resolved epigenomic profiling of single cells in complex tissues

The recent development of spatial omics methods has enabled single-cell profiling of the transcriptome and 3D genome organization with high spatial resolution. Expanding the repertoire of spatial omics tools, a spatially resolved single-cell epigenomics method will accelerate understanding of the spatial regulation of cell and tissue functions. Here, we report a method for spatially resolved epigenomic profiling of single cells using in situ tagmentation and transcription followed by multiplexed imaging. We demonstrated the ability to profile histone modifications marking active promoters, putative enhancers, and silent promoters in individual cells, and generated high-resolution spatial atlas of hundreds of active promoters and putative enhancers in embryonic and adult mouse brains. Our results suggested putative promoter-enhancer pairs and enhancer hubs regulating developmentally important genes. We envision this approach will be generally applicable to spatial profiling of epigenetic modifications and DNA-binding proteins, advancing our understanding of how gene expression is spatiotemporally regulated by the epigenome.

Immediate protein expression from exogenous mRNAs in embryonic brain

mRNA vaccines for SARS-CoV-2 have been widely used and saving millions of people in the world. How efficiently proteins are produced from exogenous mRNAs in the embryonic brain, however, is less known. Here we show that protein expression occurs highly efficiently in neural stem cells, in a very narrow time window after mRNA electroporation in the embryonic mouse brain, where plasmids have been successfully transfected. Protein expression is detected 1 h and 12 h after the electroporation of mRNAs and plasmids, respectively. The delivery of exogenous mRNAs may be useful for not only vaccines but also functional analysis in the brain.

Breasi-CRISPR: an efficient genome-editing method to interrogate protein localization and protein-protein interactions in the embryonic mouse cortex.

In developing tissues, knowing the localization and interactors of proteins of interest is key to understanding their function. Here, we describe the Breasi-CRISPR approach (Brain Easi-CRISPR), combining Easi-CRISPR with in utero electroporation to tag endogenous proteins within embryonic mouse brains. Breasi-CRISPR enables knock-in of both short and long epitope tag sequences with high efficiency. We visualized epitope-tagged proteins with varied expression levels, such as ACTB, LMNB1, EMD, FMRP, NOTCH1 and RPL22. Detection was possible by immunohistochemistry as soon as 1 day after electroporation and we observed efficient gene editing in up to 50% of electroporated cells. Moreover, tagged proteins could be detected by immunoblotting in lysates from individual cortices. Next, we demonstrated that Breasi-CRISPR enables the tagging of proteins with fluorophores, allowing visualization of endogenous proteins by live imaging in organotypic brain slices. Finally, we used Breasi-CRISPR to perform co-immunoprecipitation mass-spectrometry analyses of the autism-related protein FMRP to discover its interactome in the embryonic cortex. Together, these data demonstrate that Breasi-CRISPR is a powerful tool with diverse applications that will propel the understanding of protein function in neurodevelopment.

Nonparametric single-cell multiomic characterization of trio relationships between transcription factors, target genes, and cis-regulatory regions.

The epigenetic control of gene expression is highly cell-type and context specific. Yet, despite its complexity, gene regulatory logic can be broken down into modular components consisting of a transcription factor (TF) activating or repressing the target gene expression through its binding to a cis-regulatory region. We propose a nonparametric approach, TRIPOD, to detect and characterize the three-way relationships between a TF, its target gene, and the accessibility of the TF's binding site using single-cell RNA and ATAC multiomic data. We apply TRIPOD to interrogate the cell-type-specific regulatory logic in peripheral blood mononuclear cells and contrast our results to detections from enhancer databases, cis-eQTL studies, ChIP-seq experiments, and TF knockdown/knockout studies. We then apply TRIPOD to mouse embryonic brain data and identify regulatory relationships, validated by ChIP-seq and PLAC-seq. Finally, we demonstrate TRIPOD on the SHARE-seq data of differentiating mouse hair follicle cells and identify lineage-specific regulation supported by histone marks and super-enhancer annotations. A record of this paper's transparent peer review process is included in the supplemental information.