Transgenic Tools Research Articles

Strength of interactions in the Notch gene regulatory network determines patterning and fate in the notochord.

Development of multicellular organisms requires the generation of gene expression patterns that determines cell fate and organ shape. Groups of genetic interactions known as Gene Regulatory Networks (GRNs) play a key role in the generation of such patterns. However, how the topology and parameters of GRNs determine patterning in vivo remains unclear due to the complexity of most experimental systems. To address this, we use the zebrafish notochord, an organ where coin-shaped precursor cells are initially arranged in a simple unidimensional geometry. These cells then differentiate into vacuolated and sheath cells. Using newly developed transgenic tools together with in vivo imaging, we identify jag1a and her6/her9 as the main components of a Notch GRN that generates a lateral inhibition pattern and determines cell fate. Making use of this experimental system and mathematical modeling we show that lateral inhibition patterning is promoted when ligand-receptor interactions are stronger within the same cell than in neighboring cells. Altogether, we establish the zebrafish notochord as an experimental system to study pattern generation, and identify and characterize how the properties of GRNs determine self-organization of gene patterning and cell fate.

Integrated Behavioral, Genetic and Brain Circuit Visualization Methods to Unravel Functional Anatomy of Zebrafish Amygdala.

The mammalian amygdala is a complex forebrain structure consisting of a heterogeneous group of nuclei derived from the pallial and subpallial telencephalon. It plays a critical role in a broad range of behaviors such as emotion, cognition, and social behavior; within the amygdala each nucleus has a distinct role in these behavioral processes. Topological, hodological, molecular, and functional studies suggest the presence of an amygdala-like structure in the zebrafish brain. It has been suggested that the pallial amygdala homolog corresponds to the medial zone of the dorsal telencephalon (Dm) and the subpallial amygdala homolog corresponds to the nuclei in the ventral telencephalon located close to and topographically basal to Dm. However, these brain regions are broad and understanding the functional anatomy of the zebrafish amygdala requires investigating the role of specific populations of neurons in brain function and behavior. In zebrafish, the highly efficient Tol2 transposon-mediated transgenesis method together with the targeted gene expression by the Gal4-UAS system has been a powerful tool in labeling, visualizing, and manipulating the function of specific cell types in the brain. The transgenic resource combined with neuronal activity imaging, optogenetics, pharmacology, and quantitative behavioral analyses enables functional analyses of neuronal circuits. Here, we review earlier studies focused on teleost amygdala anatomy and function and discuss how the transgenic resource and tools can help unravel the functional anatomy of the zebrafish amygdala.

Characterizing the Drosophila Orthologs of Phosphoglycolate Phosphatase and Pyridoxal Phosphate Phosphatase

Phosphoglycolate phosphatase (PGP) and pyridoxal phosphate phosphatase (PDXP) are members of the haloacid dehalogenase (HAD) family of non‐protein phosphatases. PGP plays an important role in central carbon metabolism by regulating glycolysis, the pentose phosphate pathway, and lipid metabolism. PGP also acts as a metabolite repair enzyme by cleaning up toxic side products from glyceraldehyde 3‐phosphate dehydrogenase, pyruvate kinase, and DNA repair. PDXP regulates pyridoxal phosphate (PLP; active vitamin B6), a cofactor for 140+ enzymes. Although previous studies have characterized PGP and PDXP in mammals, their fly orthologs remain unidentified. Here, we use mutagenesis and transgenic tools to investigate the putative PGP and PDXP orthologs in the fruit fly D. melanogaster, CG5567 and CG5577. We found that CG5567 mutants survive to adulthood, unlike mice lacking PGP, but they are sensitive to ethylene glycol and have increased triglyceride levels, consistent with previous studies showing that mammalian PGP de‐phosphorylates phosphoglycolate and glycerol‐3‐phosphate. CG5567 mutants are also more resistant to starvation. CG5577 mutants also survive to adulthood but do not share these phenotypes. However, flies overexpressing CG5577 die as late third instar larvae. We are currently testing if we can rescue the CG5577 overexpression phenotype by increasing PLP levels. Additionally, we are using mass spectrometry to analyze substrate and product levels to confirm that CG5567 and CG5577 are functioning as PGP and PDXP orthologs. Our results demonstrate that D. melanogaster is a useful model for investigating the function and evolution of PGP and PDXP.

Transgenic tools for targeted chromosome rearrangements allow construction of balancer chromosomes in non-melanogaster Drosophila species.

Perhaps the most valuable single set of resources for genetic studies of Drosophila melanogaster is the collection of multiply inverted chromosomes commonly known as balancer chromosomes. Balancers prevent the recovery of recombination exchange products within genomic regions included in inversions and allow perpetual maintenance of deleterious alleles in living stocks and the execution of complex genetic crosses. Balancer chromosomes have been generated traditionally by exposing animals to ionizing radiation and screening for altered chromosome structure or for unusual marker segregation patterns. These approaches are tedious and unpredictable, and have failed to produce the desired products in some species. Here, I describe transgenic tools that allow targeted chromosome rearrangements in Drosophila species. The key new resources are engineered reporter genes containing introns with yeast recombination sites and enhancers that drive fluorescent reporter genes in multiple body regions. These tools were used to generate a doubly inverted chromosome 3R in Drosophila simulans that serves as an effective balancer chromosome.

Regeneration of the larval sea star nervous system by wounding induced respecification to the Sox2 lineage.

The ability to restore lost body parts following traumatic injury is a fascinating area of biology that challenges current understanding of the ontogeny of differentiation. The origin of new cells needed to regenerate lost tissue, and whether they are pluripotent or have de- or trans-differentiated, remains one of the most important open questions . Additionally, it is not known whether developmental gene regulatory networks are reused or whether regeneration specific networks are deployed. Echinoderms, including sea stars, have extensive ability for regeneration, however, the technologies for obtaining transgenic echinoderms are limited and tracking cells involved in regeneration, and thus identifying the cellular sources and potencies has proven challenging. In this study, we develop new transgenic tools to follow the fate of populations of cells in the regenerating larva of the sea star Patiria miniata. We show that the larval serotonergic nervous system can regenerate following decapitation. Using a BAC-transgenesis approach we show that expression of the pan ectodermal marker, sox2, is induced in previously sox2 minus cells , even when cell division is inhibited. sox2+ cells give rise to new sox4+ neural precursors that then proceed along an embryonic neurogenesis pathway to reform the anterior nervous systems. sox2+ cells contribute to only neural and ectoderm lineages, indicating that these progenitors maintain their normal, embryonic lineage restriction. This indicates that sea star larval regeneration uses a combination of existing lineage restricted stem cells, as well as respecification of cells into neural lineages, and at least partial reuse of developmental GRNs to regenerate their nervous system.

The crustacean model Parhyale hawaiensis.

Arthropods are the most abundant and diverse animals on earth. Among them, pancrustaceans are an ancient and morphologically diverse group, comprising a wide range of aquatic and semi-aquatic crustaceans as well as the insects, which emerged from crustacean ancestors to colonize most terrestrial habitats. Within insects, Drosophila stands out as one of the most powerful animal models, making major contributions to our understanding of development, physiology and behavior. Given these attributes, crustaceans provide a fertile ground for exploring biological diversity through comparative studies. However, beyond insects, few crustaceans are developed sufficiently as experimental models to enable such studies. The marine amphipod Parhyale hawaiensis is currently the best established crustacean system, offering year-round accessibility to developmental stages, transgenic tools, genomic resources, and established genetics and imaging approaches. The Parhyale research community is small but diverse, investigating the evolution of development, regeneration, aspects of sensory biology, chronobiology, bioprocessing and ecotoxicology.

A novel gene trap line for visualization and manipulation of erbb3b+ neural crest and glial cells in zebrafish

Glia are a diverse and essential cell type in the vertebrate nervous system. Transgenic tools and fluorescent reporter lines are critical resources to investigate how glial subtypes develop and function. However, despite the many lines available in zebrafish, the community still lacks the ability to label all unique stages of glial development and specific subpopulations of cells. To address this issue, we screened zebrafish gene and enhancer trap lines to find a novel reporter for peripheral glial subtypes. From these, we generated the gSAIzGFFD37A transgenic line that expresses GFP in neural crest cells and central and peripheral glia. We found that the gene trap construct is located within an intron of erbb3b, a gene essential for glial development. Additionally, we confirmed that GFP+ ​cells express erbb3b along with sox10, a known glial marker. From our screen, we have identified the gSAIzGFFD37A line as a novel and powerful tool for studying glia in the developing zebrafish, as well as a new resource to manipulate erbb3b+ ​cells.

Stress-regulated elements in Lotus spp., as a possible starting point to understand signalling networks and stress adaptation in legumes.

Although legumes are of primary economic importance for human and livestock consumption, the information regarding signalling networks during plant stress response in this group is very scarce. Lotus japonicus is a major experimental model within the Leguminosae family, whereas L. corniculatus and L. tenuis are frequent components of natural and agricultural ecosystems worldwide. These species display differences in their perception and response to diverse stresses, even at the genotype level, whereby they have been used in many studies aimed at achieving a better understanding of the plant stress-response mechanisms. However, we are far from the identification of key components of their stress-response signalling network, a previous step for implementing transgenic and editing tools to develop legume stress-resilient genotypes, with higher crop yield and quality. In this review we scope a body of literature, highlighting what is currently known on the stress-regulated signalling elements so far reported in Lotus spp. Our work includes a comprehensive review of transcription factors chaperones, redox signals and proteins of unknown function. In addition, we revised strigolactones and genes regulating phytochelatins and hormone metabolism, due to their involvement as intermediates in several physiological signalling networks. This work was intended for a broad readership in the fields of physiology, metabolism, plant nutrition, genetics and signal transduction. Our results suggest that Lotus species provide a valuable information platform for the study of specific protein-protein (PPI) interactions, as a starting point to unravel signalling networks underlying plant acclimatation to bacterial and abiotic stressors in legumes. Furthermore, some Lotus species may be a source of genes whose regulation improves stress tolerance and growth when introduced ectopically in other plant species.

Characterization and manipulation of Corticothalamic neurons in associative cortices using Syt6‐Cre transgenic mice

Corticothalamic interactions between associative cortices and higher order thalamic nuclei are involved in high-cognitive functions such as decision-making and working memory. Corticothalamic neurons (CTn) in the prefrontal cortex and other associative areas have been much less studied than their counterparts in the primary sensory areas. The availability of characterized transgenic tools to study CTn in associative areas will facilitate their study and contribute to overcome the scarcity of data about their properties, network dynamics, and contribution to cognitive functions. Here, we characterized the Syt6-Cre (KI148Gsat/Mmud) transgenic mouse line, by tracking expression of a Cre-mediated reporter. In this line, Cre-reporter is strongly expressed in the prefrontal, motor, cingulate, and retrosplenial cortices, as well as in other brain areas including the cerebellum and the olfactory tubercle. Cortical expression starts embryonically and reaches the adult expression pattern by postnatal day 15. In the cortex, Cre-reporter is expressed by layer 6-CTn and by layer 5-CTn to a lesser extent. We quantified Syt6-Cre+ CTn axon varicosities to estimate the distribution and density of putative corticothalamic driver and modulator inputs to thalamic nuclei in the medial, midline, intralaminar, anterior, and motor groups. Also, we characterized the effect of optogenetic stimulation of Syt6-Cre+ neurons in the activity of the prefrontal cortex. CTn stimulation in the prefrontal cortex induces an oscillatory activity in the local field potential that resembles the cortical downstates typically observed during slow-wave sleep or quiet wake.

Expression of Microbial Enzymes in Mammalian Astrocytes to Modulate Lactate Release.

Astrocytes support and modulate neuronal activity through the release of L-lactate. The suggested roles of astrocytic lactate in the brain encompass an expanding range of vital functions, including central control of respiration and cardiovascular performance, learning, memory, executive behaviour and regulation of mood. Studying the effects of astrocytic lactate requires tools that limit the release of lactate selectively from astrocytes. Here, we report the validation in vitro of novel molecular constructs derived from enzymes originally found in bacteria, that when expressed in astrocytes, interfere with lactate handling. When lactate 2-monooxygenase derived from M. smegmatis was specifically expressed in astrocytes, it reduced intracellular lactate pools as well as lactate release upon stimulation. D-lactate dehydrogenase derived from L. bulgaricus diverts pyruvate towards D-lactate production and release by astrocytes, which may affect signalling properties of lactate in the brain. Together with lactate oxidase, which we have previously described, this set of transgenic tools can be employed to better understand astrocytic lactate release and its role in the regulation of neuronal activity in different behavioural contexts.

Cocaine-induced projection-specific and cell type-specific adaptations in the nucleus accumbens.

Cocaine craving, seeking, and relapse are mediated, in part, by cocaine-induced adaptive changes in the brain reward circuits. The nucleus accumbens (NAc) integrates and prioritizes different emotional and motivational inputs to the reward system by processing convergent glutamatergic projections from the medial prefrontal cortex, basolateral amygdala, ventral hippocampus, and other limbic and paralimbic brain regions. Medium spiny neurons (MSNs) are the principal projection neurons in the NAc, which can be divided into two major subpopulations, namely dopamine receptor D1- versus D2-expressing MSNs, with complementing roles in reward-associated behaviors. After cocaine experience, NAc MSNs exhibit complex and differential adaptations dependent on cocaine regimen, withdrawal time, cell type, location (NAc core versus shell), and related input and output projections, or any combination of these factors. Detailed characterization of these cellular adaptations has been greatly facilitated by the recent development of optogenetic/chemogenetic techniques combined with transgenic tools. In this review, we discuss such cell type- and projection-specific adaptations induced by cocaine experience. Specifically, (1) D1 and D2 NAc MSNs frequently exhibit differential adaptations in spinogenesis, glutamatergic receptor trafficking, and intrinsic membrane excitability, (2) cocaine experience differentially changes the synaptic transmission at different afferent projections onto NAc MSNs, (3) cocaine-induced NAc adaptations exhibit output specificity, e.g., being different at NAc-ventral pallidum vs. NAc-ventral tegmental area synapses, and (4) the input, output, subregion, and D1/D2 cell type may together determine cocaine-induced circuit plasticity in the NAc. In light of the projection and cell-type specificity, we also briefly discuss ensemble and circuit mechanisms contributing to cocaine craving and relapse after drug withdrawal.

Imaging the Efficiency of Poly(3,4-ethylenedioxythiophene) Doped with Acid-Functionalized Carbon Nanotube and Iridium Oxide Electrode Coatings for Microstimulation.

Electrical microstimulation has shown promise in restoring neural deficits in humans. Electrodes coated with materials like the conducting polymer poly(3,4‐ethylenedioxythiophene) doped with acid‐functionalized carbon nanotubes (PEDOT/CNTs, or PC) exhibit superior charge injection than traditional metals like platinum. However, the stimulation performance of PC remains to be fully characterized. Advanced imaging techniques and transgenic tools allow for real‐time observations of neural activity in vivo. Herein, microelectrodes coated with PC and iridium oxide (IrOx) (a commonly used high‐charge‐injection material) are implanted in GCaMP6s mice and electrical stimulation is applied while imaging neuronal calcium responses. Results show that PC‐coated electrodes stimulate more intense and broader GCaMP responses than IrOx. Two‐photon microscopy reveals that PC‐coated electrodes activate significantly more neuronal soma and neuropil than IrOx‐coated electrodes in constant‐voltage stimulation and significantly more neuronal soma in constant‐current stimulation. Furthermore, with the same injected charge, both materials activate more spatially confined neural elements with shorter pulses than longer pulses, providing a means to tune stimulation selectivity. Finite element analyses reveal that the PC coating creates a denser and nonuniform electric field, increasing the likelihood of activating nearby neural elements. PC coating can significantly improve energy efficiency for electrical stimulation applications.

Slc1a3-2A-CreERT2 mice reveal unique features of Bergmann glia and augment a growing collection of Cre drivers and effectors in the 129S4 genetic background

Genetic variation is a primary determinant of phenotypic diversity. In laboratory mice, genetic variation can be a serious experimental confounder, and thus minimized through inbreeding. However, generalizations of results obtained with inbred strains must be made with caution, especially when working with complex phenotypes and disease models. Here we compared behavioral characteristics of C57Bl/6—the strain most widely used in biomedical research—with those of 129S4. In contrast to 129S4, C57Bl/6 demonstrated high within-strain and intra-litter behavioral hyperactivity. Although high consistency would be advantageous, the majority of disease models and transgenic tools are in C57Bl/6. We recently established six Cre driver lines and two Cre effector lines in 129S4. To augment this collection, we genetically engineered a Cre line to study astrocytes in 129S4. It was validated with two Cre effector lines: calcium indicator gCaMP5g-tdTomato and RiboTag—a tool widely used to study cell type-specific translatomes. These reporters are in different genomic loci, and in both the Cre was functional and astrocyte-specific. We found that calcium signals lasted longer and had a higher amplitude in cortical compared to hippocampal astrocytes, genes linked to a single neurodegenerative disease have highly divergent expression patterns, and that ribosome proteins are non-uniformly expressed across brain regions and cell types.

Organization and emergence of a mixed GABA-glycine retinal circuit that provides inhibition to mouse ON-sustained alpha retinal ganglion cells

SUMMARYIn the retina, amacrine interneurons inhibit retinal ganglion cell (RGC) dendrites to shape retinal output. Amacrine cells typically use either GABA or glycine to exert synaptic inhibition. Here, we combined transgenic tools with immunohistochemistry, electrophysiology, and 3D electron microscopy to determine the composition and organization of inhibitory synapses across the dendritic arbor of a well-characterized RGC type in the mouse retina: the ON-sustained alpha RGC. We find mixed GABA-glycine receptor synapses across this RGC type, unveiling the existence of “mixed” inhibitory synapses in the retinal circuit. Presynaptic amacrine boutons with dual release sites are apposed to ON-sustained alpha RGC postsynapses. We further reveal the sequence of postsynaptic assembly for these mixed synapses: GABA receptors precede glycine receptors, and a lack of early GABA receptor expression impedes the recruitment of glycine receptors. Together our findings uncover the organization and developmental profile of an additional motif of inhibition in the mammalian retina.

A suite of new Dre recombinase drivers markedly expands the ability to perform intersectional genetic targeting.

The use of the dual recombinase-mediated intersectional genetic approach involving Cre-loxP and Dre-rox has significantly enhanced the precision of invivo lineage tracing, as well as gene manipulation. However, this approach is limited by the small number of Dre recombinase driver constructs available. Here, we developed more than 70 new intersectional drivers to better target diverse cell lineages. To highlight their applicability, we used these new tools to study the invivo adipogenic fate of perivascular progenitors, which revealed that PDGFRa+ but not PDGFRa-PDGFRb+ perivascular cells are the endogenous progenitors of adult adipocytes. In addition to lineage tracing, we used members of this new suite of drivers to more specifically knock out genes in complex tissues, such as white adipocytes and lymphatic vessels, that heretofore cannot be selectively targeted by conventional Cre drivers alone. In summary, these new transgenic tools expand the intersectional genetic approach while enhancing its precision.

Identification of oxygen-sensitive neuroepithelial cells through an endogenous reporter gene in larval and adult transgenic zebrafish.

In teleost fish, specialized oxygen (O2) chemoreceptors, called neuroepithelial cells (NECs), are found in the gill epithelium in adults. During development, NECs are present in the skin before the formation of functional gills. NECs are known for retaining the monoamine neurotransmitter, serotonin (5-HT) and are conventionally identified through immunoreactivity with antibodies against 5-HT or synaptic vesicle protein (SV2). However, identification of NECs in live tissue and isolated cell preparations has been challenging due to the lack of a specific marker. The present study explored the use of the transgenic zebrafish, ETvmat2:GFP, which expresses green fluorescent protein (GFP) under the control of the vesicular monoamine transporter 2 (vmat2) regulatory element, to identify NECs. Using immunohistochemistry and confocal microscopy, we confirmed that the endogenous GFP in ETvmat2:GFP labelled serotonergic NECs in the skin of larvae and in the gills of adults. NECs of the gill filaments expressed a higher level of endogenous GFP compared with other cells. The endogenous GFP also labelled intrabranchial neurons of the gill filaments. Flow cytometric analysis demonstrated that filamental NECs could be distinguished from other dissociated gill cells based on high GFP expression alone. Acclimation to 2weeks of severe hypoxia (PO2=35mmHg) induced an increase in filamental NEC frequency, size and GFP gene expression. Here we present for the first time a transgenic tool that labels O2 chemoreceptors in an aquatic vertebrate and its use in high-throughput experimentation.

Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing

Autonomous regulation of the intestine requires the combined activity of functionally distinct neurons of the enteric nervous system (ENS). However, the variety of enteric neuron types and how they emerge during development remain largely unknown. Here, we define a molecular taxonomy of twelve enteric neuron classes within the myenteric plexus of the mouse small intestine using single cell RNA-sequencing. We present cell-cell communication features, histochemical markers for motor, sensory, and interneurons together with transgenic tools for class-specific targeting. Transcriptome analysis of embryonic ENS uncovers a novel principle of neuronal diversification, where two neuron classes arise through a binary neurogenic branching, and all other identities emerge through subsequent post-mitotic differentiation. We identify generic and class-specific transcriptional regulators and functionally connect Pbx3 to a post-mitotic fate transition. Our results offer a conceptual and molecular resource for dissecting ENS circuits, and predicting key regulators for directed differentiation of distinct enteric neuron classes.

A Subpopulation of Prefrontal Cortical Neurons Is Required for Social Memory

BackgroundThe medial prefrontal cortex (mPFC) is essential for social behaviors, yet whether and how it encodes social memory remains unclear. MethodsWe combined whole-cell patch recording, morphological analysis, optogenetic/chemogenetic manipulation, and the TRAP (targeted recombination in active populations) transgenic mouse tool to study the social-associated neural populations in the mPFC. ResultsFos-TRAPed prefrontal social-associated neurons are excitatory pyramidal neurons with relatively small soma sizes and thin-tufted apical dendrite. These cells exhibit intrinsic firing features of dopamine D1 receptor–like neurons, show persisting firing pattern after social investigation, and project dense axons to nucleus accumbens. In behaving TRAP mice, selective inhibition of prefrontal social-associated neurons does not affect social investigation but does impair subsequent social recognition, whereas optogenetic reactivation of their projections to the nucleus accumbens enables recall of a previously encountered but “forgotten” mouse. Moreover, chemogenetic activation of mPFC-to-nucleus accumbens projections ameliorates MK-801–induced social memory impairments. ConclusionsOur results characterize the electrophysiological and morphological features of social-associated neurons in the mPFC and indicate that these Fos-labeled, social-activated prefrontal neurons are necessary and sufficient for social memory.

Dysregulation of the Synaptic Cytoskeleton in the PFC Drives Neural Circuit Pathology, Leading to Social Dysfunction

Psychiatric disorders are highly heritable pathologies of altered neural circuit functioning. How genetic mutations lead to specific neural circuit abnormalities underlying behavioral disruptions, however, remains unclear. Using circuit-selective transgenic tools and a mouse model of maladaptive social behavior (ArpC3 mutant), we identify a neural circuit mechanism driving dysfunctional social behavior. We demonstrate that circuit-selective knockout (ctKO) of the ArpC3 gene within prefrontal cortical neurons that project to the basolateral amygdala elevates the excitability of the circuit neurons, leading to disruption of socially evoked neural activity and resulting in abnormal social behavior. Optogenetic activation of this circuit in wild-type mice recapitulates the social dysfunction observed in ArpC3 mutant mice. Finally, the maladaptive sociability of ctKO mice is rescued by optogenetically silencing neurons within this circuit. These results highlight a mechanism of how a gene-to-neural circuit interaction drives altered social behavior, a common phenotype of several psychiatric disorders.

The elephant in the lung: Integrating lineage-tracing, molecular markers, and single cell sequencing data to identify distinct fibroblast populations during lung development and regeneration

During lung development, the mesenchyme and epithelium are dependent on each other for instructive morphogenic cues that direct proliferation, cellular differentiation and organogenesis. Specification of epithelial and mesenchymal cell lineages occurs in parallel, forming cellular subtypes that guide the formation of both transitional developmental structures and the permanent architecture of the adult lung. While epithelial cell types and lineages have been relatively well-defined in recent years, the definition of mesenchymal cell types and lineage relationships has been more challenging. Transgenic mouse lines with permanent and inducible lineage tracers have been instrumental in identifying lineage relationships among epithelial progenitor cells and their differentiation into distinct airway and alveolar epithelial cells. Lineage tracing experiments with reporter mice used to identify fibroblast progenitors and their lineage trajectories have been limited by the number of cell specific genes and the developmental timepoint when the lineage trace was activated. In this review, we discuss major developmental mesenchymal lineages, focusing on time of origin, major cell type, and other lineage derivatives, as well as the transgenic tools used to find and define them. We describe lung fibroblasts using function, location, and molecular markers in order to compare and contrast cells with similar functions. The temporal and cell-type specific expression of fourteen “fibroblast lineage” genes were identified in single-cell RNA-sequencing data from LungMAP in the LGEA database. Using these lineage signature genes as guides, we clustered murine lung fibroblast populations from embryonic day 16.5 to postnatal day 28 (E16.5-PN28) and generated heatmaps to illustrate expression of transcription factors, signaling receptors and ligands in a temporal and population specific manner.