Mouse Heart

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B. Mouse heart at E12.5. Scale bar = 550 micrometers. ‘RV’ is right ventricle, ‘LV is left ventricle’, and the arrowhead indicates the muscular ventricular septum. JacobyRO FJG, Davisson (2002) Biology and diseases of mice. In: Fox JG, Anderson LC, Lorw FM, Quimby FW (eds) Laboratory animal medicine. Elsevier Academic Press, San Diego, pp 35–120 We were interested in how non-CMs contribute to ventricular CMs hyperplasic to hypertrophic growth during the fetal to neonatal transitional stages. To do so, we used Nichenet 59 whereby we considered genes that are differentially expressed between E17.5 and P0 Ven_CMs as target genes, receptors expressed in Ven_CMs, and ligands expressed in non-CMs. Interestingly, we found multiple ligands that are expressed in different cell types to have the potential to regulate the same set of genes (Fig. 7Ci, ii and Fig. S26). For example, we found that Fb-expressed ligand MANF, Epi-expressed ligand EFNA5, and EndoEC-derived ligand WNT11 can regulate the same group of genes, including Ler3, Actb, Atf3, Cited2, Per1, Pfn1, Ctgf, and Fhl2. Besides the genes targeted by all three ligands, MANF and EFNA5 have the potential to regulate another set of genes, including Eno3, Klf9, Ranbp1, and Rps2. These genes are from broad pathways associated with the fetal to neonatal transitions and include cell maturation ( Klf9) 60, circadian rhythm ( Per1) 61, and metabolic switching ( Eno3, Atf3) 62, 63. To understand the function of these signaling interactions, a detailed comparison of their roles under normal and abnormal conditions is important. Wilms’ tumor 1 ( Wt1) and T-Box Transcription Factor 18 ( Tbx18) are two critical transcription factors in heart development. They are highly expressed in epicardial cells but have been shown to express in other cardiac lineages at early stages 39, 40, 41, 42 as well. Homozygous Wt1 null mice die after E14.5 due to heart deficiency 43, and Tbx18 mutants die within 24 h of birth as a result of skeletal and respiratory failure 44. Heart development in Tbx18 mutants is controversial and has been reported to either result in no defects of any cardiac cell lineages or severe defects in vasculature development 45, 46. A detailed analysis of Tbx18 mutants at the single-cell level will help elucidate these findings. The single-cell assessment of Wt1 and Tbx18 mutants will provide insights into the function of the epicardium throughout heart development. After sample demultiplexing based on MULTI-seq barcodes (Fig. S5) and quality control based on sequencing reads, the number of expressed genes, and percentage of mitochondria genes (Fig. S6A, B), we integrated the different batches together and observed no obvious batch differences (Fig. S7A–C). In the CD1 dataset, we captured 65,020 cells consisting of 8987 doublets, 12,313 negatives, and 43,720 singlets. After filtering, 29,001 singlets remained that were distributed throughout the 72 samples (402 cells per sample on average) (Fig. S6C). In the C57BL/6 dataset, we captured 66,171 single cells that included 13,364 doublets, 12,086 negatives, and 40,721 singlets. After filtering, we had 25,605 singlets left across 68 samples (376 cells per sample on average) (Fig. S6C). Through unsupervised clustering analysis of the filtered cells, we found that CD1 and C57BL/6 cells were grouped into 24 and 27 clusters, respectively (Figs. 1C, D, S8). Each cluster has a varied number of cells (Fig. S9, 10). Identification of cell types in the scRNA-seq data

The evil cat queen of the tunnels, she holds a nasty reputation with the citizens of Atlantia and the Mūs. Previous scRNA-seq analyses of the developing heart were generated from a few stages with low cell numbers, limiting their usage for downstream analyses. Additionally, samples from different stages were profiled separately, which can cause confounding by batch effects. Using sample multiplexing 47, we were able to profile 72 samples from CD1 mice and 68 samples from C57BL/6 mice. Most samples were processed simultaneously and loaded into the single cell pipeline together. Sample overlap between experiments enabled evaluation and showed that our multiplexing strategy efficiently guarded against batch effects. Note that MULTI-seq has the advantage of multiplexing samples, but it can also waste many sequencing reads as some sequenced cells need to be discarded for not having unique MULTI-seq barcodes. Considering that the ventricular are larger than atrial and that hearts at later stages are larger than early stages, our datasets have better coverages in early-hearts and atrial than late-staged hearts and ventricular. Additionally, the hypertrophic growth of ventricular CMs at the neonatal stage makes them too big to fit with the 10X chromium, leading to fewer late neonatal stage ventricular CMs being sampled in our datasets. To profile the ventricular CMs at late neonatal and adult stages, single cell nuclei sequencing would be a better option.

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To identify stage-specific molecular features in each cell type, we first identified the genes that were differentially expressed at the pseudotime stages (Fig. S15). Furthermore, we analyzed gene expression modules consisting of genes with similar expression patterns. Interestingly, we found that most lineage-defining gene modules were enriched at adjacent stages and grouped into two categories: early embryonic or a combination of late embryonic/neonatal stages. Additionally, we found that modules at early stages were enriched with genes in lineage development and morphogenesis, and the modules at neonatal stages mainly included genes involved in lineage maturation and cellular function. In Atrial_CMs, we identified eight gene modules, with module 8 (M8) being expressed at early embryonic stages with an enrichment of genes in development and morphogenesis pathways like cardiac atrium morphogenesis and muscle cell differentiation (e.g., Isl1, Shox2, Bmp2). Module 2 was expressed at neonatal stages and included genes like Ttn, Pln, and Myom2, which are known to be involved in heart muscle contraction and muscle cell differentiation (Fig. 2Bi and Supplementary Data 2). In Ven_CMs, we found nine gene modules. Module 4 was expressed in early embryonic stages and was enriched with genes in the heart developmental pathways, such as heart morphogenesis and septum morphogenesis (e.g., Tbx2, Tbx3, Gata5, and Wnt2). Module 2 in Ven_CMs was expressed during late embryonic and neonatal stages and contained genes enriched for CM maturation-related pathways, such as oxidative phosphorylation and ATP metabolic process electron transport chain (e.g., Atp5pb, Cox7b, and Ndufs2) (Fig. 2Bii). In Epi cells, we identified eight gene modules. Module 8 was expressed at early stages and had genes in the vascular formation and morphogenesis pathways such as kidney vasculature morphogenesis and glomerular capillary formation (e.g., Tcf21, Nrp1, Bmp4, and Pdgfra). Module 1 was expressed at late embryonic and neonatal stages with genes from the extracellular matrix-related pathways such as extracellular structure organization and collagen fibril organization (e.g., Col3a1, Cav1, and Col1a1) (Fig. 2Biii). In Vas_ECs, we found seven gene modules. Module 6 was expressed at early stages with genes notably expressed in ribosome biogenesis and rRNA processing (e.g., Rps2, Rps10, Rpsa). Module 3 was expressed at neonatal stages with genes associated with vascular development pathways such as the regulation of angiogenesis and vasculature development pathways (e.g., Aplnr, Cldn5, Klf2, Klf4) (Fig. 2Biv). In Fb_like, Endo_ECs, and mural cells, we found the same patterns as described above. However, neonatal immune cells have gene modules for each specific day (Fig. S14B and Supplementary Data 2), which can be attributed to the highly diverse types of immune cells, each known to have their own specific transcriptional profiles.

Dietschy JM, Turley SD (2002) Control of cholesterol turnover in the mouse. J Biol Chem 277(6):3801–3804. https://doi.org/10.1074/jbc.R100057200 Embryos were harvested and fixed with 10% formalin. Embryos were dehydrated using serial exposure to 70, 95, and 100% alcohol with differing incubation times according to the age of the embryo. We soaked the embryos 2–3 times in xylene for 15 min each, followed by an ~5 h soak in 70.4% paraffin wax (containing 24.9% Vybar, 4.4% stearic acid, and 0.4% aniline dye Sudan IV). The paraffin wax was changed twice during the incubation, once every 30 min and then incubated for 4 h without disturbance. The wax changes eradicated any residual alcohol or xylene. Following this, we embedded embryos in a metal mold filled with 70.4% paraffin wax and cooled for 1 h at 21 °C.

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Kuijpers MH, van de Kooij AJ, Slootweg PJ (1996) Review article. The rat incisor in toxicologic pathology. Toxicol Pathol 24(3):346–360. https://doi.org/10.1177/019262339602400311 A. Mouse heart at E11.5. Scale bar = 400 micrometers. ‘RA’ is the right atrium, ‘RV’ is the right ventricle, ‘LA’ is the left atrium, and ‘LV’ is the left ventricle. Shakibi JG, Diehl AM . Postnatal development of the heart in normal Swiss-Webster mice. Lab Anim Sci 1972; 22:668–83. Restivo A, Piacentini G, Placidi S, Saffirio C, Marino B . Cardiac outflow tract: a review of some embryogenetic aspects of the conotruncal region of the heart. Anat Rec A Discov Mol Cell Evol Biol 2006; 288:936–43.