Miquerol L, Meysen S, Mangoni M et al (2004) Architectural and functional asymmetry of the his-Purkinje system of the murine heart. Cardiovasc Res 63(1):77–86. https://doi.org/10.1016/j.cardiores.2004.03.007 No major differences in outflow tract septation between mouse and human species were identified in this study. The processes of spiraling, fusion with endocardial cushions, and thinning of plate-like structures to form semilunar valves were comparable. In our study, two ridges were identified in the outflow tract of the mouse and human and intercalated ridges were not seen. Sophisticated molecular analysis studies in the human have also recently confirmed that the process of septation is similar in mice and human species, with formation of walls in the outflow tracts occurring by addition of ISL1-positive mesenchymal cells distally ( 26). Distinction in Ventricular Morphology The confidence of standing on this firm causal foundation could help us refine both behavioral therapies and medication induced therapies, because now you can envision targets that are peripheral, that are in the body as strategies for modulating brain states. Of course, this is a very early days, and I couldn't be certain that such an effect would be clinically useful, but in terms of opening our eyes to a new way of looking at things, this sort of discovery is, I think, illuminating. Inhibiting activity of this area reduced the effects of increased heart rate on anxiety-like behavior in the mice. Taken together, the results suggest that, in certain situations, heart rate can affect anxiety, in mice at least, and that the insular cortex is an important mediator to this happening. Anna Beyeler from the French National Institute of Health and Medical Research works on the neural circuits of behavior and has written a News and Views article about the research. She thinks that these findings provide insights into how emotions work, but there are more questions to be answered. Interviewee: Anna Beyeler 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.

Keyte A, Hutson MR . The neural crest in cardiac congenital anomalies. Differentiation 2012; 84:25–40. Episcopic fluorescence image capture (EFIC) was performed on 66 wild-type mouse embryos from embryonic day (E) 9.5 to birth; 2-dimensional and 3-dimensional datasets were compared with EFIC and magnetic resonance images from a study of 52 human fetuses (Carnegie stage 13–23). Results: Rosenthal J, Mangal V, Walker D, Bennett M, Mohun TJ, Lo CW . Rapid high resolution three dimensional reconstruction of embryos with episcopic fluorescence image capture. Birth Defects Res C Embryo Today 2004; 72:213–23. It's a demonstration that just changing one parameter — the heart rate — can induce anxiety, so this is quite important. And then I think it's super interesting that they identify the posterior insula as a key relay for that behavior. At least to me what the most interesting next question would be is the timing of this interaction from the heart to the brain and then to emotion. Here the increase heart rate is 36% above normal frequency, so they have shown that it impacts straightaway, so if we do only a 10% increase, but constantly, how does that impact behavior?Lueschow SR, McElroy SJ (2020) The paneth cell: the curator and defender of the immature small intestine. Front Immunol 11:587. https://doi.org/10.3389/fimmu.2020.00587

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 minutes each, followed by an approximately 5 hour 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 minutes and then incubated for four hours 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 hour at 21°C. First up on the show this week, we know that emotions can induce changes in the body, but new research is showing how changes in the body might be inducing emotions. Benjamin Thompson is here with the story. Time course of atrial, ventricular, and outflow septation were outlined and followed a similar sequence in both species. Bilateral venae cavae and prominent atrial appendages were seen in the mouse fetus; in human fetuses, atrial appendages were small, and a single right superior vena cava was present. In contrast to humans with separate pulmonary vein orifices, a pulmonary venous confluence with one orifice enters the left atrium in mice. Conclusion: 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. Pan H, Deutsch GH, Wert SE et al (2019) Comprehensive anatomic ontologies for lung development: a comparison of alveolar formation and maturation within mouse and human lung. J Biomed Semant 10:18. https://doi.org/10.1186/s13326-019-0209-1

Advance Praise

Weninger WJ, Mohun T . Phenotyping transgenic embryos: a rapid 3-D screening method based on episcopic fluorescence image capturing. Nat Genet 2002; 30:59–65.

Goldberg M, Kellermann O, Dimitrova-Nakov S, Harichane Y, Baudry A (2014) Comparative studies between mice molars and incisors are required to draw an overview of enamel structural complexity. Front Physiol 5:359. https://doi.org/10.3389/fphys.2014.00359 F. Human heart at EGA 8 weeks (CS 18). Scale bar = 1350 micrometers. ‘LVOT’ is the left ventricular outlow, ‘RV’ is the right ventricle, ‘LV’ is the left ventricle, and ‘PA’ is the pulmonary artery. 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

Wessels A, Sedmera D (2003) Developmental anatomy of the heart: a tale of mice and man. Physiol Genomics 15(3):165–176. https://doi.org/10.1152/physiolgenomics.00033.2003 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 Single cell mRNA sequencing (scRNA-seq) is a powerful approach to studying heart development at the single cell level. Using Mesp1-based lineage tracing mice, Lescroart et al. 24 isolated and analyzed the cardiac mesoderm cells with scRNA-seq and identified distinct populations of progenitors committed to different cardiac lineages and regions of the heart. Ivanovitch et al. 25 analyzed the cells in the heart fields using a T-based lineage tracing mouse line and found that cardiac progenitors were spatially prepatterned within the primordial streak. Jia et al. 26 profiled the two heart field progenitors after isolation via Nkx2-5 and Isl1 expression identifying novel cell populations. From our previous work profiling early staged murine hearts after microdissection into small zones, we identified zone-specific molecular signatures 27. Parallel to our investigations, DeLaughter et al. 28 profiled heart cells at five stages (E9.5, E11.5, E14.5, P0, and P21) and found temporal-specific genes. Additionally, several studies have profiled single cells and nuclei at neonatal and adult stages to understand heart maturation and regeneration 29, 30, 31, 32. However, the caveat to many of the listed studies is that the number of profiled cells was small, with most cells being CMs, thus limiting downstream analyses for non-CM lineages. The published datasets are useful for studying early heart development or heart regeneration, but they are missing key developmental timepoints and cell lineages crucial to gaining a better understanding. Reynolds RP, Kinard WL, Degraff JJ, Leverage N, Norton JN (2010) Noise in a laboratory animal facility from the human and mouse perspectives. J Am Assoc Lab Anim Sci: JAALAS 49(5):592–597

Hoyt RF, Hawkins JV, St Clair MB, Kennett MJ (2007) Mouse physiology. In: Fox JG, Barthold SW, Davisson MT, Newcomer CE, Quimby FW, Smith A (eds) The mouse in biomedical research, normative biology, husbandry, and models, vol 3, 2nd edn. Academic Press, San Diego, CA, pp 23–90 Mouse mutants are used to model human congenital cardiovascular disease. Few studies exist comparing normal cardiovascular development in mice vs. humans. We carried out a systematic comparative analysis of mouse and human fetal cardiovascular development. Methods: G. Human heart at EGA 8 (CS 18). Scale bar = 1500 micrometers. The arrowhead shows the outlet ventricular septum. Peirson SN, Brown LA, Pothecary CA, Benson LA, Fisk AS (2018) Light and the laboratory mouse. J Neurosci Methods 300:26–36. https://doi.org/10.1016/j.jneumeth.2017.04.007 Mitchell SC, Korones SB, Berendes HW . Congenital heart disease in 56,109 births. Incidence and natural history. Circulation 1971; 43:323–32.Riera CE, Tsaousidou E, Halloran J et al (2017) The sense of smell impacts metabolic health and obesity. Cell Metab 26(1):198–211.e5. https://doi.org/10.1016/j.cmet.2017.06.015 Schwob JE (2002) Neural regeneration and the peripheral olfactory system. Anat Rec 269(1):33–49. https://doi.org/10.1002/ar.10047 Moorman A, Webb S, Brown NA, Lamers W, Anderson RH . Development of the heart: (1) formation of the cardiac chambers and arterial trunks. Heart 2003; 89:806–14. McElhinney DB, Marshall AC, Wilkins-Haug LE, Brown DW, et al. Predictors of technical success and postnatal biventricular outcome after in utero aortic valvuloplasty for aortic stenosis with evolving hypoplastic left heart syndrome. Circulation 2009; 120:1482–90. So now we can outfit mice with a little vest with a light source, and we can pace their heart precisely. Primary direct control on the heart contraction, and we were able to ask the question – does this affect the emotional state? And it turned out, it did, but with a twist.