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Identification of astroglia-like cardiac nexus glia that are critical regulators of cardiac development and function

['Nina L. Kikel-Coury', 'Department Of Biological Sciences', 'University Of Notre Dame', 'Notre Dame', 'Indiana', 'United States Of America', 'Center For Stem Cells', 'Regenerative Medicine', 'Jacob P. Brandt', 'Isabel A. Correia']

Date: 2021-11

Glial cells are essential for functionality of the nervous system. Growing evidence underscores the importance of astrocytes; however, analogous astroglia in peripheral organs are poorly understood. Using confocal time-lapse imaging, fate mapping, and mutant genesis in a zebrafish model, we identify a neural crest–derived glial cell, termed nexus glia, which utilizes Meteorin signaling via Jak/Stat3 to drive differentiation and regulate heart rate and rhythm. Nexus glia are labeled with gfap, glast, and glutamine synthetase, markers that typically denote astroglia cells. Further, analysis of single-cell sequencing datasets of human and murine hearts across ages reveals astrocyte-like cells, which we confirm through a multispecies approach. We show that cardiac nexus glia at the outflow tract are critical regulators of both the sympathetic and parasympathetic system. These data establish the crucial role of glia on cardiac homeostasis and provide a description of nexus glia in the PNS.

Funding: This work was supported by the University of Notre Dame, the Elizabeth and Michael Gallagher Family (CJS), Centers for Zebrafish Research and Stem Cells Regenerative Medicine at the University of Notre Dame (CJS), the Alfred P. Sloan Foundation (FG-2017-9531)(CJS) and the National Institute of Health (DP2NS117177)(CJS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Using analysis from zebrafish, mouse, and human tissue, we searched for astroglial-like cells in the heart. Here, we describe a population of gfap + glia in the heart with integral roles in cardiac development and function. Analysis of zebrafish, mouse, and human tissue reveals conservation of this gfap + population between species. Further analysis of published single-cell sequencing of human embryonic and mouse adult hearts also reveals an abundant up-regulation of glial-associated genes. Focusing on the gfap + population in zebrafish, we discovered that these cells arise from the hindbrain neural crest and migrate to the heart, where they utilize Meteorin signaling through Jak/STAT3 to drive differentiation. Once present, cardiac glia inhibit neuronal innervation and regulate heart rate and rhythm via sympathetic and parasympathetic regulation and additionally show that OT-located cardiac glia control heart rate. In the adult, these cells localize with neurons in a net-like morphology and interact with synapses. Due to their morphology and glial-like molecular identity, we have termed these gfap + cells cardiac nexus glia (CNG). Together, these data provide evidence of a new peripheral glial population that regulates cardiac function.

Recent work has described cells in the adult heart that respond to injury after catheter ablation treatment for atrial fibrillation and express the astroglial marker, s100b, underscoring the importance of potential glial populations in the heart [ 34 ]. Additionally, the developing heart is seeded by an underdescribed population of neural crest cells at the outflow tract (OT) [ 35 , 36 ], which is intriguing, given that most peripheral glial populations are neural crest derived [ 10 ]. These cells are required for formation, remodeling, and blood vessel patterning of the OT and are highly associated with congenital heart disease (CHD), given that OT abnormalities account for 30% of CHD morbidity and mortality [ 37 – 40 ]. Despite the essential role these cells have in cardiac development, the identity of this population remains a mystery. Taken together, more research is needed to understand the presence, development, and primary function of glia within the cardiac system.

In analogous complex networks that are present in the brain, astroglia populations aid in the integration process between neurons [ 19 – 24 ]. Similarly, enteric glia express common astroglia markers and function in the gut by controlling neuronal synapses or through direct interactions with intestinal muscle [ 25 – 27 ]. Satellite glia have also been described as glial cells of the PNS that regulate neuronal activity. These cells have primarily been researched in the context of somatosensory neurons [ 28 ]. However, other astroglia-like cell types localized within organs have yet to be identified and characterized. This leaves current models of PNS construction and function lacking a critical cell type in the nervous system. While it seems possible astroglia do not extensively populate organs, there have been reports of glial-like cells in the spleen [ 29 ], pancreas [ 30 ], lungs [ 31 ], and skin [ 32 ], and electron microscopy studies have identified glial-like processes in the heart [ 33 ]. Together, these data support the conclusion we may have yet to uncover important glial-like cell types.

The PNS is an integrated network of nerves that control everyday health [ 10 , 11 ]. In addition to the widely studied nerve system that controls skeletal muscle movement, the PNS has profound control over every organ [ 12 ]. This control is enacted through a hierarchy of neuronal processes, with the PNS extending parasympathetic and sympathetic neurons to regulate the autonomic nervous system (ANS) responses known as the “rest-and-digest” and “fight-or-flight” responses, respectively [ 13 ]. These neurons then synapse with internal nerve networks that are specialized to each organ, which allows for fast modulation of resident organ-specific cell types to maintain homeostasis [ 14 , 15 ]. For example, this hierarchy is especially critical to the heart, which must constantly maintain a rhythmic beating for blood circulation [ 16 ]. The internal nerve network of the heart is known as the intracardiac nervous system (ICNS) and modulates either cardiomyocytes or the ANS to tightly control heart rate and rhythm [ 14 , 17 , 18 ]. While the role of neurons in organ control has been characterized, current research is lacking an understanding of glial involvement.

Throughout the vertebrate nervous system, glial cells are developmentally and functionally essential [ 1 – 3 ]. The existence of diverse populations of glia allows for integral support to neurons, including modulation of neuronal development [ 2 ], signal propagation [ 4 ], synaptic support [ 5 ], metabolic aid [ 6 ], and phagocytic clearance of debris [ 7 ]. These roles are divided by the specialized glial populations, largely segregated into ensheathing glia, astroglia, and microglia [ 4 , 8 , 9 ]. These glia are further divided into cell types based on their central (CNS) or peripheral (PNS) nervous system residence. As the roles for each of the cell types expands in the CNS, our understanding of specialized glia remains a major barrier to understanding the full functionality of the PNS, especially in the context of organ function.

Results

Meteorin acts as a molecular determinant of cardiac nexus glia through Jak/Stat3 signaling We next asked if metrn is a determinant of CNGs. Using CRISPR/Cas9, we generated a mutant with a 4-bp deletion that putatively truncates the Metrn protein to 38 amino acids versus 303 amino acids in wild-type animals (Fig 7A). We validated this mutant by assaying mRNA with the metrn RNAscope probe, which targets the translated region of exon 1 and observed a decrease in mRNA puncta in metrn−/− animals (n = 3) (Fig 7B). While 100.00% of wild-type (n = 10 animals) and heterozygous animals (n = 6 animals) survived to early adulthood, we noted a decrease to 33.33% survival by 12 months postfertilization (mpf) in heterozygous siblings, while wild-type animals had an 80.00% survival (Fig 7C). We did not detect adult homozygous siblings from that experiment. However, in other crosses that were grown to adulthood, homozygous animals could be identified (n = 5 out of 36 animals, 14%, chi-squared test to mendelian ratios, p = 0.2946). PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 7. Meteorin acts as a molecular determinant of CNG. (A) Schematic representation of metrn CRISPR mutation (red). (B) Confocal z-sections of metrn, id1, and cdh11 mRNA in the ventricle (white dotted outline) at 6 dpf in wild-type and metrn−/− embryos. (C) Quantification of percent survival of adult wild-type and metrn+/− animals. (D) Confocal maximum z-projection of nucGFP+ number (white arrowheads) in the ventricle (white dotted outline) at 6 dpf in Tg(gfap:nucGFP) wild-type and metrn−/− embryos. (E) Quantification of Tg(gfap:nucGFP)+ number at 6 dpf over various manipulations of the differentiation pathway. (F) Quantification of Tg(gfap:nucGFP)+ number after wash-in/wash-out of S31-201 at different time points. Crossover between the curves indicates a functional role between 4–5 dpf. Data are represented as mean ± SEM. Scale bar equals 10 μm. Statistics summarized in S1 Table. See S1 Data for raw data. aa, amino acid; CNG, cardiac nexus glia; dpf, days postfertilization; mpf, months postfertilization; V, ventricle. https://doi.org/10.1371/journal.pbio.3001444.g007 We next scored the abundance of CNG in wild-type, metrn+/−, and metrn−/− animals at 6 dpf in Tg(gfap:nucGFP). metrn+/− (n = 20 hearts) and metrn−/− (n = 16 hearts) animals displayed overall decreased number of nucGFP+ cells in the heart from an average of 10.29 ± 1.05 cells in the wild-type group (n = 14 hearts) to an average of 3.75 ± 0.80 (p < 0.0001, one-way ANOVA with Tukey’s post hoc test) and 3.75 ± 0.86 nucGFP+ cells (p < 0.0001, one-way ANOVA with Tukey’s post hoc test), respectively (Fig 7D and 7E). To further confirm that CNGs were disrupted and not just down-regulating gfap, metrn−/− animals also displayed less cdh11 and id1 (Fig 7B), consistent with the hypothesis that metrn impacts CNG. In astrocytes, metrn has been reported to function through the Jak/Stat3 pathway [70]. To test in vivo if metrn signaling pathway impacts CNG differentiation, we treated Tg(gfap:nucGFP) animals with either a Stat3 (S31-201) or Jak1 (CAS-457061-03-7) inhibitor and compared the abundance of nucGFP+ cells to DMSO-treated animals. Such treatments were done at 4 dpf, to avoid disrupting Stat3 or Jak1 during early heart development [72]. Both Stat3 (n = 6 hearts) and Jak1 (n = 6 hearts) inhibition reduced the abundance of nucGFP+ cells to an average of 1.00 ± 0.37 (DMSO = 11.83, n = 6, p < 0.0001, one-way ANOVA followed by Tukey’s post hoc test) and 0.83 ± 0.31 (DMSO = 11.00, n = 6, p < 0.0001, one-way ANOVA followed by Tukey’s post hoc test) cells (Fig 7E). However, Jak/Stat3 signaling is not activated through recruitment of its receptor, gp130, as previously found [70] (Fig 7E; DMSO = 9.17 ± 0.79 cells, n = 6, Sc144 = 13.17 ± 1.35 cells, n = 6 hearts, p = 0.5015, one-way ANOVA followed by Tukey’s post hoc test). metrn−/− animals treated with the Stat3 inhibitor (n = 3 hearts) also displayed a decrease in nucGFP+ cells to an average of 2.00 ± 0.58 cells, comparable to untreated metrn−/− animals (Fig 7E; p = 0.9968, one-way ANOVA followed by Tukey’s post hoc test), suggesting that Stat3 functions in the same genetic pathway as metrn to impact CNG differentiation. To further probe this molecular mechanism, we tested the temporal requirement of Jak/Stat3 signaling. Our data suggest that sox10+ neural crest cells seed the OT by 48 hpf then differentiate into gfap+ cells by 6 dpf (Fig 4I and 4J). If metrn signaling drives differentiation of CNG, then it should also be required from 3 to 6 dpf in zebrafish when we visualize seeding of gfap+ cells. To test this, we performed a “wash-in” drug treatment experiment, in which the drug is introduced to the egg water at time points before, during, or after the signaling is potentially necessary for the differentiation of a cell. Conversely a “wash-out” was also performed in which the drug is introduced prior to the differentiation of a cell and is replaced with normal egg water either during or after the necessary signaling. The number of cells is then scored and plotted for both the “wash-in/wash-out” treatments at each time point, and the overlap of the curves indicates the time that the signaling is most likely required. For this experiment, the Stat3 inhibitor was either washed into the egg water at 3, 4, or 5 dpf, or treated at 3 dpf and washed out at 4 or 5 dpf. The abundance of nucGFP+ cells was scored at 6 dpf. The overlap of these 2 curves occurred at 4.5 dpf (wash-in: n = 5 hearts, wash-out: n = 5 hearts), indicating that Stat3 signaling likely occurs at 4.5 dpf (Fig 7F). This developmental timing is in line with when we first can detect gfap+ cells that have transitioned from sox10+ cells.

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