Transcriptome profile of the zebrafish atrioventricular canal reveals molecular signatures of pacemaker and valve mesenchyme

The atrioventricular canal (AVC) is an essential feature of the heart, which separates the atrium from the ventricle. During heart morphogenesis, it is a hub of molecular processes necessary for distinguishing heart regions; most importantly, for the formation of the AV conduction system and cardiac valves. To better understand the molecular processes underlying AVC development and function, we utilized the transgenic zebrafish line sqet31Et with EGFP expression in the AVC region to isolate this cell population by FACS and profiled its transcriptome by RNA-seq at 48 and 72 hours post fertilization (hpf). Compared to the rest of the heart, the AVC is enriched for the expression of molecular markers associated with mammalian AVC and AV node, including cx36.7 and cx45 which encode connexins forming low conductance gap junctions. Using the transgenic line Tg(myl7:mermaid) encoding the voltage-sensitive fluorescent protein, we showed that loss of function of Isl1 abolished the pacemaker-containing sinoatrial ring (SAR) and resulted in an erratic spread of excitation pattern from the SAR to AVC, indicating the dysfunction of the primary pacemaker. Concurrently, ectopic excitation in the AVC region was observed, suggesting that the zebrafish AVC possesses inherent automaticity although insufficient to replace the primary pacemaking activity of the SAR. Comparisons between the SAR and AVC transcriptomes revealed partially overlapping expression profiles of various ion channels and gap junction proteins which reflects their diversified functions. Lastly, we observed dynamic expression of epithelial-to-mesenchymal transition markers, as well as components of TGF-β, Notch, and Wnt signaling pathways, which have been implicated in the formation of AVC conduction and cardiac valves. Our results uncovered the molecular hallmarks of the developing AVC region and demonstrated its role in the structural and electrophysiological separation between the atrium and ventricle. Author summary The atrioventricular canal is a structure in the embryonic heart which separates the atrium from the ventricle. It gives rise to the AV node and cardiac valves - two important structures which ensure unidirectional blood flow between heart chambers. The AV node serves to regulate the propagation of electrical impulses between the two chambers, such that they contract consecutively. Using the zebrafish as model organism, we performed gene expression profiling and characterized electrical conduction patterns between the sinoatrial primary pacemaker and AVC. We discovered that the zebrafish AVC possesses similar features to the mammalian AV node, including slow conduction, inherent pacemaking activity, and the expression of conserved developmental genes. The molecular profile of the AVC is distinct from that of the sinoatrial pacemaker, which reflects their distinct roles. In addition, we found that genes regulating cardiac valve development were also expressed in the AVC, illustrating the importance of this region for establishing both electrophysiological and structural separation between the heart chambers. Besides establishing conserved aspects between zebrafish and mammalian conduction system, the data generated in this study constitutes a valuable resource for studying AVC development and discovery of novel candidate genes implicated in regulating cardiac rhythm and cardiac valve formation.


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The atrioventricular canal (AVC) serves two critical purposes in cardiac development Given its important role in the formation of major cardiac structures, disruptions to AVC 147 development can result in various forms of septal defects, as well as valve abnormalities leading 148 to heart failure. In addition, defects of the AV node may lead to various degrees of AV block, 149 which gives rise to cardiac arrhythmia [42]. However, despite its importance, our knowledge function. This data constitutes a valuable resource for the study of AVC development 186 and function and identification of candidate genes implicated in these processes.
imaging of the AVC region revealed that at the surface of the AVC, the EGFP and mRFP 195 expression overlapped, confirming the myocardial nature of the EGFP-expressing cells 196 (Fig. 1C). At 72 hpf, two additional groups of ~3 cuboidal-shaped cells were detected 197 at the deeper layer facing the cardiac lumen (Fig. 1D). These cells appear to be a part of the To characterize the molecular profile of the GFP+ cell population in sqet31Et, we isolated 208 these cells using fluorescence-activated cell sorting (FACS) at 48 hpf and 72 hpf ( Fig. 2A) 209 and profiled their transcriptome by RNA-seq. The rest of the heart cells, which did not express 210 EGFP, were also collected (GFP-). Average sequencing reads mapping to the egfp sequence 211 were considerably higher in GFP+ compared to GFP-samples, confirming the high 212 representation of the EGFP-expressing cell population in the GFP+ samples (S1 Figure B).  To identify genes enriched in the GFP+ cell population compared to the rest of the heart, 217 we performed differential expression analysis of the GFP+ against GFP-fractions. In both developmental stages, a total of 3798 and 2777 genes were differentially expressed at 48 hpf 219 and 72 hpf, respectively (absolute log2FC > 2, padj < 0.05), of which 1492 were common 220 for both stages (Fig. 2C, F, S2 Table). GO and KEGG pathway enrichment analyses at 48 hpf 221 revealed that the set of genes overexpressed in GFP+ compared to GFP-cells (enriched genes) 222 was overrepresented for functional terms related to cardiac muscle development and function 223 ("cardiac muscle contraction", "adrenergic signaling in cardiomyocytes", "cardiac muscle 224 development", "cardiac muscle differentiation", and "calcium signaling pathway"), 225 which supports the myocardial identity of the GFP+ fraction (Fig. 2D, E, S3 Table). 226 On the other hand, functional terms related to cell-cell adhesion ("cell adhesion molecules", 227 "cell-cell adhesion") were overrepresented among transcripts overexpressed in the GFP-cell 228 population. At 72 hpf, similar functional terms were overrepresented with the addition 229 of "vascular smooth muscle contraction" term ( Fig. 2G, H, S3 Table), which likely corresponds 230 to the initiation of EGFP expression in the BA at this stage.

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To assess whether the sorted GFP+ fraction contained AVC cells, we explored the presence of 232 known markers of AVC in our dataset. We established a set of AVC marker genes for zebrafish 233 and mammals by retrieving genes annotated with the term "atrioventricular canal" from  Besides delaying electrical conduction between the atrium and ventricle, the mammalian 270 AV node also possesses intrinsic pacemaker activity [3,6]. We thus searched amongst the AVC-271 enriched gene list for those known to be expressed in the AV node or associated with pacemaker 272 development and function (S6 Table;    was not AVC-enriched. It has been shown that nkx2.5 is expressed in all myocardium, 280 but slightly higher in the AV conduction system [64]. Similarly, we found that nkx2.5 281 was enriched in GFP+ cells compared to GFP-at 48 hpf (S3 Figure, S6 Table). Taken together, 282 the transcriptome of AVC myocardium reveals conserved features to that of the mammalian 283 AV node in terms of expression of genes linked to slow conductivity, automaticity, 284 and molecular mechanism for AV node specification. Therefore, our results support the notion 285 that the AVC is a homologous structure to the mammalian AV node and suggest that the core 286 network leading to the specification of the AV conduction system is conserved between 287 mammals and fish. The expression of hcn4 and other markers in AVC, suggesting its homology to the mammalian 291 AV node, led us to question whether the zebrafish AVC possesses inherent pacemaking activity 292 as does its mammalian counterpart. To this end, we utilized the isl1 K88X mutant (isl1 sa29 ), 293 which has been shown to exhibit a defective SAR pacemaker function that manifests as sinus 294 pauses and bradycardia [21,25]. We observed that, apart from losing the expression of fhf2a 295 (Fig. 4A, B), bmp4 (Fig. 4C, D), and the pacemaker marker hcn4 (Fig. 4E, F) in the sinus 296 venosus, the isl1 mutant was also devoid of EGFP-positive cells at the SAR, but not the AVC, 297 as shown by analysis of sqet33mi59BEt and sqet31Et, respectively (Fig. 4G, H). Hence, we   This suggests the existence of alternative origins of automaticity that triggers the initiation 306 of cardiac contractions. To investigate whether the AVC could generate electrical impulses 229±6.6 bpm at 72 hpf, (n=18)) ( Fig. 5A). Increased variability in heartbeat duration was noted 315 in the Isl1 morphants as well. 316 Optical mapping showed that in control hearts, the excitation wave front travelled uniformly 317 across the atrium from the SAR towards the AVC (Supplementary movie 1; Fig. 5B).

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In contrast, in Isl1 morphants, atrial excitation, although predominantly originating from   Table), obtaining a total of 1516 AVC-unique and 701 SAR-unique genes, and 450 genes common 339 between the two (S7 Table). Notably, several genes encoding proteins involved in ion transport, 340 cell junction formation, and extracellular matrix were differentially enriched between the AVC 341 and SAR.

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Interestingly, the expression of hcn4 was significantly higher in the SAR, while not 343 significantly higher in the AVC, compared to the rest of the heart. This may reflect the role 344 of SAR as the dominant pacemaker (S7 Table). In addition to hcn4, several other transcripts

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Despite having some common properties, the SA and AV nodes perform distinct functions.

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The SA node serves a primarily pacemaking function, while the AV node is mainly specialized  Table).

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On the other hand, cx43.4, a paralog of Cx45, was enriched in both the SAR and AVC 362 (S7 Table). Low electrical coupling is also a necessary property within the definitive pacemaker cells of the SA node to prevent inhibitory interference from the surrounding working 364 myocardium, which is more hyperpolarized [11]. Collectively, the overall differences in ion  On the other hand, jag1a, jag2a, her6, jagn1b, notch2, and notch3 were enriched at either stage 385 (S2 Table). In contrast, valve endocardial markers notch1b and its ligand dll4  Table). Notably, GO terms related to TGF-β, Wnt, and Notch signaling pathways 407 were overrepresented at 72 hpf (S10 Table). Many members of these three signaling pathways 408 exhibited dynamic expression between 48 hpf and 72 hpf (S5 Figure; S11 Table). Collectively, 409 our observations uncover the dynamic expression of various components of the TGF-β, 410 canonical Wnt, and Notch signaling pathways in the AVC myocardium, which likely reflects 411 their role in the ongoing AVC patterning and valve development.
AVC-enriched genes are associated with human congenital heart defects related to CCS, 413 valves and septa 414 We identified the human orthologues of AVC-enriched genes and interrogated them for any 415 association with clinical phenotypes related to ClinVar terms: "arrhythmia", "AV block", "long 416 QT syndrome", and "conduction". Our analysis revealed a total of 91 and 60 unique genes 417 associated with these four ClinVar terms at 48 hpf and 72 hpf stages, respectively (S12 Table). As the AVC is also the site where the endocardial cushion and valve develops, we expected to 426 find associations between AVC-enriched genes and human valve and septal defects. 427 We searched the human orthologues of AVC-enriched for overlap with ClinVar terms 428 containing "tricuspid valve", "AV valve", "mitral valve", and "valve in general". In total, 429 115 and 93 unique genes were associated with these terms at 48 hpf and 72 hpf, respectively 430 (S13 Table). In addition, we also searched for those associated with the ClinVar term 431 "septal defect" and obtained 66 and 55 unique genes associated with the term at each stage, 432 respectively (Supplementary Table 14). In the adult human heart, the AV node is embedded 433 into the interatrial septum [3]. Given that the endocardial cushions are involved in the formation 434 of the AV valves and septa, it comes as no surprise that the defects of interatrial septum could 435 be linked to defects in cardiac conduction. In fact, a number of genes were commonly associated 436 with ClinVar terms "cardiac conduction" and "valve" (S14 Table). For example, tbx5a, whose human orthologue TBX5 causes the Holt-Oram syndrome characterized by congenital heart our results suggest that the 72 hpf transcriptome of GFP+ cells defines genes expressed in the 463 AVC and BA. Nevertheless, the distinctive tissue composition of the BA compared to AVC 464 myocardium allows for segregation based on this criterion. The expression of EGFP in the AVC 465 and BA of sqet31Et transgenic line adds to a list of common markers of these cell lineages. We are grateful to the zebrafish core facility of the IIMCB Warsaw for excellent fish care. 708 We thank Dr. Y. Okamura for the Tol2-myl7:mermaid construct and Dr. Natascia Tiso  Competing interests 713 The authors declare that they have no competing interests.      3   notch3  gata3  id4  cntn2  spock1  shox2  foxn4  scn3b  pdcd4b  kcnq3  wnt5a  msx2b  nkx2.3  foxf1  dchs1b  lama3  postnb  tbx1  fn1b  egr1  ccn1  prss23  twist1b