Heart Development in the Lizards (Varanidae) with the Greatest Extent of Ventricular 2 Septation
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bioRxiv preprint doi: https://doi.org/10.1101/563767; this version posted February 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Heart development in the lizards (Varanidae) with the greatest extent of ventricular 2 septation 3 Short title: Ventricular septum evolution 4 Jermo Hanemaaijer1,#, Martina Gregorovicova2,6#, Jan M. Nielsen3, Antoon FM Moorman1, Tobias 5 Wang4, R. Nils Planken5, Vincent M Christoffels1, David Sedmera2,6, Bjarke Jensen1,* 6 7 1University of Amsterdam, Amsterdam UMC, Department of Medical Biology, Amsterdam 8 Cardiovascular Sciences, Meibergdreef 15, 1105AZ, Amsterdam, The Netherlands. 9 2Department of Developmental Cardiology, Institute of Physiology, Academy of Sciences of the 10 Czech Republic, Prague, Czech Republic. 11 3Department of Cardiology, Institute of Clinical Medicine, Aarhus University Hospital, Skejby, 12 Aarhus, Denmark 13 4Department of Bioscience, Zoophysiology, Aarhus University, Denmark 14 5Department of Radiology, Academic Medical Center, University of Amsterdam, the Netherlands. 15 6Charles University, First Faculty of Medicine, Institute of Anatomy, U Nemocnice 3, Prague, 16 Czech Republic. 17 #These authors contributed equally 18 *corresponding author: Bjarke Jensen, Ph.D. 19 Amsterdam UMC 20 Department of Medical Biology 21 Room L2-106 22 Meibergdreef 15 23 1105AZ Amsterdam 24 The Netherlands 25 Phone: +31 205664659 26 Fax: not available 27 Mobile: +31 626450696 28 email: [email protected] 29 30 Keywords; ventricular septum, evolution, embryo, conduction system, transcription factors, 31 monitor lizard, 1 bioRxiv preprint doi: https://doi.org/10.1101/563767; this version posted February 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 32 Abstract 33 Among lizards, only monitor lizards (Varanidae) have a functionally divided cardiac ventricle. This 34 enables them to sustain higher systemic blood pressures and higher metabolic rates than other 35 reptiles of similar size. The division results from the concerted action of three partial septa, which 36 may have homology to the full ventricular septum of mammals and archosaurs. Homology, 37 however has only been inferred from anatomical comparisons of hearts of adult monitors whereas 38 gene expression during heart development has not been studied. We show in developing monitors 39 that the partial septa that separate the left and right ventricle, the ‘muscular ridge’ and 40 ‘bulbuslamelle’, express the evolutionary conserved transcription factors Tbx5, Irx1 and Irx2, 41 orthologues of which mark the full ventricular septum. Compaction of embryonic trabeculae 42 contributes to the formation of these septa. The septa are positioned, however, to the right of the 43 atrioventricular junction and they do not partake in the separation of incoming atrial blood streams. 44 Instead, the ‘vertical septum’ within the left ventricle separates the atrial blood streams. It expresses 45 Tbx3 and Tbx5, which orchestrate the formation of the electrical conduction axis of the full 46 ventricular septum. These patterns of expression are more pronounced in monitors than in other 47 lizards, and are associated with a deep electrical activation near the vertical septum, contrasting the 48 primitive base-to-apex activation of other lizards. We conclude that current concepts of ventricular 49 septum formation apply well to the monitor septa and that there is evolutionary conservation of 50 ventricular septum formation among amniote vertebrates. 51 2 bioRxiv preprint doi: https://doi.org/10.1101/563767; this version posted February 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 52 Introduction 53 The evolution of land-living vertebrates was facilitated by breathing of air with lungs (Perry & 54 Sander, 2004). A dedicated vascular bed, the pulmonary circulation, enables the perfusion of the 55 lungs by blood and the oxygenated blood of this circulation returns to the left side of the heart 56 (Burggren & Johansen, 1986). The oxygen-rich blood from the lungs, however, may mix with 57 oxygen-poor blood returning from the systemic circulation if septal structures are absent in the 58 ventricle of the heart. In amphibians, which include the earliest land-living vertebrates, the ventricle 59 is without a septum whereas in mammals and in archosaurs (crocodylians and birds) the ventricle is 60 divided into the left and right ventricle by a full ventricular septum (R.E. Poelmann et al., 2014). 61 Mammals evolved from reptile-like ancestors and archosaurs evolved within reptiles (Laurin & 62 Reisz, 1995), and among the extant non-archosaur reptiles there is substantial variation in the 63 degree of ventricular septation (B. Jensen, Moorman, & Wang, 2014). 64 The single cardiac ventricle of extant non-archosaur reptiles is partially divided by three septa 65 named the “vertical septum”, the “muscular ridge” and the “bulbuslamelle”. The ‘vertical septum’ 66 is a prominent sheet of trabecular myocardium positioned beneath the atrioventricular valve (Fig. 67 1). It separates the oxygen-rich blood from oxygen-poor blood coming from the right atrium and its 68 function therefore resembles that of the ventricular septum of mammals and archosaurs (B. Jensen 69 et al., 2014). The muscular ridge is the most prominent septal structure, but is not involved in the 70 separation of the atrial inflows because it is positioned to the right of the atrioventricular canal (R. 71 E. Poelmann et al.). The ventricular septum of mammals and archosaurs, in contrast, is positioned 72 immediately below the atrioventricular canal (Cook et al.; Greil, 1903; Webb, Heatwole, & Bavay) 73 (Fig. 1). During ejection, the muscular ridge, however, forms an important boundary between the 74 pulmonary compartment, the cavum pulmonale, and the cavum venosum to separate blood flows 75 into the aortae and the pulmonary artery (Fig. 1). The third septum, the ‘bulbuslamelle’, is 76 positioned opposite to the muscular ridge and presses against the free edge of the muscular ridge 77 during contraction. It has been shown experimentally that this coming together of the two septa 78 prevents mixing of oxygen-rich blood and oxygen-poor blood during cardiac contraction (White, 79 1959). The full ventricular septum of mammals, birds and crocodylians has a pronounced left-right 80 gradient of expression of the transcription factor Tbx5 (B. Jensen et al.; Koshiba-Takeuchi et al., 81 2009; R.E. Poelmann et al., 2014). Analyses of Tbx5 gradients suggested that the vertical septum of 82 a turtle (Trachemys), but not the vertical septum of a lizard (Norops), shows homology to the full 3 bioRxiv preprint doi: https://doi.org/10.1101/563767; this version posted February 28, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 83 ventricular septum (Koshiba-Takeuchi et al.). Studies on other species of turtles and squamate 84 reptiles, however, show that both the muscular ridge and the vertical septum display a gradient of 85 Tbx5 expression (R.E. Poelmann et al., 2014). The seemingly conflicting results in non-crocodylian 86 reptiles could be resolved if the expression of Tbx5 identifies more than one septal structure in 87 animals without a full ventricular septum. 88 Because the cardiac anatomy of the last common ancestor is likely to remain unknown, heart 89 fossilization is extremely rare (Maldanis et al., 2016), we can only provide insight into the evolution 90 of the ventricular septum through studies of the cardiac anatomy in extant reptiles. Among these, 91 the monitor lizards (Varanidae) have ventricular septa that are more developed than in any other 92 lizards (B. Jensen et al., 2014; Webb et al., 1971). Further, monitors are exceptional among lizards 93 because their cardiac ventricle is functionally divided, such that the left side of the ventricle (cavum 94 arteriosum and venosum) generates high systemic blood pressure whilst the cavum pulmonale 95 supplies the lungs at much lower pressure (Burggren & Johansen, 1982). These mammal-like blood 96 pressures support the metabolic rates that are higher than in other reptiles (Thompson & Withers, 97 1997) and the sequencing of the Komodo dragon genome suggests that many mitochondrial genes 98 of monitors have undergone positive selection (Lind et al., 2019). The greater state of development 99 of the septa could associate with more pronounced patterns of gene expression and the monitor 100 ventricle may then be the most suitable model to test the evolutionary origin of the full ventricular 101 septum. 102 To investigate whether the ventricle of monitors is divided by structures that are homologous 103 to the full ventricular septum of mammals and archosaurs, we studied heart development and 104 function in monitors (Varanus acanthurus, V. exanthematicus, V. indicus, and V. salvator) and 105 compared them to phylogenetically distant lizards with typical lizard hearts, the Brown Anole 106 (Norops sagrei) and the Leopard Gecko (Eublepharis macularius). To assess homology between 107 amniote cardiac structures, we studied the expression of anole orthologues of genes associated with 108 the formation of the ventricular septum of mammals and birds, specifically Irx1, Irx2, Tbx3, Tbx5 109 and Myh6 (Aanhaanen et al., 2010; Boukens & Christoffels, 2012; Bruneau et al., 1999; 110 Christoffels, Keijser, Houweling, Clout, & Moorman, 2000; de Groot et al., 1987; Hoogaars et al., 111 2004; A.