Astyanax mexicanus: surface fish and Pachón Methods: • Cardiac cryoinjury • Histochemistry analysis • RNAsequencing • Real-time quantitative polymerase chain reaction (qPCR) • RNAscope • QTL analysis RESULTS (CONTINUED) MATERIALS AND METHODS SUMMARY FINANCIAL SUPPORT RESULTS BACKBROUND Distinct dynamics of the extracellular matrix during heart regeneration in different Astyanax mexicanus populations Zhilian Hu1, Madeleine E. Lemieux2, Mathilda T. M. Mommersteeg1 1. Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK 2. Bioinfo, Plantagenet, Ontario, Canada The cardiac regenerative capacity of Astyanax mexicanus populations is completely distinct in response to the injury. Surface fish can regenerate the ablated heart tissue, similar to zebrafish, while Pachón cannot and instead forms a permanent scar with excess extracellular matrix (ECM), similar to human. ECM is a dynamic three-dimension network of extracellular macromolecules including collagen and non-collagenous components. Its content, relative proportion, and spatial arrangement play a pivotal role during tissue regeneration. Any changes in ECM deposition can have a big impact on the ability to regenerate. Astyanax mexicanus allows us to identify the differences in ECM deposition and removal between a degradable scar in a regenerative setting and a permanent scar in a non-regenerative setting. Our research indicates ECM components and their regulating Matrix metalloproteinases (MMPs) are dynamically and differentially expressed during heart regeneration between surface fish and Pachón. Next, we will link the key scarring differences directly to both the genome and the ability for heart regeneration using new and prior Quantitative Trait Loci analyses. This will allow to find the most fundamental molecular mechanisms directing the wound healing process towards regeneration versus scarring. Result 1: Differential regenerative capacity of the heart after cryoinjury between surface fish and Pachón Figure 1. A-A’, Adult Astyanax mexicanus surface fish (A) and cave-dwelling Pachón (B). B-C’, AFOG staining of the cardiac ventricle after cryoinjury (Fibrin in red, intact muscle in orange and collagen in blue). The hearts are similar in fibrin formation at 3 days post cryoinjury (dpci) (B-B’), followed by intermingling of fibrin with collagen fibrils at 14 dpci (C-C’). Resolution of fibrotic scar in surface fish (D), but permanent scar in Pachón (D’) at 100 dpci. E, Quantification of the scar area at 100 dpci shows a large scar is still present in Pachón, while the surface fish restored the injured ventricular apex. Scar bar: 100 µm. Result 2:Excess deposition of collagens and disorganisation of collagen fibrils in the wound area of Pachón heart Figure 2. A-B, Picro Sirius red (PSR) staining of the ventricle at 14 dpci for both fish (collagen in bright red under light microscopy). ven, ventricle. A’-B’, Imaging of collagen fibrils under a polarised microscope shows remarkable high deposition of collagens and disorganization of collagen fibrils in Pachón (Green-thin fibrils in green and thick fibrils in yellow-orange- red). Result 3: Deregulation of collagen and non-collagenous extracellular matrix encoding genes after cryoinjury Figure 3. A-A’, Diagram of the workflow of RNAseq. B, Example of candidate genes validated by qPCR at six time points (6 hpci, 24 hpci, 3 dpci, 7 dpci, 14 dpci and 30 dpci). Expression levels of non-collagen fibronectin (fn1a) and FACIT collagen 12 (col12) are dynamically and differentially expressed between both fish during the regenerative process. C-D”, RNAscope revealed that col12 is primarily expressed in the outside wall of wound. E-F”, Multiplexin collagen 18 (col18) is undetectable in the wound region of surface fish (D-D”) but is expressed in the Pachón scar (E-E”). These results indicate the composition of the scar is different between both fish. FACIT, Fibril Associated Collagens with Interrupted Triple Helices; Mu