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Clotting protein – An extracellular matrix (ECM) protein involved in crustacean hematopoiesis
Accepted Manuscript Clotting protein – An extracellular matrix (ECM) protein involved in crustacean hematopoiesis Kingkamon Junkunlo, Kenneth Söderhäll, Irene Söderhäll PII:
S0145-305X(17)30346-4
DOI:
10.1016/j.dci.2017.09.017
Reference:
DCI 2991
To appear in:
Developmental and Comparative Immunology
Received Date: 30 June 2017 Revised Date:
19 September 2017
Accepted Date: 19 September 2017
Please cite this article as: Junkunlo, K., Söderhäll, K., Söderhäll, I., Clotting protein – An extracellular matrix (ECM) protein involved in crustacean hematopoiesis, Developmental and Comparative Immunology (2017), doi: 10.1016/j.dci.2017.09.017. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Clotting protein – an extracellular matrix (ECM) protein involved in crustacean hemato-
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poiesis1
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Kingkamon Junkunlo, Kenneth Söderhäll and Irene Söderhäll
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Department of Comparative Physiology, Uppsala University, Norbyvägen 18A, 752 36 Uppsala,
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Sweden
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Corresponding author: Irene Söderhäll, Irene. Söderhä[email protected]
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Given his role as Editor in Chief, Kenneth Söderhäll had no involvement in the peer-review of this article and has no access to information regarding its peer-review. Full responsibility for the editorial process for this article was delegated to Mirodrag Belosevic
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Abstract Hematopoietic progenitor cells in crustaceans are organized in lobule-like structures sur-
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rounded by different types of cells and extracellular matrix (ECM) protein in a Hematopoietic
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tissue (HPT). Here we show that the clotting protein (CP) is part of the ECM in HPT and is se-
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creted during HPT cell culture. The formation of a filamentous network of CP was observed in
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HPT cell culture. A high amount of CP protein was detected at the surfaces of undifferentiated
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cells (round-shaped) compared with migrating cells (spindle shaped). Co-localization of the CP
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protein and TGase activity was observed on the cell surface and filamentous network between
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cells. A role for CP together with collagen was revealed in a 3D culture in which a collagen-I
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matrix was immobilized with CP or supplemented with CP. The results showed possible func-
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tions of CP, collagen, TGase and cytokine Ast1 in the regulation of HPT progenitor cell behav-
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ior. This is the first study to provide insight into the role of CP, which probably not only partici-
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pates in clot formation but also functions as an ECM component protein controlling hematopoi-
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etic stem cell behavior.
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Keywords: clotting protein, ECM, hematopoiesis, crustacean
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Abbreviations: Ast1, Astakine 1; CP, clotting protein; ECM, extracellular matrix; Hg, Hedgehog; HPT,
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hematopoietic tissue; TGase, transglutaminase; Trol, heparin sulfate proteoglycan Terribly Reduced Optic
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Lobes
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1. Introduction
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Blood coagulation or hemolymph clotting plays a crucial role in innate immune responses
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especially in invertebrates which have open circulatory systems (Vazquez et al., 2009). Clotting
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formation act as a physical barrier that prevent loss of hemolymph and infection by invaders
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(Cerenius and Söderhäll, 2013; Hall et al., 1999). In crustaceans, coagulation is caused by cova-
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lent crosslink interaction of clotting protein (CP), which is present in hemolymph, and transglu-
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taminase (TGase) enzyme (Hall et al., 1999; Kopácek et al., 1993). In the freshwater crayfish,
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Pacifastacus leniusculus, CP was originally isolated from plasma by precipitation at low ionic
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strength at pH 6.0. CP is a lipoglycoprotein that consists of two identical 210-kDa disulfide2
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bonded subunits (Kopácek et al., 1993). CP is a known substrate of TGase in hemolymph clot
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formation. Each of the 210-kDa subunits has both lysine and glutamine sidechains that are rec-
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ognized and become covalently linked to each other by TGase (Wang et al., 2001). Clot for-
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mation is induced when TGase is released from hemocytes or HPT becomes activated by the
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Ca2+ in plasma, and starts crosslinking the plasma CP molecules into large aggregates (Hall et
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al., 1999). Interestingly, we have recently detected high expression of CP mRNA in crayfish
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HPT in transcriptome analysis (BioProject ID: PRJNA259594). Furthermore, CP is also involved
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in the crosslinking process that stabilizes collagen in HPT (Junkunlo et al., 2016).
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Hematopoiesis is a complex process by which immature hematopoietic progenitor cells
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are produced and developed before being released from the HPT into the circulation (Lin and
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Söderhäll, 2011; Söderhäll, 2016). Cell homeostasis is controlled by specific microenvironments
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called niches, and the ECM plays an essential role in this regulation. The ECM can directly or
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indirectly modulate stem cell behavior such as that occurring in proliferation, self-renewal and
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differentiation (Ahmed and Ffrench-Constant, 2016). Different types of ECM molecules have
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regulatory functions for specific types of stem cells by which ECM protein can be adjusted and
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reformed to provide the most suitable environment for stem cells (Gattazzo et al., 2014). Func-
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tional studies of ECM component protein in controlled stem cell behavior are well established in
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vertebrates, but there are very few studies of ECM protein characterization and their role in con-
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trolled stem cell activity in invertebrates.
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In Drosophila, the loss of Trol (heparan sulfate proteoglycan Terribly Reduced Optic
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Lobes), an ECM protein component in hematopoiesis, causes a dramatic conformational change
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in the ECM and induces premature hemocyte differentiation (Grigorian et al., 2013). In verte-
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brates, a matrilin family protein has been reported as an extracellular adaptor protein that bind to
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different types of collagen and other ECM proteins. Matrilins support matrix assembly by con-
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necting ECM components and mediating physiological interactions between cells and the ECM
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(Uckelmann et al., 2016). Furthermore, Matrilin-1 is required for zebrafish development by facil-
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itating collagen II secretion and deposition (Neacsu et al., 2014). In crayfish, the production of
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hemocytes in crayfish occurs in the hematopoietic tissue (HPT). The immature progenitor cells
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are found in lobule-like structures inside HPT surrounded by different types of cells and connec-
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tive tissue such as collagen (Chaga and Söderhäll, 1995). These progenitor cells can differentiate
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into mature hemocytes and are released into the circulation (Söderhäll et al., 2003).
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Changes in the structure of ECM associated with extracellular signaling dispersion have
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been reported in Drosophila, and an altered state of the ECM in Trol mutant lymph glands can
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inhibit Hedgehog (Hg) signaling (Grigorian et al., 2013). The cytokine astakine (Ast1) has been
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isolated and studied in crayfish as a hematopoietic growth factor that induces the proliferation
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and differentiation of HPT cells (Lin and Söderhäll, 2011; Söderhäll et al., 2005). Furthermore,
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Ast1 acts by interfering with the ECM structure by the regulation of extracellular TGase activity.
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Previously, we have demonstrated the importance of extracellular TGase activity to control hem-
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atopoiesis through the interaction of hematopoietic cells and the ECM and have shown that high
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extracellular TGase activity is required to maintain the immature HPT cells inside the hemato-
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poietic tissue (Junkunlo et al., 2016). In addition, Ast1 functions as an inhibitor of the enzymatic
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crosslinking activity of the TGase enzyme and Ast1 treatment also led to impaired TGase-
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mediated crosslinking of CP (Sirikharin et al., 2017).
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In the present study, we focused on the potential roles of CP as a part of the ECM in the control of hematopoiesis in the crayfish P. leniusculus.
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2. Materials and Methods
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2.1 Experimental Animals
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Freshwater crayfish, P. leniusculus, were acquired from Lake Erken, Sweden. The ani-
mals were maintained in aquaria with aeration at 10°C. Healthy and intermolt male crayfish were used for the experiments.
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2.2 Purification of CP from crayfish plasma
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Hemolymph, approximately 15 ml, was withdrawn from 9 crayfish into 15 ml of ice-cold anticoagulant buffer (0.14 M NaCl, 0.1 M glucose, 30 mM trisodium citrate, 26 mM citric acid,
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pH 4.6) (Söderhäll and Smith, 1983). The hemocyte pellets were removed by centrifugation
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(800 x g, 10 min 4 C). The supernatant, called crayfish plasma, was used for purification of CP,
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as described previously (Hall et al., 1999). Crayfish plasma was dialyzed against 10 mM sodium
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phosphate, pH 6.0, containing 1 mM EDTA overnight at 4° C. The CP precipitated after dialysis
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was collected by centrifugation at 2500 x g for 10 min 4°C. The resulting supernatant without CP
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was removed and is denoted CP-depleted plasma. The orange-colored pellets of CP were re-
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suspended in 500 µl of 0.2 M sodium phosphate buffer, pH 6.0, containing 1 mM EDTA. The
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purity of CP was analyzed by SDS-PAGE and was compared with SDS-PAGE of CP-depleted
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plasma and whole crayfish plasma.
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2.3 HPT cell preparation and culture
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HPT was dissected from the dorsal side of the stomach. Subsequently, the HPT cells were isolated and cultured as previously described (Söderhäll et al., 2005). Briefly, after dissection,
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the HPT was washed with crayfish phosphate-buffered saline (CPBS; 10 mM Na2HPO4, 10 mM
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KH2PO4, 150 mM NaCl, 10 mM CaCl2 and 10 mM MnCl2, pH 6.8). Next, the HPT was incubat-
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ed at room temperature for 20 min in 800 µl of collagenase type I and type IV (Sigma-Aldrich,
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USA) at 0.1% in CPBS. The collagenase solution was removed by centrifugation at 3000 x g for
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5 min. The resulting cell pellet was washed twice with 1 ml of CPBS and was resuspended in L-
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15 medium (Sigma-Aldrich, USA) supplemented with 1 mM phenylthiourea, 60 mg/ml penicil-
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lin, 50 mg/ml streptomycin, 50 mg/ml gentamicin (Sigma-Aldrich, USA), and 2 mM L-
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glutamine. The cells were cultured in 96-well plates at a density of 4 x 104 cells/well at 16°C.
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For HPT culture, after dissection, the undigested HPT after collagenase treatment were cultured
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in 24-well plates containing 300 µl of L-15 medium supplemented as above. Crude Ast1 from
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crayfish plasma was prepared as previously described (Söderhäll et al., 2005) and was added to
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each well at a concentration of 2% (v/v). One-third of the medium was changed and supplement-
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ed with crude Ast1 from plasma every second day.
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2.4 Preparation of 3D collagen-I matrix culture
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For 3D collagen-I matrix culture, a collagen-I solution was prepared by diluting 10.6 mg/ml collagen-I stock solution (BD bioscience, USA) to 0.106 mg/ml with phosphate buffer
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(137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 2 mM KH2PO4, pH 7.4) as previously de-
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scribed (Noonin et al., 2012). Next, 150 µl of the diluted solution was added into 96-well plates
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and was allowed to polymerize at 37°C for 1h and 30 min. The 3D collagen-I matrix-gel plate
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was immediately used after preparation.
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2.5 CP addition in HPT cell culture
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To examine the effect of clotting protein on HPT cells, in vitro experiments were con-
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ducted in 3D collagen-I matrix culture. In a 3D collagen-I matrix gel culture, four different ex-
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perimental groups were evaluated; 1) CM- (which consisted of collagen I matrix without addi-
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tional proteins), 2) CMCP- (in which CP-depleted plasma was added to collagen-I solution and
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was allowed to polymerize together with collagen-I), 3) CMCP+ (in which CP was added to col-
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lagen-I solution and was allowed to polymerize with collagen-I), and 4) CMCP (in which CP was
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added to the culture medium during HPT cell preparation). In treatment 2 (collagen-I immobi-
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lized with CP-depleted plasma), the collagen-I solution was mixed with 10 µg of CP-depleted
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plasma per well and 3 (Collagen-I immobilized with CP), the collagen-I solution was mixed with
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10 µg of CP per well before being added to 96-well plates for polymerization. After polymeriza-
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tion, HPT cells at a density of 4 x 104 cells/well in 150 µl of supplemented L15 medium were
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carefully dropped onto the surface of the collagen-I matrix gel. For treatment with collagen-I
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supplemented with CP, 10 µg of CP was added into the well after the cells were plated. To study
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the effect of the combination of crude Ast1 (crayfish plasma) and CP to HPT cell morphology,
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crude Ast1, to a final concentration of 2%, was supplemented in different CP treatments as de-
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scribed above. The cell morphology was observed at 24 h, 48 h, 56 h and 72 h in culture. All cell
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cultures were maintained at 16°C, and one-third of the medium was changed (with or without
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crude Ast1 from plasma) every second day.
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2.6 Immunostaining of CP in HPT Cell Cultures
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HPT cells were cultured and maintained in 96-well plates. The medium was removed and cells were fixed with 4% paraformaldehyde in PBS for 1 h at room temperature. The HPT cells
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were subsequently washed 5 times with PBS and blocked with 10% BSA in PBS for 1 h at room
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temperature. Next, the cells were incubated for 3 h with rabbit anti-crayfish clotting protein
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(1:10000). Polyclonal antibodies against the CP were commercially raised in rabbits at the Bio-
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medical Technology Research Center, Chiang Mai University, Thailand. After being washed 5
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times with PBST (0.5% Tween 20 in PBS buffer), HPT cells were incubated for 1 h with FITC-
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conjugated anti-rabbit IgG (1:1000) and Hoechst 33258 dye at a concentration of 1 µg/ml to stain
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the nuclei at room temperature. After cells were washed 5 times with PBST, the clotting protein
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was observed under a fluorescence microscope.
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2.7 Detection of CP and TGase activity
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Partially digested HPT was cultured as described above. After 48 h in culture, 1 mM of 5-
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(biotinamido)-pentylamine (Pierce, USA), a substrate for TGase, was added to the tissue cultures
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and was incubated for 18 h. Subsequently, the tissues were fixed according to (Lin et al., 2008).
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Briefly, the medium was removed, and the tissues were fixed with 4% paraformaldehyde in PBS
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for 1 h at room temperature. Next, 25 mM glycine in PBS was added to the wells, and the cells
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were incubated for 30 min. The cells were subsequently washed 5 times with PBST and were
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blocked with 10% BSA in PBST for 1 h. The primary antibody, rabbit anti-crayfish clotting pro-
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tein (1:10000), was added and incubated for 3 h at room temperature. After being washed 5 times
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with PBST, HPT was incubated for 1 h with Alexa Fluor® 594-conjugated anti-rabbit IgG (Life
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Technologies, USA) (1:1000) together with streptavidin-FITC conjugate (GE Healthcare, USA)
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(1:200) diluted with 1% BSA (w/v) in PBST for double labeling at room temperature. The tis-
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sues were incubated with Hoechst 33258 dye at a concentration of 1 µg/ml to stain the nuclei.
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After tissues were washed 5 times with PBST, the localization of CP and TGase activity was
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observed under a fluorescence microscope.
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2.8 CP detection and double labeling of CP and Collagen IV in HPT
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Crayfish were injected with 4% paraformaldehyde in PBS before immediately soaked in
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4% paraformaldehyde overnight at 4°C. The whole stomach covered with HPT was dissected
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from crayfish and washed with PBS, and then was carefully removed from the stomach. The
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HPT was blocked with 10% BSA in PBS for 1 h at room temperature. The primary antibody,
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rabbit anti-crayfish clotting protein (1:10000), was added and incubated for 3 h at room tempera-
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ture. After being washed 5 times with PBST, HPT was incubated for 1 h with FITC-conjugated
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anti-rabbit IgG (Sigma-Aldrich, USA) (1:1000) for CP detection in Figure 6. For double labeling
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in Figure 7, the primary antibody, rabbit anti-crayfish clotting protein (1:10000) was added to-
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gether with mouse anti-collagen type IV antibody (Sigma-Aldrich, USA) (1:100). Next the sec-
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ondary antibody Alexa Fluor® 594-conjugated anti-rabbit IgG (Life Technologies) (1:1000) was
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used in CP detection together with FITC-conjugated anti-mouse IgG (Sigma-Aldrich, USA)
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(1:500) at room temperature. The tissues were incubated with Hoechst 33258 dye at a concentra-
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tion of 1 µg/ml to stain the nuclei. After tissues were washed 5 times with PBST, The localiza-
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tion of CP or collagen IV together with CP was observed under a fluorescence microscope.
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3. Results
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3.1 CP forms an extracellular filamentous network in HPT cell culture Because we observed a high expression of CP in the hematopoietic tissue in our P. leniusculus transcriptome data (Bioproject ID PRJNA 259594), we decided to study the im-
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portance of CP in crayfish HPT. Primary HPT cells were cultured for 5 days, and then im-
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munostaining of CP was performed. By using a polyclonal CP antibody, we observed the secre-
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tion and formation of a filamentous network between cells in culture (Figure. 1). CP is known as
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a substrate for TGase during clot formation (Hall et al., 1999), and a recent study has shown that
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Ast1 had a direct inhibitory effect on the crosslink formation of TGase (Sirikharin et al., 2017).
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The effect of crude Ast1 from plasma on cell spreading is shown in Figure 2. When cells were
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supplemented with crude Ast1, cell spreading was clearly observed at 72 h (Figure 2A). Without
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crude Ast1, the HPT cell morphology did not change during culture (Figure 2B). We have previ-
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ously detected high extracellular TGase activity in undifferentiated cells and have found that this
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activity is decreased during cell migration out of the HPT in culture (Lin et al., 2008). Interest-
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ingly, we also found that the CP protein was present at a higher level on the surfaces of round-c
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ells than migrating cells (spindle-shaped cells) (Figure 2A and 2B). Co-localization of CP and
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TGase activity was found on the HPT cell surface (Figure 2A and 2B), and the co-localization of
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CP and TGase activity was also found as a filamentous network between cells (Figure 2A).
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From these results, we hypothesized that CP may function as part of the ECM and that it may be
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involved in the regulation of hematopoiesis in crustaceans.
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3.2 Effects of CP on HPT cell morphology in 3D collagen-I matrix culture
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Collagen is shown as a component of the ECM proteins in HPT, and it is present on HPT
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cell surfaces (Junkunlo et al., 2016). To further elucidate the function of CP as a constituent of
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the ECM and to study the HPT cell behavior, we performed an in vitro experiment using 3D col-
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lagen-I matrix culture (Figure 3). Without the addition of crude Ast1, HPT cells did not show
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any signs of spreading after CP addition (CMCP+ or CMCP) compared with the control group-
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i.e., in CM- or CMCP- (Figure 3B). HPT cells in CM- or CMCP- form a monolayer of round-
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shaped cells at 24 h, and the cell morphology did not change after 48 h or even 7 days in the ab-
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sence of crude Ast1 (Supplement Figure S1). When crude Ast1 was added into the CM- or
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CMCP- cultures, the cells did not change in morphology after 24 h, and the HPT cells were
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round and formed small clusters (Figure 3A). However, the addition of crude Ast1 to CMCP+ or
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CMCP cultures dramatically induced cell spreading after 24 h (Figure 3A).
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In CMCP+ cultures containing crude Ast1, the cells lost their spreading capacity after 48
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h; however in the CMCP cultures with crude Ast1, the spreading of cells was more elongated and
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strengthened and branches were formed between cells at 48 h (Figure 4A) and after 72 h (Figure
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4B) or even 7 days (data not shown). The formation of a filamentous network between cells was
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observed only in CMCP cultures (Figure 4A and 4B). These results suggested that CP might func-
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tion as an ECM component together with collagen-I and regulate the spreading/migration of HPT
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cells.
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The above results indicated that culture in a collagen matrix with the addition of CP and
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crude Ast1 clearly stimulated the spreading of the HPT cells. In addition, the cross-linked func-
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tion of TGase with CP was shown in 3D collagen-I matrix culture. In CMCP+ cultures contain-
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ing crude Ast1, CP was immobilized with collagen before HPT cells was added into culture.
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Without TGase activity from HPT cells, the effect of CP together with Ast1 and collagen in-
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duced HPT cell spreading but it was less effected compared with CMCP cultures. In CMCP cul-
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tures containing crude Ast, CP was added during HPT cell preparation. TGase activity from HPT
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cells promoted the cross-linking of CP with collagen and provided a proper environment for cell
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spreading. For these reasons, more cell spreading and filamentous network formation was ob-
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served in CMCP cultures
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To test the important roles of CP and collagen in controlling HPT progenitor cell behavior, we then tested whether the collagen-I-coated surface had an effect on cell morphology. First,
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we found that the addition of CP to HPT cell culture did not induce the spreading of HPT cells
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with or without crude Ast1 (Supplementary Figure S2). Moreover, the knockdown of CP mRNA
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expression in the HPT cells in ordinary culture (plastic well plates) and in 3D collagen-I matrix
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culture had no effect on cell morphology (Supplementary Figure S3). Thus, CP addition ap-
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peared to require the presence of collagen to affect cell morphology. Accordingly, we asked next
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whether collagen-I together with CP and crude Ast1 could induce the spreading of HPT cells on
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wells coated with collagen-I, i.e., without the formation of a 3D matrix. Coating the well surface
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with collagen-I did not induce similar cell morphology as shown for culture in the 3D matrix
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made of collagen, with the addition of either CP or Ast1 (Supplementary Figure S4). An interest-
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ing observation was that, in uncoated wells, the addition of Ast1 induced cell attachment and
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spreading; however after collagen-I coating, the cells instead formed small cell clusters in the
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presence of Ast1 (Supplementary Figure S4). These results showed that the regulation of progen-
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itor cells activity are tightly regulated. To provide a proper environment for progenitor cell dif-
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ferentiation, CP together with crude Ast1 and collagen are required to collaborate in the 3D di-
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mension, which is similar to the environment in the HPT.
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3.3 CP as a part of the ECM in HPT
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Within the HPT, hematopoietic progenitor cells are enclosed in lobule-like structures with different types of cells and the ECM. This specific microenvironment is essential for stimu-
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lating or maintaining stem cell behavior. Because CP was found to be abundant in crayfish HPT,
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the localization of CP in HPT was studied (Figure 5). The localization and structure of crayfish
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are shown in Figure 5A. Whole-mount immunostaining of CP showed that this protein was
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mainly found at the edges of the HPT when compared with the central parts of HPT (Figure 5B-
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D). CP protein was clearly distributed around the surfaces of the HPT cells or covered the lob-
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ules. This result indicated that CP may act as a supportive structure of the HPT cells inside the
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lobules. CP was also detected in the anterior HPT and APC but the largest amount was found in
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the posterior HPT parts (Figure 5B and 5D). To emphasize the role of CP as a part of the ECM
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in HPT issue, co-localization of CP and collagen type IV, a component of the ECM, was investi-
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gated. At the edges of posterior HPT where CP protein is mainly localized, co-localization of CP
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with collagen type IV was observed around cells and in lobules (Figure 6). Co-localization of
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these two proteins was also found in other HPT areas (data not show). These results suggested
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that CP is a constituent of the ECM component protein in crayfish HTP.
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4. Discussion
Hematopoiesis or blood cell production in crayfish is tightly regulated and controlled in a complex environment in HPT tissue. The microenvironments or niches consist of several cell
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types and a multiple-component ECM. The ECM not only plays the role of supporting a hemato-
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poietic niche but also participates in binding different growth factors and cytokines (Schultz and
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Wysocki, 2009). Cytokines are essential signaling molecules that are transported through the
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ECM, reach the stem cell niche and ultimately activate stem cell proliferation and differentiation
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(Mercier, 2016). Our previous studies have shown that Ast1 is a key survival factor that induces
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the proliferation and differentiation of progenitor cells (Lin and Söderhäll, 2011; Söderhäll et al.,
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2005). Moreover, TGase has been shown to play a role in maintaining HPT cells in an undiffer-
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entiated form (Lin et al., 2008), and recent studies have shown that Ast1 affects TGase activity
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directly (Sirikharin et al., 2017). CP is known as a secreted protein that is abundant in plasma
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and is a substrate of TGase during clot formation (Hall et al., 1999; Wang et al., 2001) In addi-
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tion, we now have found that CP is an important protein in the crayfish HPT, as is TGase (Lin et
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al., 2008). The CP mRNA was highly expressed in HPT, while very low expression was detected
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in circulating hemocytes. For these reasons, we hypothesize that CP may participate not only in
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clot formation but also may be involved in hematopoietic regulation.
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We first investigated the role of CP in HPT cell culture. When HPT cells start to migrate,
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TGase activity was decreased (Lin et al., 2008). Immunostaining of CP in HPT cells showed that
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CP was also mainly expressed in round, non-spreading cells compared with spreading cells.
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These results indicated the possibility that CP may act together with TGase function in control-
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ling progenitor cell activity. We also observed the secretion of CP protein and the production of
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CP filaments with variable thickness, often forming branches, and connecting cells to other cells
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in the culture. Similar observations have been reported in primary embryonic mouse fibroblasts
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cell cultures (Klatt et al., 2001) and Swarm rat chondrosarcoma cell cultures (Klatt et al., 2000).
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Furthermore, the extracellular network formation of Matrilin, an ECM adaptor protein, has been
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detected in cultured cells (Klatt et al., 2011). In crayfish, Ast1 induces the proliferation and dif-
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ferentiation of progenitor cells inside HPT and causes the release of mainly semi-granular hemo-
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cytes into the circulation (Lin et al., 2010). This process can be observed in an in vitro culture
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system as spreading of the HPT cells (Lin and Söderhäll, 2011).The co-localization of TGase
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activity and CP protein observed on the HPT cell surface and in the filamentous network during
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cell spreading indicated the possible roles of CP, TGase activity and Ast1 in hematopoietic regu-
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lation.
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Collagen has been characterized as a part of ECM protein in crayfish HPT. Collagenase type I and IV enzymes are used in HPT cell culture preparation (Söderhäll et al., 2005). Previ-
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ously, we have shown co-localization of extracellular TGase and collagen IV on HPT cell sur-
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faces (Junkunlo et al., 2016). Thus, we hypothesized that CP may function together with collagen
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in controlling progenitor cell behavior. Three-dimensional (3-D) cell culture can be used to mim-
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ic the environment in the tissue under in vivo conditions, and a 3D collagen-I matrix culture can
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be used to enhance the interactions between cells and between cells and the environment (Ed-
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mondson et al., 2014). In Human Wharton’s jelly mesenchymal stem cells (HWJMSCs) develop
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various morphologies on a collagen-I-coated and in a 3D collagen matrix. On collagen-coated
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surface the HWJMSC cells show a fibroblast-like morphology, in contrast to the 3D collagen
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matrix in which they show a star-like morphology (Khodabandeh et al., 2016). Similarly, we
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observed different morphologies of the cell cultured on 3D collagen matrix compared with a col-
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lagen-coated surface and naked plastic. The 3D collagen matrix, supplemented with CP and
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crude astakine, induced a spreading morphology, and a network of fibers of CP was formed be-
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tween the cells, whereas a collagen-coated surface did not induce any morphology change. In
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addition, HPT cells were elongated and gathered into packed colonies. The elongated cells
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formed chains of cells through the surrounding ECM with other cells or connected with other
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colonies. In a previous paper, we have similarly shown that HPT cells cultured in a collagen ma-
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trix can be induced to express mRNA for a specific Kazal proteinase inhibitor that is a marker
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for semi-granular hemocytes (Noonin et al., 2012), thus again showing that a 3D collagen matrix
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culture may indeed provide a more suitable milieu and is more similar to in vivo conditions. A
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proper environment for the HPT cells is needed, and collagen, CP, TGase and Ast1 appeared to
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be required to form such an environment. Thus, our new results indicate a role for CP during
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hematopoiesis, and Ast1 might be required to suppress crosslink formation of CP by the TGase
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enzyme. This inhibition may result in less crosslinking of CP to other ECM proteins such as col-
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lagen, and finally stimulate the differentiation and release of new hemocytes, as shown earlier
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(Lin et al., 2010).
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Crucial roles of collagen in controlling stem cell behavior in 3D culture have been studied in many cell types. For example, a breast tumor cell line (MDA-MB-231) grows more slowly
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in 3D collagen-I matrix culture than on a 2D collagen-coated surface (Kim et al., 2015). In addi-
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tion, osteoblast differentiation is regulated by the presence of collagen type I in the ECM, and
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TGase is required for collagen secretion and extracellular deposition, which control the osteo-
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blast differentiation (Piercy-Kotb et al., 2012). In Drosophila, the loss of Trol, an ECM protein,
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induces the release of plasmatocytes into the hemolymph (Grigorian et al., 2013).
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To confirm a function of CP as a part of the ECM in crayfish HTP, we continued to in-
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vestigate the structural localization of CP in HPT in vivo. By immunostaining we found that CP
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was present outside the cells in the HPT, but was much less abundant in the APC area. The high-
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est levels of CP were found at the posterior parts of HPT compared with the middle parts. We
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also observed co-localization of CP and collagen IV in HPT in vivo. The co-localization of ECM
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protein and collagen has been reported in vertebrates. Syndecan-2, an adaptor protein, exhibits a
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fibrillary pattern over the cell surface and is deposited into the ECM (Filla et al., 2004). Further-
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more, Syndecans-2 has also been found to co-localize with ECM proteins, for example fibron-
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ectin, laminin and type IV collagen in Human ciliary muscle (HCM) cells and HSC endothelium
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cells. Thus, the localization of CP and co-localization of CP with collagen IV in HPT and TGase
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activity in HPT cells support the potential roles of CP as an ECM protein in hematopoietic regu-
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lation.
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5. Conclusion
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Although CP is known to be involved in clot formation after wounding or infection, we now show a potential role for CP as an ECM protein controlling the differentiation and prolifera-
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tion of progenitor cells in HPT. CP may function together with collagen in producing a suitable
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environment for hematopoietic tissue in crayfish.
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Figure Legends
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Fig. 1. Secretion and formation of clotting protein (CP) filaments in HPT cell culture. HPT
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cells were cultured for 5 days without the addition of crude Ast1. The localization of clotting
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protein (CP) was detected by immunostaining with anti-CP antibody (green), and nuclei were
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stained with Hoechst 33258 (blue).
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Fig. 2. The CP protein and TGase activity are co-localized at the surfaces of round cells
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compared with migrating cells (spindle shaped cells). Co-localization of CP and TGase activi-
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ty was determined after incubation of the partially digested HPT with the TGase substrate 5-
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(biotinamido)-pentylamine for 18 h. The cross-linking of substrate was visualized after streptavi-
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din-FITC (green) addition together with Alexa Fluor® 594-conjugated anti-rabbit IgG (red) to
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CP antibody. Hoechst 33258 (blue) was used as a nuclear stain. A) Double staining for TGase
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activity and CP was performed on partially digested HPT culture for 72 h without crude Ast1. B)
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Double staining for TGase activity and CP was performed on partially digested HPT culture for
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72 days with crude Ast1. All experiments were repeated 3 times.
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Fig. 3. CP addition together with crude Ast1 cause HPT cell spreading in 3D collagen-I ma-
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trix culture. Four different experimental treatments were performed: 1) Collagen-I matrix only
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= CM-, 2) Collagen-I immobilized together with CP-depleted plasma = CMCP-, 3) Collagen-I
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matrix immobilized together with 10 µg CP = CMCP+, and 4) Collagen-I supplemented with CP
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after polymerization = CMCP A) HPT cells at a density of 4 x 104 cells per well were cultured in
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the four different matrixes supplemented with crude Ast1. B) HPT cells at a density of 4 x 104
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cells per well were cultured in the four different matrixes without crude Ast1. The cell morphol-
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ogy was observed after 24 h in culture and all experiments were repeated 5 times.
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Fig. 4. CP promotes the elongation and clustering of HPT cells after culture in a 3D colla-
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gen-I matrix supplemented with crude Ast1. HPT cells cultured in CMCP+ or CMCP together
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with crude Ast1. A) HPT cell morphology after 48 h in culture. B) HPT cell morphology after 72
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h in culture. All experiments were repeated at least 3 times.
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Fig. 5. CP as a part of the ECM in HPT. A) Localization and structure of crayfish HPT, APC
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and different parts of HPT. HPT is divided into two parts - the posterior HPT (green) and anteri-
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or HPT (yellow). APC (blue) is a special small area in the anterior part of the HPT. Letters with
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squares in A) indicate areas where images in B), C) and D) were captured. (B-D) Whole mount
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immunohistochemistry of clotting protein in posterior HPT labeled with the primary antibody,
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rabbit anti-crayfish clotting protein (1:10000) together with the secondary antibody, FITC-
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conjugated anti-rabbit IgG (Sigma-Aldrich) (1:1000) (green). Hoechst 33258 (blue) was used as
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a nuclear stain. All experiments were at least repeated 3 times.
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Fig. 6. Co-localization of collagen type IV and CP in HPT. Double staining for CP (red) and
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collagen IV (green) was performed in whole HPT labeled with the primary antibody, rabbit anti-
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crayfish clotting protein (1:10000) together with mouse anti-collagen type IV antibody (Sigma,
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1:100). The secondary antibody Alexa Fluor® 594-conjugated anti-rabbit IgG (red) (Life Tech-
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nologies) (1: 1000) was used in CP detection together with FITC-conjugated anti-mouse IgG
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(green) (Sigma-Aldrich) (1: 500). Arrows indicate the co-localization of CP and collagen IV. All
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experiments were repeated at least 3 times.
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Figures
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Fig. 6.
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Acknowledgments
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This work was supported by the Swedish Science Research Council (VR 621-2012-2416 to KS)
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Conflicts of Interest
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The authors declare that they have no conflicts of interest with the contents of this article.
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Author Contributions
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KJ performed experiments, analyzed results, prepared figures and wrote the paper. KJ, KS and IS
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contributed to study design, interpreted the data and participated in writing the paper. All authors
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reviewed the results and approved the final version of the manuscript.
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Highlights
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Clotting protein (CP) is part of the ECM in hematopoietic tissue (HPT) CP forms an extracellular filamentous network in HPT cell culture CP together with crude Ast1 effect HPT cell morphology in 3D collagen-I matrix culture CP, collagen, TGase and Ast1 regulate HPT progenitor cell behavior
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S0145-305X(17)30346-4
DOI:
10.1016/j.dci.2017.09.017
Reference:
DCI 2991
To appear in:
Developmental and Comparative Immunology
Received Date: 30 June 2017 Revised Date:
19 September 2017
Accepted Date: 19 September 2017
Please cite this article as: Junkunlo, K., Söderhäll, K., Söderhäll, I., Clotting protein – An extracellular matrix (ECM) protein involved in crustacean hematopoiesis, Developmental and Comparative Immunology (2017), doi: 10.1016/j.dci.2017.09.017. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Clotting protein – an extracellular matrix (ECM) protein involved in crustacean hemato-
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poiesis1
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Kingkamon Junkunlo, Kenneth Söderhäll and Irene Söderhäll
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Department of Comparative Physiology, Uppsala University, Norbyvägen 18A, 752 36 Uppsala,
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Sweden
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Corresponding author: Irene Söderhäll, Irene. Söderhä[email protected]
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Given his role as Editor in Chief, Kenneth Söderhäll had no involvement in the peer-review of this article and has no access to information regarding its peer-review. Full responsibility for the editorial process for this article was delegated to Mirodrag Belosevic
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Abstract Hematopoietic progenitor cells in crustaceans are organized in lobule-like structures sur-
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rounded by different types of cells and extracellular matrix (ECM) protein in a Hematopoietic
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tissue (HPT). Here we show that the clotting protein (CP) is part of the ECM in HPT and is se-
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creted during HPT cell culture. The formation of a filamentous network of CP was observed in
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HPT cell culture. A high amount of CP protein was detected at the surfaces of undifferentiated
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cells (round-shaped) compared with migrating cells (spindle shaped). Co-localization of the CP
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protein and TGase activity was observed on the cell surface and filamentous network between
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cells. A role for CP together with collagen was revealed in a 3D culture in which a collagen-I
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matrix was immobilized with CP or supplemented with CP. The results showed possible func-
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tions of CP, collagen, TGase and cytokine Ast1 in the regulation of HPT progenitor cell behav-
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ior. This is the first study to provide insight into the role of CP, which probably not only partici-
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pates in clot formation but also functions as an ECM component protein controlling hematopoi-
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etic stem cell behavior.
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Keywords: clotting protein, ECM, hematopoiesis, crustacean
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Abbreviations: Ast1, Astakine 1; CP, clotting protein; ECM, extracellular matrix; Hg, Hedgehog; HPT,
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hematopoietic tissue; TGase, transglutaminase; Trol, heparin sulfate proteoglycan Terribly Reduced Optic
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Lobes
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1. Introduction
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Blood coagulation or hemolymph clotting plays a crucial role in innate immune responses
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especially in invertebrates which have open circulatory systems (Vazquez et al., 2009). Clotting
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formation act as a physical barrier that prevent loss of hemolymph and infection by invaders
40
(Cerenius and Söderhäll, 2013; Hall et al., 1999). In crustaceans, coagulation is caused by cova-
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lent crosslink interaction of clotting protein (CP), which is present in hemolymph, and transglu-
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taminase (TGase) enzyme (Hall et al., 1999; Kopácek et al., 1993). In the freshwater crayfish,
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Pacifastacus leniusculus, CP was originally isolated from plasma by precipitation at low ionic
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strength at pH 6.0. CP is a lipoglycoprotein that consists of two identical 210-kDa disulfide2
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bonded subunits (Kopácek et al., 1993). CP is a known substrate of TGase in hemolymph clot
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formation. Each of the 210-kDa subunits has both lysine and glutamine sidechains that are rec-
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ognized and become covalently linked to each other by TGase (Wang et al., 2001). Clot for-
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mation is induced when TGase is released from hemocytes or HPT becomes activated by the
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Ca2+ in plasma, and starts crosslinking the plasma CP molecules into large aggregates (Hall et
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al., 1999). Interestingly, we have recently detected high expression of CP mRNA in crayfish
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HPT in transcriptome analysis (BioProject ID: PRJNA259594). Furthermore, CP is also involved
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in the crosslinking process that stabilizes collagen in HPT (Junkunlo et al., 2016).
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Hematopoiesis is a complex process by which immature hematopoietic progenitor cells
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are produced and developed before being released from the HPT into the circulation (Lin and
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Söderhäll, 2011; Söderhäll, 2016). Cell homeostasis is controlled by specific microenvironments
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called niches, and the ECM plays an essential role in this regulation. The ECM can directly or
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indirectly modulate stem cell behavior such as that occurring in proliferation, self-renewal and
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differentiation (Ahmed and Ffrench-Constant, 2016). Different types of ECM molecules have
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regulatory functions for specific types of stem cells by which ECM protein can be adjusted and
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reformed to provide the most suitable environment for stem cells (Gattazzo et al., 2014). Func-
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tional studies of ECM component protein in controlled stem cell behavior are well established in
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vertebrates, but there are very few studies of ECM protein characterization and their role in con-
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trolled stem cell activity in invertebrates.
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In Drosophila, the loss of Trol (heparan sulfate proteoglycan Terribly Reduced Optic
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Lobes), an ECM protein component in hematopoiesis, causes a dramatic conformational change
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in the ECM and induces premature hemocyte differentiation (Grigorian et al., 2013). In verte-
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brates, a matrilin family protein has been reported as an extracellular adaptor protein that bind to
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different types of collagen and other ECM proteins. Matrilins support matrix assembly by con-
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necting ECM components and mediating physiological interactions between cells and the ECM
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(Uckelmann et al., 2016). Furthermore, Matrilin-1 is required for zebrafish development by facil-
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itating collagen II secretion and deposition (Neacsu et al., 2014). In crayfish, the production of
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hemocytes in crayfish occurs in the hematopoietic tissue (HPT). The immature progenitor cells
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are found in lobule-like structures inside HPT surrounded by different types of cells and connec-
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tive tissue such as collagen (Chaga and Söderhäll, 1995). These progenitor cells can differentiate
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into mature hemocytes and are released into the circulation (Söderhäll et al., 2003).
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Changes in the structure of ECM associated with extracellular signaling dispersion have
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been reported in Drosophila, and an altered state of the ECM in Trol mutant lymph glands can
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inhibit Hedgehog (Hg) signaling (Grigorian et al., 2013). The cytokine astakine (Ast1) has been
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isolated and studied in crayfish as a hematopoietic growth factor that induces the proliferation
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and differentiation of HPT cells (Lin and Söderhäll, 2011; Söderhäll et al., 2005). Furthermore,
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Ast1 acts by interfering with the ECM structure by the regulation of extracellular TGase activity.
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Previously, we have demonstrated the importance of extracellular TGase activity to control hem-
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atopoiesis through the interaction of hematopoietic cells and the ECM and have shown that high
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extracellular TGase activity is required to maintain the immature HPT cells inside the hemato-
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poietic tissue (Junkunlo et al., 2016). In addition, Ast1 functions as an inhibitor of the enzymatic
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crosslinking activity of the TGase enzyme and Ast1 treatment also led to impaired TGase-
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mediated crosslinking of CP (Sirikharin et al., 2017).
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In the present study, we focused on the potential roles of CP as a part of the ECM in the control of hematopoiesis in the crayfish P. leniusculus.
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2. Materials and Methods
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2.1 Experimental Animals
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Freshwater crayfish, P. leniusculus, were acquired from Lake Erken, Sweden. The ani-
mals were maintained in aquaria with aeration at 10°C. Healthy and intermolt male crayfish were used for the experiments.
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2.2 Purification of CP from crayfish plasma
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Hemolymph, approximately 15 ml, was withdrawn from 9 crayfish into 15 ml of ice-cold anticoagulant buffer (0.14 M NaCl, 0.1 M glucose, 30 mM trisodium citrate, 26 mM citric acid,
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pH 4.6) (Söderhäll and Smith, 1983). The hemocyte pellets were removed by centrifugation
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(800 x g, 10 min 4 C). The supernatant, called crayfish plasma, was used for purification of CP,
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as described previously (Hall et al., 1999). Crayfish plasma was dialyzed against 10 mM sodium
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phosphate, pH 6.0, containing 1 mM EDTA overnight at 4° C. The CP precipitated after dialysis
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was collected by centrifugation at 2500 x g for 10 min 4°C. The resulting supernatant without CP
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was removed and is denoted CP-depleted plasma. The orange-colored pellets of CP were re-
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suspended in 500 µl of 0.2 M sodium phosphate buffer, pH 6.0, containing 1 mM EDTA. The
113
purity of CP was analyzed by SDS-PAGE and was compared with SDS-PAGE of CP-depleted
114
plasma and whole crayfish plasma.
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2.3 HPT cell preparation and culture
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HPT was dissected from the dorsal side of the stomach. Subsequently, the HPT cells were isolated and cultured as previously described (Söderhäll et al., 2005). Briefly, after dissection,
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the HPT was washed with crayfish phosphate-buffered saline (CPBS; 10 mM Na2HPO4, 10 mM
121
KH2PO4, 150 mM NaCl, 10 mM CaCl2 and 10 mM MnCl2, pH 6.8). Next, the HPT was incubat-
122
ed at room temperature for 20 min in 800 µl of collagenase type I and type IV (Sigma-Aldrich,
123
USA) at 0.1% in CPBS. The collagenase solution was removed by centrifugation at 3000 x g for
124
5 min. The resulting cell pellet was washed twice with 1 ml of CPBS and was resuspended in L-
125
15 medium (Sigma-Aldrich, USA) supplemented with 1 mM phenylthiourea, 60 mg/ml penicil-
126
lin, 50 mg/ml streptomycin, 50 mg/ml gentamicin (Sigma-Aldrich, USA), and 2 mM L-
127
glutamine. The cells were cultured in 96-well plates at a density of 4 x 104 cells/well at 16°C.
128
For HPT culture, after dissection, the undigested HPT after collagenase treatment were cultured
129
in 24-well plates containing 300 µl of L-15 medium supplemented as above. Crude Ast1 from
130
crayfish plasma was prepared as previously described (Söderhäll et al., 2005) and was added to
131
each well at a concentration of 2% (v/v). One-third of the medium was changed and supplement-
132
ed with crude Ast1 from plasma every second day.
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2.4 Preparation of 3D collagen-I matrix culture
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For 3D collagen-I matrix culture, a collagen-I solution was prepared by diluting 10.6 mg/ml collagen-I stock solution (BD bioscience, USA) to 0.106 mg/ml with phosphate buffer
138
(137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 2 mM KH2PO4, pH 7.4) as previously de-
139
scribed (Noonin et al., 2012). Next, 150 µl of the diluted solution was added into 96-well plates
140
and was allowed to polymerize at 37°C for 1h and 30 min. The 3D collagen-I matrix-gel plate
141
was immediately used after preparation.
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2.5 CP addition in HPT cell culture
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To examine the effect of clotting protein on HPT cells, in vitro experiments were con-
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ducted in 3D collagen-I matrix culture. In a 3D collagen-I matrix gel culture, four different ex-
147
perimental groups were evaluated; 1) CM- (which consisted of collagen I matrix without addi-
148
tional proteins), 2) CMCP- (in which CP-depleted plasma was added to collagen-I solution and
149
was allowed to polymerize together with collagen-I), 3) CMCP+ (in which CP was added to col-
150
lagen-I solution and was allowed to polymerize with collagen-I), and 4) CMCP (in which CP was
151
added to the culture medium during HPT cell preparation). In treatment 2 (collagen-I immobi-
152
lized with CP-depleted plasma), the collagen-I solution was mixed with 10 µg of CP-depleted
153
plasma per well and 3 (Collagen-I immobilized with CP), the collagen-I solution was mixed with
154
10 µg of CP per well before being added to 96-well plates for polymerization. After polymeriza-
155
tion, HPT cells at a density of 4 x 104 cells/well in 150 µl of supplemented L15 medium were
156
carefully dropped onto the surface of the collagen-I matrix gel. For treatment with collagen-I
157
supplemented with CP, 10 µg of CP was added into the well after the cells were plated. To study
158
the effect of the combination of crude Ast1 (crayfish plasma) and CP to HPT cell morphology,
159
crude Ast1, to a final concentration of 2%, was supplemented in different CP treatments as de-
160
scribed above. The cell morphology was observed at 24 h, 48 h, 56 h and 72 h in culture. All cell
161
cultures were maintained at 16°C, and one-third of the medium was changed (with or without
162
crude Ast1 from plasma) every second day.
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2.6 Immunostaining of CP in HPT Cell Cultures
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HPT cells were cultured and maintained in 96-well plates. The medium was removed and cells were fixed with 4% paraformaldehyde in PBS for 1 h at room temperature. The HPT cells
168
were subsequently washed 5 times with PBS and blocked with 10% BSA in PBS for 1 h at room
169
temperature. Next, the cells were incubated for 3 h with rabbit anti-crayfish clotting protein
170
(1:10000). Polyclonal antibodies against the CP were commercially raised in rabbits at the Bio-
171
medical Technology Research Center, Chiang Mai University, Thailand. After being washed 5
172
times with PBST (0.5% Tween 20 in PBS buffer), HPT cells were incubated for 1 h with FITC-
173
conjugated anti-rabbit IgG (1:1000) and Hoechst 33258 dye at a concentration of 1 µg/ml to stain
174
the nuclei at room temperature. After cells were washed 5 times with PBST, the clotting protein
175
was observed under a fluorescence microscope.
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2.7 Detection of CP and TGase activity
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Partially digested HPT was cultured as described above. After 48 h in culture, 1 mM of 5-
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(biotinamido)-pentylamine (Pierce, USA), a substrate for TGase, was added to the tissue cultures
181
and was incubated for 18 h. Subsequently, the tissues were fixed according to (Lin et al., 2008).
182
Briefly, the medium was removed, and the tissues were fixed with 4% paraformaldehyde in PBS
183
for 1 h at room temperature. Next, 25 mM glycine in PBS was added to the wells, and the cells
184
were incubated for 30 min. The cells were subsequently washed 5 times with PBST and were
185
blocked with 10% BSA in PBST for 1 h. The primary antibody, rabbit anti-crayfish clotting pro-
186
tein (1:10000), was added and incubated for 3 h at room temperature. After being washed 5 times
187
with PBST, HPT was incubated for 1 h with Alexa Fluor® 594-conjugated anti-rabbit IgG (Life
188
Technologies, USA) (1:1000) together with streptavidin-FITC conjugate (GE Healthcare, USA)
189
(1:200) diluted with 1% BSA (w/v) in PBST for double labeling at room temperature. The tis-
190
sues were incubated with Hoechst 33258 dye at a concentration of 1 µg/ml to stain the nuclei.
191
After tissues were washed 5 times with PBST, the localization of CP and TGase activity was
192
observed under a fluorescence microscope.
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2.8 CP detection and double labeling of CP and Collagen IV in HPT
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Crayfish were injected with 4% paraformaldehyde in PBS before immediately soaked in
197
4% paraformaldehyde overnight at 4°C. The whole stomach covered with HPT was dissected
198
from crayfish and washed with PBS, and then was carefully removed from the stomach. The
199
HPT was blocked with 10% BSA in PBS for 1 h at room temperature. The primary antibody,
200
rabbit anti-crayfish clotting protein (1:10000), was added and incubated for 3 h at room tempera-
201
ture. After being washed 5 times with PBST, HPT was incubated for 1 h with FITC-conjugated
202
anti-rabbit IgG (Sigma-Aldrich, USA) (1:1000) for CP detection in Figure 6. For double labeling
203
in Figure 7, the primary antibody, rabbit anti-crayfish clotting protein (1:10000) was added to-
204
gether with mouse anti-collagen type IV antibody (Sigma-Aldrich, USA) (1:100). Next the sec-
205
ondary antibody Alexa Fluor® 594-conjugated anti-rabbit IgG (Life Technologies) (1:1000) was
206
used in CP detection together with FITC-conjugated anti-mouse IgG (Sigma-Aldrich, USA)
207
(1:500) at room temperature. The tissues were incubated with Hoechst 33258 dye at a concentra-
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tion of 1 µg/ml to stain the nuclei. After tissues were washed 5 times with PBST, The localiza-
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tion of CP or collagen IV together with CP was observed under a fluorescence microscope.
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3. Results
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3.1 CP forms an extracellular filamentous network in HPT cell culture Because we observed a high expression of CP in the hematopoietic tissue in our P. leniusculus transcriptome data (Bioproject ID PRJNA 259594), we decided to study the im-
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portance of CP in crayfish HPT. Primary HPT cells were cultured for 5 days, and then im-
216
munostaining of CP was performed. By using a polyclonal CP antibody, we observed the secre-
217
tion and formation of a filamentous network between cells in culture (Figure. 1). CP is known as
218
a substrate for TGase during clot formation (Hall et al., 1999), and a recent study has shown that
219
Ast1 had a direct inhibitory effect on the crosslink formation of TGase (Sirikharin et al., 2017).
220
The effect of crude Ast1 from plasma on cell spreading is shown in Figure 2. When cells were
221
supplemented with crude Ast1, cell spreading was clearly observed at 72 h (Figure 2A). Without
222
crude Ast1, the HPT cell morphology did not change during culture (Figure 2B). We have previ-
223
ously detected high extracellular TGase activity in undifferentiated cells and have found that this
224
activity is decreased during cell migration out of the HPT in culture (Lin et al., 2008). Interest-
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ingly, we also found that the CP protein was present at a higher level on the surfaces of round-c
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ells than migrating cells (spindle-shaped cells) (Figure 2A and 2B). Co-localization of CP and
227
TGase activity was found on the HPT cell surface (Figure 2A and 2B), and the co-localization of
228
CP and TGase activity was also found as a filamentous network between cells (Figure 2A).
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From these results, we hypothesized that CP may function as part of the ECM and that it may be
230
involved in the regulation of hematopoiesis in crustaceans.
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3.2 Effects of CP on HPT cell morphology in 3D collagen-I matrix culture
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Collagen is shown as a component of the ECM proteins in HPT, and it is present on HPT
234
cell surfaces (Junkunlo et al., 2016). To further elucidate the function of CP as a constituent of
236
the ECM and to study the HPT cell behavior, we performed an in vitro experiment using 3D col-
237
lagen-I matrix culture (Figure 3). Without the addition of crude Ast1, HPT cells did not show
238
any signs of spreading after CP addition (CMCP+ or CMCP) compared with the control group-
239
i.e., in CM- or CMCP- (Figure 3B). HPT cells in CM- or CMCP- form a monolayer of round-
240
shaped cells at 24 h, and the cell morphology did not change after 48 h or even 7 days in the ab-
241
sence of crude Ast1 (Supplement Figure S1). When crude Ast1 was added into the CM- or
242
CMCP- cultures, the cells did not change in morphology after 24 h, and the HPT cells were
243
round and formed small clusters (Figure 3A). However, the addition of crude Ast1 to CMCP+ or
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CMCP cultures dramatically induced cell spreading after 24 h (Figure 3A).
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In CMCP+ cultures containing crude Ast1, the cells lost their spreading capacity after 48
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h; however in the CMCP cultures with crude Ast1, the spreading of cells was more elongated and
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strengthened and branches were formed between cells at 48 h (Figure 4A) and after 72 h (Figure
249
4B) or even 7 days (data not shown). The formation of a filamentous network between cells was
250
observed only in CMCP cultures (Figure 4A and 4B). These results suggested that CP might func-
251
tion as an ECM component together with collagen-I and regulate the spreading/migration of HPT
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cells.
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The above results indicated that culture in a collagen matrix with the addition of CP and
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crude Ast1 clearly stimulated the spreading of the HPT cells. In addition, the cross-linked func-
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tion of TGase with CP was shown in 3D collagen-I matrix culture. In CMCP+ cultures contain-
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ing crude Ast1, CP was immobilized with collagen before HPT cells was added into culture.
258
Without TGase activity from HPT cells, the effect of CP together with Ast1 and collagen in-
259
duced HPT cell spreading but it was less effected compared with CMCP cultures. In CMCP cul-
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tures containing crude Ast, CP was added during HPT cell preparation. TGase activity from HPT
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cells promoted the cross-linking of CP with collagen and provided a proper environment for cell
262
spreading. For these reasons, more cell spreading and filamentous network formation was ob-
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served in CMCP cultures
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To test the important roles of CP and collagen in controlling HPT progenitor cell behavior, we then tested whether the collagen-I-coated surface had an effect on cell morphology. First,
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we found that the addition of CP to HPT cell culture did not induce the spreading of HPT cells
268
with or without crude Ast1 (Supplementary Figure S2). Moreover, the knockdown of CP mRNA
269
expression in the HPT cells in ordinary culture (plastic well plates) and in 3D collagen-I matrix
270
culture had no effect on cell morphology (Supplementary Figure S3). Thus, CP addition ap-
271
peared to require the presence of collagen to affect cell morphology. Accordingly, we asked next
272
whether collagen-I together with CP and crude Ast1 could induce the spreading of HPT cells on
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wells coated with collagen-I, i.e., without the formation of a 3D matrix. Coating the well surface
274
with collagen-I did not induce similar cell morphology as shown for culture in the 3D matrix
275
made of collagen, with the addition of either CP or Ast1 (Supplementary Figure S4). An interest-
276
ing observation was that, in uncoated wells, the addition of Ast1 induced cell attachment and
277
spreading; however after collagen-I coating, the cells instead formed small cell clusters in the
278
presence of Ast1 (Supplementary Figure S4). These results showed that the regulation of progen-
279
itor cells activity are tightly regulated. To provide a proper environment for progenitor cell dif-
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ferentiation, CP together with crude Ast1 and collagen are required to collaborate in the 3D di-
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mension, which is similar to the environment in the HPT.
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3.3 CP as a part of the ECM in HPT
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Within the HPT, hematopoietic progenitor cells are enclosed in lobule-like structures with different types of cells and the ECM. This specific microenvironment is essential for stimu-
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lating or maintaining stem cell behavior. Because CP was found to be abundant in crayfish HPT,
288
the localization of CP in HPT was studied (Figure 5). The localization and structure of crayfish
289
are shown in Figure 5A. Whole-mount immunostaining of CP showed that this protein was
290
mainly found at the edges of the HPT when compared with the central parts of HPT (Figure 5B-
291
D). CP protein was clearly distributed around the surfaces of the HPT cells or covered the lob-
292
ules. This result indicated that CP may act as a supportive structure of the HPT cells inside the
293
lobules. CP was also detected in the anterior HPT and APC but the largest amount was found in
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the posterior HPT parts (Figure 5B and 5D). To emphasize the role of CP as a part of the ECM
295
in HPT issue, co-localization of CP and collagen type IV, a component of the ECM, was investi-
296
gated. At the edges of posterior HPT where CP protein is mainly localized, co-localization of CP
297
with collagen type IV was observed around cells and in lobules (Figure 6). Co-localization of
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these two proteins was also found in other HPT areas (data not show). These results suggested
299
that CP is a constituent of the ECM component protein in crayfish HTP.
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4. Discussion
Hematopoiesis or blood cell production in crayfish is tightly regulated and controlled in a complex environment in HPT tissue. The microenvironments or niches consist of several cell
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types and a multiple-component ECM. The ECM not only plays the role of supporting a hemato-
305
poietic niche but also participates in binding different growth factors and cytokines (Schultz and
306
Wysocki, 2009). Cytokines are essential signaling molecules that are transported through the
307
ECM, reach the stem cell niche and ultimately activate stem cell proliferation and differentiation
308
(Mercier, 2016). Our previous studies have shown that Ast1 is a key survival factor that induces
309
the proliferation and differentiation of progenitor cells (Lin and Söderhäll, 2011; Söderhäll et al.,
310
2005). Moreover, TGase has been shown to play a role in maintaining HPT cells in an undiffer-
311
entiated form (Lin et al., 2008), and recent studies have shown that Ast1 affects TGase activity
312
directly (Sirikharin et al., 2017). CP is known as a secreted protein that is abundant in plasma
313
and is a substrate of TGase during clot formation (Hall et al., 1999; Wang et al., 2001) In addi-
314
tion, we now have found that CP is an important protein in the crayfish HPT, as is TGase (Lin et
315
al., 2008). The CP mRNA was highly expressed in HPT, while very low expression was detected
316
in circulating hemocytes. For these reasons, we hypothesize that CP may participate not only in
317
clot formation but also may be involved in hematopoietic regulation.
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We first investigated the role of CP in HPT cell culture. When HPT cells start to migrate,
320
TGase activity was decreased (Lin et al., 2008). Immunostaining of CP in HPT cells showed that
321
CP was also mainly expressed in round, non-spreading cells compared with spreading cells.
322
These results indicated the possibility that CP may act together with TGase function in control-
323
ling progenitor cell activity. We also observed the secretion of CP protein and the production of
324
CP filaments with variable thickness, often forming branches, and connecting cells to other cells
325
in the culture. Similar observations have been reported in primary embryonic mouse fibroblasts
326
cell cultures (Klatt et al., 2001) and Swarm rat chondrosarcoma cell cultures (Klatt et al., 2000).
327
Furthermore, the extracellular network formation of Matrilin, an ECM adaptor protein, has been
328
detected in cultured cells (Klatt et al., 2011). In crayfish, Ast1 induces the proliferation and dif-
329
ferentiation of progenitor cells inside HPT and causes the release of mainly semi-granular hemo-
330
cytes into the circulation (Lin et al., 2010). This process can be observed in an in vitro culture
331
system as spreading of the HPT cells (Lin and Söderhäll, 2011).The co-localization of TGase
332
activity and CP protein observed on the HPT cell surface and in the filamentous network during
333
cell spreading indicated the possible roles of CP, TGase activity and Ast1 in hematopoietic regu-
334
lation.
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Collagen has been characterized as a part of ECM protein in crayfish HPT. Collagenase type I and IV enzymes are used in HPT cell culture preparation (Söderhäll et al., 2005). Previ-
338
ously, we have shown co-localization of extracellular TGase and collagen IV on HPT cell sur-
339
faces (Junkunlo et al., 2016). Thus, we hypothesized that CP may function together with collagen
340
in controlling progenitor cell behavior. Three-dimensional (3-D) cell culture can be used to mim-
341
ic the environment in the tissue under in vivo conditions, and a 3D collagen-I matrix culture can
342
be used to enhance the interactions between cells and between cells and the environment (Ed-
343
mondson et al., 2014). In Human Wharton’s jelly mesenchymal stem cells (HWJMSCs) develop
344
various morphologies on a collagen-I-coated and in a 3D collagen matrix. On collagen-coated
345
surface the HWJMSC cells show a fibroblast-like morphology, in contrast to the 3D collagen
346
matrix in which they show a star-like morphology (Khodabandeh et al., 2016). Similarly, we
347
observed different morphologies of the cell cultured on 3D collagen matrix compared with a col-
348
lagen-coated surface and naked plastic. The 3D collagen matrix, supplemented with CP and
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crude astakine, induced a spreading morphology, and a network of fibers of CP was formed be-
350
tween the cells, whereas a collagen-coated surface did not induce any morphology change. In
351
addition, HPT cells were elongated and gathered into packed colonies. The elongated cells
352
formed chains of cells through the surrounding ECM with other cells or connected with other
353
colonies. In a previous paper, we have similarly shown that HPT cells cultured in a collagen ma-
354
trix can be induced to express mRNA for a specific Kazal proteinase inhibitor that is a marker
355
for semi-granular hemocytes (Noonin et al., 2012), thus again showing that a 3D collagen matrix
356
culture may indeed provide a more suitable milieu and is more similar to in vivo conditions. A
357
proper environment for the HPT cells is needed, and collagen, CP, TGase and Ast1 appeared to
358
be required to form such an environment. Thus, our new results indicate a role for CP during
359
hematopoiesis, and Ast1 might be required to suppress crosslink formation of CP by the TGase
360
enzyme. This inhibition may result in less crosslinking of CP to other ECM proteins such as col-
361
lagen, and finally stimulate the differentiation and release of new hemocytes, as shown earlier
362
(Lin et al., 2010).
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Crucial roles of collagen in controlling stem cell behavior in 3D culture have been studied in many cell types. For example, a breast tumor cell line (MDA-MB-231) grows more slowly
366
in 3D collagen-I matrix culture than on a 2D collagen-coated surface (Kim et al., 2015). In addi-
367
tion, osteoblast differentiation is regulated by the presence of collagen type I in the ECM, and
368
TGase is required for collagen secretion and extracellular deposition, which control the osteo-
369
blast differentiation (Piercy-Kotb et al., 2012). In Drosophila, the loss of Trol, an ECM protein,
370
induces the release of plasmatocytes into the hemolymph (Grigorian et al., 2013).
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To confirm a function of CP as a part of the ECM in crayfish HTP, we continued to in-
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vestigate the structural localization of CP in HPT in vivo. By immunostaining we found that CP
374
was present outside the cells in the HPT, but was much less abundant in the APC area. The high-
375
est levels of CP were found at the posterior parts of HPT compared with the middle parts. We
376
also observed co-localization of CP and collagen IV in HPT in vivo. The co-localization of ECM
377
protein and collagen has been reported in vertebrates. Syndecan-2, an adaptor protein, exhibits a
378
fibrillary pattern over the cell surface and is deposited into the ECM (Filla et al., 2004). Further-
379
more, Syndecans-2 has also been found to co-localize with ECM proteins, for example fibron-
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380
ectin, laminin and type IV collagen in Human ciliary muscle (HCM) cells and HSC endothelium
381
cells. Thus, the localization of CP and co-localization of CP with collagen IV in HPT and TGase
382
activity in HPT cells support the potential roles of CP as an ECM protein in hematopoietic regu-
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lation.
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5. Conclusion
386 387
Although CP is known to be involved in clot formation after wounding or infection, we now show a potential role for CP as an ECM protein controlling the differentiation and prolifera-
389
tion of progenitor cells in HPT. CP may function together with collagen in producing a suitable
390
environment for hematopoietic tissue in crayfish.
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Figure Legends
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Fig. 1. Secretion and formation of clotting protein (CP) filaments in HPT cell culture. HPT
394
cells were cultured for 5 days without the addition of crude Ast1. The localization of clotting
395
protein (CP) was detected by immunostaining with anti-CP antibody (green), and nuclei were
396
stained with Hoechst 33258 (blue).
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Fig. 2. The CP protein and TGase activity are co-localized at the surfaces of round cells
399
compared with migrating cells (spindle shaped cells). Co-localization of CP and TGase activi-
400
ty was determined after incubation of the partially digested HPT with the TGase substrate 5-
401
(biotinamido)-pentylamine for 18 h. The cross-linking of substrate was visualized after streptavi-
402
din-FITC (green) addition together with Alexa Fluor® 594-conjugated anti-rabbit IgG (red) to
403
CP antibody. Hoechst 33258 (blue) was used as a nuclear stain. A) Double staining for TGase
404
activity and CP was performed on partially digested HPT culture for 72 h without crude Ast1. B)
405
Double staining for TGase activity and CP was performed on partially digested HPT culture for
406
72 days with crude Ast1. All experiments were repeated 3 times.
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Fig. 3. CP addition together with crude Ast1 cause HPT cell spreading in 3D collagen-I ma-
409
trix culture. Four different experimental treatments were performed: 1) Collagen-I matrix only
410
= CM-, 2) Collagen-I immobilized together with CP-depleted plasma = CMCP-, 3) Collagen-I
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matrix immobilized together with 10 µg CP = CMCP+, and 4) Collagen-I supplemented with CP
412
after polymerization = CMCP A) HPT cells at a density of 4 x 104 cells per well were cultured in
413
the four different matrixes supplemented with crude Ast1. B) HPT cells at a density of 4 x 104
414
cells per well were cultured in the four different matrixes without crude Ast1. The cell morphol-
415
ogy was observed after 24 h in culture and all experiments were repeated 5 times.
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Fig. 4. CP promotes the elongation and clustering of HPT cells after culture in a 3D colla-
418
gen-I matrix supplemented with crude Ast1. HPT cells cultured in CMCP+ or CMCP together
419
with crude Ast1. A) HPT cell morphology after 48 h in culture. B) HPT cell morphology after 72
420
h in culture. All experiments were repeated at least 3 times.
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Fig. 5. CP as a part of the ECM in HPT. A) Localization and structure of crayfish HPT, APC
423
and different parts of HPT. HPT is divided into two parts - the posterior HPT (green) and anteri-
424
or HPT (yellow). APC (blue) is a special small area in the anterior part of the HPT. Letters with
425
squares in A) indicate areas where images in B), C) and D) were captured. (B-D) Whole mount
426
immunohistochemistry of clotting protein in posterior HPT labeled with the primary antibody,
427
rabbit anti-crayfish clotting protein (1:10000) together with the secondary antibody, FITC-
428
conjugated anti-rabbit IgG (Sigma-Aldrich) (1:1000) (green). Hoechst 33258 (blue) was used as
429
a nuclear stain. All experiments were at least repeated 3 times.
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Fig. 6. Co-localization of collagen type IV and CP in HPT. Double staining for CP (red) and
432
collagen IV (green) was performed in whole HPT labeled with the primary antibody, rabbit anti-
433
crayfish clotting protein (1:10000) together with mouse anti-collagen type IV antibody (Sigma,
434
1:100). The secondary antibody Alexa Fluor® 594-conjugated anti-rabbit IgG (red) (Life Tech-
435
nologies) (1: 1000) was used in CP detection together with FITC-conjugated anti-mouse IgG
436
(green) (Sigma-Aldrich) (1: 500). Arrows indicate the co-localization of CP and collagen IV. All
437
experiments were repeated at least 3 times.
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Fig. 5.
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Fig. 6.
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Acknowledgments
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This work was supported by the Swedish Science Research Council (VR 621-2012-2416 to KS)
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Conflicts of Interest
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The authors declare that they have no conflicts of interest with the contents of this article.
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Author Contributions
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KJ performed experiments, analyzed results, prepared figures and wrote the paper. KJ, KS and IS
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contributed to study design, interpreted the data and participated in writing the paper. All authors
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reviewed the results and approved the final version of the manuscript.
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Highlights
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Clotting protein (CP) is part of the ECM in hematopoietic tissue (HPT) CP forms an extracellular filamentous network in HPT cell culture CP together with crude Ast1 effect HPT cell morphology in 3D collagen-I matrix culture CP, collagen, TGase and Ast1 regulate HPT progenitor cell behavior
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