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Organization of the neuroepithelial actin cytoskeleton is regulated by heparan sulfation during neurulation
Neuroscience Letters 533 (2013) 77–80
Contents lists available at SciVerse ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Organization of the neuroepithelial actin cytoskeleton is regulated by heparan sulfation during neurulation Ya-Jun Wu a,b , Grace Shu-Xian Leong b,1 , Zhen-Min Bao a , George W. Yip b,∗ a b
Division of Life Science and Technology, Ocean University of China, 5 Yushan Road, Qingdao, Shandong 266003, China Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, 4 Medical Drive, Block MD 10, Singapore 117597, Singapore
h i g h l i g h t s Blocking heparan sulfation accelerated posterior neuropore closure Loss of heparan sulfation disrupted apical cytoskeletal actin organization Disorganized actin perturbed neuroepithelial bending.
a r t i c l e
i n f o
Article history: Received 5 September 2012 Received in revised form 22 October 2012 Accepted 26 October 2012 Keywords: Actin Cytoskeleton Heparan sulfate Neuroepithelium Neurulation
a b s t r a c t Heparan sulfate and cytoskeletal actin microfilaments have both been shown to be important regulators of neural tube closure during embryonic development. To determine the functional relationship of these two molecules in formation of the spinal neural tube, we cultured ARC mouse embryos at embryonic day E8.5 in the presence of chlorate, a competitive inhibitor of glycosaminoglycan sulfation, and examined the effects on organization of actin microfilaments in the neuroepithelium. Compared against embryos cultured under control conditions, chlorate-treated embryos had shortened posterior neuropore, a loss of median hinge point formation and increased bending at the paired dorsolateral hinge points. Furthermore, apical organization of actin microfilaments in the neuroepithelial cells was absent, and this was associated with convex bending of the neuroepithelium. The results suggest that heparan sulfate is an important determinant of cytoskeletal actin organization during spinal neurulation, and that its biological action is dependent on sulfation of the heparan molecule. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Formation of the central nervous system begins with embryonic neurulation. In this process, the surface ectoderm thickens and differentiates to become the neuroepithelium, which then undergoes elevation, apposition and fusion in the midline dorsally to give rise to the neural tube [10]. Closure of the neural tube starts at sites of de novo initiation of neural fold fusion, and continues in a zipper-like manner along the neuraxis [22]. In spinal neurulation, three modes of neural tube closure are seen, which are differentiated based on the presence of various hinge points [39]. In Mode 1, a midline bend occurs at the median hinge point in the neuroepithelium. In Mode 2, bends occur at the median hinge point as well as paired dorsolateral hinge points. In contrast, only
∗ Corresponding author. Tel.: +65 6516 3206; fax: +65 6778 7643. E-mail addresses: [email protected] (G.S.-X. Leong), [email protected] (G.W. Yip). 1 Tel: +65 6516 3200; fax: +65 6778 7643. 0304-3940/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2012.10.058
paired dorsolateral hinge points are present in Mode 3. Failure of neural tube closure leads to congenital neural tube defects such as spina bifida and myelomeningocele [10,13,17]. Glycosaminoglycans are long, unbranched polysaccharides made up of repeating disaccharide subunits of an amino sugar and an uronic acid [26,43,48]. In addition to having a structural role in the extracellular matrix, glycosaminoglycans help to regulate growth factor signaling and influence cellular behavior [15,42,48,49]. The major sulfated glycosaminoglycans synthesized during spinal neurulation are heparan sulfate and chondroitin sulfate [47]. Both molecules have been shown to play important roles during neurulation [28,30,40,47]. In spinal neurulation, inhibition of heparan sulfation was found to result in accelerated closure of the posterior neuropore as well as suppression of bending at the median hinge point [47]. Actin microfilaments are present in neuroepithelial cells, and have been hypothesized to be important for neural tube closure [1,21,35]. Syndecan-1, a heparan sulfate proteoglycan, has been shown to be associated with the actin cytoskeleton in Schwann cells [7]. Further, changes in glycosaminoglycan sulfation have been
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reported to be associated with cellular activity and growth factor signaling [5,33,38]. Thus, in this study, we aimed to determine if heparan sulfation is involved in organization of the cytoskeletal actin microfilaments in the neuroepithelium during spinal neural tube closure.
with Alexa Fluor 488-phalloidin (1:50 dilution) for 20 min at room temperature as previously described [16]. Cell nuclei were counterstained using DAPI. The fluorescence images were examined using an Olympus FluoView FV1000 laser scanning confocal microscope. 2.3. Statistical analysis
2. Materials and methods 2.1. Culture of whole embryos Six-week old randomly bred ARC(S) mice were mated overnight. The day of finding a copulation plug was designated embryonic day (E) 0.5. Embryos at E8.5 were explanted and cultured in 100% rat serum for 24 h in a roller incubator at 37 ◦ C as previously described [9,47]. Six-hourly gassing using a mixture of 5% carbon dioxide, 5% oxygen and 90% nitrogen was performed. At the end of the culture period, each embryo was removed from the surrounding membranes and examined under a Nikon SMZ1500 stereomicroscope. Development of the cultured embryos was assessed by counting the number of somites present as well as by using the morphological appearance of various embryonic features [6]. The posterior neuropore length, defined as the distance between the cranial end of the posterior neuropore and the tip of the tail bud, was measured using an eyepiece graticule. All animal experiments were conducted in accordance with institutional animal care and use guidelines of the National University of Singapore. 2.2. Phalloidin staining Embryos were fixed overnight in 4% paraformaldehyde, washed twice with phosphate-buffered saline (PBS), and cryoprotected in 30% sucrose prior to being embedded in OCT (Leica). Cryosections of 10-m thickness were then cut transversely through the closing posterior neuropore region and mounted onto gelatin-coated glass slides. Actin filaments were stained by incubating the sections
Experiments consisted of at least nine replicates per group. Statistical comparison of measured embryonic parameters between two groups was performed by Student’s t-test using Prism v5.04 (GraphPad Software). Statistical significance was defined as a p value of below 0.05. 3. Results Explanted E8.5 mouse embryos were cultured for 24 h in the presence of either 30 mM sodium chlorate (treatment group) or PBS (control group). Sodium chlorate is an inhibitor of glycosaminoglycan sulfation, and has been extensively used in cell and organ cultures at up to 30 mM with no significant effects on cell viability or protein synthesis [8,12,14,16,27,47]. As shown in our previous study, treatment with 30 mM chlorate did not affect growth and development of the cultured embryos [47]. Regular heartbeats were observed in both chlorate-treated and control embryos, and vigorous blood circulation was seen in the yolk sacs. Morphological features of chlorate-treated embryos were similar to those of control embryos (Fig. 1A,B). Furthermore, there was no statistically significant difference in the number of somites in the two groups of embryos (Fig. 1C, p > 0.05). To determine if inhibition of glycosaminoglycan sulfation affected closure of the posterior neuropore, we compared the posterior neuropore length of chlorate-treated embryos against embryos in the control group. Determination of the posterior length was performed by measuring the distance between the cranial end of the posterior neuropore and the tip of the tail bud (Fig. 1A and
Fig. 1. Morphology of cultured whole mouse embryos treated with either phosphate-buffered saline (A) or 30 mM chlorate (B). The average number of somites in the two groups of embryos is shown in (C), while the mean posterior neuropore length is shown in (D). Error bars represent standard errors, with nine embryos in each treatment group. *, posterior neuropore. Scale bars, 0.4 mm.
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Fig. 2. Transverse sections of the closing posterior neuropore of phosphate-buffered saline-treated embryos (A, B) or chlorate-treated embryos (C, D). Tissues were stained either with Alexa Fluor 488-phalloidin (A, C) or DAPI (B, D). Arrows, paired dorsolateral hinge points; arrowhead, median hinge point; *, absent median hinge point. Scale bars, 0.1 mm.
B). As shown in Fig. 1D, blocking glycosaminoglycan sulfation led to a significant increase in the rate of posterior neuropore closure (p < 0.005). The posterior neuropore length was 39.9% shorter in the chlorate-treated group compared against embryos in the control group. To investigate if sulfation of glycosaminoglycans is essential for organization of actin microfilaments in the neuroepithelium, we stained the actin fibers using phalloidin in transverse cryosections of the closing posterior neuropore (Fig. 2). Apical actin microfilaments were abundantly present in neuroepithelial cells in the closing posterior neuropore region of the control embryos, including the median hinge point and the paired dorsolateral hinge points. In contrast, the neuroepithelium showed very faint staining for actin microfilaments in the chlorate-treated group. This was associated with an absence of the median hinge point, loss of apical neuroepithelial cell wedging, and convex bending of the neuroepithelium, possibly due to floppines. In addition, increased bending was observed in the dorsolateral hinge points.
4. Discussion We have investigated the biological function of glycosaminoglycans on spinal neural tube closure by treating cultured whole embryos with chlorate. Chlorate is a structural analog of sulfate, and acts as a competitive inhibitor of glycosaminoglycan sulfation [8,14]. As in an earlier report [47], chlorate treatment in this study led to a statistically significant reduction in the length of the closing posterior neuropore without any adverse effects on the morphology of the developing embryos or in the number of somites compared against saline-treated control embryos. Although chlorate inhibits sulfation of both heparan and chondroitin, its effects on spinal neural tube closure are mainly due to a loss of heparan sulfation [47]. Addition of heparan sulfate, but not chondroitin sulfate,
to the culture medium could abolish the chlorate-induced changes in the closing spinal neural tube. Using phalloidin, we confirmed the presence of actin microfilaments in the apical region of neuroepithelial cells [1,21,29,31,35,46]. The loss of these actin microfilaments and apical wedging in the neuroepithelial cells after chlorate treatment together with the absence of the median hinge point support the hypothesis that contraction of these microfilaments regulates neural tube closure [20,21]. Interestingly, a similar phenomenon has been reported in Xenopus animal caps [19]. Degradation of heparan sulfate by heparinase treatment led to a reduction in actin gene expression. In contrast, addition of exogenous heparan sulfate to the heparinase-treated tissues restored actin transcript levels [19]. Heparan sulfate has been reported to be critical for polarization of epithelial cells [37]. Formation of the lung lumen requires interaction between heparan sulfate and laminin. Blocking this interaction through the use of an anti-laminin antibody or exogenous heparan sulfate led to inhibition of epithelial cell polarization and hindered lumen formation [37]. The axon-dendrite polarity of neurons has also been shown to be regulated by heparan sulfate [23,24,32]. Integrins help in signal transmission from the extracellular matrix to the cytoskeleton. Activation of integrins by heparan sulfate chains in syndecans, a family of transmembrane heparan sulfate proteoglycans, initiates signaling through the Rho cytoskeletal G-proteins and ␣-actinin and thereby regulates the formation of actin stress fibers and focal adhesion [2–4,11,18,36]. Heparan sulfate chains are needed to target syndecans to specific domains on the cell surface [45]. Blocking antibodies to integrin or syndecan disrupted the assembly of actin stress fibers and focal adhesion formation [36]. Inhibiting glycosaminoglycan sulfation by chlorate treatment could reduce integrin-heparan sulfate interaction and thus perturb downstream Rho signaling [34,44]. Indeed, variations in heparan sulfation patterns are known to
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differentially affect cellular activities and the binding of heparan sulfate to fibroblast growth factors and other signaling molecules [16,25,33,41,42]. This has led to the emerging field of heparanomics. In conclusion, we have demonstrated that heparan sulfate regulates the organization of cytoskeletal actin microfilaments in the murine neuroepithelium during spinal neural tube formation. This organization is dependent on heparan sulfation, and may play a part in formation of the median hinge point and maintenance of the rigidity of the neuroepithelium during neurulation. Acknowledgments The authors are grateful to Prof. Andrew J. Copp for helpful discussions on the study, and Dr. Chunhua Guo and Ms. Song-Lin Bay for their technical assistance in this project. The work was supported by Grant R-181-000-095-112 (to G.W.Y.) from the Academic Research Fund, Ministry of Education, Singapore. G.S.-X.L. is the recipient of a graduate research scholarship from the National University of Singapore. References [1] P.C. Baker, T.E. Schroeder, Cytoplasmic filaments and morphogenetic movement in the amphibian neural tube, Developmental Biology 15 (1967) 432–450. [2] D.M. Beauvais, A.C. Rapraeger, Syndecan-1-mediated cell spreading requires signaling by alphavbeta3 integrins in human breast carcinoma cells, Experimental Cell Research 286 (2003) 219–232. [3] M. Bernfield, M. Gotte, P.W. Park, O. Reizes, M.L. Fitzgerald, J. Lincecum, M. Zako, Functions of cell surface heparan sulfate proteoglycans, Annual Review of Biochemistry 68 (1999) 729–777. [4] M. Bernfield, R. Kokenyesi, M. Kato, M.T. Hinkes, J. Spring, R.L. Gallo, E.J. Lose, Biology of the syndecans: a family of transmembrane heparan sulfate proteoglycans, Annual Review of Cell Biology 8 (1992) 365–393. [5] Y.G. Brickman, M.D. Ford, J.T. Gallagher, V. Nurcombe, P.F. Bartlett, J.E. Turnbull, Structural modification of fibroblast growth factor-binding heparan sulfate at a determinative stage of neural development, Journal of Biological Chemistry 273 (1998) 4350–4359. [6] N.A. Brown, S. Fabro, Quantitation of rat embryonic development in vitro: a morphological scoring system, Teratology 24 (1981) 65–78. [7] D.J. Carey, K.M. Bendt, R.C. Stahl, The cytoplasmic domain of syndecan-1 is required for cytoskeleton association but not detergent insolubility. Identification of essential cytoplasmic domain residues, Journal of Biological Chemistry 271 (1996) 15253–15260. [8] H.E. Conrad, Heparin-Binding Proteins, Academic Press, San Diego, 1998. [9] A. Copp, P. Cogram, A. Fleming, D. Gerrelli, D. Henderson, A. Hynes, M. KolatsiJoannou, J. Murdoch, P. Ybot-Gonzalez, Neurulation and neural tube closure defects, Methods in Molecular Biology 136 (2000) 135–160. [10] A.J. Copp, N.D. Greene, Genetics and development of neural tube defects, Journal of Pathology 220 (2010) 217–230. [11] J.R. Couchman, Transmembrane signaling proteoglycans, Annual Review of Cell and Developmental Biology 26 (2010) 89–114. [12] J. Davies, M. Lyon, J. Gallagher, D. Garrod, Sulphated proteoglycan is required for collecting duct growth and branching but not nephron formation during kidney development, Development 121 (1995) 1507–1517. [13] P. de Marco, E. Merello, S. Mascelli, V. Capra, Current perspectives on the genetic causes of neural tube defects, Neurogenetics 7 (2006) 201–221. [14] H. Greve, Z. Cully, P. Blumberg, H. Kresse, Influence of chlorate on proteoglycan biosynthesis by cultured human fibroblasts, Journal of Biological Chemistry 263 (1988) 12886–12892. [15] M. Guerrini, M. Hricovini, G. Torri, Interaction of heparins with fibroblast growth factors: conformational aspects, Current Pharmaceutical Design 13 (2007) 2045–2056. [16] C.H. Guo, C.Y. Koo, B.H. Bay, P.H. Tan, G.W. Yip, Comparison of the effects of differentially sulphated bovine kidney- and porcine intestine-derived heparan sulphate on breast carcinoma cellular behaviour, International Journal of Oncology 31 (2007) 1415–1423. [17] M.J. Harris, D.M. Juriloff, An update to the list of mouse mutants with neural tube closure defects and advances toward a complete genetic perspective of neural tube closure, Birth Defects Research Part A: Clinical and Molecular Teratology 88 (2010) 653–669. [18] T. Ishikawa, R.H. Kramer, Sdc1 negatively modulates carcinoma cell motility and invasion, Experimental Cell Research 316 (2010) 951–965. [19] K. Itoh, S.Y. Sokol, Heparan sulfate proteoglycans are required for mesoderm formation in Xenopus embryos, Development 120 (1994) 2703–2711.
[20] S.D. Joshi, M. von Dassow, L.A. Davidson, Experimental control of excitable embryonic tissues: three stimuli induce rapid epithelial contraction, Experimental Cell Research 316 (2010) 103–114. [21] P. Karfunkel, The mechanisms of neural tube formation, International Review of Cytology 38 (1974) 245–271. [22] Z. Kibar, V. Capra, P. Gros, Toward understanding the genetic basis of neural tube defects, Clinical Genetics 71 (2007) 295–310. [23] F. Lafont, A. Prochiantz, C. Valenza, M. Petitou, M. Pascal, M. Rouget, A. Rousselet, Defined glycosaminoglycan motifs have opposite effects on neuronal polarity in vitro, Developmental Biology 165 (1994) 453–468. [24] F. Lafont, M. Rouget, A. Triller, A. Prochiantz, A. Rousselet, In vitro control of neuronal polarity by glycosaminoglycans, Development 114 (1992) 17–29. [25] W.C. Lamanna, I. Kalus, M. Padva, R.J. Baldwin, C.L. Merry, T. Dierks, The heparanome – the enigma of encoding and decoding heparan sulfate sulfation, Journal of Biotechnology 129 (2007) 290–307. [26] C. Malavaki, S. Mizumoto, N. Karamanos, K. Sugahara, Recent advances in the structural study of functional chondroitin sulfate and dermatan sulfate in health and disease, Connective Tissue Research 49 (2008) 133–139. [27] H.Q. Miao, R. Ishai-Michaeli, R. Atzmon, T. Peretz, I. Vlodavsky, Sulfate moieties in the subendothelial extracellular matrix are involved in basic fibroblast growth factor sequestration, dimerization, and stimulation of cell proliferation, Journal of Biological Chemistry 271 (1996) 4879–4886. [28] G. Morriss-Kay, F. Tuckett, Immunohistochemical localisation of chondroitin sulphate proteoglycans and the effects of chondroitinase ABC in 9- to 11-day rat embryos, Development 106 (1989) 787–798. [29] G. Morriss-Kay, F. Tuckett, The role of microfilaments in cranial neurulation in rat embryos: effects of short-term exposure to cytochalasin D, Journal of Embryology and Experimental Morphology 88 (1985) 333–348. [30] G.M. Morriss-Kay, B. Crutch, Culture of rat embryos with beta-d-xyloside: evidence of a role for proteoglycans in neurulation, Journal of Anatomy 134 (1982) 491–506. [31] R.G. Nagele, H.Y. Lee, Studies on the mechanism of neurulation in the chick: microfilament-mediated changes in cell shape during uplifting of neural folds, Journal of Experimental Zoology 213 (1980) 391–398. [32] K. Nishimura, M. Ishii, M. Kuraoka, K. Kamimura, N. Maeda, Opposing functions of chondroitin sulfate and heparan sulfate during early neuronal polarization, Neuroscience 169 (2010) 1535–1547. [33] V. Nurcombe, M.D. Ford, J.A. Wildschut, P.F. Bartlett, Developmental regulation of neural response to FGF-1 and FGF-2 by heparan sulfate proteoglycan, Science 260 (1993) 103–106. [34] J.A. Roper, R.C. Williamson, M.D. Bass, Syndecan and integrin interactomes: large complexes in small spaces, Current Opinion in Structural Biology 22 (2012) 583–590. [35] T.W. Sadler, D. Greenberg, P. Coughlin, J.L. Lessard, Actin distribution patterns in the mouse neural tube during neurulation, Science 215 (1982) 172–174. [36] S. Saoncella, F. Echtermeyer, F. Denhez, J.K. Nowlen, D.F. Mosher, S.D. Robinson, R.O. Hynes, P.F. Goetinck, Syndecan-4 signals cooperatively with integrins in a Rho-dependent manner in the assembly of focal adhesions and actin stress fibers, Proceedings of the National Academy of Sciences of the United States of America 96 (1999) 2805–2810. [37] L. Schuger, A.P. Skubitz, K. Gilbride, R. Mandel, L. He, Laminin and heparan sulfate proteoglycan mediate epithelial cell polarization in organotypic cultures of embryonic lung cells: evidence implicating involvement of the inner globular region of laminin beta 1 chain and the heparan sulfate groups of heparan sulfate proteoglycan, Developmental Biology 179 (1996) 264–273. [38] O.J. Scully, P.J. Chua, K.S. Harve, B.H. Bay, G.W. Yip, Serglycin in health and diseases, Anatomical Record (Hoboken) 295 (2012) 1415–1420. [39] A.S. Shum, A.J. Copp, Regional differences in morphogenesis of the neuroepithelium suggest multiple mechanisms of spinal neurulation in the mouse, Anatomy and Embryology 194 (1996) 65–73. [40] F. Tuckett, G.M. Morriss-Kay, Heparitinase treatment of rat embryos during cranial neurulation, Anatomy and Embryology 180 (1989) 393–400. [41] J.E. Turnbull, Heparan sulfate glycomics: towards systems biology strategies, Biochemical Society Transactions 38 (2010) 1356–1360. [42] J.E. Turnbull, R.L. Miller, Y. Ahmed, T.M. Puvirajesinghe, S.E. Guimond, Glycomics profiling of heparan sulfate structure and activity, Methods in Enzymology 480 (2010) 65–85. [43] A. Varki, R.D. Cummings, J.D. Esko, H.H. Freeze, P. Stanley, C.R. Bertozzi, G.W. Hart, M.E. Etzler, Essentials of Glycobiology, Cold Spring Harbor Laboratory Press, New York, 2008. [44] X. Xian, S. Gopal, J.R. Couchman, Syndecans as receptors and organizers of the extracellular matrix, Cell and Tissue Research 339 (2010) 31–46. [45] Y. Yang, M. Borset, J.K. Langford, R.D. Sanderson, Heparan sulfate regulates targeting of syndecan-1 to a functional domain on the cell surface, Journal of Biological Chemistry 278 (2003) 12888–12893. [46] P. Ybot-Gonzalez, A.J. Copp, Bending of the neural plate during mouse spinal neurulation is independent of actin microfilaments, Developmental Dynamics 215 (1999) 273–283. [47] G.W. Yip, P. Ferretti, A.J. Copp, Heparan sulphate proteoglycans and spinal neurulation in the mouse embryo, Development 129 (2002) 2109–2119. [48] G.W. Yip, M. Smollich, M. Gotte, Therapeutic value of glycosaminoglycans in cancer, Molecular Cancer Therapeutics 5 (2006) 2139–2148. [49] L. Zhang, Glycosaminoglycan (GAG) biosynthesis and GAG-binding proteins, Progress in Molecular Biology and Translational Science 93 (2010) 1–17.
Contents lists available at SciVerse ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Organization of the neuroepithelial actin cytoskeleton is regulated by heparan sulfation during neurulation Ya-Jun Wu a,b , Grace Shu-Xian Leong b,1 , Zhen-Min Bao a , George W. Yip b,∗ a b
Division of Life Science and Technology, Ocean University of China, 5 Yushan Road, Qingdao, Shandong 266003, China Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, 4 Medical Drive, Block MD 10, Singapore 117597, Singapore
h i g h l i g h t s Blocking heparan sulfation accelerated posterior neuropore closure Loss of heparan sulfation disrupted apical cytoskeletal actin organization Disorganized actin perturbed neuroepithelial bending.
a r t i c l e
i n f o
Article history: Received 5 September 2012 Received in revised form 22 October 2012 Accepted 26 October 2012 Keywords: Actin Cytoskeleton Heparan sulfate Neuroepithelium Neurulation
a b s t r a c t Heparan sulfate and cytoskeletal actin microfilaments have both been shown to be important regulators of neural tube closure during embryonic development. To determine the functional relationship of these two molecules in formation of the spinal neural tube, we cultured ARC mouse embryos at embryonic day E8.5 in the presence of chlorate, a competitive inhibitor of glycosaminoglycan sulfation, and examined the effects on organization of actin microfilaments in the neuroepithelium. Compared against embryos cultured under control conditions, chlorate-treated embryos had shortened posterior neuropore, a loss of median hinge point formation and increased bending at the paired dorsolateral hinge points. Furthermore, apical organization of actin microfilaments in the neuroepithelial cells was absent, and this was associated with convex bending of the neuroepithelium. The results suggest that heparan sulfate is an important determinant of cytoskeletal actin organization during spinal neurulation, and that its biological action is dependent on sulfation of the heparan molecule. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Formation of the central nervous system begins with embryonic neurulation. In this process, the surface ectoderm thickens and differentiates to become the neuroepithelium, which then undergoes elevation, apposition and fusion in the midline dorsally to give rise to the neural tube [10]. Closure of the neural tube starts at sites of de novo initiation of neural fold fusion, and continues in a zipper-like manner along the neuraxis [22]. In spinal neurulation, three modes of neural tube closure are seen, which are differentiated based on the presence of various hinge points [39]. In Mode 1, a midline bend occurs at the median hinge point in the neuroepithelium. In Mode 2, bends occur at the median hinge point as well as paired dorsolateral hinge points. In contrast, only
∗ Corresponding author. Tel.: +65 6516 3206; fax: +65 6778 7643. E-mail addresses: [email protected] (G.S.-X. Leong), [email protected] (G.W. Yip). 1 Tel: +65 6516 3200; fax: +65 6778 7643. 0304-3940/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2012.10.058
paired dorsolateral hinge points are present in Mode 3. Failure of neural tube closure leads to congenital neural tube defects such as spina bifida and myelomeningocele [10,13,17]. Glycosaminoglycans are long, unbranched polysaccharides made up of repeating disaccharide subunits of an amino sugar and an uronic acid [26,43,48]. In addition to having a structural role in the extracellular matrix, glycosaminoglycans help to regulate growth factor signaling and influence cellular behavior [15,42,48,49]. The major sulfated glycosaminoglycans synthesized during spinal neurulation are heparan sulfate and chondroitin sulfate [47]. Both molecules have been shown to play important roles during neurulation [28,30,40,47]. In spinal neurulation, inhibition of heparan sulfation was found to result in accelerated closure of the posterior neuropore as well as suppression of bending at the median hinge point [47]. Actin microfilaments are present in neuroepithelial cells, and have been hypothesized to be important for neural tube closure [1,21,35]. Syndecan-1, a heparan sulfate proteoglycan, has been shown to be associated with the actin cytoskeleton in Schwann cells [7]. Further, changes in glycosaminoglycan sulfation have been
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reported to be associated with cellular activity and growth factor signaling [5,33,38]. Thus, in this study, we aimed to determine if heparan sulfation is involved in organization of the cytoskeletal actin microfilaments in the neuroepithelium during spinal neural tube closure.
with Alexa Fluor 488-phalloidin (1:50 dilution) for 20 min at room temperature as previously described [16]. Cell nuclei were counterstained using DAPI. The fluorescence images were examined using an Olympus FluoView FV1000 laser scanning confocal microscope. 2.3. Statistical analysis
2. Materials and methods 2.1. Culture of whole embryos Six-week old randomly bred ARC(S) mice were mated overnight. The day of finding a copulation plug was designated embryonic day (E) 0.5. Embryos at E8.5 were explanted and cultured in 100% rat serum for 24 h in a roller incubator at 37 ◦ C as previously described [9,47]. Six-hourly gassing using a mixture of 5% carbon dioxide, 5% oxygen and 90% nitrogen was performed. At the end of the culture period, each embryo was removed from the surrounding membranes and examined under a Nikon SMZ1500 stereomicroscope. Development of the cultured embryos was assessed by counting the number of somites present as well as by using the morphological appearance of various embryonic features [6]. The posterior neuropore length, defined as the distance between the cranial end of the posterior neuropore and the tip of the tail bud, was measured using an eyepiece graticule. All animal experiments were conducted in accordance with institutional animal care and use guidelines of the National University of Singapore. 2.2. Phalloidin staining Embryos were fixed overnight in 4% paraformaldehyde, washed twice with phosphate-buffered saline (PBS), and cryoprotected in 30% sucrose prior to being embedded in OCT (Leica). Cryosections of 10-m thickness were then cut transversely through the closing posterior neuropore region and mounted onto gelatin-coated glass slides. Actin filaments were stained by incubating the sections
Experiments consisted of at least nine replicates per group. Statistical comparison of measured embryonic parameters between two groups was performed by Student’s t-test using Prism v5.04 (GraphPad Software). Statistical significance was defined as a p value of below 0.05. 3. Results Explanted E8.5 mouse embryos were cultured for 24 h in the presence of either 30 mM sodium chlorate (treatment group) or PBS (control group). Sodium chlorate is an inhibitor of glycosaminoglycan sulfation, and has been extensively used in cell and organ cultures at up to 30 mM with no significant effects on cell viability or protein synthesis [8,12,14,16,27,47]. As shown in our previous study, treatment with 30 mM chlorate did not affect growth and development of the cultured embryos [47]. Regular heartbeats were observed in both chlorate-treated and control embryos, and vigorous blood circulation was seen in the yolk sacs. Morphological features of chlorate-treated embryos were similar to those of control embryos (Fig. 1A,B). Furthermore, there was no statistically significant difference in the number of somites in the two groups of embryos (Fig. 1C, p > 0.05). To determine if inhibition of glycosaminoglycan sulfation affected closure of the posterior neuropore, we compared the posterior neuropore length of chlorate-treated embryos against embryos in the control group. Determination of the posterior length was performed by measuring the distance between the cranial end of the posterior neuropore and the tip of the tail bud (Fig. 1A and
Fig. 1. Morphology of cultured whole mouse embryos treated with either phosphate-buffered saline (A) or 30 mM chlorate (B). The average number of somites in the two groups of embryos is shown in (C), while the mean posterior neuropore length is shown in (D). Error bars represent standard errors, with nine embryos in each treatment group. *, posterior neuropore. Scale bars, 0.4 mm.
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Fig. 2. Transverse sections of the closing posterior neuropore of phosphate-buffered saline-treated embryos (A, B) or chlorate-treated embryos (C, D). Tissues were stained either with Alexa Fluor 488-phalloidin (A, C) or DAPI (B, D). Arrows, paired dorsolateral hinge points; arrowhead, median hinge point; *, absent median hinge point. Scale bars, 0.1 mm.
B). As shown in Fig. 1D, blocking glycosaminoglycan sulfation led to a significant increase in the rate of posterior neuropore closure (p < 0.005). The posterior neuropore length was 39.9% shorter in the chlorate-treated group compared against embryos in the control group. To investigate if sulfation of glycosaminoglycans is essential for organization of actin microfilaments in the neuroepithelium, we stained the actin fibers using phalloidin in transverse cryosections of the closing posterior neuropore (Fig. 2). Apical actin microfilaments were abundantly present in neuroepithelial cells in the closing posterior neuropore region of the control embryos, including the median hinge point and the paired dorsolateral hinge points. In contrast, the neuroepithelium showed very faint staining for actin microfilaments in the chlorate-treated group. This was associated with an absence of the median hinge point, loss of apical neuroepithelial cell wedging, and convex bending of the neuroepithelium, possibly due to floppines. In addition, increased bending was observed in the dorsolateral hinge points.
4. Discussion We have investigated the biological function of glycosaminoglycans on spinal neural tube closure by treating cultured whole embryos with chlorate. Chlorate is a structural analog of sulfate, and acts as a competitive inhibitor of glycosaminoglycan sulfation [8,14]. As in an earlier report [47], chlorate treatment in this study led to a statistically significant reduction in the length of the closing posterior neuropore without any adverse effects on the morphology of the developing embryos or in the number of somites compared against saline-treated control embryos. Although chlorate inhibits sulfation of both heparan and chondroitin, its effects on spinal neural tube closure are mainly due to a loss of heparan sulfation [47]. Addition of heparan sulfate, but not chondroitin sulfate,
to the culture medium could abolish the chlorate-induced changes in the closing spinal neural tube. Using phalloidin, we confirmed the presence of actin microfilaments in the apical region of neuroepithelial cells [1,21,29,31,35,46]. The loss of these actin microfilaments and apical wedging in the neuroepithelial cells after chlorate treatment together with the absence of the median hinge point support the hypothesis that contraction of these microfilaments regulates neural tube closure [20,21]. Interestingly, a similar phenomenon has been reported in Xenopus animal caps [19]. Degradation of heparan sulfate by heparinase treatment led to a reduction in actin gene expression. In contrast, addition of exogenous heparan sulfate to the heparinase-treated tissues restored actin transcript levels [19]. Heparan sulfate has been reported to be critical for polarization of epithelial cells [37]. Formation of the lung lumen requires interaction between heparan sulfate and laminin. Blocking this interaction through the use of an anti-laminin antibody or exogenous heparan sulfate led to inhibition of epithelial cell polarization and hindered lumen formation [37]. The axon-dendrite polarity of neurons has also been shown to be regulated by heparan sulfate [23,24,32]. Integrins help in signal transmission from the extracellular matrix to the cytoskeleton. Activation of integrins by heparan sulfate chains in syndecans, a family of transmembrane heparan sulfate proteoglycans, initiates signaling through the Rho cytoskeletal G-proteins and ␣-actinin and thereby regulates the formation of actin stress fibers and focal adhesion [2–4,11,18,36]. Heparan sulfate chains are needed to target syndecans to specific domains on the cell surface [45]. Blocking antibodies to integrin or syndecan disrupted the assembly of actin stress fibers and focal adhesion formation [36]. Inhibiting glycosaminoglycan sulfation by chlorate treatment could reduce integrin-heparan sulfate interaction and thus perturb downstream Rho signaling [34,44]. Indeed, variations in heparan sulfation patterns are known to
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differentially affect cellular activities and the binding of heparan sulfate to fibroblast growth factors and other signaling molecules [16,25,33,41,42]. This has led to the emerging field of heparanomics. In conclusion, we have demonstrated that heparan sulfate regulates the organization of cytoskeletal actin microfilaments in the murine neuroepithelium during spinal neural tube formation. This organization is dependent on heparan sulfation, and may play a part in formation of the median hinge point and maintenance of the rigidity of the neuroepithelium during neurulation. Acknowledgments The authors are grateful to Prof. Andrew J. Copp for helpful discussions on the study, and Dr. Chunhua Guo and Ms. Song-Lin Bay for their technical assistance in this project. The work was supported by Grant R-181-000-095-112 (to G.W.Y.) from the Academic Research Fund, Ministry of Education, Singapore. G.S.-X.L. is the recipient of a graduate research scholarship from the National University of Singapore. References [1] P.C. Baker, T.E. Schroeder, Cytoplasmic filaments and morphogenetic movement in the amphibian neural tube, Developmental Biology 15 (1967) 432–450. [2] D.M. Beauvais, A.C. Rapraeger, Syndecan-1-mediated cell spreading requires signaling by alphavbeta3 integrins in human breast carcinoma cells, Experimental Cell Research 286 (2003) 219–232. [3] M. Bernfield, M. Gotte, P.W. Park, O. Reizes, M.L. Fitzgerald, J. Lincecum, M. Zako, Functions of cell surface heparan sulfate proteoglycans, Annual Review of Biochemistry 68 (1999) 729–777. [4] M. Bernfield, R. Kokenyesi, M. Kato, M.T. Hinkes, J. Spring, R.L. Gallo, E.J. Lose, Biology of the syndecans: a family of transmembrane heparan sulfate proteoglycans, Annual Review of Cell Biology 8 (1992) 365–393. [5] Y.G. Brickman, M.D. Ford, J.T. Gallagher, V. Nurcombe, P.F. Bartlett, J.E. Turnbull, Structural modification of fibroblast growth factor-binding heparan sulfate at a determinative stage of neural development, Journal of Biological Chemistry 273 (1998) 4350–4359. [6] N.A. Brown, S. Fabro, Quantitation of rat embryonic development in vitro: a morphological scoring system, Teratology 24 (1981) 65–78. [7] D.J. Carey, K.M. Bendt, R.C. Stahl, The cytoplasmic domain of syndecan-1 is required for cytoskeleton association but not detergent insolubility. Identification of essential cytoplasmic domain residues, Journal of Biological Chemistry 271 (1996) 15253–15260. [8] H.E. Conrad, Heparin-Binding Proteins, Academic Press, San Diego, 1998. [9] A. Copp, P. Cogram, A. Fleming, D. Gerrelli, D. Henderson, A. Hynes, M. KolatsiJoannou, J. Murdoch, P. Ybot-Gonzalez, Neurulation and neural tube closure defects, Methods in Molecular Biology 136 (2000) 135–160. [10] A.J. Copp, N.D. Greene, Genetics and development of neural tube defects, Journal of Pathology 220 (2010) 217–230. [11] J.R. Couchman, Transmembrane signaling proteoglycans, Annual Review of Cell and Developmental Biology 26 (2010) 89–114. [12] J. Davies, M. Lyon, J. Gallagher, D. Garrod, Sulphated proteoglycan is required for collecting duct growth and branching but not nephron formation during kidney development, Development 121 (1995) 1507–1517. [13] P. de Marco, E. Merello, S. Mascelli, V. Capra, Current perspectives on the genetic causes of neural tube defects, Neurogenetics 7 (2006) 201–221. [14] H. Greve, Z. Cully, P. Blumberg, H. Kresse, Influence of chlorate on proteoglycan biosynthesis by cultured human fibroblasts, Journal of Biological Chemistry 263 (1988) 12886–12892. [15] M. Guerrini, M. Hricovini, G. Torri, Interaction of heparins with fibroblast growth factors: conformational aspects, Current Pharmaceutical Design 13 (2007) 2045–2056. [16] C.H. Guo, C.Y. Koo, B.H. Bay, P.H. Tan, G.W. Yip, Comparison of the effects of differentially sulphated bovine kidney- and porcine intestine-derived heparan sulphate on breast carcinoma cellular behaviour, International Journal of Oncology 31 (2007) 1415–1423. [17] M.J. Harris, D.M. Juriloff, An update to the list of mouse mutants with neural tube closure defects and advances toward a complete genetic perspective of neural tube closure, Birth Defects Research Part A: Clinical and Molecular Teratology 88 (2010) 653–669. [18] T. Ishikawa, R.H. Kramer, Sdc1 negatively modulates carcinoma cell motility and invasion, Experimental Cell Research 316 (2010) 951–965. [19] K. Itoh, S.Y. Sokol, Heparan sulfate proteoglycans are required for mesoderm formation in Xenopus embryos, Development 120 (1994) 2703–2711.
[20] S.D. Joshi, M. von Dassow, L.A. Davidson, Experimental control of excitable embryonic tissues: three stimuli induce rapid epithelial contraction, Experimental Cell Research 316 (2010) 103–114. [21] P. Karfunkel, The mechanisms of neural tube formation, International Review of Cytology 38 (1974) 245–271. [22] Z. Kibar, V. Capra, P. Gros, Toward understanding the genetic basis of neural tube defects, Clinical Genetics 71 (2007) 295–310. [23] F. Lafont, A. Prochiantz, C. Valenza, M. Petitou, M. Pascal, M. Rouget, A. Rousselet, Defined glycosaminoglycan motifs have opposite effects on neuronal polarity in vitro, Developmental Biology 165 (1994) 453–468. [24] F. Lafont, M. Rouget, A. Triller, A. Prochiantz, A. Rousselet, In vitro control of neuronal polarity by glycosaminoglycans, Development 114 (1992) 17–29. [25] W.C. Lamanna, I. Kalus, M. Padva, R.J. Baldwin, C.L. Merry, T. Dierks, The heparanome – the enigma of encoding and decoding heparan sulfate sulfation, Journal of Biotechnology 129 (2007) 290–307. [26] C. Malavaki, S. Mizumoto, N. Karamanos, K. Sugahara, Recent advances in the structural study of functional chondroitin sulfate and dermatan sulfate in health and disease, Connective Tissue Research 49 (2008) 133–139. [27] H.Q. Miao, R. Ishai-Michaeli, R. Atzmon, T. Peretz, I. Vlodavsky, Sulfate moieties in the subendothelial extracellular matrix are involved in basic fibroblast growth factor sequestration, dimerization, and stimulation of cell proliferation, Journal of Biological Chemistry 271 (1996) 4879–4886. [28] G. Morriss-Kay, F. Tuckett, Immunohistochemical localisation of chondroitin sulphate proteoglycans and the effects of chondroitinase ABC in 9- to 11-day rat embryos, Development 106 (1989) 787–798. [29] G. Morriss-Kay, F. Tuckett, The role of microfilaments in cranial neurulation in rat embryos: effects of short-term exposure to cytochalasin D, Journal of Embryology and Experimental Morphology 88 (1985) 333–348. [30] G.M. Morriss-Kay, B. Crutch, Culture of rat embryos with beta-d-xyloside: evidence of a role for proteoglycans in neurulation, Journal of Anatomy 134 (1982) 491–506. [31] R.G. Nagele, H.Y. Lee, Studies on the mechanism of neurulation in the chick: microfilament-mediated changes in cell shape during uplifting of neural folds, Journal of Experimental Zoology 213 (1980) 391–398. [32] K. Nishimura, M. Ishii, M. Kuraoka, K. Kamimura, N. Maeda, Opposing functions of chondroitin sulfate and heparan sulfate during early neuronal polarization, Neuroscience 169 (2010) 1535–1547. [33] V. Nurcombe, M.D. Ford, J.A. Wildschut, P.F. Bartlett, Developmental regulation of neural response to FGF-1 and FGF-2 by heparan sulfate proteoglycan, Science 260 (1993) 103–106. [34] J.A. Roper, R.C. Williamson, M.D. Bass, Syndecan and integrin interactomes: large complexes in small spaces, Current Opinion in Structural Biology 22 (2012) 583–590. [35] T.W. Sadler, D. Greenberg, P. Coughlin, J.L. Lessard, Actin distribution patterns in the mouse neural tube during neurulation, Science 215 (1982) 172–174. [36] S. Saoncella, F. Echtermeyer, F. Denhez, J.K. Nowlen, D.F. Mosher, S.D. Robinson, R.O. Hynes, P.F. Goetinck, Syndecan-4 signals cooperatively with integrins in a Rho-dependent manner in the assembly of focal adhesions and actin stress fibers, Proceedings of the National Academy of Sciences of the United States of America 96 (1999) 2805–2810. [37] L. Schuger, A.P. Skubitz, K. Gilbride, R. Mandel, L. He, Laminin and heparan sulfate proteoglycan mediate epithelial cell polarization in organotypic cultures of embryonic lung cells: evidence implicating involvement of the inner globular region of laminin beta 1 chain and the heparan sulfate groups of heparan sulfate proteoglycan, Developmental Biology 179 (1996) 264–273. [38] O.J. Scully, P.J. Chua, K.S. Harve, B.H. Bay, G.W. Yip, Serglycin in health and diseases, Anatomical Record (Hoboken) 295 (2012) 1415–1420. [39] A.S. Shum, A.J. Copp, Regional differences in morphogenesis of the neuroepithelium suggest multiple mechanisms of spinal neurulation in the mouse, Anatomy and Embryology 194 (1996) 65–73. [40] F. Tuckett, G.M. Morriss-Kay, Heparitinase treatment of rat embryos during cranial neurulation, Anatomy and Embryology 180 (1989) 393–400. [41] J.E. Turnbull, Heparan sulfate glycomics: towards systems biology strategies, Biochemical Society Transactions 38 (2010) 1356–1360. [42] J.E. Turnbull, R.L. Miller, Y. Ahmed, T.M. Puvirajesinghe, S.E. Guimond, Glycomics profiling of heparan sulfate structure and activity, Methods in Enzymology 480 (2010) 65–85. [43] A. Varki, R.D. Cummings, J.D. Esko, H.H. Freeze, P. Stanley, C.R. Bertozzi, G.W. Hart, M.E. Etzler, Essentials of Glycobiology, Cold Spring Harbor Laboratory Press, New York, 2008. [44] X. Xian, S. Gopal, J.R. Couchman, Syndecans as receptors and organizers of the extracellular matrix, Cell and Tissue Research 339 (2010) 31–46. [45] Y. Yang, M. Borset, J.K. Langford, R.D. Sanderson, Heparan sulfate regulates targeting of syndecan-1 to a functional domain on the cell surface, Journal of Biological Chemistry 278 (2003) 12888–12893. [46] P. Ybot-Gonzalez, A.J. Copp, Bending of the neural plate during mouse spinal neurulation is independent of actin microfilaments, Developmental Dynamics 215 (1999) 273–283. [47] G.W. Yip, P. Ferretti, A.J. Copp, Heparan sulphate proteoglycans and spinal neurulation in the mouse embryo, Development 129 (2002) 2109–2119. [48] G.W. Yip, M. Smollich, M. Gotte, Therapeutic value of glycosaminoglycans in cancer, Molecular Cancer Therapeutics 5 (2006) 2139–2148. [49] L. Zhang, Glycosaminoglycan (GAG) biosynthesis and GAG-binding proteins, Progress in Molecular Biology and Translational Science 93 (2010) 1–17.