title: Integration of shallow gradients of Shh and Netrin-1 guides commissural axons.
abstract: During nervous system development, gradients of Sonic Hedgehog (Shh) and Netrin-1 attract growth cones of commissural axons toward the floor plate of the embryonic spinal cord. Mice defective for either Shh or Netrin-1 signaling have commissural axon guidance defects, suggesting that both Shh and Netrin-1 are required for correct axon guidance. However, how Shh and Netrin-1 collaborate to guide axons is not known. We first quantified the steepness of the Shh gradient in the spinal cord and found that it is mostly very shallow. We then developed an in vitro microfluidic guidance assay to simulate these shallow gradients. We found that axons of dissociated commissural neurons respond to steep but not shallow gradients of Shh or Netrin-1. However, when we presented axons with combined Shh and Netrin-1 gradients, they had heightened sensitivity to the guidance cues, turning in response to shallower gradients that were unable to guide axons when only one cue was present. Furthermore, these shallow gradients polarized growth cone Src-family kinase (SFK) activity only when Shh and Netrin-1 were combined, indicating that SFKs can integrate the two guidance cues. Together, our results indicate that Shh and Netrin-1 synergize to enable growth cones to sense shallow gradients in regions of the spinal cord where the steepness of a single guidance cue is insufficient to guide axons, and we identify a novel type of synergy that occurs when the steepness (and not the concentration) of a guidance cue is limiting.
authors.affiliation: Molecular Biology of Neural Development, Institut de Recherches Cliniques de Montréal (IRCM), Montreal, Quebec, Canada; Division of Experimental Medicine, McGill University, Montreal, Quebec, Canada; Program in Neuroengineering, McGill University, Montreal, Quebec, Canada.
authors.forename: Tyler F W
authors.initials: TF
authors.lastname: Sloan
authors.affiliation: Program in Neuroengineering, McGill University, Montreal, Quebec, Canada; Division of Engineering, New York University Abu Dhabi, Abu Dhabi, United Arab Emirates.
authors.forename: Mohammad A
authors.initials: MA
authors.lastname: Qasaimeh
authors.affiliation: Program in Neuroengineering, McGill University, Montreal, Quebec, Canada; Department of Biomedical Engineering, McGill University, Montreal, Quebec, Canada; McGill University and Genome Quebec Innovation Centre, McGill University, Montreal, Quebec, Canada.
authors.forename: David
authors.initials: D
authors.lastname: Juncker
authors.affiliation: Molecular Biology of Neural Development, Institut de Recherches Cliniques de Montréal (IRCM), Montreal, Quebec, Canada.
authors.forename: Patricia T
authors.initials: PT
authors.lastname: Yam
authors.affiliation: Molecular Biology of Neural Development, Institut de Recherches Cliniques de Montréal (IRCM), Montreal, Quebec, Canada; Division of Experimental Medicine, McGill University, Montreal, Quebec, Canada; Program in Neuroengineering, McGill University, Montreal, Quebec, Canada; Department of Anatomy and Cell Biology, Department of Biology, McGill University, Quebec, Canada; Department of Medicine, University of Montreal, Montreal, Quebec, Canada.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Charron
mesh_terms: D000818:Animals; D002633:Chemotaxis / Q000502:physiology; D004622:Embryo, Mammalian; D018507:Gene Expression Regulation, Developmental; D020439:Growth Cones / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D053823:Hedgehog Proteins / Q000172:deficiency / Q000235:genetics / Q000494:pharmacology*; D056656:Lab-On-A-Chip Devices; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D057054:Molecular Imaging; D009414:Nerve Growth Factors / Q000172:deficiency / Q000235:genetics / Q000494:pharmacology*; D000075388:Netrin-1; D061251:Primary Cell Culture; D015398:Signal Transduction; D013116:Spinal Cord / Q000187:drug effects* / Q000254:growth & development / Q000378:metabolism / Q000648:ultrastructure; D025521:Tumor Suppressor Proteins / Q000172:deficiency / Q000235:genetics / Q000494:pharmacology*; D019061:src-Family Kinases / Q000235:genetics* / Q000378:metabolism
chemical_list: D053823:Hedgehog Proteins; D009414:Nerve Growth Factors; C000617353:Ntn1 protein, mouse; C465570:Shh protein, mouse; D025521:Tumor Suppressor Proteins; D000075388:Netrin-1; D019061:src-Family Kinases
references.citation: J Comp Neurol. 1982 Mar 10;205(4):371-82
references.pmid: 7096626
references.citation: Am J Pathol. 2013 Sep;183(3):673-80
references.pmid: 23831296
references.citation: Science. 1992 Jan 24;255(5043):472-5
references.pmid: 1734526
references.citation: Cell. 1994 Aug 12;78(3):409-24
references.pmid: 8062384
references.citation: Cell. 1994 Aug 12;78(3):425-35
references.pmid: 8062385
references.citation: Cell. 1995 May 5;81(3):445-55
references.pmid: 7736596
references.citation: Int J Dev Biol. 1995 Oct;39(5):809-16
references.pmid: 8645565
references.citation: Cell. 1996 Oct 18;87(2):175-85
references.pmid: 8861902
references.citation: Cell. 1996 Dec 13;87(6):1001-14
references.pmid: 8978605
references.citation: Nature. 1997 Apr 24;386(6627):796-804
references.pmid: 9126737
references.citation: J Neurosci. 2005 Aug 17;25(33):7669-81
references.pmid: 16107653
references.citation: J Neurosci. 2006 Aug 23;26(34):8866-74
references.pmid: 16928876
references.citation: Nature. 2006 Nov 16;444(7117):369-73
references.pmid: 17086203
references.citation: Nat Protoc. 2006;1(3):1322-8
references.pmid: 17406418
references.citation: Lab Chip. 2008 Feb;8(2):227-37
references.pmid: 18231660
references.citation: J Neurosci Methods. 2008 May 30;170(2):220-8
references.pmid: 18313760
references.citation: Proc Natl Acad Sci U S A. 2009 Mar 17;106(11):4254-9
references.pmid: 19246386
references.citation: Neuron. 1999 Sep;24(1):127-41
references.pmid: 10677032
references.citation: Cell. 2003 Apr 4;113(1):11-23
references.pmid: 12679031
references.citation: Neuron. 2003 May 8;38(3):389-401
references.pmid: 12741987
references.citation: Nat Neurosci. 2004 Jun;7(6):678-82
references.pmid: 15162167
references.citation: Nat Neurosci. 2004 Nov;7(11):1222-32
references.pmid: 15494732
references.citation: Neuron. 2009 May 14;62(3):349-62
references.pmid: 19447091
references.citation: J Neurosci. 2009 Jun 10;29(23):7450-8
references.pmid: 19515913
references.citation: Proc Natl Acad Sci U S A. 2009 Jun 23;106(25):10296-301
references.pmid: 19541606
references.citation: Proc Natl Acad Sci U S A. 2010 Mar 16;107(11):5202-7
references.pmid: 20194766
references.citation: J Vis Exp. 2010;(39). pii: 1773. doi: 10.3791/1773
references.pmid: 20505653
references.citation: Curr Biol. 2010 Dec 7;20(23):2150-6
references.pmid: 21109439
references.citation: Neural Comput. 2011 Feb;23(2):336-73
references.pmid: 21105826
references.citation: Neuron. 2011 Jun 9;70(5):966-78
references.pmid: 21658588
references.citation: Top Curr Chem. 2011;304:27-68
references.pmid: 21526435
references.citation: Cell. 2012 Feb 3;148(3):568-82
references.pmid: 22304922
references.citation: Development. 2013 Jan 15;140(2):333-42
references.pmid: 23250207
references.citation: Trends Neurosci. 2013 May;36(5):295-304
references.pmid: 23485451
references.citation: Development. 1990 Jun;109(2):435-47
references.pmid: 2205466