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First published online 1 October 2003
doi: 10.1242/dev.00765

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Axolotl pronephric duct migration requires an epidermally derived, laminin 1-containing extracellular matrix and the integrin receptor {alpha}6ß1

Andrea R. Morris1,*,{dagger}, Julie Drawbridge2 and Malcolm S. Steinberg1

1 Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA
2 Department of Biology, Rider University, Lawrenceville, NJ 08648, USA



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  		Fig. 1. ECM collection and carrier transplantation techniques. Polycarbonate carriers were cut and placed under donor epidermis, which was allowed to secrete ECM onto the carrier overnight. This `ECM-conditioned' carrier was then placed, with or without rotation, under an epidermal flap along either the left or the right PND migration pathway of the host embryo. Carriers remained in embryos overnight, followed by fixation, removal of epidermis and analysis of PND location.

 


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  		Fig. 2. Epidermal ECM provides directional cues to the PND. Epidermally secreted ECM was collected onto polycarbonate carriers, then presented in various orientations to the PND pathway of same-stage hosts (Fig. 1). (A) A control embryo undergoing normal PND migration. The PND migrates just below the somites and its tip is indicated by the arrow. (B) The PND has migrated onto somitic mesoderm – dorsal to its normal migratory pathway – following the introduction of a 90° dorsally rotated ECM-bearing carrier. The PND tip (arrow) has here traveled more than halfway up the sixth somite. (C) A 90° ventral rotation of an ECM-conditioned carrier prior to transplantation has redirected PND migration ventrally. The PND tip (arrow) is located ventral to the normal migration path, on lateral mesoderm. (A-C) Anterior is to the right and dorsal to the top.

 


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  		Fig. 3. Expression of {alpha}6 and ß1 integrins and laminin 1 in axolotl embryos. (A) A stage 24 albino embryo was treated as described in Materials and Methods, with an anti-{alpha}6 integrin antibody (GoH3) followed by antibody detection (top). Antibody staining is evident along the somites, the pronephros (arrow) and the migrating PND. A control embryo to which no GoH3 antibody was added (bottom) did not show staining. (B) A stage 26 albino embryo was fixed and the epidermis overlying the lateral flank was removed. The embryo was then treated with an anti-ß1 integrin primary antibody as described in Materials and Methods. Staining was evident throughout the lateral mesoderm, including the somites, the pronephros and the PND (arrow), indicating broad expression of ß1-containing integrins. (C) The extracellular localization of laminin was observed following transverse sectioning through an axolotl embryo that had been previously treated with an anti-laminin-1 antibody. Strong staining was evident within the outermost epidermal layers (arrow and bracket) and highlights its expected localization to the basal lamina.

 


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  		Fig. 4. Inhibition of PND migration following injection of function-blocking antibodies. Stage 21 embryos were injected with Fabs either against BSA or that specifically block integrin subunit function. (A) A BSA-injected control embryo in which the PND was found to have migrated normally. The PND tip (arrow) is at LS MINUS 1 in this embryo. (B) Following exposure to {alpha}6-integrin-function-blocking Fabs, PND migration has been inhibited, such that the PND tip (arrow) is at LS MINUS 3.5. (C) An embryo that has been coinjected with {alpha}6- and ß1-integrin-function-blocking Fab fragments. These antibodies caused a significant inhibition of PND migration, such that the tip is located at LS MINUS 7 rather than its normal position of about LS MINUS 1 to LS MINUS 2.

 


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  		Fig. 5. Summary of PND migratory inhibition. The effects of injecting function-blocking versus non-blocking agents are summarized here. BSA-injected embryos appear to undergo normal PND migration (LS MINUS 1 to LS MINUS 3) and are shown in blue for each graph. Blocking {alpha}6ß1 integrins and laminin 1 causes inhibition of PND migration (anterior to LS MINUS 3), as indicated in yellow.

 

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© The Company of Biologists Ltd 2003