QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

doi: 10.1242/10.1242/dev.00117

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Irving, C. Articles by Mason, I. Search for Related Content PubMed PubMed Citation Articles by Irving, C. Articles by Mason, I.

Establishing the trochlear motor axon trajectory: role of the isthmic organiser and Fgf8

MRC Centre for Developmental Neurobiology, King's College London, Guy's Campus, 4^th Floor New Hunt's House, London SE1 1UL, UK

View larger version (50K): [in a new window]   Fig. 3. Isthmic tissue is a source of a diffusible attractant for trochlear motor axons. (A) A schematic representation of how the collagen gel co-culture was assembled, showing the dissected MHB tissue explant containing trochlear neurons (orange) and the dorsal isthmus (blue) explant. Mb, midbrain; r1-r4, rhombomeres 1-4. (B) Scoring system for detecting deflection of axons towards a source (isthmus tissue, blue sphere). The number of axons were counted growing in two sectors (1, 2) of a T-bar placed parallel and central to the initial trajectory of the extending nerve. (C-F) Rat E11.5 explants into collagen gels that were cultured for 48 hours and stained with F84.1 antibody. (C) Trochlear axons grow from an isolated MHB explant defasciculate upon entering the gel but do not deviate greatly from their original trajectories. (D,E) MHB explants cultured at a distance from a piece of isthmus tissue: axons turn towards the isthmus tissue (i) and grow within it. (F) MHB explant cultured at a distance from a piece of posterior rhombomere 1 (rl) tissue: axons are not affected by the r1 tissue.

View larger version (45K): [in a new window]   Fig. 1. Trochlear motor neurons are located both within and posterior to the isthmic organiser. (A-D) Chick hindbrains stained for Isl1/2 (brown) and Fgf8 (blue) by combined immunohistochemistry and in situ hybridisation. All preparations are `open-book' flat mounts i.e. cut along the dorsal midline and opened such that the floor plate is medial in the preparation and dorsal regions are lateral. (A) HH25 chick embryo showing the position of trochlear motor nuclei within the anterior hindbrain. Cell bodies are detected ventrally in rostral r1, both within and adjacent to the Fgf8 expression domain. They appear more scattered than oculomotor neurons in the midbrain and most of r1 is essentially devoid of Isl1/2+ motor neurons. (B) The first trochlear motor neurons are detected at HH17 within and ventral to the Fgf8 expression domain. (C) At HH19 trochlear cell bodies are detected both within and posterior to the isthmus. (D) By HH25 the trochlear nuclei are positioned in a dense cluster in anterior r1 whose anterior extent is coincident with that of Fgf8 expression: anterior boundary of r1. III, oculomotor nucleus; IV, trochlear nucleus; V, trigeminal nucleus; i, isthmus; MB, midbrain; r1, rhombomere 1.

View larger version (93K): [in a new window]   Fig. 2. Trochlear axons project towards and extend within the isthmic organiser. (A-F) Flat mount preparations of chick hindbrains stained for the SC1 antigen. In all cases anterior is towards the top of the image. (A) HH25 chick hindbrain. Trochlear axons project dorsally forming 3-4 main fascicles. (B) Short, SC1-positive trochlear axons (arrows) are first weakly detected at HH18 extending from the anterior cells of the nucleus. (C) By HH19 pioneer axons have reached the roof plate. Axon extension appears to proceed in an anteroposterior wave with axons extending from more anterior cell bodies reach the roof plate before those emerging from posteriorly positioned cell bodies within r1. (D) At HH20 anteriorly positioned cell bodies have reached the roof plate extending dorsally within the isthmus, perpendicular to the floor plate. By contrast, axons from more posterior neurons follow a route that leads them both anteriorly towards the isthmus and dorsally towards the roof plate. (E,F) At HH25 axons are still just forming from posterior trochlear motor neurons and their growth cones are visible at higher magnification (arrows, F). (G-J) Chick hindbrain at HH25 stained for both Isl1/2 (G; green) and SC1 (H; red) and these images are combined in I and J with the trochlear nucleus (boxed in I) being shown at higher magnification in J.

View larger version (84K): [in a new window]   Fig. 4. Fgf8 is a chemoattractant for trochlear motor axons in vitro. (A) Diagramatic representation of the experiment. (B) MHB explants cultured for 48 hours together with an Affi-gel bead soaked in Fgf8b. Trochlear axons turn and grow towards the FGF beads. (C) Trochlear axons are not attracted towards a control bead soaked in PBS. (D) Staining for neurofilament to reveal all axons present shows that growth towards an Fgf8 source is not a general feature of axons from r1 neurons. (E) Turning influences of Fgf8 occur both within and outside the explant. The original trajectory of the trochlear axons extends from the top left corner of the picture (out of focus because of the thickness of the explant tissue above it). As they approach the periphery of the explant, axons turn slightly towards the Fgf8 beads, however, further reorientation towards the closest bead (i) occurs outside the explant (arrowheads). Some of the axons stall at that bead, but others (arrows) extend further and appear to be reorienting towards a second, more distant Fgf8 bead (ii).

View larger version (41K): [in a new window]   Fig. 5. Ectopic Fgf8 redirects trochlear motor axons in vivo. (A) Diagrammatic representation of the experimental manipulation. (B-G) Flat-mounted hindbrains of HH25 chick embryos stained for SC1 antigen following bead implantation at HH11-12. In all cases anterior is towards the top of the image. Individual motor nuclei are labelled where present on the image: III (oculomotor); IV, trochlear; V, trigeminal (stains only weakly for SC1) and VI, facial. Deflected and misrouted axons or fascicles are indicated by arrows and, where visible, the bead implant is indicated by the red arrowheads. (B) Control embryo showing that implanted PBS beads have no effect on trochlear nerve axon pathways. (C-G) Implanted FGF-soaked beads result in 4 classes of axonal defect. (C) Type 1 phenotype: axons extend from the floor plate dorsally but posterior axons have lost their anterior trajectory towards the isthmus and follow a direct dorsal path. (D) Type 2 phenotype: the most anterior fascicle has turned through 90° within r1 and extends towards the ectopic source of Fgf8 at the r1/2 boundary. (E,F) Low and high magnification images showing the Type 3 phenotype. The trochlear nerve is bisected; the arrow indicates fascicles that have turned towards the ectopic source of Fgf8. (G) Ectopic structures with the morphology of an ectopic isthmic region in posterior r1 induced by an Fgf8 bead are associated with ectopic SC1-positive axons that grow towards the bead.

View larger version (38K): [in a new window]   Fig. 6. Fgf8 is required for guidance of trochlear motor axons in vivo. (A) Diagram of explant culture. (B-G) MHB explants cultured for 48 hours and stained with F84.1 antibody. (B) Control explants cultured in the presence of DMSO alone. Note the formation of a single dorsal projection within the explant (C,D). Explant cultured in the presence of the FGFR inhibitor SU5402 at 10 µM; axons fail to navigate correctly within the explant. (E-G) Explants cultured in the presence of a neutralising anti-Fgf8 antiserum. Note that axonal misrouting occurs in both dorsal (arrow in E, arrowhead in F) and ventral (arrows in G) regions of explants. (F) Some appear to follow random or even circular trajectories within the explant (red arrowhead) around the bead. When axons did exit from the explant they did so over a wide region of its dorsal edge (E,G).

View larger version (58K): [in a new window]   Fig. 7. A model for trochlear axon guidance. Trochlear cell bodies (green) arise in a cluster in ventral anterior r1. Extending axons (green arrows) are exposed to a number of both positive and negative chemotropic signals within the neural tube. Initially, a strong repulsive signal from the floor plate (netrin 1; dark blue) initiates a dorsal trajectory. Additional repulsive signals are present in posterior r1 (Sema3F; light blue). In combination with an attractive signal from the isthmus (Fgf8, red), a net positive signal towards the dorsal isthmic region is produced.