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

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 Google Scholar Google Scholar Articles by Georgiou, M. Articles by Tear, G. Search for Related Content PubMed PubMed Citation Articles by Georgiou, M. Articles by Tear, G.

Commissureless is required both in commissural neurones and midline cells for axon guidance across the midline

Molecular Neurobiology Group, MRC Centre for Developmental Neurobiology, Guy’s Hospital Campus, King’s College, London SE1 1UL, UK

View larger version (141K): [in a new window]   Fig. 1. The commissureless mutant phenotype is not significantly rescued by expression of Commissureless at the midline. Axon tracts within the ventral nerve cord of (A) wild-type and (B) comm mutant embryos labelled with mAb BP102 at stage 15. In the wild-type embryo, the axons are organised into two longitudinal tracts, which are connected via two commissural tracts per segment. In comm mutants, the commissures do not form. (C) A wild-type embryo stained with a polyclonal Comm antibody. Comm protein accumulates at the commissure and is absent from the longitudinals. (D-F) Comm expression is driven at the midline using either the sim- (D) or slit- (E,F) GAL4 drivers and the resultant axon scaffold visualised with mAb BP102. (D,E) Driving wild-type Comm expression at the midline leads to a partial rescue of commA490 loss-of-function mutants. However there is no rescue of the null allele, commE39 (F). All panels are a view of the dorsal surface of the CNS and are shown anterior upwards.

View larger version (135K): [in a new window]   Fig. 2. Comm-GFP is not transferred from midline cells. (A,B) UAS-Comm-GFP, expressed at the midline using both sim- and slit-GAL4 drivers and visualised using polyclonal GFP at stage 15. (A) Comm-GFP (green) expressed in midline cells is unable to transfer onto commissural axons. mAb BP102 (red) highlights the axon scaffold. (B) When large amounts of Comm-GFP are driven at the midline, individual axons from the midline-derived neurones are decorated with Comm-GFP protein (black arrows). No GFP staining is observed at the commissure (white arrow). The panels show a view of the dorsal surface of the CNS and are oriented with the anterior of the embryo towards the left.

View larger version (102K): [in a new window]   Fig. 3. comm-HP specifically disrupts comm expression in vitro and in vivo but has no effect on sensory axon guidance. (A) Drosophila S2 cells expressing Comm-GFP in which Comm localises to intracellular vesicles (inset). (B) When comm-HP is co-expressed with comm-GFP, the number of fluorescing cells is very low. Most cells that do fluoresce show either low levels of fluorescence or a disrupted localisation (inset). (C) robo and comm-HP co-transfected cells. comm-HP does not affect the expression level or numbers of Robo-transfected cells. (D) When comm-HP is driven throughout the nervous system using elav-GAL4, sensory axon extension and guidance is unaffected. The elav-GAL4 driver was used to drive both UAS-comm-HP and UAS-GFP and axons were visualised with GFP. (E) Eagle-expressing neurones revealed with anti-Eagle antibody (green); these neurones also express Comm (F, yellow). (G) When comm-HP is driven in the Eg-positive neurones, Comm levels are reduced in these cells (arrows). However not all Eg-positive neurones are equally affected, as Comm protein remains within a subset of the Eg-positive neurones (arrowhead in G). The embryo in D shows a view of the ventral region of the PNS at stage 16, the embryo is oriented with anterior to the left and dorsal upwards. (E-G) show views of ventral regions of the CNS in stage 13 embryos oriented anterior upwards.

View larger version (150K): [in a new window]   Fig. 4. Expression of comm-HP in either neurones or midline cells leads to guidance defects at the midline. (A) Sema2b neurones in the wild-type stage 16 embryo. (B) When comm-HP is driven in midline cells, the Sema2b neurones make crossing errors. The commissures are thinner and occasionally fail to form, as do sections of the longitudinal tract. Growth cone stalling at the midline is also seen (arrow). (C) When comm-HP is driven in neurones, the commissures are more severely affected and longitudinal sections can be completely absent. (D,E) Expression of comm-HP in neurones in a comm heterozygous background results in an enhanced phenotype where crossing errors are frequent and stalling occurs (arrow). Commissures and longitudinal sections are often absent or significantly reduced. (F-J) BP102 staining of the ventral nerve cord at (F-H) stage 13 or (I,J) stage 16. Arrows indicate thinner commissures, arrowheads indicate axon stalling. (F) Wild-type embryo. (G) When comm-HP is expressed in neurones, some axon stalling around the commissures is evident (arrowhead) together with thinner longitudinals and occasional thinner commissures (arrow). (H) This phenotype is enhanced when comm-HP is expressed in neurones in a comm heterozygous background. At later stages in development, some axon stalling and thinner longitudinals are observed when comm-HP is expressed in (I) neurones or (J) midline cells. All panels show a dorsal view of the CNS and are oriented with anterior upwards.

View larger version (196K): [in a new window]   Fig. 5. comm expression is required in commissural but not ipsilateral neurones for midline crossing. (A-C) eagle-expressing neurones in stage 13 embryos, assayed using the UAS-GFP reporter and eagle-GAL4 driver and stained with an anti-GFP antibody. (A) Wild-type embryo showing axons crossing in both anterior (AC) and posterior (PC) commissures. (B) When one copy of comm-HP is driven in these neurones, the medial-most axons occasionally fail to cross (arrow). (C) When two copies are driven, crossing errors are more frequent and lateral axons also fail to cross on occasion. (D) When UAS-GFP and comm-HP is driven in the eagle-expressing neurones and at the midline by eg-Gal4 and slit-GAL4 the number of crossing errors remains similar to that seen when comm-HP is driven in the eagle-expressing neurones alone. However embryos with increased numbers of crossing failures are occasionally observed. Arrows show axon bundles turning away from the midline. (E,F) When comm-HP is driven in ipsilaterally projecting neurones their outgrowth is unaffected. Line 15J2 (E) was used to drive both comm-HP and a GFP reporter in the vMP2 and dMP2 interneurones. Line 15J2, MZ465 (F) additionally drives expression in pCC, aCC and RP2. All panels show dorsal views of the CNS oriented with anterior towards the left.

View larger version (76K): [in a new window]   Fig. 6. Comm protein is observed in commissural but not ipsilateral cell bodies. (A,B) Comm protein (red), visualised with anti-Comm, co-localises with the cell bodies of the Eagle-positive neurones visualised using eg-GAL4 driving UAS-GFP and stained with anti-GFP (green). Longitudinal view of a stage 16, anterior towards the left, dorsal upwards. (C,D) Comm protein (green), visualised with anti-Comm, colocalises with the cell bodies of the Sema2b neurones (red), visualised with anti-Myc at stage 16. Anterior is upwards. (E,F) Comm protein (red) does not colocalise with the cell bodies of the ipsilateral vMP2 and dMP2 neurones (green) identified by the 15J2 GAL4 line at stage 16. Arrows indicate Comm colocalisation. Anterior is towards the left.