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First published online May 17, 2004
doi: 10.1242/10.1242/dev.01147

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The Drosophila ARF6-GEF Schizo controls commissure formation by regulating Slit

Institut für Neurobiologie, Universität Münster, Badestrasse 9, D-48149 Münster, Germany

View larger version (138K): [in a new window]   Fig. 1. schizo is required for commissure formation. Frontal views of dissected central nervous system (CNS) preparations of stage-16 embryos. (A-C) Stained for the presence of all CNS axons using Mab BP102. (D-F) Stained for the presence of the Myc epitope using Mab 9E10. (C) The midline glial cells are labeled by ß-galactosidase expression using the AA142 enhancer. Anterior is up. (A) Wild-type embryos are characterized by a regular arrangement of longitudinal connectives (lc) and segmental commissures (ac, pc). (B) In homozygous schizoC1-28 mutant embryos the formation of commissures is reduced (arrow). The longitudinal connectives are thinner. (C) Homozygous schizoU112 embryos display a stronger commissural phenotype. Most frequently the anterior commissure is affected (arrow). In neuromeres with reduced commissures the midline glial cells migrate toward the connectives (arrowhead). (D) In wild-type embryos the sema-myc marker is expressed in only a few neurons in each hemineuromer. The corresponding axons cross the midline in one fascicle and turn anterior within the longitudinal connective. (E,F) In mutant schizoU112/C1-28 embryos the sema-myc marker cannot be detected in about 50% of the commissures. Within the longitudinal connectives we noted a defasciculation of the Myc positive axon bundles (arrow).

View larger version (135K): [in a new window]   Fig. 3. schizo affects the balance between repulsive and attractive signaling. Frontal views of dissected central nervous system (CNS) preparations of stage-16 embryos stained for the presence of all CNS axons using Mab BP102. Anterior is up. (A) Wild-type embryo. Two commissures form in each neuromere. (B) schizo mutant embryo lacks many commissural tracts. (C) Expression of netrinB in all CNS midline cells of schizo mutant restores the commissural pattern. (D) Embryos homozygous for the hypomorphic slit550 allele develop a characteristic fused commissure phenotype, which is similar to the loss of function robo1 phenotype (E). (F,G) The schizo mutant phenotype is reverted by reducing the dose of slit or robo. (H) Overexpression of schizo using a translocation chromosome in a heterozygous slit mutant background also led to a fused commissure phenotype.

View larger version (83K): [in a new window]   Fig. 2. schizo interacts with frazzled and netrin. Whole-mount antibody staining of stage-15/16 embryos using BP102 and subsequent HRP immunohistochemistry. Anterior is to the left. (A) Wild-type embryo. (B) Homozygous schizoU112 mutant embryos have reduced commissures. (C) Df(1)netrin/schizoU112 double-mutant embryo lacks most of the commissures compared with schizoU112 mutants. (D) In a frazzled23H9/schizoU112 double-mutant embryo the axons cannot respond to the attractive signal secreted by the midline cells and almost all commissures are missing, indicating that schizo acts independently of the netrin pathway.

View larger version (52K): [in a new window]   Fig. 4. schizo encodes two Arf-GEF-like proteins. (A) Schematic drawing of the schizo locus. Two transcripts are represented by EST clones identified by the BDGP (t1 RE44556, t2 LP01489). Transcription is from left to right. The location of two P-element insertions is indicated, both l(3)3 and P224 lead to a strong schizo mutant phenotype (data not shown). (B) The two deduced Schizo proteins are characterized by three sequence domains: an IQ domain, which might confer Calmodulin binding, a Sec7 and a Plecstrin homology (PH) domain, which are a signature of guanine exchange factors. (C) schizo is expressed maternally and appears to be negatively regulated by segmentation genes (D). (E,F) schizo is broadly expressed during early development. (G) At the extended germ band stage expression of schizo in the ectoderm is reduced. (H) schizo continues to be expressed in the central nervous system (CNS). The arrow denotes expression in the CNS midline.

View larger version (85K): [in a new window]   Fig. 5. schizo acts in the central nervous system (CNS) midline cells. Frontal views of dissected CNS preparations of stage-16 embryos stained for the presence of all CNS axons using Mab BP102. Anterior is up. (A) Wild-type embryo: note the regular arrangement of the Fas2-positive longitudinal fascicles. (B) The EMS-induced schizoU112 mutation leads to a lack of commissures. (C) Following expression of schizot2 in all midline cells of a homozygous mutant schizo embryo, the mutant phenotype is rescued and commissures form normally.

View larger version (135K): [in a new window]   Fig. 6. Commissure formation requires endocytosis. Frontal views of dissected central nervous system (CNS) preparations of stage-16 embryos stained for the presence of all CNS axons using Mab BP102. Anterior is up. (A) Wild-type embryo. (B) Overexpression of schizo in all midline cells of a heterozygous slit embryo leads to a fused commissure phenotype. (C) Expression of a dominant negative form of Shibire in all CNS midline cells blocks endocytosis and leads to a schizo-like CNS phenotype. (D) Expression of a dominant negative form of Shibire in all CNS midline cells of a heterozygous slit animal does not impair commissure formation. (E) Expression of a dominant negative Arf6 protein in all CNS midline cells lead to schizo phenocopy.