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Evidence for differential and redundant function of the Sox genes Dichaete and SoxN during CNS development in Drosophila

Paul M. Overton1,*, Lisa A. Meadows2,*,{dagger}, Joachim Urban2 and Steven Russell1,{ddagger}

1 Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, UK
2 Institute of Genetics, University of Mainz, Becherweg 32, D-55099, Mainz, Germany
* These authors contributed equally to this work
{dagger} Present address: Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, UK



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  		Fig. 1. Nervous system defects in SoxNeuro mutants. Flat preparations (A-E,G,H) or a whole-mount (F) of stage 16 (A,B,E-H) and stage 11 (C,D) wild-type (A,C,E,G) and SoxNeuroU6–35 (B,D,F,H) embryos stained with anti-Eagle (blue) and anti-Engrailed (brown) (A,B), anti-Eagle (blue) and anti-Ems (brown) (C,D), monoclonal antibody mAbBP102 (E,F) and anti-Fasciclin II (G,H). (A,B) In wild-type embryos, Eagle staining is observed in progeny of the NB2-4, NB3-3, NB7-3 and thoracic NB6-4 lineages. In SoxNeuroU6–35 embryos, no Eagle staining is seen in these lineages. Eagle expression is still seen in cells in the gnathal midline. (C,D) Ems-expressing progeny of the NB3-5 and NB4-4 and NB3-3 lineages are absent in more than 96% of hemisegments in SoxNeuroU6–35 embryos. Note that in embryos in which one of these cells is observed, we are unable to unambiguously identify which of the three neuroblasts is present. Tracheal Ems expression is still present. (E,F) In SoxNeuroU6–35 embryos, longitudinal BP102 staining is absent in 60% of hemisegments (arrowheads in F); in addition commissures fail to separate correctly in 52% of hemisegments. (G,H) In SoxNeuroU6–35 embryos, the regular axonal fasciculation pattern is disrupted and many axons cross the midline inappropriately (arrow).

 


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  		Fig. 2. Lateral neuroblast lineages are absent in SoxNeuro mutants. Flat preparations of late stage 8 embryos stained with anti-Hunchback and anti-Engrailed. (A) Wild-type, Hunchback-expressing neuroblasts form an orthogonal array of four neuroblasts per hemisegment in each of the medial and lateral (large white arrowhead) columns, with a single cell in the intermediate column. (B) Lateral column neuroblasts are lost in between 20% and 70% of hemisegments in SoxNeuroU6–35 mutant embryos (large white arrowhead), for example, NB3-5 (black arrowhead). Medial column neuroblasts are less frequently affected; NB1-1 (white arrow) and NB7-1 (white arrowhead) are present in over 88% of hemisegments. (C,D) Flat preparations of stage 16 embryos stained with anti-Even skipped. (C) Wild-type and (D) SoxNeuroU6–35. The NB4-2 lineage RP2 motorneurones (arrows) and the NB3-3 lineage lateral cluster (arrowheads) are absent in SoxNeuroU6–35; however, aCC/pCC and CQ cells are unaffected. (E) Stage 16 whole-mount SoxNeuroU6–35 embryo stained with anti-Even skipped (black) and anti-Engrailed (brown). The spacing within the CNS is greatly increased between segments A3 and A4 and reduced between A2 and A3; notice that both Eve- and En-expressing neurones are no longer aligned with Engrailed expression in the epidermis (arrow). (F) Stage 16 whole-mount SoxNU6–35 /SoxNU6–35; KrGAL4/UASSoxN embryo. Within the domain of KrGAL4 expression, RP2 motorneurones (arrow) and ELC cells (arrowhead) are present in 67% and 33% of hemisegments, respectively.

 


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  		Fig. 3. Lateral Achaete expression is lost in SoxNeuro mutant embryos. Late stage 8 (A,B) and stage 9 (C,D) whole-mount views of wild-type (A,C) and SoxNeuroU6–35 (B,D) embryos stained with anti-Achaete. In wild-type embryos, Achaete is expressed in proneural clusters of five to seven cells that give rise to the medial MP2 and NB7-1 and the lateral NB3-5 and NB7-4 neuroblasts. In SoxNeuroU6–35 embryos, Achaete protein is undetectable laterally in 70% of rows (white arrowhead); however, Achaete is still observed in 80% of rows medially (white arrow); asterisk in D shows absence of Achaete in one NB7-1 neuroblast. (E) Achaete expression is greatly reduced in both medial and lateral columns in SoxNeuroU6–35. The SoxNU6–35/SoxNU6–35 embryo to the right shows a much lower level of Achaete expression than its SoxNU6–35/Cyo sibling of the same stage from the same staining reaction.

 


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  		Fig. 4. SoxNeuro; Dichaete double mutant embryos are far more severely affected than either single mutant. Whole-mount ventral views of stage 16 (A-D), stage 8 (E) and stage 9 (F) SoxNeuroU6–35; Dr72 double mutant embryos stained with BP102 (A,B), anti-Eve (C,D), anti-Achaete (E,F) and anti-Hb (H,I). (A,B) Double mutant embryos show a variable severe hypoplasia; longitudinal axons are almost totally absent and there are frequent gaps in the neuropil. (C,D) As in SoxNeuroU6–35, RP2 and ELC staining is variably absent in double mutant embryos; in addition, we see a loss of aCC/pCC and CQ cells in 15% of hemisegments (arrowheads). The defects in D are much more extreme than can be accounted for by the Dichaete segmentation phenotype, suggesting that many segments fail to express Eve in any cells. (E,F) Achaete protein is absent laterally in SoxNeuro; Dichaete embryos as in SoxNeuroU6–35; however, Achaete is now undetectable in 54% of medial clusters (arrowheads); Achaete staining is completely absent in 21% of segments (arrowheads). (G) Diagrammatic representation of S1 neuroblasts at late stage 8. Neuroblasts are arranged in an orthogonal array of four rows and three columns [medial (M), intermediate (I) and lateral (L)]. (H,I) Late stage 8 D72 (H) and SoxNU6–35; Dr72 (I) embryos. S1 neuroblasts are barely affected in Dichaete mutant embryos [e.g. NB5-3 (arrowhead) is missing in 2% of hemisegments]. As in SoxNU6–35, lateral NBs are frequently absent in double mutant embryos; in addition, within the intermediate column we observe Hb-expressing cells in only 21% of hemisegments compared with 48% in SoxN mutants (arrowheads). In I, we are unable to unambiguously identify these cells as NB 5-3.

 


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  		Fig. 5. Dichaete expression is variably reduced in SoxNeuroU6–35 mutant embryos. Whole-mount ventral (A,C) and dorsal (B,D) views of stage 9 wild-type (A,B) and SoxNeuroU6–35 (C,D) embryos stained with anti-Dichaete. In half of mutant embryos, Dichaete expression is reduced in the anterior region of the neuroectoderm but appears normal posteriorly. Note that expression of D in the midline and brain is unaffected.

 

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