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Formation of neuroblasts in the embryonic central nervous system of Drosophila melanogaster is controlled by SoxNeuro

¹ MRC Centre for Developmental Neurobiology, King’s College London, Guy’s Campus, New Hunts House, London SE1 1UL, UK
² Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609, Republic of Singapore

View larger version (90K): [in a new window]   Fig. 1. Mutations in SoxN result in the loss of neurons/GMCs and cause multiple morphological defects. (A-D) Immunostaining with anti-Eve antibody. Dorsal view of (A) wild type and (B) SoxN dissected stage 16 embryos. Arrows indicate the RP2 neuron (A) or the RP2 neuron position (B); arrowheads mark the EL neuron cluster (A) or the position of the EL neuron cluster (B). Ventral view of (C) wild-type and (D) SoxN whole-mount embryos. Arrows indicate GMC4-2a (C) or the GMC4-2a position (D). (E) Wild-type and (F) SoxN stage 11 embryos stained with anti-Ftz antibody. Brackets encompass one hemisegment each. Note the drastic loss of neurons/GMCs in intermediate and lateral regions of the CNS in F. (G,H) Ventral view of the cuticle of first instar larva, (G) wild-type and (H) SoxN. Arrowheads indicate a denticle belt. Note the reduction of the denticle belts along the AP axis in H. (I,J) Lateral view of stage17 whole-mount embryos. (I) Wild type and (J) SoxN. Note the defects in head and gut formation in J. Anterior is towards the left.

View larger version (74K): [in a new window]   Fig. 2. SoxN mutation leads to the loss of lateral and intermediate neuroblasts. (A-E) Immunostaining with anti-Wor antibody. (A) Wild-type and (B) SoxN stage 9 embryos; (A',B') higher magnification of two consecutive segments of A,B. The broken line indicates the ventral midline; (v) ventral, (i) intermediate and (l) lateral column of NBs. The arrowhead marks NB5-3 (A') or the position of NB5-3 (B'); the asterisk indicates the position of NB3-5. (C) Quantification of the SI NB phenotype: the percentages of samples showing loss of each NB are given. Note that the loss of NBs predominantly occurs in the intermediate and lateral column. (D) Wild-type and (E) SoxN stage 11 embryos. Note the drastic loss of NBs in E. (F) Wild-type and (G) SoxN stage 11 embryos stained with anti-Eagle antibody. Note the complete absence of Eagle-positive NBs in G. (H) Wild-type and (I) SoxN stage 11 embryos stained with anti-ß-gal to detect hkb-lacZ (5953) expression (black) and anti-Engrailed (brown) to facilitate the identification of positions along the AP axis. (I) Note the strong reduction of hkb-lacZ expression in the intermediate and lateral regions of the neuroectoderm. (H',I') Higher magnification of one hemisegment of H,I. Note the loss of NB2-4 in row 2 and the complete absence of hkb-lacZ expression in row 4. Ventral views with anterior towards the left.

View larger version (15K): [in a new window]   Fig. 3. The C463 allele contains an internal deletion. The C463 allele was found to have an internal deletion of 311 bp (position 1373-1684), which introduces a frame-shift. The deduced C463 234 amino acid polypeptide shares the first 215 amino acids with wild-type SoxN protein followed by 19 amino acids of novel sequence.

View larger version (80K): [in a new window]   Fig. 4. SoxN RNA and protein expression pattern. (A-D) RNA in situ with a SoxN-specific probe. (E-J) Immunostaining with anti-SoxN antibody. (A,E) Stage 8: SoxN is expressed in the entire neuroectoderm with exception of the ventral midline. (B,F) Late stage 10: the staining has become metameric. (C,G) Stage 12: SoxN is expressed in ectodermal stripes that extend laterally. Strong expression is seen in the ventral midline. (D,H) Ectodermal stripes extend the entire circumference of the embryos. SoxN is expressed in a subset of CNS and PNS cells. (I) At stage10, SoxN levels are high in neuroectodermal cells and low in delaminated NBs. (H) Stage 10: SoxN protein distribution in the neuroectoderm is ubiquitous but not uniform. Ventral views with anterior towards the left are shown except I, which is a lateral view.

View larger version (114K): [in a new window]   Fig. 5. SoxN and Dichaete both contribute to the formation of ventral S1 neuroblasts. (A-D) Whole-mount stage 9 embryos stained with anti-Wor antibody. Each panel shows one segment. Anterior is upwards. The bracket encompasses the ventral NBs. v, ventral; i, intermediate; l, lateral. (A) Wild type; (B) Dichaete87; (C) SoxNGA1192; and (D) Dichaete87; SoxNGA1192

View larger version (97K): [in a new window]   Fig. 6. SoxN genetically interacts with vnd and ind. (A-D) Whole-mount stage 9 embryos stained with anti-Wor antibody. Each panel shows one segment. Anterior is upwards; the broken line indicates the midline. The arrowhead indicates the position of NB5-3 in B,D; the arrow indicates the position of NB1-1 in B,C. v, ventral; i, intermediate. (A) Wild-type; (B) SoxNC2139; (C) vnd /+; SoxNC2139; and (D) SoxNC2139;ind/+. The percentages of samples showing loss of each NB are given; 50 hemisegments were counted for each NB.

View larger version (86K): [in a new window]   Fig. 7. SoxN is required for the upregulation of Ac expression and the initiation of Ase expression. (A) Wild-type and (B) SoxN whole-mount early stage 8 embryos stained with anti-L’sc antibody. (C) Wild-type and (D) SoxN dissected late stage 8 embryos stained with anti-Ac antibody. Note the slight reduction of Ac expression in lateral clusters. (E) Wild-type and (F) SoxN dissected late stage 9 embryos stained with anti-Ac antibody. Note the strong reduction of Ac expression in the lateral column. (G) Wild-type and (H) SoxN whole-mount stage 9 embryos stained with anti-Ase antibody. Anterior is towards the left.

View larger version (86K): [in a new window]   Fig. 8. SoxN does not act to antagonize productive Notch signaling. (A-D) Immunostaining of stage 9 embryos with anti-Wor antibody. Anterior is upwards. Each panel shows the SI NB pattern of one segment. Anterior is upwards. The arrow indicates one NB in the 3-5 position in wild type (A), multiple NBs in the 3-5 position in the E(spl) mutant (B), and the lack of a NB in the 3-5 position in SoxN (C) and SoxN/E(spl) double mutant (D).