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First published online February 18, 2004
doi: 10.1242/10.1242/dev.00962

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robo2 and robo3 interact with eagle to regulate serotonergic neuron differentiation

Jessica A. Couch, John Chen^*, Heather I. Rieff^*,, Ellen M. Uri and Barry G. Condron

Department of Biology, 071 Gilmer Hall, University of Virginia, Charlottesville, VA 22903, USA

View larger version (73K): [in a new window]   Fig. 5. robo2 mutants lose eagle expression. Stage 16 wild-type (A), egMz360 hypomorph (B), robo2 loss-of-function (C) and (EP2582/EP2582; egMz360/egMz360) (D) nerve cords. Serotonin uptake, red; Eg, green; yellow, co-localization. M indicates the position of the ventral midline. (A) The wild-type nerve cord has two serotonergic neurons per hemisegment, both positive for SerT activity and for the transcription factor Eagle. (B,C) An egMz360 and a robo2 mutant show a loss of SerT activity in a percentage of hemisegments. In the egMz360 (B) CNS, all Eg staining is absent although some SerT remains, while in the robo2 mutant (C), Eg is lost only in those cells that lack SerT activity. (D) When two copies of robo2 (EP2582, Robo gain of function++) are overexpressed in an egMz360 (hypomorph) background, both Eg expression and SerT activity are rescued despite the fact that expression of robo2 prevents axons from crossing the midline. Scale bar: 5 µm.

View larger version (153K): [in a new window]   Fig. 1. The onset of SerT expression is correlated with axon guidance across the midline. (A,B) Staining for serotonin uptake in red and ß-galactosidase in green (UAS-tau-lacZ driven by egGal4). egGal4 expression of lacZ is seen in all four progeny of NB 7-3, which includes the serotonergic neurons, two other neurons and one pair of lateral neurons that arise from a different lineage. (A) SerT activity was never seen before growth cones crossed the midline in stage 14 ventral nerve cords (10:20-11:20 hours development), but (B) always seen after axons reached the contralateral neuropil at late stage 15 (11:20-13:00 hours). (C) In situ hybridization for SerT mRNA shows robust expression by late stage 15. Staining for the axon scaffold of the ventral nerve cord is shown in brown with mAb BP102. Scale bar: 10 µm.

View larger version (95K): [in a new window]   Fig. 2. Initial screen of midline axon guidance mutants did not reveal a loss of SerT expression. (A-F) stage 16 ventral nerve cords are stained for serotonin uptake in red (A-D) or in white (E,F). (A-D) show the axon scaffold stained with mAb BP102 in green. (A) The wild-type CNS is organized into two longitudinal axon tracts connected by two commissures per segment. Serotonergic axons are seen crossing in the posterior commissure. Loss-of-function mutants (B) commissurelesse39, (C) slit2and (D) roboGA285 were identified based on CNS phenotype. In comm mutant embryos, the commissures do not form, while in slit mutants, axons fail to leave the midline and in robo mutants serotonergic axons are disorganized as a result of ectopic midline crossing throughout the CNS. (E,F) eagleGal4 drives expression of (E) robo (UAS-robo) and (F) robo2 (EP2582) in the serotonergic neurons. A gain-of-function in robo2, but not robo, prevents the crossing of the midline by serotonergic neurons. Despite defects in serotonergic neuronal axon guidance seen in these embryos, SerT activity remains normal. Scale bar: 10 µm.

View larger version (97K): [in a new window]   Fig. 4. robo2 and robo3 are required for normal SerT expression. (A-C) Stage 16 ventral nerve cords from robo2x123 (A,B) and robo31 (C) loss-of-function mutant embryos stained for serotonin uptake in red (A,C) and white (B). The slightly disorganized axon tracts of the mutant CNS are labeled in green with mAb BP102. robo2 and robo3 loss-of-function nerve cords show a random loss of SerT activity in approximately half of hemisegments. (B) SerT activity is lost in both a single hemisegment (arrow) and an entire segment (arrowhead). (D) In situ hybridization for SerT mRNA in a stage 16 robo2 mutant indicates a loss of SerT transcription in the same pattern as the loss of SerT activity. Mutant nerve cords were identified by axonal morphology or, in the case of D, irregular width. (E,F) Serotonin uptake is shown in red and eagle-lacZ is shown in green in wild type (E) and a robo2x123 mutant (F). (E) In a wild-type cord, both serotonergic neurons in each hemisegment express eg and therefore stain for lacZ. (F) Although robo2 mutants lack SerT activity in many neurons, lacZ expression remains normal. Serotonergic cell bodies positive for lacZ but not SerT activity are highlighted in the insets below (asterisk). Scale bar: 20 µm for D-F; 10 µm for A-C.

View larger version (45K): [in a new window]   Fig. 3. Physical or genetic disruption of midline contact affects SerT activity in wild type, Robo gain-of-function and Unc5 gain-of-function (but not Robo2 gain-of-function) mutants. (A-C) Staining of ventral nerve cords for serotonin uptake (red in left panel and white in right) and for engrailed (green in left panel). A cut (line of stars) was made in isolated stage 16 cords asymmetrically down the midline such that one set of cell bodies was separated from the midline. The tissue preparations were allowed to develop for 2 hours prior to fixation. (A) In 15 out of 20 wild-type preparations, SerT activity was lost from the cell bodies on the cut side (arrow). (B) Similar cuts in embryos overexpressing Robo in serotonergic neurons (Robo gain of function; UAS-robo with eagleGal4) also showed a loss of SerT activity in the midline-minus side (arrow; three out of three). (C) However, when Robo2 was overexpressed (eagleGal4), only one out of 20 preparations showed a loss of SerT activity after a midline cut. (D) Expression of Unc5 with eagleGal4 specifically prevents the crossing of the midline by serotonergic neurons and also causes a partial loss of SerT activity as seen by staining for serotonin uptake (red/white), but does not affect whole CNS axon guidance (BP102, green). Arrows (left panel) indicate hemisegments where SerT expression is lost and yellow stars (right panel) indicate axons that extends towards the midline despite expression of Unc5. Scale bar: 10 µm.

View larger version (17K): [in a new window]   Fig. 7. Average loss of SerT activity between genotypes. Error bars represent the standard deviation of the mean. Genotypes were scored for the percent of hemisegments negative for SerT activity/total hemisegments per VNC. n represents the number of total hemisegments scored. At least 10 cords were averaged for each genotype. Wild-type cords have 0% loss of SerT. (a,b) P<0.001 (One-way ANOVA, Tukey comparison), showing that SerT activity is rescued by (a) Eg gain of function in an robo2 loss of function and (b) Robo2 gain of function in an eg hypomorph. No significant difference (ns, P>0.05) was found between robo2 loss of function and the double robo2 loss of function; egMz360 mutants. robo2 loss of function score represents data from both robo2x123 and robo2x135 alleles, as phenotypes were indistinguishable.

View larger version (60K): [in a new window]   Fig. 6. Overexpression of Robo2 rescues an eagle hypomorphic mutation and overexpression of Eg rescues a robo2 loss of function. (A) Wild-type stage 16 nerve cord stained for serotonin uptake showing two serotonergic cell bodies per hemisegment with axons extending across the midline. (B) Embryos homozygous for the hypomorphic egMz360 allele (also an eagleGal4) show 30% loss of SerT-positive cells as well as axon guidance defects. (C) Expression of one copy of Robo2 (EP2582, Robo gain of function) with one copy of egGal4 (heterozygous egMz360) prevents axons from crossing the midline but does not disrupt SerT activity. (D) Expression of Robo2 with two copies of the hypomorphic egGal4 allele (homozygous egMz360) disrupts axon guidance, but also causes a loss of SerT as seen in B. (E) However, expression of two copies of Robo2 (Robo2 gain of function++) with two copies of egGal4 (homozygous egMz360) rescues the loss of SerT phenotype. (F) Conversely, expression of Eg (egGal4) rescues both SerT activity and the serotonergic axon guidance phenotype observed in robo2 mutants (compare with Fig. 4B). Scale bar: 10 µm.

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