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First published online 24 September 2003
doi: 10.1242/dev.00772

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Retrograde Gbb signaling through the Bmp type 2 receptor Wishful Thinking regulates systemic FMRFa expression in Drosophila

Guillermo Marqués1,{dagger}, Theodore E. Haerry1,§, M. Lisa Crotty1, Mingshan Xue2, Bing Zhang2 and Michael B. O'Connor1,¶

1 Department of Genetics, Cell Biology and Development, Howard Hughes Medical Institute, University of Minnesota, Minneapolis, MN55455, USA
2 The University of Texas at Austin, Section of Neurobiology Institute for Cellular and Molecular Biology, 227 Patterson Laboratories, Austin, TX78712, USA



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  		Fig. 1. Mutations in wit eliminate systemic expression of FMRFa. (A) Wild-type staining of global FMRFamide expression in crawling third instar larval brain and ventral ganglia as revealed by an anti-FMRFamide antibody. A DIC image is overlaid onto the fluorescent image produced by an anti-rabbit Alexa 568-coupled secondary antibody. Two of the three dorsal neurohemal organs (NHO) are identified by white arrows. Three of the six bilaterally symmetric Tv neurons that innervate the NHO are marked with yellow arrows, while the two subesophageal neurons (SE2) are highlighted by light blue arrows. (B) wit mutant showing loss of FMRFamide staining in the NHO and Tv neurons. Note that expression in the SE2 neurons is unaffected. In C-F, expression of a FMRFa/lacZ transgene (Benveniste and Taghert, 1999Go) is illustrated in wild-type (C,E) or wit mutant animals (D,F). (G,H) {tau}lacZ labeling of the NHO of wit mutants (H: w; P{UAS>{tau}lacZ, w+}/P{ap>Gal4, w+}; witA12, st/witB11, st) or control animals (G: w; P{UAS>{tau}lacZ, w+}/P{ap>Gal4, w+}; wit, st/TM6B) showing that the Tv neurons properly innervate the NHO. Particular cells and structures are marked as above. Scale bars: in A 100 µm for A-D; in E 100 µm for E,F; in G, 20 µm for G,H.

 


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  		Fig. 2. Expression of Wit in Tv neurons but not the NHO is able to rescue FMRFa expression. Rescue of a wit null heteroallelic animal by elav>Gal4 (A, w; P{UAS>wit, w+}/P{elav>Gal4, w+}; witA12, st/witB11, st) or C929>Gal4 (B, w; P{UAS>wit, w+}/P{C929>Gal4, w+}; witA12, st/witB11, st) driving UAS>wit. Note rescue of FMRFa expression in the NHO and Tv neurons. (A,B) The third pair of Tv neurons is not in the plane of focus. (C-F) Expression of nuclear lacZ using the 929>Gal4 driver is shown in green, while FaRP is in red. (C) Focused on the NHO. Note the lack of lacZ staining in the NHO. (D) Tv neurons showing FMRFa expression (E) The same plane of focus as in D showing nuclear lacZ. Yellow arrows identify Tv neuron nuclei. (F) The merged image showing that the C929>Gal4 driver expresses in Tv neurons. Scale bars: in A 100 µm for A,B; panel C 30 µm; panel D (for D-F) 50 µm.

 


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  		Fig. 3. Cell-autonomous rescue of FMRFamide expression by Wit. Larvae of the genotype w; P{UAS>witGFP, w+}/P{OK6>Gal4, w+}; witA12, st/witB11, st were stained for FMRFamide and Wit (GFP). z-stacks are shown. (A-C) Dorsal view with anterior on top, showing the chimeric rescue of FMRFamide expression in Tv neurons. The three (out of six possible) FMRFamide-positive Tv neurons are marked by yellow arrows (A). Note the predominantly motoneuron GFP staining that reports driver expression. (D-F) High magnification view of the frame in B. Note co-expression (yellow in E) of WitGFP (F) and FMRFamide (D). (B,E) Merged images. Scale bar: 100 µm in A-C; 20 µm in D-F. Dorsal view, anterior upwards.

 


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  		Fig. 4. Mutations in wit do not affect apterous expression or Tv neuron viability. Third instar larval brains are shown with GFP in green and anti-FMRFamide in red. FMRFamide staining in Tv and subesophageal neurons is indicated by yellow and blue arrows, respectively. (A-F) w; P{aP>Gal4, w+}/{UAS>GFP, w+}; wit/TM6B. (D-F) Higher magnification of the area framed in B. (G-L) w; P{aP>Gal4, w+}/{UAS>GFP, w+}; witA12/witB11 animals. (J) High-magnification view of the area framed in G. (K,L) High-magnification views of Ap-positive clusters in two other mutant animals. Scale bar: 100 µm for A-C,G-I; 20 µm for D-F,J-L.

 


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  		Fig. 5. Bmp signals are received by Tv neurons. (A) A single plane of focus showing P-Mad accumulation in subsets of neurons in the thoracic region of the ventral ganglion. Two Tv neurons are highlighted with yellow arrows. (B) The same image as in A except FMRFa expression is overlaid to highlight the two Tv neuron cell bodies. (C) A compressed stack of optical images through the thoracic region of the ventral ganglion showing all P-Mad accumulating cells. Most of these are motoneurons as revealed by double staining with other markers (data not shown). (D) An equivalent stack to that in C but through a witB11/witA12 mutant animal. Note the absence of P-Mad accumulation in all cells of the ventral ganglion. (E) FMRFa expression in a sax3/saxDf Hr–1 mutant animal. (F) Rescue of FMRFa expression in the Tv neurons (yellow arrows) and the NHO (white arrows) of witA12/witB11 animals ubiquitously expressing the WitEC/TkvIC and TkvEC/WitIC pair of chimeric receptors. The schematic above F depicts, from left to right, the situation in wild-type animals in which a heterodimer of Wit (black) and Tkv (red) binds Gbb (blue) resulting in activation of the pathway (red arrow); wit mutants in which there is no ligand binding nor pathway activation; and wit mutants supplemented with the reciprocal chimeras. In this case, the intracellular domains of Wit and Tkv are brought together by Gbb binding to the extracellular domains of the receptors, resulting in pathway activation analogous to the wild-type situation. See Feng et al. (Feng et al., 1995Go) for more details. (G) FMRFa/lacZ expression in the brain and ventral ganglia of a baboP1164 homozygous larva. Scale bars: in A, 50 µm for A-D; in E, 50 µm for E-G.

 


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  		Fig. 6. Gbb is required for FMRF expression. (A) in situ hybridization of third instar brain and ventral ganglia with a gbb probe. The arrows indicate the NHO. (B-E) FMRF expression in (B) wild-type, (C) gbb1/gbb4 hypomorph and (D) gbb1/gbb2 null mutant animals. (E) Rescue of FaRP expression in a gbb-null background by expression of Gbb using the neuron specific elav>Gal4 driver (w; P{UAS>gbb, w+}, gbb1/gbb2; P{elav>Gal4, w+}/+), (F) Lack of FaRP rescue in a gbb null background using the muscle specific G14>Gal4 driver to express Gbb (w; P{UAS>gbb, w+}, gbb1/gbb2, P{G14>Gal4, w+}). Scale bars: in A, 50 µm for A; in B, 100 µm for B-F Anterior towards the left, dorsal views.

 


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  		Fig. 7. Expression of Gbb in the NHO rescues FMRFa expression in Tv neurons. Nuclear lacZ accumulation from a UAS nuclacZ driven by 24B>Gal4 is shown in green, while FaRP expression is in red. The 24B driver is not expressed in the Tv neurons (A-C) but it is expressed in the NHO cells (D-F). (G) Rescue of FaRP expression in Tv neurons (yellow arrows) using the 24B>Gal4 driver to drive UAS>gbb (w; P{UAS>gbb, w+}, gbb1/gbb2; P{24B>Gal4, w+}/+). Dorsal view in all panels. (A-F) Anterior upwards; (G) anterior towards the left. Scale bars: in A, 50 µm for A-F; in G, 50 µm for G.

 


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  		Fig. 9. Expression of FMRFa in neuroendocrine cells enables wit mutants to eclose without rescuing the primary defects in synaptic transmission or synapse morphology. (A) Wild-type Oregon R. (B-D) Partial rescue of wit adults in which FMRFa is expressed in neuroendocrine cells using the C929 driver (w; P{UAS>FMRFa, w+}/P{C929>Gal4, w+}; witA12, st/witB11, st). (B) Partial eclosion, (C) full eclosion but no wing inflation, and (D) full eclosion and full wing inflation. (E) Lack of wing inflation in adult expressing {Delta}Gl in neuroendocrine cells. (F) Wild-type synapse at muscles 6/7 visualized by anti-Csp staining. (G) Small synapse at muscles 6/7 of witA12/witB11 animals expressing FMRFa with the C929 driver. (H) Representative traces of evoked junctional potentials of wild type, wit mutant and wit mutant expressing FMRFa. (I) Histogram representation of 5 recordings. Scale bar: 20 µm.

 


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  		Fig. 8. Disruption of the Dynein motor complex selectively interferes with FMRFa expression in Tv neurons. (A) FaRP expression in third instar larval brain and ventral ganglia in which {Delta}Gl is produced in neuroendocrine cells using the C929 driver. Note lack of staining in the NHO and Tv neurons, SE2 unaffected (light-blue arrows). (B) Expression of {Delta}Gl in the NHO using the 24B driver does not interfere with FaRP staining in Tv neurons (yellow arrows) or NHO (white arrow). (C) FaRP staining in animals expressing p50/Dynamitin using the C929 driver. P-Mad accumulation in the midgut of Y45>Gal4; UAS-{Delta}Gl (D) and wild-type embryos (E). P-Mad accumulation in future amnioserosa cells of da>Gal4; UAS-{Delta}Gl (F) and wild-type embryos (G). Scale bars: in A, 50 µm for A-C; in D, 200 µm for D-G. Anterior leftwards in all panels. (A-C,F,G) Dorsal views; (D,E) lateral views.

 

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© The Company of Biologists Ltd 2003