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doi: 10.1242/10.1242/dev.00438

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An urbilaterian origin of the tripartite brain: developmental genetic insights from Drosophila

Frank Hirth1,*,{dagger}, Lars Kammermeier1,*, Erich Frei2, Uwe Walldorf3, Markus Noll2 and Heinrich Reichert1

1 Institute of Zoology, Biozentrum/Pharmazentrum, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland
2 Institute for Molecular Biology, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland
3 Department of Anatomy and Cell Biology, University of Saarland, Building 61, D-66421 Homburg/Saar, Germany



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  		Fig. 1. Expression of engrailed (en) delimits the neuromeric subdivisions of the embryonic brain and ventral nerve cord. Laser confocal microscopy of stage 13/14 wild-type embryos, reconstructions of optical sections. (A,C) Frontal views; (B,D) lateral views. The bracket in B indicates the extend of optical sections used to reconstruct the frontal views of A,C. (A,B) Immunolabelling with a neurone-specific anti-HRP antibody (red). (C,D) Double-immunolabelling with anti-HRP (red) and anti-EN (green, yellow). The embryonic brain can be subdivided into the protocerebrum (PC or b1), deutocerebrum (DC or b2) and tritocerebrum (TC or b3) of the supraesophageal ganglion and the mandibular (S1), maxillary (S2) and labial (S3) neuromeres of the sub-oesophageal ganglion. en-expressing cells delimit the posterior boundaries of the PC (en-b1), DC (en-b2) and TC (en-b3), and of the mandibular (en-S1), maxillary (en-S2) and labial (en-S3) neuromeres.

 


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  		Fig. 2. Whole-mount in situ hybridization of Pax2 transcripts in wild-type embryos. (A,B,B') Lateral views, (C,D,D') dorsal views; anterior is towards the left. (A) By stage 9/10, Pax2 expression is detectable in a segmentally reiterated pattern of the developing procephalic and ventral neuroectoderm, with its anteriormost expression domain located at the future deutocerebral-tritocerebral boundary (arrow). (B-D) At stage 14, Pax2 transcripts are most prominent in a longitudinal stripe at the medial part of the protocerebrum (arrows) and in a transversal stripe (arrowheads) at the posterior border of the deutocerebrum. Asterisks in C,D indicate Pax2 expression in cells associated with the developing hypopharyngeal organ outside the CNS (see also Fig. 3A). (B') Simplified summary scheme of B with brain-specific Pax2 expression domains (grey) shown in relation to protocerebral and deutocerebral neuromere boundaries (unbroken lines). (D') Simplified summary scheme of D with brain-specific Pax2 expression domains (grey) shown in relation to protocerebral and deutocerebral neuromere boundaries (unbroken lines).

 


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  		Fig. 3. Pox neuro (Poxn) and Pax2 are expressed in distinct domains in the embryonic brain and ventral nerve cord. Laser confocal microscopy of stage 13/14 embryos, reconstructions of optical sections. (A-I) P{lacZ}Pax2 {Delta}122/+. (A,D,G) Frontal views, (B,E,H) lateral views, (C,F,I) ventral views. The bracket in B indicates the extend of optical sections used to reconstruct the frontal views of A,D,G, the dotted line demarcates the deutocerebral/tritocerebral neuromere boundary. (A-C) Double-immunolabelling with anti-HRP (red) and anti-ß-gal (green, yellow). (D-F) Double-immunolabelling with anti-HRP (red) and anti-POXN (blue). (G-I) Triple-immunolabelling with anti-HRP (red), anti-ß-gal (green, yellow) and anti-POXN (blue, white); arrows in G,H indicate Pax2 and Poxn expression at the same anteroposterior position along the neuraxis in the posterior deutocerebrum where they define a transversal domain. This transversal domain of adjacent Pax2/5/8 orthologue expression is located anterior to the deutocerebral/tritocerebral neuromere boundary. Note that expression of Pax2 and Poxn are never observed in the same cells. The apparent co-expression of Pax2 and Poxn shown in G (white) is an artefact caused by superimposed optical sections. Asterisks in A indicate Pax2 expression in cells associated with the developing hypopharyngeal organ outside the CNS.

 


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  		Fig. 4. otd and unpg expression domains in the embryonic brain and ventral nerve cord. Laser confocal microscopy of stage 13/14 embryos, reconstructions of optical sections. (A-C) Wild type, (D-I) P{lacZ}unpgf85/+. (A,D,G) Frontal views, (B,E,H) lateral views, (C,F,I) dorsal views. The brackets in B,E,H indicate the extend of optical sections used to reconstruct the frontal views of A,D,G, respectively. (A-C) Double-immunolabelling with anti-HRP (red) and anti-OTD (green, yellow). (D-F) Double-immunolabelling with anti-HRP (red) and anti-ß-gal (green, yellow). (G-I) Double-immunolabelling with anti-OTD (red) and anti-ß-gal (green, yellow); arrows indicate interface of otd and unpg expression at the anterior border of the deutocerebral-tritocerebral boundary (DTB) region. Note that a direct interface of the otd and unpg expression domains only occurs on the ventral (according to neuraxis) side of the brain; on the opposing side, a small gap between the two expression domains is seen (H, arrow).

 


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  		Fig. 5. Brain-specific expression of otd, unpg, en and Poxn. Laser confocal microscopy of stage 13/14 embryos, reconstructions of optical sections, frontal views. The extend of optical sections used to reconstruct the frontal views is more dorsal (according to neuraxis) than for Fig. 4A,D,G. (A,B) Wild type, (C,D) P{lacZ}unpgf85/+. (A) Double-immunolabelling with anti-OTD (red) and anti-EN (green, yellow). (B) Double-immunolabelling with anti-OTD (red) and anti-POXN (green, yellow). (C) Double-immunolabelling with anti-ß-gal (red) and anti-EN (green, yellow). (D) Double-immunolabelling with anti-ß-gal (red) and anti-POXN (green, yellow). Arrowheads indicate en-b1 stripes, arrows indicate en-b2 stripes. Asterisks in A-C indicate non-neuronal expression domains of OTD and EN in the foregut.

 


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  		Fig. 8. Tripartite organization of the Drosophila, mouse and ascidian brain, based on expression patterns of orthologous genes. The expression of otd/Otx2, unpg/Gbx2, Pax2/5/8 and Hox1 gene orthologues in the developing CNS of (A) stage 13/14 Drosophila embryo, (B) stage E10 mouse embryo (Wurst and Bally-Cuif, 2001Go) and (C) neurula ascidian embryo (Wada et al., 1998Go). In all cases, a Pax2/5/8-expressing domain is located between an anterior otd/Otx2-expressing region and a posterior Hox-expressing region in the embryonic brain. (Note that in Drosophila, the Pax2/5/8 orthologues Pax2 and Poxn also show a segmentally reiterated expression pattern as outlined with striped red boxes in A.) Moreover, in Drosophila, as in mouse, a Pax2/5/8-expressing domain is positioned at the interface between the otd/Otx2 expression domain and a posteriorly abutting unplugged/Gbx2 expression domain. This otd/Otx2-unpg/Gbx2 interface displays similar developmental genetic features in both Drosophila and mouse (see text for details).

 


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  		Fig. 6. Altered expression patterns of unpg, en and Poxn in brains of otd–/– embryos. Laser confocal microscopy of stage 13/14 embryos, reconstructions of optical sections. (A) Frontal view, (B,C) lateral views. (A,B) otdJA101; P{3'lacZ}unpgr37/+, (C) otdJA101. The extend of optical sections used to reconstruct the frontal view in A is the same as in Fig. 3B. Broken rectangles in the summary schemes indicate the region of the embryonic brain shown at higher magnification in B,C. (A,B) Triple-immunolabelling with anti-HRP (red), anti-ß-gal (green, yellow) and anti-EN (blue, white). Arrows indicate the anterior shift of brain-specific unpg expression into the anterior deutocerebrum (compare with Fig. 4D,E). Note that the protocerebrum is missing in otd mutants (Hirth et al., 1995Go), resulting in the absence of en-b1-expressing cells, whereas en-b2-expressing cells delimiting the deuto-tritocerebral boundary are present (A,B, arrowheads). (C) Double-immunolabelling with anti-HRP (red) and anti-POXN (green, yellow). Arrow indicates the normal position of the posterior POXN stripe in the posterior deutocerebrum, which is absent in the otd-null mutant embryo. PC, protocerebrum; DC, deutocerebrum; TC, tritocerebrum; Sub, sub-oesophageal ganglion; VNC, ventral nerve cord.

 


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  		Fig. 7. Altered expression patterns of otd and lab in brains of unpg–/– embryos. Laser confocal microscopy of stage 13/14 embryos, reconstructions of optical sections. (A,C,D) Frontal views, (B,E,F) lateral views. (A-D,F) P{3'lacZ}unpgr37, (E) P{3'lacZ}unpgr37/+. The extend of optical sections used to reconstruct the frontal views in A,C,D is the same as in Fig. 3B; this results in increased visualization of gut-specific otd and en expression. Broken rectangles in the summary schemes indicate the region of the embryonic brain shown as magnified view in B,F. (A,B) Double-immunolabelling with anti-OTD (red) and anti-ß-gal (green, yellow). Arrows indicate the posterior shift of brain-specific otd expression into the anteriormost unpg expression domain. (C) Double-immunolabelling with anti-OTD (red) and anti-EN (green, yellow). Arrows indicate the posterior shift of brain-specific otd expression up to the deutocerebral en-b2 stripe into the posterior deutocerebrum (compare with Fig. 5A). Broken line indicates non-neuronal expression domains of OTD and EN in the foregut. (D) Double-immunolabelling with anti-OTD (red) and anti-POXN (green, yellow). Arrows indicate that the posterior border of brain-specific otd expression extends posteriorly up to the Poxn expression domain of the DTB (compare with Fig. 5B). Broken line indicates non-neuronal expression domains of OTD and POXN in the foregut. (E,F) Double-immunolabelling with anti-LAB (red) and anti-ß-gal (green, yellow) in unpg+/– versus unpg–/– embryos; the broken line demarcates the tritocerebral/mandibular neuromere boundary. Arrowheads indicate the anterior shift of lab expression from the posterior tritocerebrum (E) (Hirth et al., 1998Go) into the anterior tritocerebrum (F). PC, protocerebrum; DC, deutocerebrum; TC, tritocerebrum; Sub, sub-oesophageal ganglion; VNC, ventral nerve cord.

 




© The Company of Biologists Ltd 2003