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A region-specific neurogenesis mode requires migratory progenitors in the Drosophila visual system

Nature Neuroscience volume 18, pages 46–55 (2015)Cite this article

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An Erratum to this article was published on 26 May 2015

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Abstract

Brain areas each generate specific neuron subtypes during development. However, underlying regional variations in neurogenesis strategies and regulatory mechanisms remain poorly understood. In Drosophila, neurons in four optic lobe ganglia originate from two neuroepithelia, the outer (OPC) and inner (IPC) proliferation centers. Using genetic manipulations, we found that one IPC neuroepithelial domain progressively transformed into migratory progenitors that matured into neural stem cells (neuroblasts) in a second domain. Progenitors emerged by an epithelial-mesenchymal transition–like mechanism that required the Snail-family member Escargot and, in subdomains, Decapentaplegic signaling. The proneural factors Lethal of scute and Asense differentially controlled progenitor supply and maturation into neuroblasts. These switched expression from Asense to a third proneural protein, Atonal. Dichaete and Tailless mediated this transition, which was essential for generating two neuron populations at defined positions. We propose that this neurogenesis mode is central for setting up a new proliferative zone to facilitate spatio-temporal matching of neurogenesis and connectivity across ganglia.

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Figure 1: The larval Drosophila optic lobe shows extensive cell streams in the IPC.
Figure 2: Cell streams in the IPC consist of progenitors.
Figure 3: Migratory progenitors arise by EMT and require escargot (esg).
Figure 4: Local Dpp signaling is required for EMT in p-IPC subdomains.
Figure 5: d-IPC progeny are generated in a defined spatio-temporal pattern.
Figure 6: Transcription factor expression patterns in d-IPC Nbs.
Figure 7: L'sc and Ase differentially promote d-IPC neuroblast supply and maturation.
Figure 8: Dichaete acts upstream of Tll to mediate the transition from Ase+ to Ato+, Dac+ Nbs.

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Change history

  • 23 December 2014

    In the version of this article initially published, there were misworded sentences in the abstract and introduction and formatting errors in the genotypes in the Online Methods. The abstract referred to “neural stem cells and neuroblasts” where it should have read “neural stem cells (neuroblasts).” The fourth paragraph began, “The IPC produces a larval neuron population known as the distal cells, whose neurites in the adult extend into either the medulla and lobula or the medulla and lamina, lobula plate neurons, whose neurites connect the lobula plate with the medulla or lobula, and lobula neurons.” The corrected sentence reads, “The IPC produces three populations: first, a larval neuron population known as the distal cells, whose neurites in the adult extend into either the medulla and lobula or the medulla and lamina; second, lobula plate neurons, whose neurites connect the lobula plate with the medulla or lobula; and third, lobula neurons.” In the Online Methods, first paragraph, the following should have been superscripted: x in brkx47 in all three instances, strII in tkvstrII in item (5) of the second numbered list, 1 in “dac1 from F. Pignoni”, IR KK100642 in UAS-fas3IR KK100642, and IR KK104691 in UAS-l′scIR KK104691. The following should not have been subscripted: sc in UAS-l′scIR TRiP.JF02399. The errors have been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank A. Baena-Lopez (MRC-NIMR), A. Carmena (CSIC University of Alicante), A. Gould (MRC-NIMR), Y.N. Jan (University of California, San Francisco), H. Jäckle (Max Planck Institute), A. Jarman (University of Edinburgh), G.X.S. Jefferis (MRC-LMB), J. Mueller (Max Planck Institute), J. Skeath (Washington University), R. Sousa-Nunes (King's College), E. Piddini (Gurdon institute), F. Pignoni (SUNY Upstate Medical University), J.P. Vincent (MRC-NIMR), U. Walldorf (University of Homburg), the Bloomington Drosophila Stock Center (US National Institutes of Health P40OD018537), the Drosophila Genomics Resource Center, the Vienna Drosophila RNAi Center and the Developmental Studies Hybridoma Bank for fly strains and antibodies. We thank A. Alifandi for help with EdU feeding experiments, A. Bailey and C. Desplan for helpful discussions, and A. Baena-Lopez, F. Guillemot, E. Ober, J.P. Vincent, B. Richier and N. Shimosako for critical reading of the manuscript. This work was supported by the Medical Research Council (U117581332).

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  1. Division of Molecular Neurobiology, MRC National Institute for Medical Research, London, UK

    Holger Apitz & Iris Salecker

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  1. Holger Apitz
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Contributions

H.A. and I.S. conceived the project, H.A. carried out the experiments, and H.A. and I.S. analyzed the data and wrote the manuscript.

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Correspondence to Iris Salecker.

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Integrated supplementary information

Supplementary Figure 1 Expression of escargot (esg) and genetic approach for achieving IPC-specific knockdown.

(a) esgMH766-Gal4 UAS-cd8GFP (green) and esg-lacZB7-2-22 (red) show similar expression patterns. Both transgenes label cells at the p-IPC neuroepithelial margins (arrowheads), as well as migratory progenitors within cell streams (arrows) of late third instar larvae (3L). (b,c) fasciclin 3 (fas3)NP1233-Gal4 drives expression of UAS-cd8GFP (green) in the IPC and its progeny, while ey3.5-Gal80 blocks Gal4 activity in R-cells (arrows, b). This approach efficiently drives expression of UAS-RNAi transgenes, as revealed by the knockdown of fas3 in p-IPC neuroepithlial cells and their progeny (asterisks, red, c). (d) In a EdU pulse-chase experiment, brains of mid third instar larvae (mid 3L) were assessed 4 hours after 2.5 hours EdU feeding. At this stage, p-IPC neuroepithelial cells extensively incorporate EdU (red) and thus are proliferative. dc, distal cells; ln, lamina neurons; lopn, lobula plate neurons; mn, medulla neurons. For genotypes and sample numbers, see Supplementary Table 2. Scale bars represent 50 ÎĽm.

Supplementary Figure 2 Validation of lethal of scute (l’sc) RNAi-mediated knockdown and characterization of phenotypes in the developing IPC.

(a) In control animals, maintained at 18°C, L’sc is expressed in neuroepithelial cells of the OPC and p-IPC (red, arrows). (b,c) Experimental animals were grown at 29°C during the third instar larval stage. IPC-specific expression of two different l’sc RNAi transgenes - l’scIR JF02399 and l’scIR KK104691 - using fasciclin 3 (fas3)NP1233-Gal4 results in efficient knockdown of L’sc expression in the p-IPC (asterisks) without affecting expression in the OPC (arrows). (d-g) IPC-specific expression of l’scIR KK104691 results in identical phenotypes as l’scIR JF02399 (cf. Fig. 7). Compared to controls, Deadpan-positive (Dpn+) neuroblasts (red, arrows, d,e) and Dachshund-positive (Dac+) progeny (red, arrows, f,g), including lobula plate neurons (lopn), are reduced. (h-k) Knockdown of l’sc does not affect esg-lacZ expression (red, h,i), p-IPC morphology (arrow, h,i), and Dichaete expression in cell streams (red, arrows, j,k). Although the d-IPC is reduced in size, Dichaete continues to be expressed (j,k). For genotypes and sample numbers, see Supplementary Table 2. Scale bars represent 50 μm.

Supplementary Figure 3 Validation of Dichaete RNAi-mediated knockdown in the IPC.

(a) In controls, Dichaete (red) is expressed in cell streams (arrow), the d-IPC and medulla neurons (mn). (b,c) IPC-specific expression of two different RNAi transgenes - DichaeteIR KK107194 and DichaeteIR GD49549 – using fasciclin 3 (fas3)NP1233-Gal4 results in efficient knockdown of Dichaete expression in progenitors in cell streams and neuroblasts in the d-IPC (asterisks), without affecting expression in medulla neurons. (d-f) Asense (Ase, red) is not expressed in cell streams (arrows) in control animals (d) and upon Dichaete knockdown (e,f). Both Dichaete RNAi transgenes show identical phenotypes in impairing d-IPC neurogenesis. Discs large (Dlg) immunolabeling is shown in blue. For genotypes and sample numbers, see Supplementary Table 2. Scale bars represent 50 μm.

Supplementary Figure 4 Knockdown of tailless (tll) affects early p-IPC development.

(a,b) Compared to controls (a), IPC-specific expression of a tll RNAi transgene using fasciclin 3 (fas3)NP1233-Gal4 strongly impairs early p-IPC neuroepithelial development, visualized by aPKC labeling at the third instar larval stage (red, b). The p-IPC is absent (asterisk) and the d-IPC is reduced (arrow). (c,d) neuralized (neur)-Gal4 mediated tll RNAi transgene expression results in knockdown of Tll (red) in the d-IPC (asterisk, d) without affecting expression in the lamina (La) and p-IPC (arrows). Low levels of Tll expression remain in the d-IPC. Discs large (Dlg) immunolabeling is shown in blue. For genotypes and sample numbers, see Supplementary Table 2. Scale bars represent 50 ÎĽm.

Supplementary Figure 5 Model illustrating two neuroblast competence stages in the d-IPC.

Migratory progenitors mature into neuroblasts (Nb) in the d-IPC, where they transition through two competence stages that are defined by the sequential expression of Asense (Ase) and Atonal (Ato)/Dachshund (Dac). These give rise to Twin of Eyeless-positive (Toy+) distal cells (dc) and Dachshund-positive (Dac+) lobula plate neurons (lopn), respectively. Dichaete (D) acts upstream of tailless (tll) to promote the transition between the two neuroblast stages. Genetic manipulations indicate that Dichaete is required to induce tll. tll is required to suppress Dichaete and ase, and to induce ato and dac.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 and Supplementary Tables 1 and 2 (PDF 4643 kb)

Supplementary Methods Checklist (PDF 382 kb)

3D model of proliferation zones and cell streams in the optic lobe.

To generate the model of a third instar larval optic lobe, the following areas were reconstructed using escargot (esg)-Gal4MH766, UAS-cd8GFP, Asense and E-cadherin as markers: Opc neuroepithelium (purple), lamina (light grey), medulla neuroblasts and ganglion mother cells (Gmc) (light purple), p-Ipc neuroepithelium (light green), progenitor cell streams (yellow green), d-Ipc neuroblasts and Gmcs (green), as well as s-Ipc derived lobula clusters (lo1 and lo2) including neuroblasts, Gmcs, postmitotic neurons and extending neurite tracts (grey). For labels see Fig. 1l. (MOV 10741 kb)

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Apitz, H., Salecker, I. A region-specific neurogenesis mode requires migratory progenitors in the Drosophila visual system. Nat Neurosci 18, 46–55 (2015). https://doi.org/10.1038/nn.3896

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