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First published online 2 January 2008
doi: 10.1242/dev.010876

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Drosophila Activin-β and the Activin-like product Dawdle function redundantly to regulate proliferation in the larval brain

¹ Department of Genetics, Cell Biology and Development, Howard Hughes Medical Institute, University of Minnesota, 6-160 Jackson Hall, Minneapolis, MN 55455, USA.
² Institute of Neuroscience, Howard Hughes Medical Institute, University of Oregon, Eugene, OR 97403, USA.
³ Department of Biological Sciences, University of Alberta, Alberta, T6G 2E9, Canada.
⁴ Howard Hughes Medical Institute, University of Oregon, Eugene OR 97403, USA.
⁵ Howard Hughes Medical Institute, University of Minnesota, 6-160 Jackson Hall, Minneapolis, MN 55455, USA.

View larger version (76K): [in this window] [in a new window]   Fig. 1. babo mutants exhibit a severe defect in photoreceptor axon targeting. (A) Wild-type yw photoreceptor axon projections in a late third-instar Drosophila larva are highlighted by staining with antibody 24B10. The growth cones of R1-R6 form a neural plexus (arrowhead) at the lamina. R7 and R8 axons project to the medulla with individual growth cones forming a lattice-like array (arrow). Structures of individual growth cones of R7/R8 are illustrated with higher magnification in the inset. (B) An early white prepupa of babo32/52 mutant (same magnification as A) showing a smaller brain lobe, reduced lamina plexus (arrowhead), abnormal R7/R8 photoreceptor axon projections (arrow) and bundled growth cones (inset). (C) A late third-instar Drosophila Smad2 (dSmad2mb388) mutant larva displaying photoreceptor axon (green, 24B10) targeting defects similar to those of babo mutants. Glia cells are stained by an anti-Repo antibody (red), and Dachshund antibody labeled the lamina neuron precursor cells (green, arrowhead) in the brain lobe and photoreceptor precursor cells in eye discs (also green, arrowhead). (D) A wild-type day 3 pupa showed normal turning (arrow) of R7/R8 axons (stained with 24B10) between lamina and medulla and a very well spaced array of R7/R8 axons in the medulla. (E) A day 3 babo32/52 pupa showing lack of turning (arrow) and highly disorganized photoreceptor axons. (F) A schematic graph shows Drosophila central nervous system of a late third-instar larva with eye disc. Most images in this paper are horizontal confocal optic sections unless otherwise stated. br, brain lobes; ed, eye disc; la, lamina; md, medulla; os, optic stalk.

View larger version (126K): [in this window] [in a new window]   Fig. 2. Characterization of the optic lobe phenotypes of Drosophila babo mutants. (A) A wild-type yw white prepupa showed a large cap structure of lamina neurons (arrowheads) and lamina neuron precursor cells in the lamina cartridge (arrow). (B) The strongest babo32/52 mutants have a very reduced number of lamina cap (arrowheads) and cartridge neurons (arrows), as revealed by Dachshund antibody (green) and Elav antibody (red) staining. (C) A wild-type yw wandering third-instar larva stained for Robo (green) and Elav (red). The arrowheads point to the lamina cap neurons, whereas the arrow points to the medulla neuropil (bracket of white dots). (D) A babo32/52 mutant displayed a small lamina cap (arrowheads) and an aberrant medulla neuropil (arrow and white dots). (E) Normal distribution of glial cells labeled by repo antibody (green) in a brain lobe of a wild-type white prepupa. (F) A brain lobe of a babo32/52 white prepupa, showing a reduced number of glial cells at both the lamina and medulla. (G,H) N-Cadherin (red) and 24B10 (photoreceptors green) staining of the optic lobe region from a yw white prepupae (G) and a babo26/32 mutant (H). (I,J) The same images as G and H but red channel (N-Cadherin) only. Arrows mark photoreceptors and arrowheads the medulla neuropil. Note that overall intensity of N-Cadherin is not changed in either the photoreceptors or medulla neuropil, but the medulla neuropil is much smaller. la-g, glial cells at lamina; me-g, glial cells at medulla.

View larger version (123K): [in this window] [in a new window]   Fig. 3. Babo is required in the developing larval brain lobes but not in eye discs for normal photoreceptor axon targeting in Drosophila. Ubiquitous expression (da>Gal4) of UAS-babob (A) but not the baboa (B) isoform rescues the photoreceptor axon targeting and small brain phenotype of babo26/52 mutants. (C) No rescue of photoreceptor axon targeting phenotypes of babo26/32 mutants by the expression UAS-baboa+b in eye discs (ey>Gal4 driver) or in glial cells (D, repo>Gal4). (E) babo52 homozygous mutant photoreceptor clones (GFP-negative) from an eye disc induced by ey>Gal4-UAS-Flp showed normal axon projections (red, anti-24B10) into a babo52 heterozygous brain lobe. Anti-Elav antibody labeled differentiated neurons (magenta). (F) Expression of the 1407 Gal4 driver in the brain lobe is highlighted in green (anti-β-gal) and neurons in red (anti-Elav). Note the lack of lacZ staining in the eye disc and prominent staining of central brain neuroblasts (arrowhead) and the OPCs. (G) Expression of both UAS-baboa+b by the 1407 driver rescued the babo26/32 mutant phenotype. Photoreceptor axons are in green (anti-24B10) and glia are in red (anti-Repo). (H) Expression of nuclear-GFP with the Wor>Gal4 driver is specific to neuroblasts and GMCs (arrowheads). Neurons are stained with Elav. Note the absence of GFP in the eye disc. The smaller OPC and IPC neuroblasts are not evident in this picture. (For additional images of Wor-Gal4 expression, see Fig. S3A-D in the supplementary material.) (I) Expression of both UAS-baboa+b by the wor>Gal4 rescues the babo26/32 mutant phenotype. Photoreceptor axons are in green (anti-24B10) and neurons in red (anti-Elav). ed, eye disc; la, lamina.

View larger version (88K): [in this window] [in a new window]   Fig. 4. Characterization of brain lobe size and proliferation rate of neuroblasts in Drosophila babo mutant larvae. (A) Brain lobe size as a function of larval stage. Larvae were dissected in PBS, mounted without coverslips, and brain lobe diameter was measured using a calibrated reticule. P-value is from Student's t-test. Error bars are standard deviation. (B,C) White prepupa (wpp) brain lobes of either wild type (B) or babo32/52 mutant (C) were labeled by anti-Dachshund (red), anti-Miranda (green) and anti-Scribbled (blue) antibodies. (B) One half wild-type wpp optic lobe; anterior is up, posterior is down, lateral is right and medial is left. Scrib outlines all cell cortices in the wpp optic lobe; Mir marks medial neuroblasts of the optic lobe; Dach marks LPCs (arrow) and central plug progenitor cells from the IPC (arrowhead). (C) Three-quarters of a much smaller babo32/52 wpp optic lobe. Much of the optic lobe remains primitive neuroepithelial cells indicative of a younger optic lobe [Scrib+, Mira- and Deadpan (Dpn), data not shown]. Dach marks the first progenitors to be born from the IPC (arrowhead). (D) Quantification of the average number of medial optic lobe (OL) neuroblasts (Nbs) per optic section of wandering third-instar larva brain (11 sections on left and right lobes for a total of 22 sections). On average, about 8-10 Miranda-positive optic lobe neuroblasts are seen per inner optic section of wild-type control brain lobes, whereas babo mutants have only about 4 Miranda-positive optic lobe neuroblasts per section. (E-G) MARCM clonal analysis of wild-type clones (arrows in E) or a babo mutant clone (arrow in F) 48 hours after heat shock. Brain lobes were stained by anti-Elav antibody (red). Mutant or wild-type clones are marked by GFP expression. The number of cells in well-defined clones within the optic lobes were counted and the quantification is shown (G). (H-J) MARCM clonal analysis of the proliferation rates of wild-type central larval brain neuroblasts (arrows in H) or a babo9 mutant central brain neuroblast clone (arrow in I) 48 hours after heat shock. Larval brain lobes were stained with anti-Prospero antibody (red) and anti-Elav antibody (blue). (J) Quantification of cell numbers derived from individual central brain neuroblasts.

View larger version (93K): [in this window] [in a new window]   Fig. 5. babo mutants display cell cycle progression defects. S-phase cells (arrows) within the developing optic lobes of a wandering third-instar yw Drosophila larva (A) or babo32/52 mutant (B) or Drosophila Smad2 (dSmad2mb388) (C) mutant were labeled with BrdU (green). M-phase cells in optic lobes of wandering third-instar larvae of wild type yw (D) and babo32/52 mutant (E) and Smad2mb388 mutant (F) revealed by staining with phosphorylated histone H3 antibody (green, p-H3). Differentiated neurons are labeled by anti-Elav staining (A-F, magenta or red). Photoreceptor axons in red are labeled by 24B10 antibody (D,E). (G) Comparison of the ratio of M-phase cells versus S-phase cells in the optic lobes of yw, babo32/52 mutant and Smad2mb388 mutants. BrdU-positive cells or p-H3 positive cells were counted from three individual and distinct optic sections at roughly the same plane of each optic lobe of the three genotypes. The ratio of p-H3 positive cells versus BrdU-positive cells was calculated for each genotype and compared. Note that both babo32/52 and Smad2mb388 mutants showed a much reduced ratio of p-H3 positive cells to BrdU-positive cells compared with that of yw control. (H) Normal expression level of Cyclin A protein is seen in both IPC and OPC of an optic lobe of a yw third-instar larva. (I) High level Cyclin A protein is present at the optic center of a babo32/52 mutant third-instar larva in both IPC and OPC stained and photographed with the same setting as wild type. (J-L) Elevated Cyclin A protein (red, arrowheads in K) is detected in a GFP-negative babo52 mutant clone (arrowheads in J) induced by heat shock from the optic lobe of babo52/+ heterozygote larva brain. (L) Merged image of J and K. (M) Heterozygosity for Cyclin A rescues babo32/52 photoreceptor axon targeting and lamina neuron phenotypes. (N) Anti-active Caspase-3 antibody staining (red) of babo52 mutant GFP-negative clones (arrowheads) in an otherwise babo52 heterozygous GFP-positive developing larval brain lobe. Caspase-3-positive apoptotic cells are indicated by arrows. (O,P) Apoptotic cells (arrows) identified by anti-active Caspase-3 antibody staining (red) of a yw wandering third-instar brain lobe and eye disc (O) compared to a babo26/32 mutant brain (P). Note that there is no overall increase in the number of apoptoic cells in the babo mutant tissue. br, brain lobe; ed, eye disc.

View larger version (83K): [in this window] [in a new window]   Fig. 6. Activin and Activin-related (daw) genes are expressed in developing larval optic lobes and required for normal optic lobe development and correct photoreceptor axon targeting in Drosophila. (A,B) In situ hybridization of wild-type yw mid-third-instar larval brain lobes with antisense probes of activin-β (A) and daw (B). The transcripts of actβ are abundantly expressed in both larval optic lobe and the central brain lobe, whereas daw gene is also expressed in the larval optic lobe (arrow) in addition to its known expression in glial cells. (C) A daw promoter-enhancer Gal4 transgene drives the expression of a uasGFP reporter in glia cells (green and yellow) in the developing larval brain lobes. Glial cells were stained by an anti-Repo antibody (red). (D) The majority (95%) of homozygous dawex32 wandering third-instar larvae do not show abnormal optic lobe development or photoreceptor axon targeting defects. (E) A minority (5%) of daw32 homozygotes show optic lobe and axon targeting defects reminiscent of babo mutants. (F) Optic lobes developed normally and photoreceptor axons target correctly in actβed80 mutant larvae. (G) Double mutants of the genotype dawex11:actβed80 exhibit altered R7 and R8 growth cone bundling and morphology. (H) The dawex32;actβed80 double mutants showed significantly enhanced penetrance of the strong optic lobe phenotype. Central bl, central brain lobe.