First published online 2 January 2008
doi: 10.1242/dev.010876
Development 135, 513-521 (2008)
Published by The Company of Biologists 2008
Drosophila Activin-β and the Activin-like product Dawdle function redundantly to regulate proliferation in the larval brain
Changqi C. Zhu1,
Jason Q. Boone2,
Philip A. Jensen1,
Scott Hanna3,
Lynn Podemski3,
John Locke3,
Chris Q. Doe2,4 and
Michael B. O'Connor1,5,*
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.
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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.
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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.
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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.
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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.
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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.
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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.
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