bifocal and pp1 interaction regulates targeting of the r-cell growth cone in drosophila

lsevier.com/locate/ydbio

Developmental Biology 2

Bifocal and PP1 interaction regulates targeting of the R-cell growth

cone in Drosophila

Kavita Babu a,*,1, Sami Bahri b, Luke Alphey c, William Chia a,2

a Temasek Life Science Laboratory and Department of Biological Sciences, 1 Research Link, National University of Singapore, 117604, Singaporeb Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos 138673, Singapore

c Department of Zoology, Oxford University, South Parks Road, Oxford, UK

Received for publication 16 February 2005, revised 7 September 2005, accepted 10 September 2005

Available online 8 November 2005

Abstract

Bifocal is a putative cytoskeletal regulator and a Protein phosphatase-1 (PP1) interacting protein that mediates normal photoreceptor

morphology in Drosophila. We show here that Bif and PP1-87B as well as their ability to interact with each other are required for photoreceptor

growth cone targeting in the larval visual system. Single mutants for bif or PP1-87B show defects in axonal projections in which the axons of the

outer photoreceptors bypass the lamina, where they normally terminate. The data show that the functions of bif and PP1-87B in either stabilizing

R-cell morphology (for Bif) or regulating the cell cycle (for PP1-87B) can be uncoupled from their function in visual axon targeting. Interestingly,

the axon targeting phenotypes are observed in both PP1-87B mutants and PP1-87B overexpression studies, suggesting that an optimal PP1

activity may be required for normal axon targeting. bif mutants also display strong genetic interactions with receptor tyrosine phosphatases,

dptp10d and dptp69d, and biochemical studies demonstrate that Bif interacts directly with F-actin in vitro. We propose that, as a downstream

component of axon signaling pathways, Bif regulates PP1 activity, and both proteins influence cytoskeleton dynamics in the growth cone of R

cells to allow proper axon targeting.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Bifocal; Protein phosphatase-1 (PP1); Axon guidance; Photoreceptors; Optic lobe; Cytoskeleton; F-actin

Introduction

Precise axon targeting is achieved by integrating signaling

events involving guidance receptors and their downstream

components (Tessier-Lavigne and Goodman, 1996). Some of

the molecules involved in axonal guidance in the Drosophila

embryo also play a role in retinotopic axonal targeting,

suggesting that a similar set of molecules governs axon guidance

and normal topographic mapping of the axons in the embryo and

the fly visual system. These include Dock (Desai et al., 1999;

Garrity et al., 1996; Hing et al., 1999; Rao and Zipursky, 1998),

Trio (Awasaki et al., 2000; Bateman et al., 2000; Luo, 2000;

0012-1606/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.ydbio.2005.09.017

* Corresponding author.

E-mail address: [email protected] (K. Babu).1 Present address: Department of Molecular Biology, Massachusetts General

Hospital, Richard B. Simches Research Building, 185 Cambridge Street, CPZN

7250, Boston, MA 02114, USA.2 Requests for reagents should be made to [email protected].

Newsome et al., 2000b), Lar (Clandinin et al., 2001; Krueger et

al., 1996; Maurel-Zaffran et al., 2001), Cadherins (Iwai et al.,

1997, 2002; Lee et al., 2001), Ephrins and their receptors

(Bossing and Brand, 2002; Dearborn et al., 2002; Palmer and

Klein, 2003; Schmucker and Zipursky, 2001; Yu and Bargmann,

2001), Protocadherins (Lee et al., 2003; Senti et al., 2003) and

Dptp69D (Desai and Purdy, 2003; Desai et al., 1996; Garrity et

al., 1999; Newsome et al., 2000a; Sun et al., 2000).

The Drosophila eye has 750–800 repeats of a basic unit

called an ommatidium and comprised of 8 photoreceptor cells

(R1–R8). Overlying these photoreceptor cells are four cone

cells and two primary pigment cells surrounding the cone cells.

The eight axons from the photoreceptors of an ommatidial unit

in the eye travel via the optic stalk into the optic lobe region of

the brain as part of an ommatidium-specific fascicle with the

axons of R1–R7 surrounding the R8 axon (Hanson, 1993).

Incoming R-cell axons induce the production and differentia-

tion of glia in the optic lobe lamina (Huang and Kunes, 1998;

Selleck and Steller, 1991; Winberg et al., 1992). Lamina

88 (2005) 372 – 386

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K. Babu et al. / Developmental Biology 288 (2005) 372–386 373

differentiation by R-cell-derived signals plays a crucial role in

matching the number of afferents to their targets. Different R

cells in the eye disc extend axons that terminate at different

levels in the optic lobe. The 6 outer photoreceptors, R1–R6, of

each ommatidium terminate their axons at the outer layer of the

optic lobe or the lamina, while the inner two photoreceptors R7

and R8 send their axons beyond the lamina into the second

optic ganglion, the medulla (Hanson, 1993). The growth cone

from an initial R8 neuron can be thought to pioneer the path to

retinotopic target destination and enters the optic lobe first,

while the growth cones of R2 and R5, R3 and R4, R1 and R6

and R7 follow in sequence along the expanding ommatidial

fiber. This process of axonal targeting is apparent by the third-

instar larval stage of Drosophila development, such that at this

stage a very precise pattern of connectivity is apparent, with R1

to R6 axons ending at the lamina and R7 and R8 axons entering

the medulla and sending their axons into two distinct

neuropiles in the medulla (Kunes and Steller, 1993; reviewed

by Kunes, 1999; Kunes et al., 1993).

Targeting errors for R-cell axons have been described for

several mutations including those of the receptor tyrosine

phosphatase receptors such as dlar, dptp69d and off-track

(Cafferty et al., 2004; Clandinin et al., 2001; Garrity et al., 1999).

Downstream components of the phosphotyrosine signaling

system, of which dock is a component, also play important

roles in axon guidance in the visual system (Garrity et al., 1996).

Targeting defects in these mutant backgrounds include irregular

and uneven lamina layer. In addition, R1–R6 axons that

normally synapse at the lamina can bypass the lamina and enter

the medulla or stop at the wrong points in the lamina or medulla

(Clandinin et al., 2001; Desai et al., 1999; Garrity et al., 1996).

Here, we show that Bifocal and PP1-87B are components of a

genetic axon guidance pathway in the visual system involving

the phosphatase receptors, Dptp69D and Dptp10D.

The Bifocal protein is expressed in neuronal cells in both the

embryonic nervous system and the larval visual system of the

fly (Bahri et al., 1997; Helps et al., 2001). Although Bif is

expressed on embryonic CNS axons, bif mutant embryos do

not show any obvious defects in axon guidance. However, Bif

has been shown to be required for the normal shape of the

R-cell rhabdomeres in the fly eye (Bahri et al., 1997) and for

anchoring of proteins to the posterior of the oocyte along with

the F-actin-binding protein, Homer (Babu et al., 2004).

In the visual system, we have previously shown that Bif is

one of the regulators of phosphatase activity, and it directly

interacts with PP1-87B via its consensus phosphatase-binding

motif, RVQF (Helps et al., 2001). We have also shown that the

Bif–PP1-87B complex is required for normal photoreceptor

rhabdomere morphogenesis (Helps et al., 2001). Furthermore,

Ruan et al. (2002) have shown that the loss of bif function can

give rise to axon guidance phenotypes in the larval optic lobe

and that in this system bif interacts with misshapen (msn), a

gene which encodes for a protein kinase that regulates normal

photoreceptor axon targeting (Ruan et al., 2002).

Although Bif has been shown to function in R-cell

guidance, several questions remained unanswered. For exam-

ples: (1) are the guidance defects an indirect consequence of a

lack of Bif function in rhabdomere morphogenesis? (2) Is

binding to PP1 essential for Bif’s axon guidance role? (3) Does

Bif form a complex with actin directly or indirectly? Here, we

demonstrate that the axon guidance defects in bif mutants are

not mere consequences of its effects on cell morphology. We

also show that the PP1-binding site of Bif is essential for its

function and that PP187B itself is also required for proper axon

guidance. We further demonstrate a dominant genetic interac-

tion between bif and PP1-87B, indicating that these two

molecules may function in the same signaling pathway during

normal photoreceptor axon targeting. Moreover, bif genetically

interacts with the receptor tyrosine phosphatases Dptp10D and

Dptp69D in the larval optic lobe, and it can directly bind to

F-actin, suggesting that Bif may contribute to the propagation

of signaling via these receptors to the actin cytoskeleton during

the process of axon guidance.

Materials and methods

Genetics

The protein phosphatase-1 mutants used in this study, pp1-87Bhs46,

pp1-87Be211 and pp1-87Be078, have been previously described by Axton et al.

(1990). We recombined the mutants onto an FRT chromosome and examined

the eye discs of transallelic larvae. These transallelic animals developed fairly

normal eye discs and were used for further examinations. The transgenic fly

lines carrying PP1 inhibitors and UAS-PP1-87B have been previously

described (Parker et al., 2002).

The bif mutants and bif transgenic lines, UAS-bif + and UAS-bif F995A, used

in this study were previously described by Bahri et al. (1997) and Helps et al.

(2001). The cDNA for the shorter Bif isoform called bif 10DA was cloned into

the pUAST vector, and fly transformants were generated using standard genetic

techniques. Ectopic expression of the various transgenes in the fly eye was

performed using GMR-GAL4 (Hay et al., 1997).

The Rotau-lacZ marker was used to mark R2R5 axons in the larval brain,

and the Rh1-slacZ and Rh4-slacZ markers were used for labeling R1–R6 and

R7 axons respectively in the adult brain (Garrity et al., 1999; Newsome et al.,

2000a). The dptp10d alleles, dptp10d1 and deletion Df(1)59 which deletes both

bif and dptp10d, were previously described (Bahri et al., 1997; Sun et al.,

2000). The dptp69d1 allele and a deletion uncovering dptp69d were obtained

from Bloomington Stock Center, and other mutant lines were obtained either

from Bloomington or as gifts from various laboratories.

Immunohistochemistry and microscopy

Larval eye discs/brain complexes were dissected and fixed in accordance

with previously published protocols with minor modifications (Wolff and

Ready, 1991). Wandering third-instar larval and 55 h pupal eye discs/brains

were dissected in phosphate-buffered saline and fixed in 4% paraformaldehyde

for 30 min on ice. The dissected brains or eye discs were washed, blocked with

BSA or goat serum and stained with primary antibody overnight. After several

washings, secondary antibodies were added, and the specimens were mounted

for microscopic observation. Several primary antibodies were used: rabbit anti-

PP1-87B (1:1500), sheep anti-Bif (1:1000), mAb 24B10 (1:3), rabbit anti-hGal(1:3000 from Cappel), mAb anti-Dac (1:500) or rabbit anti-Repo (1:500).

Either fluorescent-conjugated secondary antibodies (FITC 1:100 dilution or cy3

1:750 dilution) or peroxidase-conjugated secondary antibodies (from Jackson

laboratories used at 1:150 dilution) were used for signal detection. To enhance

the HRP signal in the adult brains, the Vectashield ABC kit was used.

Fluorescent-stained specimens were mounted in Vectashield and visualized

using confocal microscopy. F-actin was visualized with tetramethyl rhodamine

isothiocyanate–phalloidin (Sigma) or Alexa Fluor 546 Phalloidin (Molecular

Probes). Fluorescent signals were detected using confocal microscopy.

Transmission electron microscopy was used to visualize rhabdomeres of the

adult eye as previously described (Helps et al., 2001).

K. Babu et al. / Developmental Biology 288 (2005) 372–386374

Actin co-sedimentation assay

The actin co-sedimentation assay was performed as previously described

(Hohaus et al., 2002) with minor changes. Full-length and different deletions of bif

were cloned into the pGADT7 vector that was used to in vitro translate Bif products.

For making the constructs containing the N-terminal 500 amino acids of Bif, a 500

bp EcoRI/ClaI fragment was isolated from bif cDNA and inserted into the

pGADT7 vector. To make other constructs, different regions of bif were amplified

by PCR, and the PCR products were then digested with EcoRI/XhoI (these sites

were engineered in the PCR products) and cloned into the pGADT7 vector.

The actin co-sedimentation assay was performed using G-actin from Sigma.

The G-actin was allowed to polymerize using salt and ATP, and the in vitro

translated and radiolabeled polypeptides (Bif or luciferase control) were then

added to the polymerized F-actin or the control solution (buffer without F-actin)

and incubated for 60 min at room temperature. The mixtures were then spun at

100 K in an airfuge for 40 min, and both supernatants and pellets were then

boiled in 2� SDS buffer and run on an SDS-PAGE gel. In each experiment, the

samples were run onto two gels, one for autoradiography to detect the labeled

protein and another for Coomassie staining to detect the actin band.

Results

The role of Bif in axon guidance and rhabdomere shape can be

uncoupled

The involvement of Bif in axon guidance of R cells and its

expression and localization in the rhabdomeres and axonal

growth cones of these cells have been previously described

(Bahri et al., 1997; Ruan et al., 2002). The results of these studies

suggested a role for Bif in rhabdomere morphogenesis and a cell

autonomous requirement in the outer R cells for their axons to be

targeted to the lamina. In bif R47 (an antigen-minus allele)

animals, most R2–5 axons bypass the lamina and terminate in

the medulla, clumping of axons as well as breaks in the lamina

surface are visible in these mutants (Figs. 1A–D; Ruan et al.,

2002), but no gross abnormalities in the optic lobe can be

detected (Figs. 3E–J). However, it is not clear how the axonal

defects might relate to the morphogenesis defects in bif mutants.

To uncouple the function of Bif in axon guidance from its

function in rhabdomere morphogenesis, we made use of the two

protein isoforms of Bif that have been previously described (Bahri

et al., 1997;Helps et al., 2001). TheseBif isoforms are generated by

alternative splicing events, with the larger (1196 amino acids) bif +

isoform produced from five exons and the smaller (1063 amino

acids) bif 10Da isoform produced from six exons. We have shown

previously that the bif + isoform driven byGMR-GAL4 rescues the

rhabdomere as well as the actin localization defects associated with

the bif mutant discs (Helps et al., 2001). A similar rescue using the

Fig. 1. The R-cell axonal phenotype of bifocal mutant larvae and rescue experimen

stained with anti-chaoptin antibody 24B10; clumping of axons at the lamina and lam

optic lobe (A; inset in A is another control bif LH114 which does not delete into the

mutant optic lobes. Panels C (control Rotau-lacZ) and D (bif R47/bif R47; Rotau-lacZ

hgal staining; R2–R5 axons terminate at the lamina in the control optic lobe (C; arr

but most of the R2–R5 axons bypass the lamina and terminate at the medulla in the

100% of the mutant optic lobes (n = 17) with 65–75% of axons bypassing the lam

(78%, n = 36) showing rescue of the bif mutant axon clumping phenotype using UA

H are representative images of the majority of preparations (89%, n = 44) showing th

the axon clumping phenotype associated with the bif mutations (arrowheads in pa

phenotype of bif mutants is rescued by UAS-bif + (I) but not UAS-bif 10Da (J; arro

which detects the LacZ expression derived from the Rotau-lacZ marker. Scale bar

bif 10Da isoform of bif was also obtained (Figs. 2A, B, I, J and data

not shown), suggesting that either isoform is sufficient to produce

normal rhabdomere morphology. To ascertain whether these bif

isoforms rescue the axon guidance defects, GMR-GAL4, which is

expressed in all post-mitotic photoreceptor cells of the Drosophila

eye (Hay et al., 1997), was used to drive the expression of UAS-

bif + or UAS-bif 10Da transgenes specifically in the eye disc but not

the optic lobe in bifmutant backgrounds. In these experiments, only

bif + and not bif 10Da was able to rescue the axonal defects of bif

eyes (Figs. 1E, F, I, J). These data revealed differential requirements

for the two bif isoforms, with bif 10Da being largely required for

normal R-cell rhabdomere morphology but not having a predom-

inant role in normal axonal projections onto the optic lobe, while

bif + is involved in both R-cell morphology and axon guidance.

The uncoupling of Bif functions in axon guidance and rhabdomere

morphology also suggested that the axon guidance phenotype of bif

mutants is not merely a secondary effect of the R-cell morpholog-

ical abnormalities. Overexpression of bif 10Da in a wild type

background did not yield any obvious axonal phenotype as

observed with mAb24B10 staining; furthermore, the photoreceptor

rhabdomeres as observed under transmission electron microscopy

were largely normal in shape and organization (Supplementary Fig.

1). It was shown by Ruan et al. (2002) that Bif + overexpression

causes premature stop of the larval axons before they reach the

target lamina. We find that the overexpression of the Bif10Da

isoform does not cause such a premature stop phenotype, and

therefore it is unlikely that the lack of rescue of the bif bypass

phenotype is due to such an effect.

Adult brains of bif mutants were also examined by 24B10

staining (Figs. 2C, D) and markers specific for either R1–R6

projections (Rh1-slacZ; Figs. 2E, F) or R7 projections (Rh4-

slacZ; Figs. 2G, H). Surprisingly, the bif mutant adult brains

showed largely normal arrays of axons, even though the

rhabdomeres of the mutant adult retina have defective morphol-

ogy (Figs. 2A, B; Bahri et al., 1997), indicating that the axonal

phenotypes observed in the mutant larval optic lobe are largely

corrected in the mutant adult brain and further demonstrating

that the axon guidance phenotype and morphological defects of

R cells can be uncoupled in bif mutants.

The PP1-binding site in Bif is required for its function in

photoreceptor axon guidance

We have previously shown that Bif can associate with

PP1-87B in vivo and can directly bind and inhibit PP1-87B

ts. Panels A (control bif R47/+ or wt) and B (bif R47/bif R47) show optic lobes

inal breaks are visible in the mutant (arrowhead in B) as compared to the control

bif coding sequence). This phenotype was observed in 100% (n = 50) of the

) show targeting of the axons of photoreceptors R2–R5 as visualized with anti-

owhead; arrow in panel C shows the Bolwigs nerve which enters the medulla),

mutant (D; arrowhead). The R2–R5 axons bypass phenotype was observed in

ina. Panels E and G are representative images of the majority of preparations

S-bif + driven by GMR-GAL4 and visualized with 24B10 staining. Panels F and

at neither UAS-bif 10Da nor UAS-bif F995A expression under GMR-GAL4 rescues

nels F and H) as visualized with 24B10 staining. The R2–R5 axons bypass

whead) or UAS-bif F995A (K; arrowhead) as visualized with anti-hgal staining,is 20 Am.


activity in vitro. Bif contains a consensus four amino acid

PP1-binding motif, RVQF, towards the C-terminus. This

RVQF motif is present in both Bif + and Bif10Da isoforms

and spans amino acids 992–995. This motif is required for

Bif to bind PP1-87B in vitro and for bif function in vivo,

and a single amino acid mutation in this motif of Bif +,

Fig. 2. bif mutants show normal axon targeting in adult optic lobes. Panels A (wt) and B (bif R47/bif R47) are EM micrographs showing defective rhabdomeres shape

of bif mutants (B) as compared to wt ommatidia (A). Panels C–H show the optic lobes of WT (C, E and G) and bif mutant (D, F and H) adults stained with markers

for all or specific subsets of photoreceptor axons; no obvious defects are observed with 24B10 staining (D; bif mutants as compared to wt in C), with the Rh1-lacZ

R1–R6 axons marker (F; bif mutants as compared to wt in E) or with the Rh4-lacZ R7 axons marker (H; bif mutants as compared to wt in G). Panels I and J are EM

micrographs showing rescue of the adult rhabdomere phenotypes of bif mutants using either UAS-bif + (I) or UAS-bif 10Da (J); rescue of abnormal pattern of

rhabdomere arrangement was observed in 90% of ommatidia preparations (n = 30), and rescue of rhabdomere shape was observed in 81% of rhabdomeres (n = 210).

Compare the rescue images in panels I and J to the bif mutants in panel B and wt in panel A. Scale bar is 500 nm.


Bif F995A, can abolish this interaction (Helps et al., 2001). In

addition, the mutant Bif F995A form cannot rescue the bif R-

cell morphological defects in vivo (Helps et al., 2001). To

test whether an intact PP1-binding site in Bif is also required

for axon guidance in the larval optic lobe, the mutant UAS-

bif F995A was driven by GMR-GAL4 in bif mutant back-

grounds (Figs. 1H, K). When compared with UAS-bif +, very

little rescue of the axonal defects was observed with this

mutant UAS-bif F995A construct as visualized with 24B10

(Figs. 1G–H) and the Rotau-lacZ marker (Figs. 1I, K). This

indicated that the PP1-binding site of Bif is essential for its

function in axon guidance, and it raised the possibility that

PP1 may also play a role in R-cell axon guidance (see

below). Consistent with this view, double heterozygous

larvae (bif/+; pp1-87B/+) showed clumping of axons at the

lamina as well as defects in axon targeting, with R2–5

Fig. 3. Genetic interaction between bif and pp1-87B and Dac and Repo staining in larval brains. Panels A–D show larval optic lobes stained with 24B10 (A, B) or

anti-hgal (C, D). Panels A and C are representative images showing the normal 24B10 staining pattern (A) and the normal R2–R5 axonal pattern (C; as visualized

with the Rotau-lacZ marker) observed in either bif R47/+ or pp1-87B/+ heterozygotes. Transheterozygous bif/+; pp1-87B/+ animals show axon clumping in the

lamina as visualized with 24B10 (B, arrow; phenotype observed in 91.67% of brains, n = 36) and mistargeting of axons to the medulla as visualized with the Rotau-

lacZ marker (D, arrow; phenotype observed in 27–30% of axons, n = 5) is mistargeted into the medulla. Panels E–J are confocal images of anti-Dac (red) and anti-

Repo (green) stainings in wt (E–G) and bif R47 mutant (H–J) optic lobes; no obvious defects are observed with either Dac or Repo staining in bif mutants as

compared to wt. Panels G and J are merged images of panels E and F and H and I respectively. Scale bar is 20 Am.


Fig. 4. Inhibition of PP1 activity causes axonal targeting defects in the larval eye disc. Overexpression of the NIPP1 (A) or the I-2PP1 (B) inhibitors by GMR-GAL4

causes no obvious abnormality in photoreceptor formation as can be visualized with mAb 22C10, but staining with mAb 24B10 reveals laminal breaks and clumping

of axons (C, NIPP1; D, I-2PP1); the axon phenotype caused by NIPP1 overexpression was observed in 100% (n = 20) of preparations, while, for the weaker I-2PP1

inhibitor,¨67% (n = 15) showed the phenotype. Using the Rotau-lacZ axonal marker, a severe defect in R2–R5 targeting is observed with NIPP1 overexpression (E;

¨55% of the axons are mistargeted to the medulla, and this phenotype is observed in 100% of optic lobes, n = 16), and a relatively milder phenotype is observed

with I-2PP1 overexpression (F; ¨5% of the axons are mistargeted to the medulla, and this phenotype is observed in 61% of optic lobes, n = 18). Scale bar is 20 Am.


axons bypassing the lamina and entering the medulla in

these optic lobes (Figs. 3C, D), whereas neither bif nor pp1-

87B single heterozygotes show a phenotype with 24B10 or

the Rotau-lacZ marker (Figs. 3A, B; data not shown). These

data further support the view that Bif and PP1 act together

for normal photoreceptor axon targeting in the larval optic

lobe.

Fig. 5. Axonal defects in pp1 mutants. Two alleles of pp1 (pp1e211 and pp1e078) wer

pp1e078/+ (A; control) and the transheterozygotes pp1e078/pp1hs46 or pp1e211/p

transheterozygous combinations show an uneven lamina as visualized with mAb 24

phenotype (97.2%; n = 36) than pp1e211/pp1hs46 (C). Using the Rotau-lacZ mark

mistargeted into the medulla (panel E is pp1e211/pp1hs46 with 91.6% of preps, n =

showing the phenotype). Panel G is a larval brain overexpressing PP1-87B and show

observed in 100% of optic lobes, n = 28); overexpression of PP1 also gives rise t

phenotype (panel I is a representative image, with 30–35% of the axons bypass the l

defects (panel J is a representative image, with more than 70% of the axons entering t

of R2–R5 axons to the lamina as visualized with the Rotau-lacZ marker. Scale bar

Overexpression of PP1-87B in the eye causes axon guidance

defects

Since Bif can inhibit the activity of PP1 (Helps et al., 2001),

we wondered whether PP1-87B overexpression in wild type

backgrounds might mimic the effects of bif loss-of-function on

axon guidance. To test this, a UAS-PP1-87B construct was

e studied in trans with the strong allele of pp1, pp1hs46. The larval eye discs of

p1hs46 (B) do not show any obvious defects with 22C10 staining. Both

B10 staining with the pp1e078/pp1hs46 (D; arrowhead) showing a more severe

er, both pp1 transheterozygous combinations show ¨50% of R2–R5 axons

24, showing the phenotype; F is pp1e078/pp1hs46 with 100% of preps, n = 17,

ing an uneven lamina (arrowhead) as visualized with 24B10 (this phenotype is

o mistargeted R2–R5 axons with the majority of preps showing mild bypass

amina into the medulla) and the remainder of the preps showing severe targeting

he medulla). Panel H is a control pp1e078/+ optic lobe showing normal targeting

is 20 Am.


driven withGMR-GAL4 in the eye disc, and the optic lobes from

these animals were stained with 24B10. Axonal breaks in the

lamina similar to those observed in bif mutants were observed

(Fig. 5G). The R2–R5 axon-specific marker also showed

mistargeting of these axons into the medulla (Figs. 5I, J).

Inhibition of PP1 activity or mutations in PP1 cause R-cell

axon targeting defects

We wanted to determine whether PP1-87B mutants

themselves exhibit any axonal phenotype in the eye, but it


was not possible to use homozygotes of the available strong

PP1-87B mutant alleles as this gene is required for normal

cell cycle progression in larval brain neuroblasts and in the

eye imaginal disc; hence, most PP1-87B mutants die at

larval or early pupal stages (Axton et al., 1990). More

importantly, these mutants have severely deformed small eye

discs due to cell division defects, making the defective

axonal projections they exhibit difficult to interpret (data not

shown). As a first step in determining PP1 function in axon

guidance, specific PP1 inhibitors, NIPP1 (Bennett et al.,

2003; Beullens et al., 1992; Parker et al., 2002; Van Eynde

et al., 1995) and I-2PP1 (Bennett et al., 1999, 2003; Helps

and Cohen, 1999; Huang and Glinsmann, 1976; reviewed in

Cohen, 2002), were driven post-mitotically in the eye disc

using pGMR-GAL4. The rationale being that by inhibiting

PP1 activity in post-mitotic cells the problems associated

with cell cycle progression should be circumvented, making

it possible to examine the effects of PP1 loss on axonal

targeting. PP1 has been shown to associate with endogenous

inhibitors, such as Inhibitor 2 (I-2) and Nuclear Inhibitor of

PP1 (NIPP1), which may help to prevent inappropriate

dephosphorylation of nonphysiological targets. It has also

been shown that ectopic expression of the inhibitors

specifically reduces PP1 activity in vivo and results in

phenotypes resembling those of PP1c mutants (Bennett et

al., 2003). Upon overexpression of the inhibitors under

GMR-GAL4, the eye disc morphology appeared essentially

normal, but the photoreceptor axons showed defects (Figs.

4A–D). The lamina formed by the axons expressing either

NIPP1 (Fig. 4C) or I-2PP1 (Fig. 4D) was discontinuous

with breaks, and the R2–R5-specific Rotau-lacZ marker

showed axon mistargeting to the medulla mainly with the

stronger NIPP1 inhibitor (Fig. 4E); the weaker I-2PP1

inhibitor showed mild or no mistargeting defect in the optic

lobes as visualized with the R2–R5-specific marker (Fig.

4F). These results suggest that PP1 activity is required for

photoreceptor axon guidance in the fly visual system.

Since NIPP1 and I-2PP1 are general PP1 inhibitors and

not specific for PP1-87B, we next used different allelic

combinations to bypass the early cell cycle defects

associated with the pp1-87B mutations. Three alleles of

pp1-87B, pp1e211, pp1e078 and pp1hs46 (Axton et al., 1990)

were used to examine the pp1-87B phenotype in the eye

disc. Using the strong pp1hs46 allele in trans with the other

2 weaker alleles allowed larval development to proceed; in

these particular transheterozygous combinations, the larval

eye discs formed fairly normally as can be visualized with

22C10 (Figs. 5A, B), presumably because there is sufficient

residual PP1 activity to enable cell divisions to occur at a

near normal level. Staining of the pp1 transheterozygotes

with 24B10 showed axons clumping at the lamina and

laminal breaks (Figs. 5C, D), and some of the R2–R5 axons

in these animals were also mistargeted to the medulla (Figs.

5E, F). Taken together, the axonal defects in the PP1-87B

mutant combinations in which cell cycle defects appear to

be minimal further strengthen the view that PP1-87B is

required for normal photoreceptor axon guidance.

Like Bif, PP1-87B is expressed in R-cell axons and in the optic

lobe

Bif expression and localization in the rhabdomeres and

axonal growth cones have been previously described (Bahri et

al., 1997; Ruan et al., 2002). However, a close examination of

Bif staining revealed widespread expression in the optic lobe

(Figs. 6, 7) which was not previously reported. Double labeling

with anti-Bif and anti-Dachshund (Garrity et al., 1999; Mardon

et al., 1994) showed that Bif localizes in the outer region of the

lamina (Figs. 6A–C), where the photoreceptor axons initially

send their projections into the optic lobe (Kunes et al., 1993).

Bif also showed co-localization with F-actin in the optic lobe

(Figs. 6J–L), similar to what has been reported in the eye disc

(Bahri et al., 1997). In these experiments, Bif staining was

stronger in the optic lobe cortex and weaker in the cortical

axons (Figs. 6M–O). Bifocal staining was weak/absent in the

eye disc and optic lobe of bif R47 mutants (Figs. 6P–R),

indicating that anti-Bif staining in these tissues is specific. On

the other hand, F-actin staining was still visible in these

mutants. Although Bif is expressed in the optic lobe, no

obvious defects were detected in bif mutants with either

Dachshund (Mardon et al., 1994), which marks the proliferat-

ing cells of the lamina, or with Reversed polarity (Halter et al.,

1995), which marks the glia (Figs. 3E–J). This indicated that

the formation of the lamina and glia appears normal in bif

mutants.

Double labeling with anti-Bif and anti PP1-87B antibodies

of wild type preparations indicated that both proteins have

overlapping expression patterns in most regions of the optic

lobe (Figs. 7A–C). Similar to the staining pattern of Bif, anti-

PP1-87B staining in the brain also showed enhanced signals in

the brain cortex and the outer layer of the lamina, while it was

considerably weaker in the axons. In addition, double staining

with Bif and PP1-87B or Bif and phalloidin showed that both

Bif and PP1-87B are expressed in the axons of the optic lobe

(Figs. 7D–I). These experiments indicated that, like Bif, PP1-

87B is expressed in R-cell axons consistent with a role in

targeting of these axons. It should be noted here that the

possibility of the PP1-87B antibody cross-reacting with any of

the other 3 isoforms of PP1 present in Drosophila cannot be

excluded. We carried out staining on pp1 mutants and found

that the mutants still show positive staining; this is most

probably due to the fact that the tissues retain residual PP1-87B

activity because the transheterozygous combination we used to

circumvent cell division defects associated with loss of PP1-

87B is not null for PP1-87B. We therefore cannot rule out the

possibility of cross-reaction of the PP1-87B antibody we used

with different PP1 isoforms.

Bif interacts genetically with receptor tyrosine phosphatases

and can directly bind F-actin

The interaction of Bif with intracellular signaling molecules

like misshapen (Ruan et al., 2002) and PP1 (this work) seems

to be important for normal R-cells axon guidance. In order to

identify additional, more upstream, components, which interact

Fig. 6. Bif expression pattern in the optic lobe. Panels A–O are confocal images of wt third-instar larval optic lobes stained with anti-Bif (green) and various other

markers. Double labeling with anti-Dac (A–C; red) shows Bif expression in the cortex of the optic lobe, and a high level of expression is seen on the outermost

region of the lamina (arrowhead in panel A, merged panel C) where Dac is expressed (B). Double labeling with 24B10 (D–I) shows a high level of Bif expression in

the axons that pass through the optic stalk (arrowhead in D, merged panel F); Bif expression in the axons when they enter the optic lobe becomes reduced when

compared to its expression in the optic stalk (D and G), whereas 24B10 staining is still seen at high levels in the optic lobe axons (H and merged panel I). Double

labeling with phalloidin (J–O) shows high expression for Bif (J) and co-localization with F-actin (K, merged panel L) in the cortex. Expression of Bif in a deeper

plane of focus (M and merged image O) in the optic lobe is less than in the cortex (compare M to J). No expression of Bif is seen in bifR47 mutant optic lobes (P),

while phalloidin staining is still seen in these mutants (Q). Panel R is a merged image of panels P and Q. Scale bar is 20 Am.


with bif, genetic interaction experiments between bif and other

molecules involved in photoreceptor axon guidance were

performed. bif R47/+ individuals are wild type (Fig. 3A) as

are dptp69d/+ or dptp10d/+ (Fig. 8A; data not shown),

however, axonal defects were observed in double transheter-

ozygous optic lobes of bif/dptp10d and bif/+; dptp69d/+

combinations (Figs. 8D, E). This phenotype was also seen in

optic lobes from females heterozygous for a deletion removing

bif and dptp10d (Fig. 8F). There was no phenotype in double

heterozygotes of bif R47 with dock, pak, dlar, ena or abl (Fig.

8B; data not shown). These genetic interaction experiments

suggested that bif preferentially interacts with the upstream

dptp10d and dtptp69d and that bif might be a component of

axon guidance pathways involving these signaling receptors.

We have used the Rotau-lacZ axonal marker on the bif/+,

dptp69d/+ and bif/dptp10d/+ double heterozygotes, but we did

not detect any mistargeting phenotype. This may indicate that

Bif does not cooperate with the receptors to influence the ‘‘stop

decision’’ of growth cones at the lamina. However, our results

with 24B10 staining showed uneven lamina only with the bif

mutant in combination with the 10D and 69D mutants, but we

did not see a similar effect on the lamina when we used genetic

Fig. 7. Bifocal and PP1 are expressed in axons. Panels A–C show co-expression of Bif (A, green) and PP1 (B, red) in the wt optic lobe; panel C is a merged image of

panels A and B. Panels D–F show double labeling of Anti-Bif (D, green) and phalloidin (E, red) in wt optic lobes; note that Bif is expressed in the axons in a manner

similar to F-actin; the arrow in panel E indicates axons stained with phalloidin, and panel F is a merged image of panels D and E. Panels G–I show double staining of

wt optic lobes with phalloidin (G, red) and anti-PP1 (H, green); note PP1 staining in axons (H, arrows). Scale bar is 20 Am.


combinations between bif and several other mutants including

dpak, dock, dlar, ena and abl. Hence, we have concluded that

bif genetically interacts with the receptor phosphatases.

It has previously been shown that Bif from embryonic fly

extracts can be pulled down using F-actin columns (Sisson et

al., 2000), indicating that Bif may be a component of an actin-

associated complex of proteins in vivo. However, the mode of

interaction between Bif and F-actin is not clear since Bif does

not have any obvious actin-binding domains (Bahri et al.,

1997). In order to determine whether the interaction of Bif with

F-actin is direct, a co-sedimentation assay was performed using

F-actin and in vitro translated Bif. In this experiment, Bif was

pulled down by F-actin, indicating that Bif and F-actin can bind

directly to each other (Fig. 9A). Further dissection of the Bif

protein mapped the F-actin-binding domain to 2 novel regions

in the last 400 amino acids of the Bif + protein (schematic in

Fig. 9B). Only one of these domains is shared by both Bif + and

Bif10Da isoforms, while the other actin-binding domain is

specific to Bif + (Fig. 9B). These experiments indicated that

Bif/F-actin complexes may form through direct binding and

that Bif may link various axon guidance signaling pathways to

the actin cytoskeleton.

Discussion

Bifocal is a protein required for normal photoreceptor

rhabdomere morphology in Drosophila (Bahri et al., 1997),

and, in this process, it functions together with PP1 (Helps et al.,

2001). Bif is also a target of the Ser/Thr kinase Misshapen, and,

together, these molecules regulate photoreceptor axon guidance

and targeting during the larval stages (Ruan et al., 2002). In this

report, we have shown that Bif function in R-cell axons can be

uncoupled from its function in maintaining R-cell shape and

morphology and that the PP1-binding site in Bif is required for

its function in axon guidance. In addition, we have shown that

the Bif-interacting partner, PP1-87B, is required for axon

guidance in the visual system and that Bif is likely to function

as a downstream component of signaling pathway(s) that

involves the receptor tyrosine phosphatases, Dptp10D and

Dptp69D. Finally, we have shown that Bif can directly bind F-

actin and thus might provide a link between axon guidance

pathways and the underlying actin cytoskeleton.

The rescue experiments utilizing the two different isoforms of

bif demonstrate that the function of Bif in photoreceptor axon

guidance can be uncoupled from its function in rhabdomere

Fig. 8. Genetic interaction between bif and receptor tyrosine phosphatases. The receptor tyrosine phosphatases dptp69d and dptp10d genetically interact with bif.

Control optic lobes from ptp10d/+ (A) and ptp69d/+ (C) do not show any defects when stained with mAb 24B10. Panel B is a bif/+; pak/+ optic lobe showing

normal axonal pattern as visualized with 24B10 staining. Staining of bif/+; ptp69d/+ optic lobes with mAb 24B10 shows clumps and breaks in the lamina (D,

arrowhead; 84% or preps show a phenotype, n = 37). A similar phenotype is seen when one copy of bif and one copy of ptp10d are simultaneously removed (E,

arrowhead; 85% of preps show a phenotype, n = 34) or in animals heterozygous for a small deletion that removes both bif and ptp10d (F, arrowhead; 89% of preps

show a phenotype, n = 38). Scale bar is 20 Am.


morphology in photoreceptor cells, and this indicates that Bif

has a dual role in the fly visual system, normal axon connectivity

in the larval stages and the formation of normal rhabdomeres in

the adult eye. It should however be noted that the rhabdomere

rescue obtained using both the bif + and the bif10Da isoforms of

bif was not complete, possibly because the expression pattern

and/or level of Bif expression in the rescue experiments are

somehow different from the wild type expression of Bif. The

larger Bif + isoform is 1196 amino acids long, whereas the

shorter Bif 10DA isoform is 1063 and is formed by an alternative

splice site between exon 4 and 5. It is somewhat surprising to

find that the two isoforms of Bif behave differently with respect

to axon guidance since both isoforms contain the PP1 and actin-

binding sites (see Fig. 9). One possible explanation is that the

additional C-terminal sequences present in Bif + are required for

its function in axon guidance. Interestingly, this region

comprises one of the actin-binding domains. The precise role

of these sequences in vivo is not clear at present, but it is possible

to envisage roles for them in protein folding or mediating

binding to other partners. In this regard, one plausible scenario is

that the two Bif isoforms may assume distinct patterns of

subcellular localization due to their different C-termini. It is

possible for future approaches to address this question by

generating specific antibodies that recognize the different

isoforms of Bif or expressing tagged versions of the two Bif

products.

Mutations in the conserved phosphatase-binding motif of

Bif, which abolish binding of Bif to PP1-87B, render the

protein less effective in axon guidance. We have previously

reported similar effects on Bif activity for these mutations in

Fig. 9. Bif binding to F-actin in vitro. Panel A shows a series of gels from co-

sedimentation assays of F-actin with the various radiolabeled Bif + products; the

supernatant fractions in the presence (+As) or absence (�As) of F-actin and the

pellet fractions in the presence (+Ap) or absence (�Ap) of F-actin are indicated

on top. The first row is co-sedimentation of an in vitro translated luciferase

control showing the labeled luciferase remains in the supernatant (+As), and it

does not pellet by F-actin (+Ap). The second row is co-sedimentation of an in

vitro translated full-length Bif + (FL Bif) showing the labeled Bif + protein

precipitated with the F-actin pellet (+Ap); the Bif + band is absent from the pellet

fraction in the absence of F-actin (�Ap). The third row is co-sedimentation of an

in vitro translated N-terminal aa500 of Bif + (N-ter Bif); this N-terminal product

is not pelleted by F-actin (+Ap). The fourth row is co-sedimentation of an in

vitro translated C-terminal aa396 of Bif + (C-ter Bif); the C-terminal product is

pelleted by F-actin (+Ap); The last row is a control gel stained with Commassie

blue to show the F-actin band. Panel B shows schematic diagrams of the two Bif

isoforms, Bif + and Bif 10Da, and the various protein regions used in the F-actin

co-sedimentation assays; (+) indicates binding to F-actin and (�) indicates lack

of binding to F-actin. The first aa1000 are common between Bif + and Bif 10Da,

but the two protein isoforms differ in their C-termini; two broad domains of Bif +

spanning aa800–950 and aa1000–1196 (in red) bind actin, while Bif 10Da

contains only the aa800–950 actin-binding domain; the last aa63 of Bif 10Da

(which are not found in Bif +) do not bind F-actin. The RVQF PP1-binding motif

in Bif + and Bif 10Da is indicated by an arrow.


the morphogenesis of rhabdomeres (Helps et al., 2001).

Another interesting aspect is that Bif can form a complex with

the cytoskeletal F-actin both in vivo and in vitro (Sisson et al.,

2000; this work). Changes in the actin cytoskeleton are known

to be essential for remodeling of the axon growth cone. These

data suggest that, in addition to its inhibitory regulatory effect

on the phosphatase activity, Bif might serve as a direct link

between PP1-87B and the actin cytoskeleton. Similar observa-

tions were reported for neurabins, which are the inhibitors of

PP1 in mammals. It has been shown that neurabins bind actin

and serve as linkers between the actin cytoskeleton and the

plasma membrane at the cadherin-based cell–cell adhesion

sites. Neurabin I is highly concentrated at the synapse of

developed neurons and in the lamellapodia of growth cones

during the development of neurons, suggesting that it could be

required for synapse formation or function (MacMillan et al.,

1999; McAvoy et al., 1999; Oliver et al., 2002; Terry-Lorenzo

et al., 2002). Although it does not share any sequence

homology with mammalian neurabins, Bif could be a

functional homologue of neurabins in the Drosophila larval

visual system by associating with PP1-87B and the actin

cytoskeleton. Such functions of Bif might consequently affect

the phosphorylation status of various actin-binding proteins

like myosin II (reviewed by Tan et al., 1992) and cofilin

(reviewed by Lawler, 1999), which are known to undergo

phosphorylation and dephosphorylation.

A role for PP1 itself in axonal connectivity in the larval optic

lobe is supported by two observations: firstly, expressing PP1

inhibitors in post-mitotic cells in the eye imaginal disc results in

axon defects; secondly, transallelic combinations of pp1-87B

mutants, which are able to bypass the earlier defects of cell cycle

arrest that are usually seen in homozygotes of strong pp1-87B

mutants, reveal axon targeting defects in the larval lamina.

Although the PP1 inhibitors are not specific to PP1-87B, they

nevertheless do support the notion that the phosphatase activity

of PP1 is required for proper axon guidance. In the transallelic

combinations of pp1 mutations, formation of the R cells of the

larval eye disc is not adversely affected, suggesting that the axon

guidance phenotype of pp1-87B mutants is not simply due to the

lack of cells in the developing eye disc. Interestingly, both pp1-

87B mutants as well as PP1 overexpression give rise to axons

bypassing the lamina and entering the medulla of the optic lobe.

This suggests that an optimal level of PP1 activity in the eye disc

and optic lobemay be required, and either increase or decrease of

PP1 levels could lead to changes in the phosphorylation status of

target proteins and subsequent defects in the growth cone

cytoskeleton and axon guidance.

PP1 is one of the most abundant eukaryotic protein

phosphatases that dephosphorylate serine and threonine residues

of target proteins. The activities of various PP1 catalytic (PP1c)

subunits are extensively regulated in various organisms and

tissue types within a single organism. PP1c binds various

regulatory molecules, which could bring the phosphatase to its

site of action (reviewed by (Cohen, 2002) or act as adaptors for

the phosphatase at its site of function. There are 4 related PP1c

subunits in Drosophila (Dombradi et al., 1990b, 1993).

Drosophila PP1s are very similar proteins encoded by different

loci in the fly genome, and they are variably regulated at

different points in development (Alphey et al., 1997; Asztalos et

al., 1993; Axton et al., 1990; Baksa et al., 1993; Bennett et al.,

1999; Carvalho et al., 2001; Dombradi and Cohen, 1992;

Dombradi et al., 1990a,b, 1993; Helps et al., 1998, 2001;

Raghavan et al., 2000; reviewed by Cohen, 2002). It is

conceivable that Bif acts as one of the regulatory subunits of

PP1 in the fly eye, and it is required either as a complex with PP1

and the actin cytoskeleton or for recruiting PP1 to subcellular

sites where the PP1 activity is required to modulate the actin

cytoskeleton. The former hypothesis seems more plausible as bif

mutant larval and pupal eye discs do not show any obvious

changes in PP1 staining (our unpublished data).


Based on the genetic interactions of bif and the receptor tyrosine

phosphatase genes,Dptp10D andDptp69D, and on the data of Bif

functional and biochemical interactions with PP1-87B and F-actin,

a possible model for Bif function in axon guidance could be put

forward: activation of receptor molecules leads to signaling events

(perhaps also involving misshapen) that activate Bif in the growth

conewhich in turn leads to inhibition of PP1 activity and changes in

the actin cytoskeleton to support axon outgrowth and guidance. In

this scenario, PP1 must have a threshold level of activity as

decreased levels of PP1-87B and general inhibition of PP1 activity

also cause axon guidance defects. Bif might participate in lowering

PP1 activity to the threshold level but must not abolish all PP1

activity in order to allow normal photoreceptor axon guidance.

Interestingly, overexpression of Bif as four copies has been shown

to induce axonal targeting defects (Ruan et al., 2002).

Finally, bif mutants affect axon targeting in the larval optic

lobe, but these defects seem to be corrected in the adult. How

this axon defect is rectified in the adult stages remains unclear,

and it probably occurs during the extensive remodeling that

takes place during the pupal stages of fly development. In

future studies, it would be interesting to determine if any of the

other molecules involved in Drosophila photoreceptor axon

guidance behave in a similar manner, and it would perhaps also

be informative to classify phenotypes into those that can be

repaired and those which cannot.

Acknowledgments

We thank the following people for providing flystocks,

antibodies and other reagents: Barry Dickson, David Van Vactor,

Graeme Marden, Larry Zipursky, Kai Zinn, Yang Rao, Nick

Harden, Nick Helps, Tricia Cohen andUlrike Heberlein.We also

thank the Bloomington stock center for providing fly stocks. Our

special thanks to Richard Tuxworth for help with the image

collection, Rachna Kaushik for critical comments on the

manuscript and the other laboratory members for discussion

and help. The Wellcome Trust UK, Temasek Life Science

Laboratory (W. Chia and K. Babu) and Institute of Molecular

and Cell Biology (S. Bahri) for financial support. This work was

supported in part by a Wellcome Trust Principal Research

Fellowship and Programme Grant to WC.

Appendix A. Supplementary data

Supplementary data associated with this article can be found

in the online version at doi:10.1016/j.ydbio.2005.09.017.

References

Alphey, L., Parker, L., Hawcroft, G., Guo, Y., Kaiser, K., Morgan, G., 1997.

KLP38B: a mitotic kinesin-related protein that binds PP1. J. Cell Biol. 138,

395–409.

Asztalos, Z., von Wegerer, J., Wustmann, G., Dombradi, V., Gausz, J., Spatz,

H.C., Friedrich, P., 1993. Protein phosphatase 1-deficient mutantDrosophila

is affected in habituation and associative learning. J. Neurosci. 13, 924–930.

Awasaki, T., Saito, M., Sone, M., Suzuki, E., Sakai, R., Ito, K., Hama, C., 2000.

The Drosophila trio plays an essential role in patterning of axons by

regulating their directional extension. Neuron 26, 119–131.

Axton, J.M., Dombradi, V., Cohen, P.T., Glover, D.M., 1990. One of the protein

phosphatase 1 isoenzymes in Drosophila is essential for mitosis. Cell 63,

33–46.

Babu, K., Cai, Y., Bahri, S., Yang, X., Chia, W., 2004. Roles of Bifocal, Homer,

and F-actin in anchoring Oskar to the posterior cortex of Drosophila

oocytes. Genes Dev. 18, 138–143.

Bahri, S.M., Yang, X., Chia, W., 1997. The Drosophila bifocal gene encodes a

novel protein which colocalises with actin and is necessary for photore-

ceptor morphogenesis. Mol. Cell. Biol. 17, 5521–5529.

Baksa, K., Morawietz, H., Dombradi, V., Axton, M., Taubert, H., Szabo, G.,

Torok, I., Udvardy, A., Gyurkovics, H., Szoor, B., et al., 1993. Mutations in

the protein phosphatase 1 gene at 87B can differentially affect suppression

of position-effect variegation and mitosis in Drosophila melanogaster.

Genetics 135, 117–125.

Bateman, J., Shu, H., Van Vactor, D., 2000. The guanine nucleotide exchange

factor trio mediates axonal development in the Drosophila embryo. Neuron

26, 93–106.

Bennett, D., Szoor, B., Alphey, L., 1999. The chaperone-like properties of

mammalian inhibitor-2 are conserved in a Drosophila homologue.

Biochemistry 38, 16276–16282.

Bennett, D., Szoor, B., Gross, S., Vereshchagina, N., Alphey, L., 2003. Ectopic

expression of inhibitors of protein phosphatase type 1 (PP1) can be used to

analyze roles of PP1 in Drosophila development. Genetics 164, 235–245.

Beullens, M., Van Eynde, A., Stalmans, W., Bollen, M., 1992. The isolation of

novel inhibitory polypeptides of protein phosphatase 1 from bovine thymus

nuclei. J. Biol. Chem. 267, 16538–16544.

Bossing, T., Brand, A.H., 2002. Dephrin, a transmembrane ephrin with a

unique structure, prevents interneuronal axons from exiting the Drosophila

embryonic CNS. Development 129, 4205–4218.

Cafferty, P., Yu, L., Rao, Y., 2004. The receptor tyrosine kinase Off-track is

required for layer-specific neuronal connectivity in Drosophila. Develop-

ment 131, 5287–5295.

Carvalho, A.B., Dobo, B.A., Vibranovski, M.D., Clark, A.G., 2001. Identifi-

cation of five new genes on the Y chromosome of Drosophila melanoga-

ster. Proc. Natl. Acad. Sci. U. S. A. 98, 13225–13230.

Clandinin, T.R., Lee, C.H., Herman, T., Lee, R.C., Yang, A.Y., Ovasapyan, S.,

Zipursky, S.L., 2001. Drosophila LAR regulates R1–R6 and R7 target

specificity in the visual system. Neuron 32, 237–248.

Cohen, P.T., 2002. Protein phosphatase 1—Targeted in many directions. J. Cell

Sci. 115, 241–256.

Dearborn Jr., R., He, Q., Kunes, S., Dai, Y., 2002. Eph receptor tyrosine kinase-

mediated formation of a topographic map in the Drosophila visual system.

J. Neurosci. 22, 1338–1349.

Desai, C., Purdy, J., 2003. The neural receptor protein tyrosine phosphatase

DPTP69D is required during periods of axon outgrowth in Drosophila.

Genetics 164, 575–588.

Desai, C.J., Gindhart Jr., J.G., Goldstein, L.S., Zinn, K., 1996. Receptor

tyrosine phosphatases are required for motor axon guidance in the

Drosophila embryo. Cell 84, 599–609.

Desai, C.J., Garrity, P.A., Keshishian, H., Zipursky, S.L., Zinn, K., 1999. The

Drosophila SH2–SH3 adapter protein Dock is expressed in embryonic

axons and facilitates synapse formation by the RP3 motoneuron. Develop-

ment 126, 1527–1535.

Dombradi, V., Cohen, P.T., 1992. Protein phosphorylation is involved in the

regulation of chromatin condensation during interphase. FEBS Lett. 312,

21–26.

Dombradi, V., Axton, J.M., Barker, H.M., Cohen, P.T., 1990. Protein phosphatase 1

activity in Drosophila mutants with abnormalities in mitosis and chromosome

condensation. FEBS Lett. 275, 39–43.

Dombradi, V., Axton, J.M., Brewis, N.D., da Cruz e Silva, E.F., Alphey, L.,

Cohen, P.T., 1990. Drosophila contains three genes that encode distinct

isoforms of protein phosphatase 1. Eur. J. Biochem. 194, 739–745.

Dombradi, V., Mann, D.J., Saunders, R.D., Cohen, P.T., 1993. Cloning of the

fourth functional gene for protein phosphatase 1 in Drosophila melanoga-

ster from its chromosomal location. Eur. J. Biochem. 212, 177–183.

Garrity, P.A., Lee, C.H., Salecker, I., Robertson, H.C., Desai, C.J., Zinn, K.,

Zipursky, S.L., 1999. Retinal axon target selection in Drosophila is

regulated by a receptor protein tyrosine phosphatase. Neuron 22, 707–717.

http:\\dx.doi.org\doi:10.1016\j.ydbio.2005.09.017


Garrity, P.A., Rao, Y., Salecker, I., McGlade, J., Pawson, T., Zipursky, S.L.,

1996. Drosophila photoreceptor axon guidance and targeting requires the

dreadlocks SH2/SH3 adapter protein. Cell 85, 639–650.

Halter, D.A., Urban, J., Rickert, C., Ner, S.S., Ito, K., Travers, A.A., Technau,

G.M., 1995. The homeobox gene repo is required for the differentiation and

maintenance of glia function in the embryonic nervous system of

Drosophila melanogaster. Development 121, 317–332.

Hanson, M.a., 1993. The development of the optic lobe. In: Martinez-Arias,

M.a. (Ed.), The Development of Drosophila melanogaster, vol. II. Cold

Spring Harbour Laboratory Press.

Hay, B.A., Maile, R., Rubin, G.M., 1997. P element insertion-dependent gene

activation in the Drosophila eye. Proc. Natl. Acad. Sci. U. S. A. 94,

5195–5200.

Helps, N.R., Cohen, P.T., 1999. Drosophila melanogaster protein phosphatase

inhibitor-2: identification of a site important for PP1 inhibition. FEBS Lett.

463, 72–76.

Helps, N.R., Vergidou, C., Gaskell, T., Cohen, P.T., 1998. Characterisation of a

novel Drosophila melanogaster testis specific PP1 inhibitor related to

mammalian inhibitor-2: identification of the site of interaction with PP1.

FEBS Lett. 438, 131–136.

Helps, N.R., Cohen, P.T., Bahri, S.M., Chia, W., Babu, K., 2001. Interaction

with protein phosphatase 1. Is essential for bifocal function during the

morphogenesis of the Drosophila compound eye. Mol. Cell. Biol. 21,

2154–2164.

Hing, H., Xiao, J., Harden, N., Lim, L., Zipursky, S.L., 1999. Pak functions

downstream of Dock to regulate photoreceptor axon guidance in Droso-

phila. Cell 97, 853–8563.

Hohaus, A., Person, V., Behlke, J., Schaper, J., Morano, I., Haase, H., 2002. The

carboxyl-terminal region of ahnak provides a link between cardiac L-type

Ca2+ channels and the actin-based cytoskeleton. FASEB J. 16, 1205–1216.

Huang, F.L., Glinsmann, W., 1976. A second heat-stable protein inhibitor of

phosphorylase phosphatase from rabbit muscle. FEBS Lett. 62, 326–329.

Huang, Z., Kunes, S., 1998. Signals transmitted along retinal axons in

Drosophila: Hedgehog signal reception and the cell circuitry of lamina

cartridge assembly. Development 125, 3753–3764.

Iwai, Y., Usui, T., Hirano, S., Steward, R., Takeichi, M., Uemura, T., 1997.

Axon patterning requires DN-cadherin, a novel neuronal adhesion receptor,

in the Drosophila embryonic CNS. Neuron 19, 77–89.

Iwai, Y., Hirota, Y., Ozaki, K., Okano, H., Takeichi, M., Uemura, T., 2002. DN-

cadherin is required for spatial arrangement of nerve terminals and

ultrastructural organization of synapses. Mol. Cell. Neurosci. 19, 375–388.

Krueger, N.X., Van Vactor, D., Wan, H.I., Gelbart, W.M., Goodman, C.S.,

Saito, H., 1996. The transmembrane tyrosine phosphatase DLAR controls

motor axon guidance in Drosophila. Cell 84, 611–622.

Kunes, S., 1999. Stop or go in the target zone. Neuron 22, 639–640.

Kunes, S., Steller, H., 1993. Topography in the Drosophila visual system. Curr.

Opin. Neurobiol. 3, 53–59.

Kunes, S., Wilson, C., Steller, H., 1993. Independent guidance of retinal axons

in the developing visual system of Drosophila. J. Neurosci. 13, 752–767.

Lawler, S., 1999. Regulation of actin dynamics: the LIM kinase connection.

Curr. Biol. 9, R800–R822.

Lee, C.H., Herman, T., Clandinin, T.R., Lee, R., Zipursky, S.L., 2001. N-

cadherin regulates target specificity in the Drosophila visual system.

Neuron 30, 437–450.

Lee, R.C., Cladinin, C.R., Lee, C.H., Chen, P.L., Meinertzhagen, I.A., Zipursky,

S.L., 2003. The protocadherin Flamingo is required for axon target selection

in the Drosophila visual system. Nat. Neurosci. 6, 557–563.

Luo, L., 2000. Trio quartet in D. melanogaster. Neuron 26, 1–2.

MacMillan, L.B., Bass, M.A., Cheng, N., Howard, E.F., Tamura, M., Strack, S.,

Wadzinski, B.E., Colbran, R.J., 1999. Brain actin-associated protein

phosphatase 1 holoenzymes containing spinophilin, neurabin, and selected

catalytic subunit isoforms. J. Biol. Chem. 274, 35845–35854.

Mardon, G., Solomon, N.M., Rubin, G.M., 1994. dachshund encodes a nuclear

protein required for normal eye and leg development in Drosophila.

Development 120, 3473–3486.

Maurel-Zaffran, C., Suzuki, T., Gahmon, G., Treisman, J.E., Dickson, B.J.,

2001. Cell-autonomous and-nonautonomous functions of LAR in R7

photoreceptor axon targeting. Neuron 32, 225–235.

McAvoy, T., Allen, P.B., Obaishi, H., Nakanishi, H., Takai, Y., Greengard, P.,

Nairn, A.C., Hemmings Jr., H.C., 1999. Regulation of neurabin I inter-

action with protein phosphatase 1 by phosphorylation. Biochemistry 38,

12943–12949.

Newsome, T.P., Asling, B., Dickson, B.J., 2000a. Analysis of Drosophila

photoreceptor axon guidance in eye-specific mosaics. Development 127,

851–860.

Newsome, T.P., Schmidt, S., Dietzl, G., Keleman, K., Asling, B., Debant, A.,

Dickson, B.J., 2000b. Trio combines with dock to regulate Pak activity

during photoreceptor axon pathfinding in Drosophila. Cell 101, 283–294.

Oliver, C.J., Terry-Lorenzo, R.T., Elliott, E., Bloomer, W.A., Li, S., Brautigan,

D.L., Colbran, R.J., Shenolikar, S., 2002. Targeting protein phosphatase 1

(PP1) to the actin cytoskeleton: the neurabin I/PP1 complex regulates cell

morphology. Mol. Cell. Biol. 22, 4690–4701.

Palmer, A., Klein, R., 2003. Multiple roles of ephrins in morphogenesis,

neuronal networking, and brain function. Genes Dev. 17, 1429–1450.

Parker, L., Gross, S., Beullens, M., Bollen, M., Bennett, D., Alphey, L., 2002.

Functional interaction between NIPP1 and PP1 in Drosophila: conse-

quences of over-expression of NIPP1 in flies and suppression by co-

expression of PP1. Biochem. J. 368 (Pt. 3), 789–797.

Raghavan, S., Williams, I., Aslam, H., Thomas, D., Szoor, B., Morgan, G.,

Gross, S., Turner, J., Fernandes, J., VijayRaghavan, K., Alphey, L., 2000.

Protein phosphatase 1beta is required for the maintenance of muscle

attachments. Curr. Biol. 10, 269–272.

Rao, Y., Zipursky, S.L., 1998. Domain requirements for the Dock adapter protein

in growth-cone signaling. Proc. Natl. Acad. Sci. U. S. A. 95, 2077–2082.

Ruan, W., Long, H., Vuong, D.H., Rao, Y., 2002. Bifocal is a downstream

target of the Ste20-like serine/threonine kinase misshapen in regulating

photoreceptor growth cone targeting in Drosophila. Neuron 36, 831–842.

Schmucker, D., Zipursky, S.L., 2001. Signaling downstream of Eph receptors

and ephrin ligands. Cell 105, 701–704.

Selleck, S.B., Steller, H., 1991. The influence of retinal innervation on

neurogenesis in the first optic ganglion of Drosophila. Neuron 6, 83–99.

Senti, K.A., Usui, T., Boucke, K., Greber, U., Uemura, T., Dickson, B.J., 2003.

Flamingo regulates R8 axon–axon and axon– target interactions in the

Drosophila visual system. Curr. Biol. 13, 828–832.

Sisson, J.C., Field, C., Ventura, R., Royou, A., Sullivan, W., 2000. Lava lamp, a

novel peripheral golgi protein, is required for Drosophila melanogaster

cellularization. J. Cell Biol. 151, 905–918.

Sun, Q., Bahri, S., Schmid, A., Chia, W., Zinn, K., 2000. Receptor tyrosine

phosphatases regulate axon guidance across the midline of the Drosophila

embryo. Development 127, 801–812.

Tan, J.L., Ravid, S., Spudich, J.A., 1992. Control of nonmuscle myosins by

phosphorylation. Annu. Rev. Biochem. 61, 721–759.

Terry-Lorenzo, R.T., Carmody, L.C., Voltz, J.W., Connor, J.H., Li, S., Smith,

F.D., Milgram, S.L., Colbran, R.J., Shenolikar, S., 2002. The neuronal actin-

binding proteins, neurabin I and neurabin II, recruit specific isoforms of

protein phosphatase-1 catalytic subunits. J. Biol. Chem. 277, 27716–27724.

Tessier-Lavigne, M., Goodman, C.S., 1996. The molecular biology of axon

guidance. Science 274, 1123–1133.

Van Eynde, A., Wera, S., Beullens, M., Torrekens, S., Van Leuven, F.,

Stalmans, W., Bollen, M., 1995. Molecular cloning of NIPP-1, a nuclear

inhibitor of protein phosphatase-1, reveals homology with polypeptides

involved in RNA processing. J. Biol. Chem. 270, 28068–28074.

Winberg, M.L., Perez, S.E., Steller, H., 1992. Generation and early differen-

tiation of glial cells in the first optic ganglion of Drosophila melanogaster.

Development 115, 903–911.

Wolff, T., Ready, D.F., 1991. The beginning of pattern formation in the

Drosophila compound eye: the morphogenetic furrow and the second

mitotic wave. Development 113, 841–850.

Yu, T.W., Bargmann, C.I., 2001. Dynamic regulation of axon guidance. Nat.

Neurosci. 4, 1169–1176.

bifocal and pp1 interaction regulates targeting of the r-cell growth cone in drosophila

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