Endocytic trafficking during Drosophila development

Marcos Gonzalez-Gaitan

Max-Planck Institute for Molecular Cell Biology and Genetics, Pfotenhauerstr. 108, Dresden D-01307, Germany

Accepted 12 June 2003

Abstract

During the last decade, many of the factors and mechanisms controlling membrane and protein trafficking in general and endocytic

trafficking in particular have been uncovered. We have a detailed understanding of the different endocytic trafficking steps: plasma

membrane budding, endocytic vesicle motility and fusion with the endosome, recycling, transcytosis and lysosomal degradation. The

kinetics and trafficking pathway of many signaling receptors and the relevance of endocytic trafficking during signaling in many mammalian

cultured cells are also well understood. However, only in recent years has the role of endocytic trafficking during cell-to-cell communication

during development, i.e. during patterning, induction and lateral inhibition, begun to be explored. The contribution of Drosophila

developmental genetics and cell biology has been fundamental in elucidating the essential role of endocytosis during these processes.

Reviewed here are some of the recent developments on the role of endocytic trafficking during long- and short-range signaling and during

lateral inhibition.

q 2003 Elsevier Ireland Ltd. All rights reserved.

Keywords: Endocytic trafficking; Morphogenetic signaling; Morphogens; Decapentaplegic; Wingless; Hedgehog; Notch; Delta

1. Endocytic trafficking during long-range

morphogenetic signaling: Dpp in the wing

1.1. Organizers and long-range morphogens

During morphogenesis, cells within developing tissues

acquire positional information in a process that involves a

particular kind of signaling molecules: the morphogens

(Turing, 1952; Wolpert, 1969). Morphogens are ligands

which are secreted from a restricted groups of cells, a

source. Upon secretion, morphogens move away from the

source to form gradients of concentration. Positional

information is encoded in the graded distribution of

morphogens: cells read their distance to the source by

computing the morphogen concentrations which surround

them. Long-range morphogens act as organizers, being

secreted from a small source and providing positional

information to cells far away from the producing cells.

Therefore morphogens move far from their source and

establish a stable gradient that lasts during several days of

development. What cellular mechanisms control the long-

range movement of morphogens? What ensures their graded

distribution? How are their slopes and ranges stably

maintained?

To answer these questions it is necessary to know: (i) the

key factors involved in the morphogenetic signaling event,

i.e. the signaling cascade from the ligand, through the

receptor and the effecting transcription factors; (ii) the

developmental properties of the signaling event: where is

the source? which are the receiving cells? which target

genes are activated at which distance from the source? and

(iii) how developing cells (as opposite to cultured cells)

control membrane and protein trafficking at the molecular

level and establish the kinetic parameters of protein

movements within and between cells in the tissues where

morphogenetic signaling is taking place. This rich pool of

interdisciplinary information is starting to be available in

the case of morphogenetic signaling in Drosophila. In

particular, the role of endocytic trafficking in three

morphogenetic signaling pathways, Dpp, Wingless and

Hedgehog, are the focus of a number of Drosophila

laboratories in recent years. The comparison of the role of

endocytosis during these three signaling events uncovers

how endocytic trafficking mediates long-range signaling

(Dpp) as well as shorter signaling events (Wg, Hh).

0925-4773/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved.

doi:10.1016/j.mod.2003.06.002

Mechanisms of Development 120 (2003) 1265–1282

www.elsevier.com/locate/modo

E-mail address: gonzalez@mpi-cbg.de (M. Gonzalez-Gaitan).

1.2. Dpp and endocytosis: spreading by planar transcytosis

Dpp acts as a long-range morphogen that provides

information along the anterior–posterior axis during wing

development (Capdevila et al., 1994). Dpp directly

activates the expression of target genes in a concentration

dependent manner (Lecuit et al., 1996; Nellen et al., 1996).

Thus, two target transcription factors, the zinc-finger

proteins Spalt and Optomotorblind are expressed in nested

domains of expression around the Dpp expressing cells

during wing development. Dpp forms a gradient spanning

40 cells diameters that activates Spalt in wing cells at a

distance of 15 cells and Omb as far as 30 cells away from

the source.

Dpp spreading to form such a long-range gradient

requires endocytosis at the receiving cells (Entchev et al.,

2000; Gonzalez-Gaitan and Jackle, 1999). Three kind of

experiments uncovered the role of endocytosis during long-

range movement of Dpp: the ‘shibire rescue’ assay, the

‘shadow’ assay and the receptor mosaic assay (Fig. 1A–D)

(Entchev et al., 2000). In a set of experiments (shibire

rescue; Fig. 1B), endocytosis was blocked in the receiving

cells using the thermosensitive Dynamin mutant shibire,

while the producing cells at the source were rescued by

expressing there a Dynamin transgene. In these developing

wings, fluid phase endocytosis was controlled by observing

dextran internalization into endosomes: endocytosis was

restricted to the source of Dpp and internalization was

tightly blocked in the receiving cells. In this scenario, Dpp

internalization into endosomes was blocked in the receiving

cells and its distribution was reduced to the first three cell

rows adjacent to the source, where it appeared as an

extracellular weak staining. These observations suggested

that long-range spreading requires Dpp endocytosis by the

receiving cells. It is, however, still possible that extra-

cellular Dpp is diluted in a larger volume than the

endosomes: low levels of Dpp would only be visible when

concentrated in the endosomes.

The shadow assay, however, shows that indeed endocy-

tosis is essential during long-range Dpp trafficking (Fig. 1C).

In the shadow assay, the progression of Dpp emanating from

the source is challenged by a patch of shibire mutant cells

which cannot perform endocytosis. The assay takes

advantage of the thermosensitivity of Dpp expression

driven by the gal4 system and the thermosensitivity of

shibire. The experiment starts at low temperature, when

(i) the shibire cells can perform endocytosis at the

permissive temperature and (ii) low levels of Dpp are

expressed and therefore it cannot be seen in the receiving

cells. Then, raising the temperature has two consequences:

(i) endocytosis is blocked in the patch of shibire cells; (ii) a

wave of Dpp secreted from the source is travelling through

the tissue. When confronted with the shibire clone of cells

the wave of Dpp cannot cross the endocytosis defective

tissue and therefore a shadow with low levels of Dpp is

formed distal to the mutant mosaic. The shadow is,

however, transient because Dpp moves rapidly and in all

directions, entering shortly thereafter (within few hours) the

shadow area.

The Dpp receptor, composed of two proteins, Punt and

Thickveins (Tkv), is required for Dpp internalization and for

its long-range movement. A patch of Tkv mutant cells is

incapable of internalizing Dpp. As a consequence Dpp

accumulates around the mutant cells. But extracellular Dpp

accumulation is restricted to the cells of the clone which

face the Dpp source (Fig. 1D). An explanation of this

behaviour of the Tkv clone is that Dpp accumulated around

the cells facing the source is unable to progress further into

the mutant territory, suggesting again a scenario in which

Dpp movement through the target tissue requires its

internalization by endocytosis, specifically by Dynamin-

dependent receptor-mediated endocytosis.

Based on these three observations, a planar transcytosis

model has been proposed in which Dpp movement through

the target tissue involves short-range extracellular diffusion

(across less than 3 cell diameters) and active transport of the

morphogen through the receiving cells by vesicular

trafficking involving the endocytosis and re-release of the

morphogen molecules (Fig. 1E) (Entchev et al., 2000). In

this scenario, endocytic block allows the short-range

extracellular diffusion of the morphogen, but impedes its

active transport through the cells and thereby its long-range

distribution in the tissue.

1.3. An alternative model: Dpp spreading by diffusion

The above three assays show that the control of receptor-

mediated endocytosis is required in order to achieve the

long-range spreading of Dpp. The planar transcytosis model

assumes that Dpp internalized in the receiving cells is re-

secreted to be internalized (and signal) in the neighbouring

cells. The event of Dpp re-secretion from the receiving cells

has, however, not yet been directly monitored. Therefore, an

alternative formal possibility is that internalization is not

required as the first step of Dpp planar transcytosis, but that

endocytosis down-regulates another critical molecule by

removing it from the plasma membrane. Thus it has been

suggested that endocytosis block causes the accumulation of

the Dpp receptor at the cell surface and this in turn traps Dpp

while moving extracellularly by free-diffusion across the

extracellular matrix (Fig. 2A) (Lander et al., 2002). This

possibility is supported by the fact that Dpp signaling range

is decreased by overexpression of the receptors at the

receiving cells (Lecuit and Cohen, 1998). In this scenario, a

proper internalization of the receptor ensures that enough

Dpp free molecules could circulate to build-up the long-

range gradient. To address this possibility, the differential

equations governing Dpp trafficking by free-diffusion

through the extracellular space were studied (Fig. 2B)

(Lander et al., 2002). The model of Lander et al. assumes:

(i) extracellular diffusion of Dpp according to the Fick’s

law; (ii) binding/release of Dpp to the receptors at the plasma

M. Gonzalez-Gaitan / Mechanisms of Development 120 (2003) 1265–12821266

membrane; (iii) endocytosis, recycling and degradation of

the free receptors and endocytosis þ degradation of the

ligand bound to the receptors; (iv) boundary conditions in

the modeling area: Dpp is degraded in a sink region of

the developing tissue and (v) before GFP-Dpp emanates

from its source, there is no Dpp in the receiving tissue.

In the model, the developing wing is simplified in its

geometry to a one-dimensional diffusion problem in which

Fig. 1. Experimental assays to study the role of endocytosis during Dpp morphogen movement. (A) Dpp long-range gradient. Dpp forms a gradient of

concentration from the source, (red cells, left) and appears graded both extracellularly and when internalized in the endosomes. (B) ‘Shi rescue assay’. It

consists of mosaic discs where the producing cells at the source (red cells, left) are wildtype (shiþ) while the receiving cells (right) are mutants for a

thermosensitive mutation in Dynamin (shits) and thereby endocytosis defective cells. Endocytosis is blocked for 6 h. Dpp, degraded, disappears from the

endosome of receiving cells, while extracellular Dpp is restricted to the cells adjacent to the source. (C) ‘Shadow assay’. It starts by producing a tissue void of

Dpp at the receiving cells. A patch of shits cells is generated at the permissive temperature. Then Dpp is pulsed from the source and endocytosis is blocked at the

restrictive temperature of shits. The front of Dpp advancing through the tissue meets the mutant clone. It cannot move across the endocytosis defective territory

and forms a shadow behind the clone. (D) ‘Receptor mosaic assay’. Consist in a mutant clone that lacks the receptor. The clone is exposed to the gradient for

many cell generations. As a consequence, Dpp, which cannot be internalized by receptor-mediated endocytosis, accumulates in between the cells of the clone

which are nearer the source. (E) A model of Dpp spreading by planar transcytosis. Dpp is secreted to an apical location below the adherens junctions from the

producing cells. At the extracellular space, it does not move a long distance by simple diffusion. Instead it is internalized by endocytosis and accumulates in an

apical endosome from where it is degraded or recycled again. This way it moves on through the next cells and thereby spreads through the target tissue forming

a gradient. Degradation in each of the receiving cells ensures the formation of a stable gradient of concentration.

M. Gonzalez-Gaitan / Mechanisms of Development 120 (2003) 1265–1282 1267

Fig. 2. Modelling the free-diffusion of morphogens. (A) Endocytosis block may cause surface receptor accumulation. (i) Internalization of receptors (II)

by endocytosis regulates the amount of surface receptors at the plasma membrane. Receptors internalized by endocytosis are targeted to degradation.

Receptors at the plasma membrane bind to the morphogen (green) and hamper its further spreading through the target tissue. (ii) In endocytosis defective

cells (grey cells) surface receptors are not down-regulated by endocytosis, thereby causing accumulation of receptors at the plasma membrane.

Accumulated at the plasma membrane, high levels of receptors block the progression of the morphogen through the receiving cells. (B)

Parameters affecting the free-diffusion of morphogens when surface receptors hamper their spreading. (C) System of differential equations which describes

the free-diffusion and titration of morphogen spreading by surface receptors. (D–F) Behaviour of the shadow assay under a model of free-diffusion of the

morphogen and spreading is impaired by surface receptors. (D) Internalized Dpp in the shadow assay (gray, shibire-clone) before the steady-state (5 h).

Note that a shadow is seen behind the clone (i), but also before the clone (ii). Also note that, in the clone where internalization is blocked internalized

Dpp is increased (iii). This is due to the fact that the levels of extracellular receptors are 10 times those of the wildtype situation and endocytosis is not

M. Gonzalez-Gaitan / Mechanisms of Development 120 (2003) 1265–12821268

the morphogen is introduced at a rate n at one location (the

source), and absorbed at another, a sink at the edge of the

gradient where the morphogen is totally degraded. While it is

clear that there is a source of Dpp in the developing wing, no

sink exists in the developing wing primordium, i.e. no region

in the disc is dedicated to the total degradation of the

remaining morphogen. The existence of such sink by itself

would generate gradients resembling the one observed for

Dpp regardless of the mechanism of transport proposed.

Within the modeling area, diffusion of Dpp would

behave according to Fick’s second law

›½L�=›t ¼ D›2½L�=›x2

where ½L� is the concentration of the morphogen, t is time, x

is distance and D the diffusion coefficient. In addition to

this, Dpp spreading is governed by Dpp/receptor binding

and dissociation (with constants kon and koff, respectively) as

well as the rates of biosynthesis, endocytosis and degra-

dation of the receptor and the receptor/ligand complexes

(Fig. 2B,C). Then, the receptor/ligand association rate

constant (kon ¼ 3 £ 105 or 1.2 £ 105 M21 s21) is introduced

from the literature (Iwasaki et al., 1995; Natsume et al.,

1997). The values of the other parameters were scanned to

fit the gradient shape and kinetics of gradient formation

observed for Dpp. The values of the different trafficking

rates selected this way were smaller than those previously

estimated experimentally for EGF and its receptor (Lauf-

fenburger and Linderman, 1993). This analysis shows that

there is a universe of parameters for which a gradient

resembling that observed for Dpp can be formed by

extracellular diffusion and receptor binding.

Lander et al. argued that another interesting conse-

quence of the model is that in a scenario of diffusion and

receptor binding as proposed by their model, shibire

clones would also generate distal shadows. This is due to

the fact that endocytosis block would cause the accumu-

lation of surface receptors at the shibire mutant cells

thereby trapping Dpp on its travel to form the gradient

(Fig. 2D–F). However, in the model, both the shadow and

the clone behave different from the experimentally

observed situation: (i) surprisingly shadows can be seen

distally (Fig. 2Di), but also proximally to the shibire clone

(Fig. 2Dii); (ii) the result of blocking internalization in the

clone is that the internalized Dpp is increased with respect

to wildtype cell (Fig. 2Diii), which was not observed in

the shibire clones: internalized Dpp disappeared in the

endocytosis-defective cells, it was not increased! and (iii)

the amount of extracellular Dpp at the clone increased by

a factor of 40 (Fig. 2Eiv), which was also not observed.

In summary, the shibire clone in a scenario of diffusion þ

receptor binding of Dpp behaves different to the observed

result (cf. Figs. 2G vs. 1B).

In addition, in the simulation of the shibire clone, in the

endocytosis defective cells, the receptor levels increase by a

factor of 10 instantaneously, instead of increasing progress-

ively according to the differential equations and the

biosynthetic rates proposed when endocytosis is blocked

in the shibire clone. As a consequence, when the Dpp

propagation front reaches the clone, it finds a concentration

of the receptor 10 times bigger than normal causing the

formation of the shadow behind. After 6 h of endocytic

block, the levels of the Dpp receptor determined exper-

imentally using antibodies do not increase by a factor of 10,

but only suffer a marginal increase (Pantazis and G-G,

unpublished results).

Another issue is the plausibility of the spreading of Dpp

by intracellular trafficking. It has been argued that if moving

by intracellular trafficking, Dpp has to be transported across

a single cell in 150 s on average in order to account for the

speed of expansion of the Dpp gradient in less than 8 h. For

various mesenchymal cells in culture, the rates of recycling

of ligands such as Transferrin or EGF imply mean transit

times of minutes to hours (Sheff et al., 1999; Shitara et al.,

1998). No data is, however, available in the case of

developing epithelial cells. Interestingly, secretion to the

apical-junctional complex in MDCK cells mediated by the

exocyst (Grindstaff et al., 1998) and accumulation of

receptors to the junctional region (Banerjee et al., 1987;

Fehon et al., 1991; Tomlinson et al., 1987) may make the

transport from cell to cell more effective than the three-

dimensional random walk predicted by Fick’s law which is

on the basis of the model. It is worth noting that Dpp

accumulates in apical endosomes (Entchev et al., 2000),

also suggesting that the trafficking routes of Dpp in the

developing epithelial wing cells might be far from random

throughout the full volume of the cell. It will be interesting

to estimate experimentally the rates of dissociation of Dpp

from its receptor and, more generally, its trafficking routes

and kinetics at the receiving cells. In the future, a

fluent crosstalk between experimental data and modeling

of non-linear systems will be necessary in order to integrate

trafficking and developmental approaches.

1.4. Endosomal dynamics during Dpp signaling

Trafficking through the endocytic pathway involves a

number of intermediate compartments, including the early

endosome, where the decision is made to target the endocytic

totally blocked in the simulation, but decreased by a factor of 10. Therefore, although endocytosis is decreased, the huge amount of surface receptors

account for increased receptor-mediated endocytosis inside the clone. This was not seen in the experimental data. (E) Surface Dpp in the shadow assay

(gray, shibire-clone) before the steady-state (5 h). Surface Dpp is increased by a factor of 40 (iv). (F) Internal Dpp after 24 h. No shadow can be seen.

This, however, does not represent the steady-state situation. (G) Schematic representations of the behaviour of the shadow assay in the diffusion/

hampering receptors model. The result differs substantially from the observed results (cf. Fig. 1C).

R

M. Gonzalez-Gaitan / Mechanisms of Development 120 (2003) 1265–1282 1269

cargo to recycling or to degradation at the late endosome/-

lysosome (reviewed in Mukherjee et al., 1997). Each

particular trafficking step through the endocytic pathway

is controlled by a small GTPase of the Rab family. Thus,

Rab5 controls the step from the plasma membrane to the

early endosome, Rab7 controls the targeting to the

degradation pathway and Rab4/Rab11, the recycling path-

way (reviewed in Novick and Zerial, 1997). Consistent with

their role in endosomal dynamics, these Rab proteins accu-

mulate in distinct endosomal compartments: Rab4/Rab5 in

the early endosome, Rab11 in a recycling endosome and

Rab7 in the late endosome. In Drosophila, these factors are

highly conserved in aminoacid sequence (between 75 and

85% identity, 83–91% similarity), localization and function

(Entchev et al., 2000 and unpublished results).

The mutant phenotype of these Rab proteins and their

localization with respect to Dpp uncovered an essential

role of endosomal dynamics during Dpp signaling

(Entchev et al., 2000). Thus, blocking Rab5 in the

receiving cells causes a reduction of Dpp signaling range,

as monitored by the distance to the source at which

target genes are activated. Conversely, Rab5 overexpres-

sion causes an expansion of the Dpp signaling range

(Fig. 3C). Similarly, enhanced degradation caused by

expression of a gain of function Rab7 mutant protein in

the receiving cells reduces the signaling range.

These results are consistent with a scenario in which

internalized Dpp is directed to degradation and recycling in

the receiving cells in a mechanism controlled by Rab

proteins which determines the efficiency of Dpp spreading

and thereby the range and slope of the gradient. In addition

to controlling Dpp spreading, endosomal dynamics may

also determine the efficiency of Dpp signal transduction.

Indeed, endosomal trafficking turned out to critically control

the signal transduction event by other TGF-beta-like ligands

in mammalian cells.

1.5. TGF-beta signal transduction and endosomal dynamics

in mammalian cells: SARA, Smurfs, Clathrin and Caveolin

TGF-beta signals through heteromeric complexes of Type

II and Type I transmembrane Ser-Thr kinase receptors

(reviewed in Massague, 1998). Ligand induces the assembly

of a heteromeric receptor complex within which Type II

receptor transphosphorylates and activates the Type I

receptor (Wrana et al., 1994). The Type I receptor in turn

phosphorylates the receptor regulated Smads, R-Smads

(Smad1,2,3,5,8) (Miyazono, 2000). Once phosphorylated,

the R-Smads bind to another Smad, the Co-Smad Smad4,

forming a complex which is imported in the nucleus

and elicits transcription of target genes. Finally the inhibitory

Smads, I-Smad (Smad6,7), negatively regulate TGF-beta

signaling by binding to the Type I receptor and recruiting the

Smurf E3 ubiquitin ligases, which direct the ubiquitin-

dependent degradation of the TGF-beta receptor–Smad7

complexes (Ebisawa et al., 2001; Kavsak et al., 2000).

Like other receptors, the TGF-beta receptors are

down-regulated by internalization both constitutively

and upon ligand binding (Massague and Kelly, 1986;

Sathre et al., 1991). Endocytosis here may affect

negatively the signal transduction event by removing

the receptors from the plasma membrane where the

ligand binds the receptors and the receptors phosphor-

ylate the transcription factor. The discovery of an

endosomal protein, SARA, as an essential adaptor

between the activated Type I receptor and the transcrip-

tion factors, Smad2/3, uncovered the endosome as a

platform during signal transduction (Tsukazaki et al.,

1998). In mammalian cells, SARA acts specifically

during Smad signaling involving Smad2/3 which mediate

TGF-beta/Activin signaling, but not Smad1/5/8 which

mediate BMP signaling (Tsukazaki et al., 1998). In

Drosophila, however, it has been suggested that Droso-

phila SARA is also involved in Dpp signaling, a BMP

pathway mediated by an Smad1 homolog, Mad (Bennett

and Alphey, 2002).

SARA is targeted to the endosome through its FYVE

finger domain (Hayes et al., 2002; Itoh et al., 2002;

Panopoulou et al., 2002), which specifically binds to PI(3)P

when in a bilayer, a situation specific of the early endosomal

membrane and the internal vesicles of the multivesicular

bodies (Gillooly et al., 2000). In addition to its role as an

adaptor, SARA itself is involved in endosomal dynamics in

a process that also requires its FYVE domain (Hayes et al.,

2002; Itoh et al., 2002; Panopoulou et al., 2002), reminiscent

of other FYVE domain proteins such as EEA1 and

Rabenosyn which are involved in endocytic vesicle

tethering and fusion with the endosome (Gillooly et al.,

2001; Wurmser et al., 1999).

What role does SARA play during Smad phosphoryl-

ation? Three possibilities have been proposed: (i) targeting

of Smad to the receptor when localized at the endosome, (ii)

Smad phosphorylation at the endosome and (iii) diversion of

activated receptors away from the degradation pathway.

Based on the fact that SARA binds directly to the Type I

receptor and Smad2 it was proposed that SARA acts by

targeting Smad to the receptor. This event would take place

at the endosome, mediated by the targeting of SARA itself

to this compartment by means of its FYVE domain (Fig. 3D)

(Tsukazaki et al., 1998). However, if endocytosis is blocked,

SARA is still found in a trimeric complex with the receptor

and Smad2, suggesting that this interaction does not need

the endosomal milieu (Penheiter et al., 2002). While

the trimeric complex forms at the plasma membrane,

phosphorylation of Smad2 by the Type I receptor is blocked

in the absence of endocytosis, suggesting that SARA

endosomal localization is essential to allow Smad2

phosphorylation.

An interesting model for the role of the endocytic

pathway during TGF-beta signal transduction has been

suggested recently by the Wrana Laboratory, which

originally discovered SARA (Fig. 3E) (Di Guglielmo et al.,

M. Gonzalez-Gaitan / Mechanisms of Development 120 (2003) 1265–12821270

2003; Tsukazaki et al., 1998). They showed that TGF-beta

receptors internalize both into Caveolin- and EEA1-positive

vesicles. These vesicular structures are the target compart-

ments of the endocytic vesicles generated by two different

molecular mechanisms: Clathrin-mediated endocytosis

generates EEA1-positive vesicles and caveolae, deep

invaginations of the membrane, generate vesicular struc-

tures containing the raft-protein Caveolin. Rafts are lipid

microdomains enriched in cholesterol which has been

proposed to play an essential role as signaling platforms

(Simons and Ikonen, 1997). However, in the case of TGF-

beta signaling, it seems that the Clathrin dependent

internalization of the receptors into the EEA1-positive

endosome, where the Smad2 anchor SARA is enriched,

promotes TGF-beta signaling, while the lipid raft-caveolar

internalization pathway is required for rapid receptor

degradation. Thus, in the Caveolin-positive compartment

the receptor colocalizes with Smad7–Smurf2, two factors

Fig. 3. Role of endocytic trafficking during TGF-beta-like signaling. (A–C) Essential and rate-limiting role of Rab5 during Dpp signaling. (A) Wild-type. Dpp

emanating from the source (green cells) activates target genes such as Spalt at a distance from the source (red cells). (B) Dominant negative Rab5 mutant in the

posterior receiving cells (dash) reduced the range of Dpp signaling: only cells adjacent to the source activate the target gene. (C) Rab5 overexpression in the

posterior receiving cells expands the range of Dpp signaling. To drive the mutant Rab5 proteins the Engrailed-gal4 driver (en < ) was used. A/P,

anterior/posterior boundary. Arrows represent the range of Dpp signaling. (D) Model of SARA-mediated TGF-beta signal transduction from the endosome.

Binding of TGF-beta to the receptor at the plasma membrane causes phosphorylation of the Type I receptor and activation of its kinase. At the endosome,

SARA brings the Smad2 transcription factor to the receptor. Then Smad2 is phosphorylated and becomes active and imported into the nucleus. (E) A model of

endosome/caveolae competition for TGF-beta signaling. CCP, Clathrin-coated pit. For details, see text.

M. Gonzalez-Gaitan / Mechanisms of Development 120 (2003) 1265–1282 1271

Fig. 4. Endocytosis, lysosomal degradation and asymmetric Wg signaling. (A–E) Embryonic segmentation of the cuticle involves four different signaling

pathways. (A) Stages 9 and 10. Mutual activation of Wg (red) and Hh (blue) in adjacent cells. Wg is distributed symmetrically at both sides of its source.

(B) Stage 11. Ser (grey) is expressed between the Hh and Wg stripes due to repression from both signaling pathways. Wg is already distributed asymmetrically.

(C) Stage 12. Rho (green) is expressed in the cells between Hh and Ser. This is because either ligand can activate Rho expression. Anterior to Hh and posterior

to Ser, Rho is not expressed due to repression by Wg. (D) Spi (green) is secreted from the Rho expressing cells and activate the expression of Svb (orange), the

master regulator of denticle differentiation. Spi activates Svb expression in a longer range towards anterior than towards posterior. This is due to long-range

M. Gonzalez-Gaitan / Mechanisms of Development 120 (2003) 1265–12821272

which are involved in receptor degradation and signaling

down-regulation. Thus, segregation of TGF-beta receptors

into distinct endocytic compartment regulates Smad acti-

vation and receptor turnover mediated by Smurf.

Smurf proteins are E3 ubiquitin ligases that inhibit TGF-

beta signaling and ubiquitinate both the TGF-beta receptors

and Smad proteins (Kavsak et al., 2000; Lin et al., 2000;

Zhu et al., 1999). Consistently, a Drosophila Smurf

mutation is unable to inhibit Dpp signaling and expands

indeed the signaling range (Podos et al., 2001). Smurf

proteins seem to act at three levels during TGF-beta

signaling inhibition: (i) ubiquitination of the R-Smads to

target it to the proteosome for degradation (Lin et al., 2000;

Zhu et al., 1999); (ii) binding to the I-Smad Smad7 to target

it to the receptor where it competes with the R-Smad for

receptor binding (Ebisawa et al., 2001; Kavsak et al., 2000)

and (iii) target the receptor–Smad7 complex for degra-

dation a process that at least partially takes place in the

lysosome, thereby down-regulating the receptor (Kavsak

et al., 2000).

2. Endocytic trafficking during short-range

morphogenetic signaling: Wg in the embryo

While Dpp signals over a long-range during wing

development across 40 cell diameter, other signaling

molecules, like Wingless, are also distributed as a gradient,

but with a short-range. During embryogenesis, the Wg

signal extends across 4 cell diameters in the developing

epidermis. While it is not clear whether Wingless acts itself

as a morphogen (Martinez Arias, 2003), its distribution as a

gradient and/or its signal transduction mechanism seems

also to be mediated by endocytic trafficking. The role of

endocytosis during Wingless signaling was best studied

during segmentation (Fig. 4A–G).

2.1. Segmentation: an array of signaling molecules

and the asymmetric spreading of Wg

Drosophila larval epidermis shows a repetitive pattern of

cuticular structures that manifest the segmental organization

of the insect body (Fig. 4E). Each epidermal segment

derives from a stripe of 12 rows of embryonic cells. On the

ventral surface of the larva, cell rows 1–5 and 12

differentiate denticles which are small hair-like structures

at the apical side of the epidermal cells, while cell rows

6–11 show a smooth surface also called ‘naked cuticle’

(Fig. 4E). During embryonic segmentation, four different

signaling pathways, (Hedgehog (Hh), Wingless (Wg),

Notch (N) and EGF-receptor) interact with one another to

give rise to the spatial information which lies on the basis of

this stereotyped spatial distribution of cuticular elements.

Early during embryogenesis, Hh and Wg are expressed in

adjacent stripes of cells (Fig. 4A). At this time, Hh and Wg

are involved in a paracrine signaling loop: Hh activates

expression of Wg in the adjacent cells and vice versa

(DiNardo et al., 1988; Ingham et al., 1988). In addition, both

Hh and Wg repress expression of a third signaling molecule,

the Notch ligand Serrate (Ser) which is thereby expressed in

the middle of the segment (rows 4–6) where neither Hh

(rows 11,12) nor Wg (row 10) reach (Fig. 4B) (Alexandre

et al., 1999; Wiellette and McGinnis, 1999). Finally a fourth

ligand, the EGF-receptor ligand Spitz, is secreted from a

stripe of cells (rows 1–3) between Hh and Ser. Restricted

secretion of Spitz is mediated by Rhomboid (Rho), which is

expressed in these cells (Golembo et al., 1996). Rho

expression is activated by both Hh and Ser and repressed by

Wg, hence the localization of the Rho stripe and restricted

Spitz secretion (Fig. 4C) (Alexandre et al., 1999; Gritzan

et al., 1999; Sanson et al., 1999).

A single master transcription factor, Shavenbaby (Svb),

controls the differentiation of the denticles in the cell rows

12/1–5 (Payre et al., 1999). Svb expression is activated by

Spitz emanating from rows 1–3 and repressed by Wg

secreted from row 10 (Fig. 4D). Wg spreading from row 10

is asymmetric, with a range of four rows towards anterior

and only one cell row towards posterior. This explains the

skewed localization of Svb at both sides of the Spitz

secreting cells. Therefore, positional information is deter-

mined in this case by an array of signaling molecules

arranged in a spatial sequence and by the asymmetric range

of spreading of one of the ligands, Wg.

2.2. Asymmetric wingless signaling in the embryo:

asymmetric degradation

Wg is distributed symmetrically at the beginning of

embryogenesis (Fig. 4A) and its posterior range of

spreading is restricted later (Fig. 4B–D) (Gonzalez et al.,

1991). Secreted Wg seems to be stabilized by its receptors at

the anterior side, since posterior restriction correlates with a

posterior reduction of the RNA levels of Wg receptors,

Frizzled and Frizzled2 (Dubois et al., 2001). Another level

repression of Svb by wingless towards anterior and short Wg repression towards posterior. As a consequence Rho expression domain is not centered with

respect to Svb. (E) Differentiation of denticles depends on Svb which is due to the interplay of four signaling pathways (Wg, Hh, Rho, Ser) distributed in an

array of cells along the anterior–posterior axis. Positioning of Svb depends on Rho/Spi and asymmetric signaling by Wg. 1–12, cell rows within the segmental

unit (delimited by vertical lines). (F) Secreted Wg is internalized into in an early endosomal structure. Wg is degraded in a lysosome where it could not be

detected. Its distribution is asymmetric. (G) HRP-Wg reveals the asymmetric degradation of Wg. HRP-Wg is symmetrically distributed. Due to the HRP

moiety, it can be detected both in the early endosome and the lysosome. It therefore uncovers enhanced lysosomal degradation in the posterior cells. As a

consequence it shows a symmetric distribution: HRP-Wg in early endosomes of anterior cells; HRP in the lysosomes of posterior cells. In posterior cells,

enhanced degradation is controlled by Spi signaling (green dots) emanating from Rho expressing cells (green nuclei).

R

M. Gonzalez-Gaitan / Mechanisms of Development 120 (2003) 1265–1282 1273

of regulation for the posterior spreading of Wg is its specific

degradation at the lysosome: Wg is degraded at higher rates

in the posterior cells than in the anterior ones (Dubois et al.,

2001).

This was shown by monitoring a functional horseradish

peroxidase (HRP)-Wgfusion(Duboisetal.,2001).HRPin the

fusion persists in the lumen of the degradative compartment

even after Wg itself has been digested. At a stage when Wg

already spreads asymmetrically from row 10 and is only seen

in the posterior cells adjacent to the source (Fig. 4F), HRP-

wingless shows a symmetric distribution and can be seen in

posterior cells distant from the source (Fig. 4G). Therefore,

the posterior restriction of Wg requires its differential

degradation in posterior cells (Fig. 4F,G). At the EM level,

HRP-Wg is indeed seen in degradative compartments:

multivesicular (MVB) and multilamellar bodies (MLB), con-

firming previous immuno-EM results (Gonzalez et al., 1991;

vandenHeuveletal.,1989).AlthoughMVB/MLBcontaining

HRP-Wg are seen in both anterior and posterior cells, Wg

seems to be more actively degraded in the posterior cells,

since HRP-Wg-labelled MVB/MLB in the posterior cells are

four times more frequent than in anterior cells.

Consistent with the idea that the asymmetric distribution

of Wg is mediated by its differential degradation, treatment

with chloroquine, a lysosomal inhibitor, or reduction of

internalization by endocytosis and lysosomal degradation

with Clathrin heavy chain and deep orange mutations

abolishes the asymmetric distribution of Wg (Dubois et al.,

2001). How is this differential degradation controlled?

Restricted Wg spreading towards posterior is under the

control of Hh: in Hh pathway mutants, Wg distribution

remains symmetric (Sanson et al., 1999). As described above,

Hh activates Rho expression inposterior cells determining the

restrictedsecretionof the EGF-receptor ligand Spitz,which in

turn elicits the transcription of an unknown factor mediating

enhanced Wg degradation in the cells that received the Spitz

signal (Fig. 4G). Ina numberofcases,EGF-receptor signaling

has been shown to control endocytic trafficking by interfering

with the Rab5 endocytic machinery (Lanzetti et al., 2000; Tall

et al., 2001), suggesting this small GTPase and its regulators/

effectors as possible targets of Spitz control.

2.3. Endocytosis and Wg signaling

What signaling step is affected by the differential

degradation of Wg? Spreading? Transduction? The fact

that HRP-Wg is seen far from the source in the posterior

cells, suggests that Wg spreading is not affected by

differential degradation. An alternative possibility is that

Wg signal transduction is affected by the degradation of Wg.

Future work will elucidate which of this two possibilities are

more plausible, and whether wg signal transduction occurs

from an intracellular locale.

Wg is internalized by Dynamin-mediated endocytosis

into vesicular structures in the target tissue (Bejsovec and

Wieschaus, 1995). In shibirets mutants, Wg is only able to

elicit signaling in cells adjacent to row 10. Therefore,

long-range Wg activity requires endocytosis. Again, is

endocytosis required for Wg release from the producing

cells, its spreading or its transduction? The answer is

unclear. On one hand, the shits embryonic phenotype is

reminiscent of a secretion-defective Wg mutant,

suggesting that endocytic block affects Wg release

(Bejsovec and Wieschaus, 1995; Gonzalez et al., 1991).

This possibility has also been proposed for Wg during

wing development (Strigini and Cohen, 1999). On the

other hand, endocytic block restricted to the receiving

cells also affects Wg signaling, consistent with a

endocytic role during either spreading or transduction of

the signal (Moline et al., 1999).

Like in the case of Dpp, a possible role of endocytosis

during the spreading of Wg is that it initiates the planar

transcytosis of the morphogen: Wg is internalized by

endocytosis, traffics through the endocytic pathway and is

re-secreted again to move further into the target tissue.

Wg recycling in this way was observed using a functional

Wg-GFP fusion (Pfeiffer et al., 2002). Fluorescence time-

lapse microscopy of Wg-GFP shows that the Wg

internalized in endocytic vesicles can return to the cell

surface. Although recycling could only be observed in the

apical surface, which is optically accessible, it is still

possible that re-secretion also occurs laterally within the

epithelium, thereby directing the ligand to the neighbour-

ing cells. Ligand recycling is a key feature of the planar

transcytosis model of transport: the ligand spreads by

repeated cycles of internalization, recycling, and presen-

tation to further cells. The observation of Wingless

recycling shows that planar transcytosis is plausible.

However, one should note that, in the above experimental

situation, very few endocytic vesicles containing GFP-

Wingless were present in non-expressing cells. Therefore,

either a small number of vesicles are sufficient for

transport or transcytosis is not an important contributor

to Wg transport.

In the wing, it has been argued that endocytosis is indeed

not required for the dispersal of the signal. This was based

on the absence of Wg shadows (Strigini and Cohen, 1999)

when performing a shadow assay as described above for

Dpp (Entchev et al., 2000). However, in the case of the Wg

experiment, the shadow assay was performed in a steady-

state situation. In this experimental condition, before Wg

disappears from the distal side of the endocytosis-defective

mutant clone, molecules of Wg emanating from the sides

will fill this distal region rapidly. As a consequence, like in

the case of Dpp, no distal Wg shadows will be formed if the

experiment is started in a steady-state situation if Wg moves

rapidly and in all directions. Wg moves rapidly: the gradient

can expand at a speed of 15 cells in 30 min. It also moves in

all directions. To observe shadows the experiment should be

done in conditions in which the propagation front of Wg

gradient expansion meets a clone of Shibire mutant cells as

performed in the Dpp shadow assay.

M. Gonzalez-Gaitan / Mechanisms of Development 120 (2003) 1265–12821274

Whatever the mechanism of Wg dispersal (diffusion vs.

planar transcytosis), is Wg transported in vesicular struc-

tures while spreading through the receiving tissue? A recent

report shows that, like Hh (see below), Wingless is

palmitoylated (Willert et al., 2003), suggesting that it

would not appear as a soluble protein in the extracellular

space, but associated to a lipidic millieu. Interestingly, Wg

has been seen in the same endocytic compartment as the

argosomes, lipidic fragments that disperse over long

distances in the wing epithelium (Greco et al., 2001). The

molecular and cellular nature of the argosomes and the

question whether they serve as lipidic carriers for signaling

molecules such as Wg or Hh remains yet to be fully

understood, but such mechanism of morphogen dispersal is

certainly tantalizing.

3. Trafficking during short-range morphogenetic

signaling: Hh in the wing and the embryo

Like Dpp and Wg, Hh forms concentration gradients

during development which instructs cells about their

positional information. Hh shows post-tranlational modifi-

cations by covalent binding to two lipid moieties:

cholesterol (Porter et al., 1996) and palmitoyl acid

(Chamoun et al., 2001; Pepinsky et al., 1998). This implies

that Hh is a membrane-tethered molecule. Indeed Hh range

of spreading is shorter than for other morphogens, such as

Dpp. In the case of the developing wing Hh elicits signaling

not farther beyond 10 cells and in the embryo, not beyond

four cells. But being membrane-tethered, how is Hh

released from the producing cells? How does it spread

through the target tissues? What are the mechanisms

underlying the transduction of the signal? These are indeed

unsolved issues.

In the producing cells, Hh family proteins undergo a

autoproteolytic cleavage under the control of the C-terminal

domains. Concomitantly, a cholesterol moiety is added to

Hh N-terminal domain at its C-terminus to give raise to the

active Hh fragment, which is thereby linked to the

membrane (Beachy et al., 1997) and is found in lipid rafts

(Rietveld et al., 1999). Strikingly, the cholesterol-modified

membrane-tethered N-terminal Hh fragment carries the

morphogenetic signaling functions both at long- and short-

range, while the C-terminal domain, secreted and soluble, is

inactive for signaling (Beachy et al., 1997). Another lipid

modification of Hh, a palmitoylation of its N-terminal

cysteine, has been shown to occur in vertebrates and

Drosophila (Chamoun et al., 2001; Pepinsky et al., 1998).

The factor that catalyses Hh palmitoylation, Skinny hedge-

hog, shares sequence homology with O-linked acyl

transferases. These are cytosolic enzymes that therefore

add the palmitoyl moiety to the cytoplasmic side of

transmembrane proteins. It is therefore intriguing how, in

the case of Hh, they catalyse the lipid modification on the

luminal side of the protein.

3.1. Hh release and spreading: cholesterol modification

What are these lipid modifications good for? In the case

of the cholesterol modification, it is controversial whether

cholesterol modification restricts or widens the range of

secreted Hh. This question was addressed using a truncated

N-terminal Hedgehog transgene which does not undergo

cholesterol modification (Burke et al., 1999; Lewis et al.,

2001). In a mesenchymal situation, the developing mouse

limb, this non-modified Hh is only able to elicit short-range

signaling, suggesting that the cholesterol modification is

essential for long-range spreading of the ligand (Lewis et al.,

2001). In contrast, in an epithelial situation, the Drosophila

developing wing, the corresponding non-modified Hh

shows a longer spreading range, leading to the opposite

proposal that the cholesterol moiety restricts Hh spreading.

In the case of the wing, absence of cholesterol modification

leads to a mistargeting of Hh release (Burke and Basler,

1996): in the wildtype, Hh is seen in the basolateral side of

the epithelium, while non-cholesterol-modified Hh is found

in the apical side of the developing wing. This raises the

possibility that cholesterol controls Hh secretion into a side

of the epithelium where the extracellular milieu might

determine the range of spreading. Thus, while the apical,

luminal side of the developing wing will allow free-

diffusion of Hh to a long distance from the source, in the

case of the mesenchymal cells, the limiting factor might not

be release/spreading, but the transduction of the signal (see

below). In another developing context, the embryonic

segmentation of the epidermis, the cholesterol modification

of Hh has also been recently shown to be responsible for its

targeting to large punctate structures, possibly an endocytic

compartment, at the apical pole of the receiving cells (Gallet

et al., 2003). The proper targeting of cholesterol-modified

Hh is mediated by a sterol-sensing domain protein,

Dispatched (Burke et al., 1999; Ma et al., 2002). It is

therefore possible that cholesterol modification endows Hh

with targeting information during trafficking in polarized

epithelial cells, a role for cholesterol which is reminiscent of

cholesterol-enriched lipid rafts in apical targeting in the

biosynthetic pathway of epithelial cells (Simons and Ikonen,

1997). Once properly targeted to the right side of the cells,

Hh is released to a extracellular space of the epithelium

where proteoglycans synthesized by the GAG transferase

enzyme Tout-velou mediate the movement of modified Hh

throughout the tissue (Bellaiche et al., 1998; Gallet et al.,

2003; The et al., 1999).

In addition to the role of cholesterol modification and

proteoglycans, the Hh receptor Patched (Ptc) also plays a

key role to restrict Hh spreading. High levels of Ptc restricts

Hh movement throughout the tissue (Chen and Struhl,

1996): Ptc overexpressing clones cast shadows behind, like

in the shadow assay. Hh is involved this way in an

autoregulatory loop that limits its spreading: Hh signaling

upregulates Ptc, which in turn binds to Hh and titrates it,

impeding its long-range movement. Subsequently, the levels

M. Gonzalez-Gaitan / Mechanisms of Development 120 (2003) 1265–1282 1275

of Ptc at the plasma membrane are regulated by Dynamin-

dependent internalization initiated by Ptc binding to Hh

(Denef et al., 2000), which lead to targeting to endocytic

vesicles and MVBs (Capdevila et al., 1994). It is, however,

unclear whether endocytosis itself plays any role during Hh

spreading and to date no data suggests that Hh movement

involves a planar transcytosis mechanism.

3.2. Hh signal transduction and the endocytic pathway

Ptc acts as a Hh receptor, but instead of eliciting signaling,

its function is to repress the signaling pathway. In the absence

of the ligand, Ptc inhibits a latent, tonic signaling activity of

another transmembrane protein, Smoothened (Smo)

(reviewed in Ingham and McMahon, 2001). Ptc binding

destabilizes Smo (Denef et al., 2000). Binding of Hh to Ptc,

releases its inhibition of Smo. This release is mediated by

Hh-induced internalization of Ptc, followed by phosphoryl-

ation of Smo, which in turn is stabilized and accumulates at

the plasma membrane (Denef et al., 2000). These obser-

vations suggested that Hh elicits signaling by diverting Ptc

into a distinct trafficking route that would release Smo from

Ptc repression. Consistent with this, a mutant in the sterol-

sensing domain of Ptc which is not affected in binding/

sequestration of Hh, is unable to repress the intrinsic

signaling activity of Smo (Chen and Struhl, 1996; Martin

et al., 2001; Strutt et al., 2001). The sterol-sensing domain

has been implicated in the targeting of proteins and lipids

through specific membrane compartments (Kuwabara and

Labouesse, 2002). Indeed, the sterol-sensing domain mutants

in Ptc are mistargeteds and do not anymore colocalize with

Smo in the endocytic compartment (Martin et al., 2001). In

summary, the role of Hh as a signaling molecule would then

be to get rid of a molecule, Ptc, that acts as a repressor of a

Smo-dependent tonic signaling.

In mammalian cells in culture, the intricacies of this

fascinating possibility have been studied in some detail by

the Roelink group (Incardona et al., 2002). Ptc and Smo

colocalize extensively in the absence of Sonic Hedgehog

(Shh), a mammalian Hh homolog. Upon ligand binding,

Shh, Ptc and Smo are internalized together, but become

subsequently segregated into different compartments: the

Ptc-Hh complex is targeted for degradation at the lysosome

while Smo is not. The sorting of Smo requires an acid

luminal endosomal environment in which the membrane is

enriched in lysobiphosphatidic acid (LBPA), suggesting a

specific trafficking pathway during Hh signaling.

3.3. Cholesterol-dependent Hh trafficking and Hh

functional diversity

Cholesterol modification of Hh and the sterol-sensing

domain in Ptc may play key roles in trafficking through this

specific pathway. An interesting possibility is that, during

Hh signaling, activation of different target genes is mediated

by Hh presentation from different membrane compartments

determined by the lipid modification of Hh (Gallet et al.,

2003). This has been suggested for Hh signaling during

epidermal embryonic segmentation (Fig. 5). As described

above, Hh is expressed in two rows of cells abutting the

parasegmental boundary in the embryonic epidermis and

from there (i) activates Wg in the cells adjacent in the

anterior direction and (ii) activates Rho in three cell rows

towards the posterior direction (Fig. 5A,B). Targeting of Hh

to an apical compartment in the receiving cells (Fig. 5A,C)

depends on cholesterol modification and the function of the

sterol-sensing domain protein Dispatched (disp), which has

being implicated previously in Hh release from the

producing cells (Burke et al., 1999; Ma et al., 2002). In

non-cholesterol-modified hh or in disp mutants, apical

targeting of Hh in the receiving cells is absent. These mutant

conditions abolish Hh signaling in the anterior adjacent

cells, but do not affect signaling towards the posterior cells,

which thereby activate Rho normally (Gallet et al., 2003).

Similarly, Tout-velu mutants affect anterior Wg expression,

but not posterior signaling. Tout-velu promotes the spread-

ing of cholesterol-modified Hh through the extracellular

space by catalyzing the synthesis of a proteoglycan that

mediates Hh movement.

In summary, anterior signaling requires Hh targeting to

the apical endosome to be properly released from the

producing cells in a process which requires Cholesterol

modification of Hh, its release in Disp-dependent manner

and the proteoglycan-dependent spreading of cholesterol-

modified Hh mediated by Tout-velu (Fig. 5C). In contrast,

posterior signaling does not require Hh modification with

cholesterol, its release or proteoglycan-mediated movement

(Fig. 5C). This uncovers a scenario in which different

cellular environments are able to elicit the expression of

different target genes depending on the lipid modification

and thereby the subcellular compartment (i.e. the apical

endosomal compartment where Hh is normally found) in

which the Hh ligand is present.

It is, however, unclear how a Hh molecule which has not

been cholesterol-modified, that cannot be released properly

(in disp mutants) or cannot spread through a long-range (in

Tout-velu mutants) is nevertheless able to elicit signaling

across 3–4 cell diameters in the posterior direction.

Conversely, it is surprising that all these cellular require-

ments, which are essential for long-range spreading of

the ligand, are essential to signal to the neighbour anterior

cell. These paradoxes reflect that probably we are missing

some essential cellular/molecular process at the basis of

asymmetric Hh signaling during the process of

segmentation.

4. Endocytic trafficking during lateral inhibition: Notch

signaling and asymmetric cell division

Dpp, Hh and Wg form gradients of concentration that

elicits signaling in cells at a distance from the source.

M. Gonzalez-Gaitan / Mechanisms of Development 120 (2003) 1265–12821276

During asymmetric cell division (ACD), the Notch signal-

ing pathway mediates lateral inhibition between two

adjacent cells contacting each other. Asymmetric Notch

signaling probably capitalizes on asymmetric endocytic

trafficking.

4.1. Asymmetric localization of Numb controls Notch

signaling during ACD

The external sense organs (ES) of the PNS are

composed of two outer cells (hair and socket), two inner

cells (neuron and sheath) and a glial cell (Fig. 6A) (Gho

et al., 1999). These four cells arise from a single sensory

organ precursor (SOP) cell in a stereotyped lineage:

the SOP divides into a posterior pIIa and an anterior pIIb

cell; pIIb generates the pIIIb and a glial cell, pIIa the hair

and the socket and pIIIb the neuron and the sheath

(Fig. 6B) (Gho et al., 1999).

Numb, a phosphotyrosine binding protein, determines

the fate of the SOP progeny (Rhyu et al., 1994; Uemura

et al., 1989): in loss of function numb mutants SOPs divide

symmetrically into two pIIa cells, which subsequently

generate two sockets (Fig. 6C). Conversely, Numb over-

expression in the SOP lead to the generation of four neurons.

Fig. 5. Cholesterol and asymmetric Hh signaling? (A,B) Hh signaling (blue) activates Wg (red) in the anterior adjacent cells and Rho (green) within a range of 3

cell diameters towards the posterior. In receiving cells, Hh is internalized into ‘large punctate structures’ (LPS) probably corresponding to an apical endosomal

structure. (A) Z section through the epithelium. (B) XY section. (C) Hh is secreted from the source (blue nucleus) by a process that requires Disp. It is

internalized into an LPS in a process that requires Ttv. Anterior signaling to activate Wg (red) requires Disp, Ttv and the cholesterol moiety (Hh-Np). Posterior

signaling to activate Rho (green) is independent of Hh release, Disp, Ttv and the cholesterol moiety.

M. Gonzalez-Gaitan / Mechanisms of Development 120 (2003) 1265–1282 1277

Fig. 6. Endocytosis and asymmetric cell division. (A) Schematic representation of an ES organ. (B) Schematic representation of the asymmetric cell divisions

that give rise to the five cells in the ES organ. (C,D) numb2/a-adaptinear (C) and Nts (D) mutant phenotypes. (E) Molecular interactions between Notch, Numb,

a-adaptin and Clathrin. (F) Notch binding of Numb recruits a-adaptin/Clathrin thereby initiating its internalization by endocytosis. (G) A model for the role of

endocytosis during asymmetric cell division. Numb distributed in a crescent in the SOP causes the asymmetric distribution of a-adaptin. Higher levels of

Numb/a-adaptin in one cell causes the enhanced down-regulation of Notch by endocytosis in that cell. This gives advantage to one of the two cells during

mutual competition for Delta/Notch signaling. This initial bias is increased until it is converted into a directional Notch signaling from one of the two cells, that

inhibits the PIIa fate in the adjacent cell.

M. Gonzalez-Gaitan / Mechanisms of Development 120 (2003) 1265–12821278

Interestingly, in the SOPs, the Numb protein concentrates in

the area of the cortex that overlies one of the two

centrosomes (Knoblich et al., 1995) and is segregated into

one of the two daughter cells upon cytokinesis (Fig. 6G)

(Rhyu et al., 1994). Therefore, Numb behaves as a

segregating determinant in the SOP lineage.

Numb controls ACD by repressing Notch signaling.

Notch is the receptor of two ligands, Delta and Serrate,

which upon binding lead to Notch cleavage. The intracellu-

lar Notch fragment is then imported into the nucleus and

converts Suppressor of Hairless, a repressor, into a

transcriptional activator. Inactivation of Notch function

during ACD lead to cell fate transformations which are the

opposite to those observed in numb mutants: SOP divides

into two pIIb which generate four neurons (Fig. 6D)

(Hartenstein and Posakony, 1989). Numb acts upstream of

Notch (Guo et al., 1996) and both molecules bind to each

other (Guo et al., 1996). A model has been suggested (Zeng

et al., 1998) in which both Delta and Serrate bind to Notch

in the SOP lineage: Notch signaling induces pIIa fate in one

daughter cell, while Numb prevents Notch signal transduc-

tion in the other, which thereby adopts the pIIb fate.

How does Numb control Notch signal transduction? A

mammalian Numb homolog localizes to endocytic vesicles

and binds to a-Adaptin, a component of the AP2 complex

(Santolini et al., 2000). AP2 initiates Clathrin coating in

endocytic vesicles by recruiting Clathrin to the endocytic

bud and triggering Clathrin polymerization into a lattice.

That prompted the possibility that Numb controls Notch

signaling by orchestrating the endocytosis of the receptor. It

was already known that Dynamin plays a key role during

Notch signaling (Seugnet et al., 1997).

4.2. Endocytosis, Notch signaling and ACD

Another endocytic factor, a-Adaptin, plays a specific

role during ACD. a-Adaptin mutants were recovered in a

screen for factors involved in ACD (Berdnik et al., 2002).

a-Adaptin is composed of a head domain, an ear domain

and a hinge domain. While loss of function mutations in

a-Adaptin blocks endocytosis and cause embryonic leth-

ality (Gonzalez-Gaitan and Jackle, 1997), mutations in the

ear domain of a-Adaptin cause a milder phenotype and

resemble Numb loss of function mutations during ES

development (Fig. 6C). The ear domain of a-Adaptin has a

regulatory role during endocytosis by binding to accessory

endocytic proteins including AP-180, Epsin, EPS-15,

Dynamin and Amphiphysin. It was also shown to bind to

mammalian Numb (Santolini et al., 2000). Furthermore, it

was shown that the ear domain of Drosophila a-Adaptin is

able to bind Numb both in vitro and in vivo (Berdnik et al.,

2002). The Numb binding domain contains a DPF domain

that was shown to be essential for Notch repression (Frise

et al., 1996).

In the asymmetrically dividing SOPs, a-Adaptin, like

Numb, is concentrated to one side of the cell cortex

during metaphase (Fig. 6G) (Berdnik et al., 2002). During

cell fate specification in the Drosophila ES lineage, a-

Adaptin acts downstream of Numb and upstream of

Notch: in numb mutants a-Adaptin fails to localize

asymmetrically. Since Numb can directly interact with

Notch intracellular domain, Numb may antagonize Notch

activity by recruiting a-Adaptin (Fig. 6E). This would in

turn initiate Notch internalization by Clathrin-mediated

endocytosis (Fig. 6F) and ultimately cause down-regu-

lation of Notch signaling in the cells that get the higher

levels of Numb/a-Adaptin (Fig. 6G). Testing this

hypothesis will require Notch internalization assays in

vivo during the development of ES.

5. Concluding remarks

Different signaling events capitalize on endocytic

trafficking in different ways: endocytic trafficking powers

the long-range spreading of Dpp, but is also used to restrict

the range of Wg signaling in the embryo; it is used to

implement Hh signaling by degrading the repressing

receptor, Ptc and trafficking it away from the tonic activator

Smo and, during Notch signaling in ACD, to have a biased

internalization of the receptor, thereby skewing a reciprocal

signaling event. These proposed models of action for

endocytosis are qualitative, while their basis is the

quantitative change in the rates of internalization, degra-

dation and recycling. To fully understand the mechanisms,

we will need to examine these processes in quantitative

detail. Therefore, future directions will include the

quantitative assessment of the rates of trafficking and

signaling.

In Drosophila, tools like the thermosensitive shibire

mutant have provided the possibility of studying phenotypes

in real time, a key tool when monitoring trafficking. The

analysis of other kinds of mutants only allows monitoring of

the effects of the mutations after the developing system

reaches its steady-state. This means that many of these

phenotypes could be indirect, the consequence of the reaction

of a system which probably has backups built in. In the future,

a useful tool to help us understand the trafficking mechanisms

during signaling would be conditional mutant situations that

allow us to study the direct cell biological effects of depleting

a gene function in real time.

Acknowledgements

I would like to thank Karen Echeverri, Christian Bokel

and Alfonso Martınez-Arias for critically reading this

manuscript. My work is supported by the Max-Planck

Society and DFG.

M. Gonzalez-Gaitan / Mechanisms of Development 120 (2003) 1265–1282 1279

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