Download Short-range positional signals in the developing

59
Development 1989 Supplement, 59-63
Printed in Great Britain © The Company of Biologists Limited 1989
Short-range positional signals in the developing Drosophila eye
ANDREW TOMLINSON
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH
Summary
Positional signals provided by immediate neighbours
appear to direct developmental decisions in the eye of
Drosophila. By a combined genetic and molecular approach the biochemical bases of the signal and reception
mechanisms are being systematically dissected. Three
key gene products have now been identified, sevenless is
a transmembrane tyrosine kinase probably transducing
positional signals that direct the R7 cell to its fate. The
bride of sevenless gene product is on the signalling side of
the mechanism and is required in R8 for R7 to develop.
The type of protein bride of sevenless encodes is not yet
known. The rough gene encodes a transcription factor
on the signalling side required in R2 and R5 for
positional signals to be transmitted to neighbouring
cells.
Introduction
Cell lineage directed developmental decisions do no
occur in assembling ommatidia (Ready et al. 1976;
Lawrence and Green, 1979) and examinations of cellfate choices occurring in this system therefore focus
upon the nature of the positional cues. The specific
questions that are being addressed are the nature and
transmission of the signals, the mechanisms the cells use
to receive them, and the molecular events that follow
reception and lead to developmental decisions.
Ommatidia develop in a monolayer epithelium, contained within the eye-antennal imaginal disc (Fig. ID),
in which cells extend from the apical surface to the basal
membrane. Each ommatidium is built from a foundation precluster made from five cells destined to form
photoreceptors R2, R3, R4, R5 and R8. The nuclei of
these cells are positioned within the apical regions of
the epithelium and the nuclei of all other cells destined
to join the ommatidium are placed in the basal regions.
Cells are systematically incorporated into the cellular
unit and the ommatidium grows in a radial manner. As
a cell joins the ommatidium it first establishes a precise
set of contacts with cluster cells in the apical regions.
Subsequently these cellular contacts extend throughout
the entire apical/basal depth of the ommatidium and
the nucleus of the cell rises from its basal position into
the apical clustering. Following this the cell shows
evidence of differentiation such as expression of neural
antigens recognisable by specific antibodies and axon
out-growth. The five cell unit grows to eight cells with
the incorporation of the cells destined to become Rl,
R6 and R7, completing the photoreceptor complement
of the ommatidium. Lens-secreting cone cells are the
next to be added and in this manner the ommatidium
carries on growing. Using antibodies that recognise
neural epitopes a developmental sequence can be
Cells within a developing organism can be directed to
their fate by positional cues. These can be long range in
nature with cells being developmentally directed by
signals from a distant source. Until recently few longrange diffusable signals had been identified, however
some good examples have now been demonstrated.
Perhaps the best example is the bicoid protein which is
translated in the anterior region of the Drosophila
embryo and redistributes posteriorly. A gradient of
protein concentration is then established along the
anterior-posterior axis of the embryo, to which the
hunchback gene is thought to respond (Driever and
Niisslein-Volhard,
1989). Many developmental
phenomena are difficult to explain with models using
long-range diffusable molecules and positional signals
which operate over smaller distances local to the source
itself are expected. Spatially restrained positional
signalling could occur when cues are communicated
between immediate neighbours. There have been few
descriptions of this but there are some good examples in
the vertebrate immune response where signals are
passed through the MHC complexes of cells contacting
each other. Strong evidence is accumulating from
analyses of ommatidial development in the compound
eye of the fruit fly for positional signals that are of shortrange nature and are presented by directly adjacent
cells.
The compound eye is made from many hundred
identical subunit ommatidia each of which is a simple
assembly of 20 cells (Fig. 1). Each cell can be identified
both by its position in the unit and its cell type, and the
many hundred fold reiteration of the structure allows
large numbers to be sampled in a single preparation.
Key words: Drosophila, compound eye, positional signals,
tyrosine kinase transmembrane receptor, homeobox.
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A. Tomlinson
Fig. 1. (A) A smooth array of 700-800 ommatidia forms a Drosophila compound eye. Anterior is to the right. Small
mechanosensory bristles project between ommatidia. (B) Tangential section of the eye; anterior is to the right. In any cross
section, ommatidia present an asymmetric, trapezoidal pattern of seven rhabdomeres. (C) A schematic ommatidium,
anterior is to the right. Below the corneal lens (cl) is a second lens element, the pseudocone (c), which is a refractile
extracellular secretion of the four underlying cone cells (cc). The accessory cone cells meet in the centre occluding the
principal cells from contact. The cone cells are collared by the two primary pigment cells (pp). Photoreceptors or retinuala
cells (re) are elongated sensory neurons that carry rhabdomeres (rh), dense stacks of rhodopsin-loaded microvilli.
Rhabdomeres of photoreceptors R1-R6 extend the depth of the ommatidium. The rhabdomere of R7 lies above that of R8
on the central axis. A sheath of secondary and tertiary pigment cells (sp, tp) optically insulates each ommatidium. (D) A
late third instar eye-antennal disc; anterior is to the top. The upper portion is the antennal disc which is folded into a series
of concentric rings, and below it the briad slightly cupped eye disc. The morphogenetic furrow (indentation visible in eye
disc) lies dorsoventrally and moves across the eye disc from posterior to anterior. Ommatidial patterning occurs posterior to
the furrow. At the posterior of the eye disc is the optic stalk which carries the axons to the brain (not shown). Reprinted
from Tomlinson and Ready (1987o), with permission.
Short-range signals in the Drosophila eye
Fig. 2. Sequential development of the ommatidium. Upper
panel shows the differentiation sequence of the-eight
photoreceptors. R8 differentiates first followed by the pair
R2 and R5, next come R3 and R4, followed by Rl and R6
and lastly R7. The cells are shown in the positions they
come to occupy rather than the position they hold at any
particular stage. Below shows where mutations have
identified gene products involved in the inductive sequence.
rough (ro) breaks the sequence after the addition of R2 and
R5 but before the determination of R3 and R4, the gene
product is required in R2 and R5 to communicate a signal
to R3 and R4. sevenless (sev) and bride ofsevenless (boss)
prevent the differentiation of R7. boss is required in R8 to
signal and sev is required in R7 to receive.
detected in the five cells of the precluster. R8 is the first
to differentiate followed by the pair R2 and R5 followed
by the pair R3 and R4. Rl and R6, and R7 follow in the
sequence as to be expected from the description above
(Fig. 2; Ready et al. 1976; Tomlinson, 1985; Tomlinson
and Ready, 1987a).
Mutants in which perfectly normal ommatidia form,
even though they are surrounded by aberrantly patterned ones indicate the positional cues directing the
cells to their fate in the developing ommatidium are
local to the ommatidium itself. Since cells differentiate
as pairs (R2/5, R3/4, Rl/6) then the positional cues
directing the cells of a pair to their fate must be
presented simultaneously on opposite sides of the
ommatidium. A model was proposed to account for
these features (Tomlinson and Ready, 1987a) in which
undetermined cells are cued to their fate by the combination of differing cell types they contact. The model
envisages that differentiating cells express cell-typespecific signals and undetermined cells, which occupy a
precise position in the developing unit, would be in
contact with a specific subset of differentiating types,
the combination of which specify the developmental
pathway of the undetermined cell (Tomlinson and
Ready, 1987a). A prediction made from this model is
that mutations should be recoverable that interfere with
cells' abilities to express signals and others that prevent
cells from receiving the signals. Both types of mutation
would be expected to cause cells to be developmentally
misdirected. In any particular mutant the pattern formation should be normal up to the point when the
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particular gene product is used and then the developmental error should occur. Once mutations have been
identified mosaic analysis can be used to assess whether
the mutation is in a gene used on the signalling or
reception side of the mechanism, and to determine in
which cells of the ommatidium the gene product is
required. Mosaic analysis is performed by inducing a
patch of mutant tissue in an otherwise wild-type eye.
Where the two tissue types meet the cells of the
different genotypes mix freely and ommatidia containing cells of both types form. The questions that can be
asked by mosaic and other analyses are: (1) Can
surrounding wild-type cells rescue mutant cells from
their inappropriate developmental pathway, indicating
a role of the gene product on the signalling side of the
mechanism? (2) Which specific cells must carry the gene
product for the ommatidium to form correctly and do
these correspond to the cells that behave wrongly in the
mutant? After cloning the gene, its nucleotide sequence
may indicate the type of protein encoded, and antibodies raised against the protein can be used to establish in which cells and where in these cells the protein is
found. The developmental analysis of the mutant, the
mosaic analysis, the nucleotide sequence of the gene
and the spatial and temporal localisation of the protein
can be collectively assessed for indications of the gene's
function in the communication mechanism.
To date there are three genes identified that have
been shown to have a role in the communication of the
developmental directives between the cells, sevenless
and bride of sevenless are genes used in determining the
R7 cell type and rough is used to establish R3 and R4.
sevenless
sevenless is a mutation that causes each ommatidium to
specifically lack R7 (Harris et al. 1976). Analysis of the
developmental phenotype showed that, although a cell
occupies the position in the developing ommatidium
that normally generates R7, it fails to differentiate as
that cell type, becoming instead a lens-secreting cone
cell. Occupation of the correct developmental position
by a cell which then fails to differentiate as the type
normal to that position is the phenotype expected of
mutations in genes used in the communication mechanism. From mosaic analysis, it has been shown that
genetically sevenless cells in the R7 position cannot be
rescued from transformation to the cone cell by surrounding wild-type cells (Harris et al. 1976; CamposOrtega et al. 1979; Tomlinson and Ready, 1987ft). This
indicates that the cell in the R7 position is supplied with
the correct positional signals but is incapable of receiving or interpreting them. The nucleotide sequence of
the gene correlates well with this, showing that the gene
encodes a putative trans membrane protein with a large
extracellular domain and an intracellular tyrosine
kinase, similar in general structure to hormone receptors such as the EGF receptor and insulin receptor
(Hafen etal. 1987). This led to the proposal that the
sevenless protein transduces signals for the R7 developmental pathway by binding of a signalling ligand to the
large extracellular domain and subsequent modulation
62
A. Tomlinson
of the tyrosine kinase activity internally within the cell.
A single amino acid substitution within the tyrosine
kinase domain of the protein (known in similar proteins
to abolish kinase activity) eliminates sevenless gene
function, indicating that the signal transduction operates through the kinase domain (Basler and Hafen,
1988). Localisation of the protein indicates it is expressed in many cells (including the presumptive R7) in
their apical plasma membranes (Bannerjee etal. 1987;
Tomlinson et al. 1987). Expression of a receptor protein
in more cells than those in which it is required is
expected of a positional signalling system since prior to
occupying a specific position a cell must be able to
differentiate as one of many cell types, including the R7
type, which requires the sevenless protein. In cells that
express the sevenless protein and contact R8, an accumulation of the protein is seen where the cells meet
R8 in the apical cell junctions (Tomlinson etal. 1987).
This suggested that a ligand for the sevenless protein is
expressed by R8, and a requirement for signals from R8
for R7 to develop will be described below. By reintroducing the sevenless gene under an inducible (heat
shock) promoter it has been demonstrated that sevenless protein is required in a short temporal window of
ommatidial development, correlating well with the few
hours that the protein is detected in the presumptive R7
by antibody analysis (Basler and Hafen, 1989). Blanket
expression of the protein using heat shock does not
interfere with the R7 developmental decision, neither
does it affect the development of the rest of the fly,
indicating that sevenless activation is achieved by a
precise spatial restriction of its ligand(s).
a wide variety of combinations can be mutant without
affecting ommatidial development. However, the developmental analysis indicates that R2 and R5 probably
develop normally in the mutant, R3 and R4 are the cells
that clearly behave inappropriately. This then indicates
a developmental communication between R2/5 and
R3/4. After R3 and R4 misbehave the ommatidia carry
on building in variable and uninterpretable ways. Cells
joining the ommatidia later than R3 and R4 may also
need R2 and R5 to have the rough gene product, but
from the analyses performed these requirements would
remain undetected.
The nucleotide sequence of the rough gene shows it
contains a homeobox which suggests a role for the
protein in DNA binding and transcriptional regulation.
Since the cells in which the protein is required appear
normal in the mutant, neighbouring cells being the ones
affected, then it is suggested that the rough protein acts
as a transcription factor regulating the expression of
signals by R2 and R5 communicated to the cells
destined to become R3 and R4 (Tomlinson et al. 1987).
From these analyses it is becoming clear that developmental cues can be presented by directly adjacent
cells. Whether these signals are held on the expressing
cells' plasma membranes or whether they are shortrange diffusable molecules is not clear. The question to
be addressed is how applicable very short-range communication is to development generally. There are a
large number of developmental phenomena that could
be explained by positional signals being passed between
directly neighbouring cells, but in general this has yet to
be demonstrated. The power of the genetic and mosaic
analyses in Drosophila has allowed this short range
communication to be detected, but finding similar
signalling mechanisms in animals lacking experimental
advantages will be difficult.
bride of sevenless
bride of sevenless is a mutation with a similar phenotype
to sevenless in that each ommatidium specifically lacks
R7. Mosaic analysis has shown that unlike sevenless, R7
can be rescued by surrounding wild-type cells. More
specifically, the bride of sevenless gene product is
required in R8, and only R8, for the ommatidium to
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