Download Boundary formation in the hindbrain

260
Review
TRENDS in Neurosciences Vol.25 No.5 May 2002
Boundary formation in the hindbrain:
Eph only it were simple…
Julie E. Cooke and Cecilia B. Moens
Segmentation of the vertebrate hindbrain into rhombomeres is a key step in
the development of a complex pattern of differentiated neurons from a
homogeneous neuroepithelium. Many of the transcription factors important
for establishing the segmental plan and assigning rhombomere identity are
now known. However, the downstream effectors that bring about the
formation of rhombomere boundaries are only just being characterized. Here
we discuss molecules that could be responsible for segregating populations of
cells from different rhombomeres. We focus on recent work demonstrating that
the Eph family of receptor tyrosine kinases and their ligands, the ephrins,
function in rhombomere-specific cell sorting and initiation of a structural
boundary. We discuss the contributions of two mechanisms – cell sorting and
plasticity – to the formation of rhombomere boundaries.
Julie E. Cooke*
Cecilia B. Moens
Howard Hughes Medical
Institute, Division of Basic
Sciences, Fred
Hutchinson Cancer
Research Center B2-152,
1100 Fairview Avenue N.,
Seattle, WA 98109, USA.
*e-mail: jcooke@
fhcrc.org
The segments, or rhombomeres, of the vertebrate
hindbrain are visible transiently during development
as a series of seven bulges in the neuroepithelium.
They underlie reiterated patterns of neuronal
differentiation [1–4] and neural crest specification
[5,6] but also develop rhombomere-specific features.
The appearance of morphologically visible
rhombomeres requires the segment-restricted
expression of genes encoding transcription factors
such as the Hox group 1–4 proteins, Krox20 and
Valentino/Kreisler/MafB [7–10]. The expression
boundaries of these transcription factors and some of
their downstream targets are initially diffuse
(Fig. 1a) but eventually sharpen [11] (Fig. 1b) and
prefigure the positions of rhombomere boundaries
(Fig. 1c). A rhombomere boundary can initially be
considered as the interface between adjacent
segments; however, a ‘boundary zone’, displaying
specific cell types, cell behaviors, histology and gene
expression, subsequently develops [1,3,12–16]
(Fig. 1d).
What mechanisms underlie rhombomere
boundary formation? The sharpening of gene
expression domains, an early step in boundary
formation, occurs by at least two mechanisms (Fig. 2):
the sorting of cells with a particular segment identity
from cells with a different identity (a cell
sorting-based mechanism), and the regulation of
segment identity in cells that find themselves on the
‘wrong’ side of a presumptive boundary (a cell
plasticity-based mechanism). Selective cell death
could also eliminate such ‘wrongly placed’ cells,
although no evidence exists for increased apoptosis at
presumptive rhombomere boundaries. Here we
review recent evidence suggesting that both cell
sorting and plasticity contribute to boundary
formation and maintenance during hindbrain
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development, and we discuss the underlying
molecular mechanisms.
Identifying the problem: how experimental embryology
defined the questions
Clonal analysis in the developing chick hindbrain
provided the first evidence that vertebrate
rhombomeres are polyclonal units of cell-lineage
restriction [17]. A cellular mechanism for lineage
restriction was suggested by grafting experiments
using chick and quail. Such experiments
demonstrated that cells from neighboring
rhombomeres, or from three rhombomeres apart,
have adhesive differences that prevent them from
mixing with each other, and that a lack of cell mixing
correlates with a tendency to form boundaries [18,19]
(Fig. 3). Selective cell mixing was also seen when cells
from different rhombomeres were dissociated, mixed
and then allowed to re-aggregate [20] (Fig. 3).
It is an oversimplification to suggest that all
odd-numbered rhombomeres share a particular
adhesive property and all even-numbered
rhombomeres share a different adhesive property, as
the degree of cell mixing varied within each category
of rhombomere combination (e.g. neighboring
rhombomeres, or three rhombomeres apart) [18–20].
However, there are clearly differences in the adhesive
properties of different rhombomeres that could
prevent cell mixing between adjacent segments once
boundaries are established. Moreover, if these
adhesive differences are established at earlier stages
they could also drive cell sorting during sharpening of
gene expression boundaries (Fig. 1a,b). An obvious
question arising from these grafting and aggregation
experiments is: what are the molecules responsible
for rhombomere-specific adhesive properties?
Candidates for a role in rhombomere-specific cell
sorting: the Eph and ephrin family
Eph family molecules are expressed in segmentally
restricted domains
Several members of the Eph family of receptor
tyrosine kinases, and their membrane-bound
ligands the ephrins, are candidate effectors for
segment-specific cell sorting: they are repellent
cell-surface molecules expressed in alternating
presumptive rhombomeres [21–25] (Box 1). For
example, Epha4 is strongly expressed from early
stages in the presumptive odd-numbered
rhombomeres r3 and r5, where it is a direct
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Review
TRENDS in Neurosciences Vol.25 No.5 May 2002
261
(i.e. odd- or even-numbered) segments [33–36]. Some
species-specific differences also exist. For example,
Efnb2 (the gene encoding ephrin-B2), has two
orthologs in the zebrafish: ephrin-B2a and
ephrin-B2b. These zebrafish genes are expressed in
r1, r4, r7 and in r1, r4, respectively [37]. Candidate
Eph–ephrin interfaces have not yet been found for all
boundaries in all species.
Overexpression of dominant-negative Epha4 in
Xenopus and zebrafish resulted in ectopic expression
of r3 and r5 markers in the adjacent even-numbered
segments [29] (Fig. 4a). Whether the effect was due to
abnormal cell sorting, a disruption of the dynamic
regulation of gene expression (cell plasticity), or both,
was not ascertained.
(a)
(b)
(c)
Functional studies reveal a role for Eph signaling in cell
sorting
(d)
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Fig. 1. Stages of rhombomere boundary formation. Schematic dorsal
views of part of the developing vertebrate hindbrain (left side of each
panel) and dorsal views of flat-mounted zebrafish embryo hindbrains at
corresponding stages (right side of each panel). Anterior is to the left;
scale bars, 50 µm. (a) Genes expressed in restricted domains
(represented in red and blue) within the anteroposterior axis of the
hindbrain initially show diffuse boundaries. For example, krox20
expression (shown on the right as blue signal following in situ
hybridization) shows diffuse boundaries in presumptive r3 and r5 at
bud stage (10 hours post-fertilization), (b) Gene expression domain
boundaries progressively sharpen to form straight interfaces.
At 18 somites (18 hours post-fertilization), domains of krox20
expression are sharply restricted in presumptive r3 and r5. (c) Gene
expression domain boundaries coincide with structural boundaries;
actin accumulation (shown on the right as red signal after alexa-redphalloidin staining) transiently delineates rhombomere boundaries
(white arrowheads). (d) Mature rhombomere boundary zones are
characterized by large intercellular spaces (white dots) and
concentrations of axons. Different types of cell differentiate at
stereotypical positions with respect to the boundary (indicated by
gradient of shading across each rhombomere). Expression of mariposa
(shown on the right as blue signal following in situ hybridization) is
localized to rhombomere boundary zones.
transcriptional target of the early patterning gene,
krox20 [26]. These expression domains are
conserved in the mouse, Xenopus, zebrafish and
chick [21,27–30]. Ephrin-B2, a ligand for EphA4, is
expressed in even-numbered rhombomeres r2, r4
and r6 in mouse and Xenopus [24,25,31,32]. Thus, in
some species, EphA4–ephrin-B2 interfaces coincide
with all boundaries from r2 to r6.
To complicate matters, a number of other Eph
family molecules are expressed in presumptive
rhombomeres, but not necessarily in alternating
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Subsequent studies in zebrafish showed that Eph
signaling is important for rhombomere-specific cell
sorting [37–39]. Cells overexpressing full-length or
truncated EphA4 could contribute to odd-numbered
rhombomeres but were preferentially distributed to
the boundaries of even-numbered rhombomeres in
mosaic embryos. Conversely, cells overexpressing
full-length or truncated ephrin-B2 contributed to
even-numbered rhombomeres but localized to the
boundaries of odd-numbered rhombomeres [38]
(Fig. 4b). The sorting of cells expressing Eph and/or
ephrin constructs occurred during the same stages as
the sharpening of gene-expression domain
boundaries [11] (Fig. 1a,b), suggesting a role for
Eph-mediated cell sorting in this process.
A cell-aggregation assay was used to evaluate
mixing between adjacent populations of cells
overexpressing combinations of full-length and
truncated Eph receptors and ephrins [39] (Fig. 4c).
In contrast to the mosaic overexpression studies [38],
unidirectional signaling was not sufficient to restrict
intermingling in this assay (Fig. 4cii); rather,
bidirectional signaling was required (Fig. 4ciii).
Perhaps additional mechanisms compensated for the
lack of bidirectional signaling within the context of the
hindbrain, allowing cell sorting to occur. However, both
unidirectional and bidirectional signaling correlated
with a loss of gap-junctional communication
(Fig. 4cii,iii), a feature of rhombomere boundaries
in vivo [13]. Intriguingly, truncated (signal
transduction-defective) forms of Eph receptors and
ephrins can exist in vivo as a result of alternative
processing [40–44]; it is not known whether such forms
are expressed or function in the developing hindbrain.
Zebrafish embryos with a null mutation in
valentino (val, homologous to mouse kreisler [45])
lack rhombomere boundaries in the caudal hindbrain
[10]. Val is required to establish complementary
domains of ephB4a and ephrin-B2a expression in the
caudal hindbrain (Fig. 4di,ii); loss of ephB4a–ephrinB2a expression domain interfaces in val mutants is
correlated with the absence of rhombomere
TRENDS in Neurosciences Vol.25 No.5 May 2002
(c)
(a)
(b)
(e)
Cell sorting
(d)
Jagged boundary of
krox20 expression
Sharp boundary of
krox20 expression
Cell plasticity
boundaries [37] (Fig. 4dii). In genetic mosaics, the
repulsion of val– donor cells from wild-type r5 and r6
(rhombomeres that normally express val and ephB4a
[10,36,45]) is inhibited when bi-directional Eph
signaling is blocked [37]. This suggests that
inappropriate Eph signaling underlies the repulsion
of val– cells from r5 and r6. Repulsion of mutant cells
that are unable to adopt the appropriate segmental
identity might be an exaggerated form of the cell
Nd
r4
Nd
r5
r6
Nd
r3
r4
r5
Nd
r6
Nd
Yes
r2
r3
Not
usually
r2
No
Fig. 2. Mechanisms of
rhombomere boundary
formation. Cells on the
wrong side of a
presumptive
rhombomere boundary
[expressing krox20 in
presumptive
rhombomere 2 in this
example; arrowed in (a),
schematized in (b)] can
either move to the other
side of the boundary [as in
(c); a cell sorting-based
mechanism] or regulate
their gene expression to
match that of their
neighbors [as in (d); a cell
plasticity-based
mechanism]. In either
case, the result is that the
boundary sharpens
(e). Scale bar, 50 µm.
Review
Boundary formation?
262
Cell mixing
Low Medium High
TRENDS in Neurosciences
sorting that normally occurs at rhombomere
boundaries. Because blocking Eph signaling alters
this cell repulsion, Eph molecules are implicated in
normal cell sorting at rhombomere boundaries.
Transplanting wild-type donor cells into the
presumptive hindbrain of a val– host causes
reconstitution of an ephB4a–ephrin-B2a interface
that co-localizes with actin accumulation, a transient
indicator of boundary formation (Fig. 4diii). Thus,
induction of an Eph–ephrin interface is correlated
with initiation of a structural boundary [37].
Signaling downstream of Eph receptors and ephrin-B
ligands results in cytoskeletal changes (Box 1) that
not only underlie the motile behavior required for cell
sorting but presumably also trigger changes in cell
shape and/or polarity required for initiation of a
structural boundary.
In summary, Eph receptor–ephrin-B signaling
influences segment-specific cell sorting, suggesting
that it has a role in sharpening of rhombomerespecific gene expression boundaries. The observations
that Eph signaling affects gap junctions and
influences cytoskeletal rearrangements further
suggest a role for Eph signaling in triggering the
cascade of cell behavioral responses that initiate
structural boundary formation, and that culminate in
the emergence of a mature boundary zone.
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Fig. 3. Boundary formation correlates with a lack of cell mixing in
grafting and cell-aggregation assays. Combinations of identical
rhombomeres (r) allow extensive cell mixing (dark blue). No boundary
is formed in this situation (dark green). Combinations of two odd- or
two even-numbered rhombomeres allow cell mixing, but to a lesser
extent than combinations of identical rhombomeres (mid-blue). For
r3 with r5, no new boundary is formed (dark green), for r2 with r4 and r4
with r6, in most cases no new boundary is formed (mid-green).
Combinations of adjacent rhombomeres or rhombomeres three
segments apart correlate with a lack of cell mixing (light blue) and new
boundary formation (light green). Abbreviation: Nd, not determined.
Data summarized from Refs [18–20].
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Could Notch–Fringe signaling participate in
rhombomere boundary formation?
Fringe-mediated modulation of the Notch signaling
pathway is important for boundary formation in
systems as diverse as the Drosophila wing disc and
the vertebrate CNS (reviewed in Refs [46,47]). Mouse
Lunatic fringe (L-fng) is sufficient to direct cell
sorting and maintain compartmental integrity of the
zona limitans intrathalamica (zli), a subdivision of
the forebrain [48].
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TRENDS in Neurosciences Vol.25 No.5 May 2002
263
Box 1. Ephs and ephrins: a handle on the molecular control of morphogenesis
Eph receptors are the largest subfamily of receptor tyrosine
kinases [a]. Their ligands, the ephrins, are membranebound, either via a glycosyl phosphatidylinositol linkage
(ephrin-A ligands) or via integral transmembrane and
intracellular domains (ephrin-B ligands) [b]. Eph receptors
are subdivided into EphA and EphB classes on the basis of
ligand-binding preference and sequence homology [b].
Binding within a class is promiscuous but not uniform;
EphA4 is currently the only receptor known to bind ephrins
of both classes [c]. Ephrins need to be membrane-bound or
artificially clustered in order to dimerize and activate Eph
receptor signaling [d].
The intracellular domains of ephrin-B ligands become
tyrosine phosphorylated on receptor binding, owing to
activity of cytoplasmic kinases [e,f]. Thus, contact of an
Eph-expressing cell with an ephrin-B-expressing cell
results in bi-directional induction of downstream signaling
events (i.e. in both cells). Signaling downstream of the
receptor is regarded as ‘forward’ signaling, whereas
ephrins are regarded as transducing ‘reverse’ signaling
events. Truncated Eph receptors and ephrin-B ligands (i.e.
those lacking their intracellular domains but retaining their
extracellular and transmembrane domains) can activate
full length ephrin-B ligands and EphB receptors,
respectively, but are themselves incapable of transducing
signals back into their own cell (resulting in uni-directional
signaling). Soluble, monomeric forms of ephrin-B ligands
block bi-directional signaling because they bind, but do not
cluster and activate, Eph receptors [d], and they
competitively inhibit binding of Eph receptors to
endogenous ephrin-B ligands [g,h].
The signaling pathways downstream of Eph receptors and
ephrins are becoming better understood [i–m]. Eph signaling
can trigger rearrangements of actin- and/or microtubulebased cytoskeletal elements [n,o]. In some cases, this is via
interactions with guanine nucleotide exchange factors that
influence activation of Rho family GTPases [p,q], providing a
mechanism for Eph-induced repulsive guidance or growthcone collapse. Activated ephrin-B ligands associate
intracellularly with Grb4, an intermediate required for ephrinB-dependent cytoskeletal regulation [l].
Expression of Eph receptors and ephrins is often in
complementary domains, [c] with interactions presumably
occurring at domain interfaces. Eph receptors and ephrins
function as repulsive guidance molecules for migrating
neuronal growth cones (e.g. in the formation of the
retinotectal topographic map [r]) and neural crest cells
[g,s,t]. However, not all Eph interactions are repulsive, and
in some contexts adhesive effects can be elicited [u–y]
(reviewed in Ref. [m]).
References
a Flanagan, J.G. and Vanderhaeghen, P. (1998) The ephrins
and Eph receptors in neural development. Annu. Rev.
Neurosci. 21, 309–345
b Eph Nomenclature Committee (1997) Unified nomenclature
for Eph family receptors and their ligands. Cell 90, 403–404
c Gale, N. et al. (1996) Eph receptors and ligands comprise two
major specificity subclasses and are reciprocally
compartmentalized during embryogenesis. Neuron 17, 9–19
d Davis, S. et al. (1994) Ligands for EPH-related receptor
tyrosine kinases that require membrane attachment or
clustering for activity. Science 266, 816–819
e Bruckner, K. et al. (1997) Tyrosine phosphorylation of
transmembrane ligands for Eph receptors. Science 275,
1640–1643
Interfaces between L-fng and Manic fringe
(M-fng)-expressing and non-expressing cells
prefigure mouse rhombomere boundaries [49]
http://tins.trends.com
f Holland, S. et al. (1996) Bidirectional signalling through the
Eph-family receptor Nuk and its transmembrane ligands.
Nature 383, 722–725
g Krull, C. et al. (1997) Interactions of Eph-related receptors
and ligands confer rostrocaudal pattern to trunk neural crest
migration. Curr. Biol. 7, 571–580
h Durbin, L. et al. (1998) Eph signalling is required for
segmentation and differentiation of the somites. Genes Dev.
12, 3096–3109
i Bruckner, K. and Klein, R. (1998) Signaling by Eph
receptors and their ephrin ligands. Curr. Opin. Neurobiol. 8,
375–382
j Holder, N. and Klein, R. (1999) Eph receptors and ephrins:
effectors of morphogenesis. Development 126, 2033–2044
k Schmucker, D. and Zipursky, S.L. (2001) Signaling
downstream of Eph receptors and ephrin ligands. Cell 105,
701–704
l Cowan, C.A. and Henkemeyer, M. (2001) The SH2/SH3
adaptor Grb4 transduces B-ephrin reverse signals. Nature
413, 174–179
m Klein, R. (2001) Excitatory Eph receptors and adhesive ephrin
ligands. Curr. Opin. Cell Biol. 13, 196–203
n Meima, L. et al. (1997) Lerk2 (ephrin-B1) is a collapsing factor
of a subset of cortical growth cones and acts by a mechanism
different from AL-1 (ephrin-A5). Mol. Cell. Neurosci. 9,
314–328
o Meima, L. et al. (1997) AL-1-induced growth cone collapse of
rat cortical neurons is correlated with REK7 expression and
rearrangement of the actin cytoskeleton. Eur. J. Neurosci. 9,
177–188
p Wahl, S. et al. (2000) Ephrin-A5 induces collapse of growth
cones by activating Rho and Rho kinase. J. Cell Biol. 149,
263–270
q Shamah, S.M. et al. (2001) EphA receptors regulate growth
cone dynamics through the novel guanine nucleotide
exchange factor ephexin. Cell 105, 233–244
r Drescher, U. et al. (1995) In vitro guidance of retinal ganglion
cell axons by RAGS, a 25 kDa tectal protein related to the
ligands for Eph receptor tyrosine kinases. Cell 82, 359–370
s Wang, H. and Anderson, D. (1997) Eph family
transmembrane ligands can mediate repulsive guidance of
trunk neural crest migration and motor axon outgrowth.
Neuron 18, 383–396
t Smith, A. et al. (1997) The EphA4 and EphB1 receptor
tyrosine kinases and ephrin-B2 ligand regulate targeted
migration of branchial neural crest cells. Curr. Biol. 7,
561–570
u Orioli, D. et al. (1996) Sek4 and Nuk receptors cooperate in
guidance of commissural axons and in palate formation.
EMBO J. 15, 6035–6049
v Holmberg, J. et al. (2000) Regulation of repulsion versus
adhesion by different splice forms of an Eph receptor. Nature
408, 203–206
w Becker, E. et al. (2000) Nck-interacting Ste20 kinase couples
Eph receptors to c-Jun N-terminal kinase and integrin
activation. Mol. Cell. Biol. 20, 1537–1545
x Gu, C. and Park, S. (2001) The EphA8 receptor regulates
integrin activity through p110 γ-phosphatidylinositol-3
kinase in a tyrosine kinase activity-independent manner. Mol.
Cell. Biol. 21, 4579–4597
y Knöll, B. et al. (2001) A role for the EphA family in the
topographic targeting of vomeronasal axons. Development
128, 895–906
and boundaries between high- and low-level
expression of the L-fng homolog, lfng, prefigure
zebrafish rhombomere boundaries [50]. Moreover, in
Review
264
(a)
r3
(c) (i)
(r4)
(b)
r5
No Eph
signaling
r3
(ii) Uni-directional
signaling
Cell mixing?
Yes
Gap junctions? Yes
(d)
r4
r4
rX
r5
r7
r6
(iii) r4
r4
r5
(iii) Bi-directional
signaling
Cell mixing?
Yes
Gap junctions? No
(i)
(ii)
TRENDS in Neurosciences Vol.25 No.5 May 2002
Cell mixing?
No
Gap junctions? No
r7
rX
r7
TRENDS in Neurosciences
Fig. 4. Eph signaling and boundary formation. Schematics of dorsal views of the hindbrain [a, b, d;
anterior is towards the left, rhombomeres (r) are numbered] and of a cell aggregation assay (c).
(a) Expression of krox20 and ephA4 (red) is normally restricted to r3 and r5. Disruption of EphA4
signaling results in ectopic expression of r3 and r5 markers (red) in r4 territory (blue). Data
summarized from Ref. [29]. (b) Mosaic activation of Eph signaling. Mosaic zebrafish embryos were
made by injecting constructs encoding full length or truncated Eph receptors or ephrins into one cell
at the eight-cell stage. The distribution of ectopically expressing cells was analyzed after rhombomere
boundaries had formed. Results of all experiments (overexpression of receptor constructs and of
ephrin constructs) are schematized in one panel, but were performed separately. Cells overexpressing
full-length or truncated EphA4 receptors are localized in odd-numbered rhombomeres or boundaries
of even-numbered rhombomeres (red ovals), cells overexpressing full-length or truncated ephrin-B2
are localized in even-numbered rhombomeres or at boundaries of odd-numbered rhombomeres
(blue ovals). Data summarized from Ref. [38]. (c) Uni-directional versus bi-directional Eph signaling.
Individual animal caps were dissected from fluorescently labeled zebrafish embryos overexpressing
full-length or truncated Eph receptor or ephrin. These were juxtaposed and cultured overnight, and
the distribution of the two cell populations was assessed by confocal microscopy. Gap junctional
communication was assessed by examining transfer of Lucifer Yellow dye between the cells. (i) In
control assays (embryos injected with fluorescent dye only), the two cell populations (represented by
red and green circles) mixed, and gap junctional communication occurred. (ii) With uni-directional
Eph signaling (through Eph receptors or ephrins), the two populations mixed, but gap junctional
communication was disrupted. (iii) With bi-directional Eph signaling (through Eph receptors and
ephrins) the two populations did not mix and a clear border was visible. Gap junctional
communication was also disrupted. Data summarized from Ref. [39]. (d) Eph signaling and Valentino
in cell sorting and boundary formation. (i) In the caudal hindbrain of wild-type zebrafish, ephB4a is
coexpressed in r5 and r6 with the valentino (val) transcription factor (red) and ephrin-B2a is expressed
in r4 and r7 (blue). The ephB4a–ephrin-B2a expression domain interfaces correspond to the r4–5 and
r6–7 boundaries (black lines represent boundaries). (ii) In val mutants, the region between r4 and r7 is
shortened by the length of one rhombomere, has a mixed identity and is referred to as rX [10]. EphB4a
expression is lost from the caudal hindbrain and ephrin-B2a expression (blue) is upregulated. The
loss of ephB4a–ephrin-B2a expression domain interfaces correlates with a loss of boundaries in the
val mutant. (iii) Wild-type donor cells expressing val and ephB4a (red) aggregate into clumps (arrows)
when transplanted into rX of a val mutant host (which expresses ephrin-B2a; blue). Actin
accumulation at the new ephB4a–ephrin-B2a interface (green) signifies formation of a structural
boundary. Data summarized from Ref. [37].
val mutants, reminiscent of the loss of
ephB4a–ephrin-B2a expression domain interfaces
(see above), a loss of the interface between high- and
low-level lfng expression domains is correlated with
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an absence of boundaries [50]. It will be interesting to
determine whether members of the Fringe–Notch
signaling pathways are required to establish and/or
maintain rhombomere boundaries, and whether they
interact with members of the Eph signaling pathway
in this process.
Other cell-surface effectors of segmentation: cadherins
and integrins?
Segmentation need not rely solely on repulsive
interactions; preferential cohesion between like cells
could also play a role. Disruption of Ca2+-dependent
adhesion abolishes rhombomere-specific cell
segregation [20], implicating cadherins (reviewed in
Refs [51,52]), and integrins (reviewed in Ref. [53]) in
rhombomere-specific cell sorting. Indeed, sorting
experiments show that cells expressing different
cadherins segregate from each other [54,55].
Furthermore, differential expression of two cadherins
is important for restricting cell movement between
adjacent territories at the cortico–striatal boundary
in the mouse forebrain [56]. However, it should be
noted that subtle differences in the levels of
expression of one particular cadherin are sufficient to
separate two cell populations [57,58].
Are any cadherins expressed appropriately for a
role in rhombomere-specific cell sorting? Mouse
cadherin-6 is a good candidate, because it is initially
expressed in the caudal hindbrain up to the r4–r5
boundary, but is subsequently expressed only in r6.
Thus, it sequentially delineates different prospective
rhombomere boundaries [59]. By virtue of its
expression pattern, cadherin-6 is a potential target of
kreisler, suggesting a direct link between a
segmentation gene and an effector of cell sorting.
A thorough expression analysis of other cadherins
could uncover further candidates [52].
Involvement of the repulsive Eph-based system
and the homotypic cadherin-based system are by no
means mutually exclusive. Indeed, the two pathways
could interact. Cadherins have been implicated in
regulation of Eph function [60,61], suggesting
coordination of the two systems. Conversely, Eph
signaling has been shown to regulate integrinmediated adhesion [62–64] (Box 1; reviewed in
Ref. [65]), although the current literature is
confusing: integrin-mediated adhesion appears to be
downregulated by EphA receptors, upregulated by
ephrin-A ligands [62–64], and both up- and
downregulated by EphB receptors [66,67]. The
relevance of Eph and/or ephrin interactions with
integrins to rhombomere boundary formation is
therefore still unclear.
A role for plasticity in sharpening boundaries?
Repulsive interactions between cells from different
segments, and adhesive interactions between cells
from the same segment, might be sufficient to provide
a lineage-restriction-based mechanism for both
establishing and maintaining rhombomere
Review
TRENDS in Neurosciences Vol.25 No.5 May 2002
265
Questions for future research
• What are the relative contributions of cell sorting and of
cell plasticity to boundary formation? The involvement of
each of these two mechanisms can be determined by
studying the behavior of individual cells that naturally
find themselves on the wrong side of a boundary, such as
the cells arrowed in Fig. 2a of the main text. Do such cells
move into r3, or do they downregulate their expression of
krox20? It will be possible to address this question using
animals expressing in vivo reporter genes in
rhombomere-specific patterns. With such tools, the
behavior of misplaced cells can be followed in living
embryos.
• What is the function of a rhombomere boundary? Is it
required to restrict cell movement, to confine gene
expression domains and/or to specify neuronal
patterning across the segment? Models in which
boundaries are disrupted tend to have altered expression
of segmental identity genes that encode transcription
factors with a large number of targets [a–e], complicating
interpretation of the phenotype. Null mutations in genes
encoding Eph receptors or ephrins that are expressed
within the hindbrain have so far failed to show a
hindbrain phenotype [f–h]. This could reflect a subtle
defect, redundancy within the Eph family or between Eph
molecules and cadherins, or a compensatory role for
plasticity. Specific ablation of the boundaries, perhaps by
simultaneously disrupting the function of a number of
Eph molecules, could help to answer these questions.
Acknowledgements
We would like to thank
Tom Schilling,
Andrew Waskiewicz
and Charles Kimmel for
helpful comments on the
manuscript. J.C. is the
recipient of a Wellcome
Trust Prize Travelling
Research Fellowship,
C.M. is an Assistant
Investigator with the
Howard Hughes Medical
Institute.
boundaries. However, experimental observations of
plasticity of segmental identity (the ability of a cell to
alter its expression of segment-specific genes) in the
hindbrain suggest that a dynamic regulation of gene
expression could also play a role in establishing sharp
boundaries [68]. Cell sorting and cell plasticity
(Fig. 2) could be redundant mechanisms, together
guaranteeing precise rhombomere boundary
formation if one mechanism fails. Alternatively, the
two mechanisms could be complementary, acting with
different temporal specificities.
Individual hindbrain cells can regulate their
expression of segment-specific genes in response to
positional cues. In a recent study, the majority of
single cells transplanted between zebrafish
rhombomeres showed plasticity with respect to
expression of two Hox genes [69]. Furthermore,
in a series of inter-rhombomere transplants
in the mouse, donor cells that remained in a coherent
group autonomously maintained Hox expression
appropriate to their position of origin (an example of a
‘community effect’), whereas individual cells that
became separated from the primary graft exhibited
plasticity with respect to Hox expression [70].
In support of a role for plasticity in boundary
formation, a recent study indicated that plasticity is
the main mechanism underlying the sharp
demarcation between Otx2-expressing midbrain cells
and Gbx2-expressing hindbrain cells in the chick [71].
The midbrain–hindbrain boundary is maintained by
labile fates and mutual repression of Otx2 and Gbx2
gene expression, rather than by cell-lineage
restriction.
http://tins.trends.com
References
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At what stages might plasticity contribute to
rhombomere boundary formation? The clonal
analysis of Fraser and colleagues [17] showed that
cells become lineage-restricted only after the
appearance of morphological boundaries, strongly
suggesting the involvement of a mechanism such as
plasticity at stages before overt boundary formation.
Consistent with this, the ability of single cells to
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Determining gene expression in boundary-violating
cells would answer some outstanding questions
concerning plasticity.
If cells normally regulate their identity even after
boundary formation, it is possible that hindbrain
266
Review
TRENDS in Neurosciences Vol.25 No.5 May 2002
compartment boundaries are regularly breached.
For the sharply defined segmental organization of
the hindbrain to be maintained under these
circumstances, cells would continually need to
re-assess their position with respect to patterning
signals in their environment. Evidence from
quail–chick and mouse–chick chimeras suggests
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identity under the influence of its immediate
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local signals. Thus, even if a strict lineage-based
mechanism is sufficient to establish and maintain
rhombomere boundaries, additional mechanisms
are available to fine-tune the segmental pattern of
the hindbrain.
It is possible that the dramatic cell-sorting
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mosaic and rhombomere grafting experiments was
exaggerated because cells were unable to change
their identity (either because of a genetic lesion in
the case of val− cells [37], or community effects in
the case of the chick grafting experiments [18,19]).
It could be that when cells are unable to change
their identity, cell sorting is the only mechanism
available for boundary formation, but that under
normal circumstances cell sorting and the
regulation of cell identity both contribute to
boundary formation.
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