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microscopic and biochemical evidence that chromatin
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40. Wolffe, A. Chromatin Structure and Function
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41. Kornberg, R. & Lorch, Y. Twenty-five years of the
nucleosome, fundamental particle of the eukaryotic
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42. Thomas, G. J., Prescott, B. & Olins, D. E. Secondary
structure of histones and DNA in chromatin. Science
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Nucleosome cores have two specific binding sites for
nonhistone chromosomal proteins HMG 14 and HMG
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44. Sandeen, G., Wood, W. I. & Felsenfeld, G. The
interaction of high mobility proteins HMG14 and 17
with nucleosomes. Nucl. Acids Res. 8, 3757–3778
(1980).
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transcription in three dimensions. Science 220,
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46. Olins, A. L., Olins, D. E. & Bazett-Jones, D. P.
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Sargent, D. F. & Richmond, T. J. Crystal structure of
the nucleosome core particle at 2.8 Å resolution.
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Acknowledgements
The authors express their gratitude to Bowdoin College and
to the German Cancer Research Center (Heidelberg) for providing stimulating intellectual and scientific environments. H.
Herrmann and P. Lichter, our generous hosts at the German
Cancer Research Center, supplied helpful comments on the
manuscript. Several anonymous referees made significant
contributions towards the improvement of this essay. The
authors dedicate this review to the memory of H. G. Davis
(formerly at the Department of Biophysics, King’s College,
London), an excellent microscopist and our good friend.
Online links
DATABASES
The following terms in this article are linked online to:
LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/
H2A | H2B | H3 | H4 | HMGN1 | HMGN2 | lamin A | lamin B1 |
lamin B2 | LBR
OMIM: http://www.ncbi.nlm.nih.gov/Omim/
Emery–Dreifuss muscular dystrophy |
Pelger–Huët anomaly
Access to this interactive links box is free online.
OPINION
Can transcription factors function as
cell–cell signalling molecules?
Alain Prochiantz and Alain Joliot
Recent data support the view that
transcription factors — in particular,
homeoproteins — can be transferred from
cell to cell and have direct non-cellautonomous (and therefore paracrine)
activities. This intercellular transfer, based
on atypical internalization and secretion, has
important biotechnological consequences.
But the real excitement stems from the
physiological and developmental
implications of this mode of signal
transduction.
Transcription factors are present in the
nucleus, and sometimes in the cytoplasm, but
on the whole they are not thought to travel
between cells. This is because of their
hydrophilic properties and the absence of a
signal peptide. But there are exceptions and,
in fact, some transcription factors travel
between cells because they contain protein
domains that allow them to do so. This is the
case for the HIV transcription factor TAT1
and for several homeoproteins, such as
Engrailed2,3, Hoxa5, Hoxb4, Hoxc8, Emx1,
Emx2, Otx2 and Pax6 (G. Mainguy, A. Maizel,
A.P. and A.J., unpublished observations). On
the basis of the conservation of the internalization and secretion signals that have been
identified in Engrailed (see below), it is anticipated that this property is shared by most
homeoproteins.
Homeoproteins are known to contribute
to cellular positioning. They were actually dis-
| OCTOBER 2003 | VOLUME 4
covered in the fly on the basis of mutations
that affect the spatial identity of segments and
appendages (for example, antennae can be
transformed into legs). Within a single structure, such as the spinal cord, specific combinations and concentrations of homeoproteins
define the anterior–posterior and dorso–ventral positions of cells. Furthermore, the
homeoprotein Engrailed can define the midbrain and the position of cells within the
anterior–posterior axis of the midbrain. It is
widely thought that homeoprotein function
involves the regulation of genes that encode
signalling molecules such as surface receptors
or growth factors. By contrast, direct
paracrine homeoprotein activity is not generally envisaged, although in theory it represents a parsimonious way for neighbouring
cells to coordinate positional information. So
the ability of homeoproteins to transfer
between cells is extremely exciting. There are
more than 400 of these proteins in mice and
humans, and they are involved in all the main
developmental decisions. Many of them also
function in the control of adult physiology.
For example, Engrailed 1 and Engrailed 2
(EN1 and EN2; collectively known as
Engrailed) are expressed in adult aminergic
nuclei that control motor behaviour, mood
and addiction4.
Because the transfer of positional information is a general phenomenon that occurs
during development and throughout adulthood, because homeoproteins contribute to
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© 2003 Nature Publishing Group
PERSPECTIVES
Homeodomain
Secretion sequence
AQELGLNESQ
Internalization sequence
(Penetratin)
RQIKIWFQNRRMKWKK
Nuclear export sequence
QSLAQELGLNESQIKI
Figure 1 | Functional domains for
homeoprotein intercellular transfer. Within the
homeodomain, three domains that are required
for secretion, internalization and nuclear export
have been characterized by loss-of-function
(deletion) or gain-of-function (synthetic peptides)
studies. The secretion sequence (in green) is part
of the nuclear export sequence (in yellow) and its
deletion blocks nuclear export. The internalization
sequence (in red) has been used as a vector to
introduce cargoes into live cells and is therefore
also known as Penetratin. It is important to note
that the secretion and internalization sequences
are distinct and that their presence within the
homeodomain makes it impossible to define
mutations that would block intercellular transfer
without modifying transcription properties.
encoding such information, and because
homeoproteins can transfer between cells
in vitro, we shall focus on homeoproteins to
outline our model that transcription factors
can function as true signalling proteins. In
support of this hypothesis is the well-established transfer of homeoproteins in higher
plants5, which occurs through intercytoplasmic bridges known as plasmodesmata. It is,
however, noteworthy that the intercellular
transfer of Knotted-1 (KN1), a maize homeoprotein, through plasmodesmata is poorly
affected by conditions that strongly affect the
passage of other types of protein6,7. So there
might be other mechanisms of transfer that
work in parallel.
Homeoprotein entry into cells
The evidence. The finding that the 60-aminoacid DNA-binding domain (homeodomain)
of the Antennapedia homeoprotein translocates across biological membranes was
serendipitous8. But, once observed, the phenomenon had to be investigated further. It was
then shown that Antennapedia homeodomain
internalization properties are conferred by its
third helix (now referred to as Penetratin9;
(FIG. 1)) and that this region absolutely requires
the tryptophan at position 48 (W48) in a basic
environment. Exogenously added fluorescein
isothiocyanate (FITC)-labelled homeodomain
and Penetratin peptides can be detected in the
nucleus of live cells. Internalization is inhibited
by the replacement of W48 by a phenylalanine.
This, and the visualization of internalized
homeodomains and homeoproteins in nonfixed cells8, precludes internalization being a
post-fixation artefact (as has recently been suggested for several basic peptides and proteins10). The property of internalization into
live cells is shared by all homeodomains that
have been tested, probably because the third
helix is highly conserved.
Potential entry mechanisms. Biophysical data
indicate that Penetratin fully translocates into
artificial lipid vesicles11, which confirms that
chiral receptors are not required for internalization. This was also shown by the internalization of a Penetratin peptide composed of
12
D-enantiomers , which cannot, therefore, recognize a protein receptor of opposite chirality
(composed of L-amino acids). Current models of internalization involve peptide binding
to the cell surface through charge interactions
and membrane destabilization after W48
insertion in the bilayer. Destabilization might
assist in the formation of inverted micelles13,
which would allow peptides to translocate
across the membrane and be released into the
cytoplasm. Full-length homeoproteins are
also internalized by live cells14 and, when
tested, the same mutations (for example, the
deletion of W48 in the homeodomain) block
the internalization of the third helix, of the
homeodomain or of the entire protein3,15. So,
it is assumed that the same mechanisms are
responsible in all cases when homeoproteins
are internalized.
identify a short sequence (∆1 sequence; FIG. 1)
that is necessary for secretion. This sequence,
which spans part of helices 2 and 3 of the
homeodomain, is also required for efficient
cEN2 nuclear export16, which indicates that
secreted cEN2 might originate from the
nucleus (FIG. 1; see below). As already mentioned, the homeodomain has a highly
conserved structure and this nuclear
export/secretion domain is also highly conserved, indicating that intercellular transfer
might be a conserved property of homeoproteins. Accordingly, intercellular passage has
been confirmed for almost all homeoproteins
that have so far been tested, including Hoxa5,
Hoxb4, Hoxc8, cEN2, Emx1, Emx2, Otx2 and
Pax6 (G. Mainguy, A. Maizel, A.J. and A.P.,
unpublished observations).
Potential secretion mechanisms. Subcellular
localization studies showed that, in cultured
cells, a pool of cEN2 was associated with membrane fractions and, in part, was protected
against proteolysis17. By contrast, the non-secretable variant (∆1 deletion) is accessible to proteases3, which indicates that there might be an
exportable pool of cEN2 (~5% of the entire
Engrailed
Cytosol
D
NLS
?
Nucleus
NES
B
?
NLS
Secretion of homeoproteins from cells
The evidence. The concept of a ‘messenger
protein’ (FIG. 2) requires that transcription factors — in this case, homeoproteins — be also
exported into the extracellular medium. This
is despite the absence of a classical signal peptide, which is also absent from other
hydrophilic proteins — in particular, interleukin-1 (IL-1) and mammalian fibroblast
growth factors (FGFs). In co-cultures of primary neurons and COS-7 cells that express
chick Engrailed 2 (cEN2), cEN2 is found —
intact — in the 100,000 g supernatant (not
in the pellet) of the culture medium and
within the neurons3. This indicates that the
protein has been secreted by the COS cells
into the surrounding medium, and has then
been taken up by the neurons.
Interestingly, the cellular export and
import pathways seem to be distinct, as
mutating W48, which is mandatory for internalization, does not affect cEN2 export.
Systematic mutagenesis has been used to
NATURE REVIEWS | MOLECUL AR CELL BIOLOGY
C
A
Figure 2 | Intercellular transfer of Engrailed
homeoprotein. At equilibrium, Engrailed is
detected in the cytosol and nucleus. In principle,
this dual distribution can result from an early
nucleocytoplasmic partition of newly synthesized
Engrailed (A) or, alternatively, from Engrailed
continuous shuttling between the nucleus and
cytosol owing to its nuclear import (NLS) and
export (NES) sequences (B). Our data favour the
latter hypothesis and indicate that nuclear export
might comprise an important step to gain access
to the secretory compartment. So, Engrailed
transport from the nucleus to the cytoplasm
(binding to transporters, dissociation from DNA)
could be a site of regulation for its secretion. Only
forms of Engrailed that can be secreted are
directed into the lumen of vesicular compartments
(C) by a mechanism that is still undefined but that
is distinct from the internalization mechanism (D).
Engrailed internalization (D) is endocytosis
independent, and requires the third helix of the
homeodomain as the driving domain.
VOLUME 4 | OCTOBER 2003 | 8 1 5
© 2003 Nature Publishing Group
PERSPECTIVES
Growing axon
hydrophilic proteins seems unlikely. In fact, in
genetically well-characterized organisms (such
as bacteria), six different unconventional
secretion pathways have been identified, even
though they usually do not co-exist in the
same organism.
Target
Cytosol
a
Stabilization
a′
b
Collapse
Nucleus
Mature axon
a
a
a′
mRNA
a′
b
Presynaptic axon
Ribosome
Nascent
polypeptide
b
Postsynaptic element
Figure 3 | Hypothetical signalling with homeoproteins. The example taken here is signalling between
either a navigating axon (growing axon) and an intermediate target, or the pre- and postsynaptic elements
of a functioning synapse (mature axon). The main advantage of this model is that, in addition to a classical
signalling activity (for example, a neurotransmitter-induced change in ionic conductance), it also confers
positional information. Indeed, because the combination of homeoproteins expressed within a cell is a
signature of its position in the organism, the cells are reciprocally informed of their topological origins. The
exchange of transcription factors between the axon (growing or mature; blue) and the target cell (green)
can be interpreted at the level of translation (a, a′) or transcription (b). For example, homeoprotein transfer
into the growth cone might regulate (up or down) the translation (a) of a messenger encoding a receptor or
an adhesion molecule, leading to stabilization or turning away (or collapse) of the growth cone.
Reciprocally, homeoprotein passage from the growth cone or nerve terminal into the target cell might
regulate the translation (a′) of messengers (for example, sub-synaptic dendritic messengers in adult
synapses) or the transcription of specific genes (b) after transfer into the target-cell nucleus. Indeed, it
cannot be precluded that, following transfer, homeoproteins show unsuspected physiological activities (for
example, activation of second messengers). Green circles represent proteins originating from the growth
cone or presynaptic axon, and the red circles represent proteins travelling in the opposite direction.
cellular pool) that exists within vesicles. These
vesicles have ‘caveolae-like’ properties, such as
high cholesterol and glycosphingolipid contents, which confer resistance to Triton and
separation in sucrose density gradients17. The
same proportion of endogenous rat EN1 and
EN2 that is expressed in the embryonic mesencephalon is resistant to proteolysis and present in these caveolae-like vesicles in vivo.
Although intercellular transfer has only been
directly visualized in vitro, the presence of EN1
and EN2 in a secretion compartment in vivo
gives strength to the idea that this phenomenon is of physiological relevance.
So how might these homeoprotein-containing vesicles arise? An interesting possibility is that homeoproteins are incorporated
into nascent vesicles as they form; exosomes,
with their inside–out membrane orientation
(their cytosolic face is inside), are ideal candidates. In fact, galectin-3 and annexin II — two
816
secreted hydrophilic proteins — have been
detected in exosomes by mass spectroscopy18.
In some cases, for example for IL-1β19 and
FGF2 (REF. 20), protein translocation channels
of the multidrug resistance (MDR) family
seem to be used for unconventional secretion.
However, the correlation between Engrailed
secretion and its localization in the lumen of
vesicles3 indicates the potential existence of an
unidentified mechanism that directs
Engrailed into secretory vesicles.
Unconventional secretion does not necessarily require cell-type-specific machineries,
and so it might reflect an ancestral mode of
secretion. For instance, MDR-dependent
secretion is found in organisms ranging from
bacteria to humans. Another example is the
unconventional secretion of thioredoxin that
is observed in bacteria21 (Helicobacter pylori),
plants22 and vertebrates23. Taken together, a
single unconventional pathway for all secreted
| OCTOBER 2003 | VOLUME 4
An active role for the nucleus? Intriguingly, a
vast number of secreted hydrophilic proteins
find their way to the cell nucleus (VP22,
galectin-3, FGFs, thioredoxin, high-mobility
group box (HMGB) proteins and homeoproteins) and some of them are capable of nucleocytoplasmic exchange24. This exchange might
control the amount of protein that is available
for secretion, as alluded to below. The nucleocytoplasmic distribution of FGF2 (including
its 18-kDa secreted isoform) is highly regulated during development25. In culture, phosphorylation modulates (directly or indirectly)
the nucleocytoplasmic distribution of both
FGF2 (REF. 26) and thioredoxin27. FGF1 accumulates in the nucleus before reaching the
cytoplasm28. Transfer of nuclear non-histone
HMGB1 protein to the cytoplasm precedes its
lysophosphatidylcholine-induced secretion29.
Both annexin II 30 and Engrailed16 contain an
active nuclear export signal.
Furthermore, secretion often correlates
with nuclear, rather than cytoplasmic, localization. The phorbol ester phorbol 12-myristate 13-acetate (PMA) promotes both
thioredoxin secretion31 and its nuclear localization27. Similarly, amino-terminal deletion
abolishes galectin-3 secretion and its nuclear
accumulation32. In the case of cEN2, the fact
that an active nuclear export sequence that
takes the protein out of the nucleus is necessary for cEN2 secretion might be seen as a
counter example. However, the addition of an
extra nuclear localization signal (NLS) does
not impair secretion (A. Maizel and A.J.,
unpublished observations) and many modifications that decrease cEN2 secretion correlate
with a pronounced cytoplasmic localization.
For example, Engrailed phosphorylation
blocks its secretion33 and favours its accumulation in the cytoplasm (A. Maizel and A.J.,
unpublished observations). So, although exit
from the nucleus is important to gain access
to the secretory compartment, a passage
through the nucleus seems necessary, therefore indicating an active role for the nucleus
in the secretion of secreted hydrophilic proteins. Whether this role is related to the
nuclear accumulation of some endocytic
proteins34 or to the nucleus–membrane
shuttling of some proteins (for example, the
scaffold proteins Ste5 (REF. 35) or regulator of
G-protein signalling 4 (REF. 36)) remains to be
investigated.
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© 2003 Nature Publishing Group
PERSPECTIVES
Transfer in other phyla
Behaviours that are indicative of transcription-factor transfer have been observed in
several phyla. This generality might indicate
that the phenomenon is important.
Yeast and bacterial functions. In many lower
eukaryotes, some homeoprotein functions are
related to intercellular communication.
Following cell fusion that accompanies mating, the homeoprotein content of each contributing cell determines the behaviour of the
resultant diploid cell38,39. Direct transfer of
Functions in higher plants. Movement proteins that undergo intercellular transfer are
commonplace in plants and some of these
proteins are transcription factors. This
explains why paracrine activity of transcription factors, including homeoproteins, has
been proposed in higher plants. In specific
cases, homeoproteins might even be used as
chaperones for protein transfer, as implied by
the observation that a single mutation antagonizes the intercellular transfer of a plantvirus movement protein and its binding to a
homeodomain protein42.
Because movement proteins traffic
through plasmodesmata, it is thought that
homeoproteins use the same pathway.
However, the homeodomain of the shuttling
plant homeoprotein KN1 can be transferred
between animal cells (which lack plasmodesmata), and the only mutation — located in
the homeodomain — that is known to
inhibit KN1 intercellular transfer in plants5
also inhibits the intercellular transfer of the
KN1 homeodomain between animal cells
(M. Tasseto, A. Maizel, A.P. and A.J., unpublished observations). This indicates a possible homology between metaphytes and
metazoans with respect to homeoprotein
transfer. In fact, it would be curious if, even
though animal proteins have efficient
import and export signals, animals are
unique in not using this powerful signalling
pathway.
In search of paracrine functions
Most secreted hydrophilic proteins have
extracellular functions that involve interactions with plasma-membrane receptors
(these could be on the cells that secrete them,
as well as on different cells). However, for
some of them, such as TAT43 and FGFs44,45,
internalization is known to be required for
them to elicit their full range of biological
effects. Homeoproteins, however, seem to be
atypical secreted hydrophilic proteins that so
far have no characterized extracellular functions in metazoans. It is therefore anticipated
that their paracrine targets are intracellular.
Because homeoproteins regulate transcription and, in some instances, translation, these
two modes of action are most likely to affect
paracrine action.
NATURE REVIEWS | MOLECUL AR CELL BIOLOGY
Transcription and translation. It seems evident that homeoproteins that have been
transferred from one cell to another should
regulate transcription in the nucleus of the
receiving cell. This is likely, but nonautonomous target genes might differ from
autonomous ones — for example, the proteins might become modified during transport, or they might take up a different role in
a
VE
Epi
VE
Otx2
Epi
Otx2 ? Otx2
VE cell Epi cell
ANE
transcription factors from pathogen to host
also happens during infection by pathogenic
bacteria40. Also, although it is not considered
to be a transcription factor, the T-DNA-binding protein VirE2 of Agrobacterium is directly
involved in the penetration of the host plasma
membrane by the T-DNA, and its subsequent
nuclear import41.
AVE
The data described above show that cEN2
and many other homeoproteins can pass
between cells by two original and distinct
mechanisms of secretion and internalization. Secretion involves a category of vesicles
that contain the protein within their luminal compartment, and EN1 and EN2 are
found in such vesicles in vivo. Finally, the
sequences that are necessary for unconventional export and import both reside within
the homeodomain and are conserved
among homeoproteins. How might this
intercellular transfer be regulated? The
absence of chiral receptors and a mode of
entry that is primarily based on the interaction
of the proteins with charged phospholipids
almost certainly preclude any regulation of
intercellular transfer at the entry site. By
contrast, the secretion of homeoproteins is
probably highly regulated.
Engrailed is a phosphoprotein in vivo and
is phosphorylated by casein kinase 2 (CK2).
In CK2–cEN2 co-transfection experiments,
CK2 blocks cEN2 secretion by phosphorylating a short serine-rich domain that is
upstream of the homeodomain33. Secretion of
proteins in which serine residues within the
serine-rich domain were mutated to alanine
residues was insensitive to CK2. By contrast,
the replacement of the same serine residues
by glutamate residues — mimicking phosphorylated serine residues — produced a
protein that was unable to be secreted.
Other putative modes to regulate secretion
control are the regulation of the nucleocytoplasmic distribution or the fusion of caveolae-like vesicles with the plasma membrane.
The latter process is known to be regulated, in
particular, through protein kinase C activation37, and therefore any mechanism regulating the fusion of these vesicles with the
plasma membrane might also regulate homeoprotein exocytosis. So, homeoprotein secretion is probably a highly regulated process
that can only happen in specific physiological
and developmental situations.
AVE
Regulation of intercellular transfer
Otx2
b
Neural tube (E 8,5)
Otx2
MHB
Anterior
Posterior
c
MHB
Wild-type
d
Otx2
e
Gbx2
MHB
Otx1–/– Otx2+/–
f
En1Otx2
MHB
+/–
Figure 4 | A model for homeoproteins as
infectious proteins. a | Signalling and inductive
activity between two layers is presented here in
the case of Otx2. Otx2 that is first expressed in
the anterior visceral endoderm (AVE; left) is
transported into the anterior epiblast (Epi), where it
activates Otx2 transcription (middle panels), which
is necessary for the formation of the anterior
neural ectoderm (ANE). This auto-induction could
be dependent of, or amplified by, co-factors.
Following its induction in the epiblast, Otx2
spreads by cell–cell transfer into the posterior part
of the neuroepithelium (b) until it encounters a
nuclear environment (such as the presence of
Gbx2) that represses its replication. The latter
model is hypothetical and supported by the
following observations: first, homeoproteins can
be secreted and internalized; second, in the wildtype (c), the mid–hindbrain border (MHB) forms
where Otx2 and Gbx2 meet; third, Gbx2 and Otx2
are auto-activators and reciprocal inhibitors (d);
fourth, downregulation of Otx expression shifts the
expression of Gbx2 and the position of the MHB
into a more anterior position (e); and, finally,
conversely forcing the expression of Otx2 in the
En1 domain represses Gbx2 and shifts the MHB
position into a more posterior position (f).
VOLUME 4 | OCTOBER 2003 | 8 1 7
© 2003 Nature Publishing Group
PERSPECTIVES
a different cellular context. But additional
functions can also be envisaged. As mentioned, homeoproteins might regulate messenger RNA stability and/or translation
(FIG. 3a). Direct evidence that a homeoprotein
can regulate translation is the repression, in
Drosophila, of caudal mRNA translation by
Bicoid46,47. The Bicoid mRNA-binding
domain has been identified48 and its conservation among homeoproteins raises the possibility that other homeoproteins bind RNA.
Indirect evidence is that Bicoid-dependent
repression of translation involves its binding to the eukaryotic translation initiation
factor 4E (eIF4E)49, a property that is shared
by proline-rich homeobox proteins and
Hox11 (REF. 50). Most importantly, the domain
of interaction with eIF4E is present in 200
other homeoproteins50.
Homeoproteins are known to have a role
in shaping neuronal arbors8,15,51,52. The development of the nervous system involves the
formation of many pathways and synapses
with an extremely precise topology. In addition, the functioning of adult neuronal networks requires that each synapse be informed
of its position within the network. So, economical mechanisms for position coding are
of primary importance in the developing and
adult nervous systems. Because homeoproteins contribute to positional information,
homeoprotein transfer would indeed be a
very economical way to combine signal transduction and topological information (FIG. 3).
We speculate that homeoproteins might
affect axonal navigation during development
and modify synaptic properties within adult
neuronal networks by regulating transcription and/or translation in the receiving compartment (FIG. 3). For example, the levels of
cell-surface receptors or cell-adhesion molecules might be changed, which, in turn, would
affect axon guidance. However, we do not
exclude other unsuspected modes of
paracrine activity, such as, for example, activating second messengers.
Could homeoproteins be morphogens? During
development, the transfer of positional information is either vertical, between layers of cells,
or tangential, within layers. A clear example of
vertical induction is the expression of the
homeoprotein Otx2 in the anterior visceral
endoderm, from where it is transported into
the anterior epiblast. Here, it induces its own
transcription, which is necessary for the formation of the anterior neural ectoderm. The
sequential expression of this anterior genetic
marker in the two layers involves a non-cellautonomous (direct or indirect) function of
Otx2 (FIG. 4a; reviewed in REF. 53).
818
Tangential induction within an epithelium
has been proposed as a patterning mechanism54. We speculate that homeoprotein tangential transfer might be involved in border
formation within the neuroepithelium,
thereby participating in the formation of the
compartments of the future brain. For example, the isthmus is an important embryonic
brain structure that forms where the expression of Otx2 meets that of Gbx2 (REF. 55; FIG. 4b),
and genetically decreasing Otx2 or Gbx2 levels anteriorizes or posteriorizes (respectively)
the position of the isthmus. The same holds
true for Emx2 and Pax6 in the compartmentalization of the cortex56, of Gsh2 and Pax6 for
the dorso–ventral patterning of the telencephalon57, or of Engrailed and Pax6 for the
mesencephalon–diencephalon border58. It is
striking that each homeoprotein in a pair activates its own transcription and antagonizes
that of its ‘partner’.
In the hypothetical models outlined in FIG. 4,
homeoproteins can be considered as ‘infectious entities’ — they invade the neuroepithelium until they encounter a nuclear
environment that is unsuitable for their own
induction, partly because of high expression
of the antagonist homeoprotein (FIG. 4b). In
fact, the protein does not have to travel far —
just one cell — but propagates rapidly by
inducing and amplifying its own transcription. All this happens as if the genes themselves were diffusible, a term that evokes the
definition of morphogens by Alan Turing in
1953 (REF. 59):
“The substances will be called morphogens, the word being intended to convey
the idea of a form producer. It is not intended
to have any very exact meaning, but is simply
the kind of substance concerned in this theory…The genes themselves may also be considered to be morphogens. But they form
rather a special class. They are quite indiffusible…”
Testing hypotheses
Homeoprotein intercellular transfer in vivo
has been observed in plants5 but not in
metazoans. Although it is possible that this is
the true scenario, it might be that homeoprotein transfer in animals is below the
threshold of detection or is highly regulated
in time and place. In favour of homeoprotein transfer is the presence of endogenous
Engrailed in the lumen of vesicles17, and that
of Emx1 in the axonal terminals of olfactory
receptors60. In addition, the existence of
highly conserved but distinct sequences that
allow secretion and capture is probably not
purely coincidental. In others words, we
think that the main question is not whether
| OCTOBER 2003 | VOLUME 4
homeoproteins are transferred between cells
but when, where and why they are.
To test the proposed hypotheses, it will be
necessary to block the intercellular transfer of
homeoproteins in vivo without modifying
their intracrine activity. A main objective
would be to identify and mutate sequences
that confer direct paracrine properties and to
insert the mutated gene into the normal locus
to produce an ‘intracrine-only’ mouse. As
shown in FIG. 1, the two sequences that are
necessary for secretion and internalization
are in the homeodomain. This is very interesting with respect to homeodomain conservation, but makes it impossible to introduce
mutations that would not modify the roles of
these proteins as transcriptional regulators.
So, the strategy that is being used at present is to design mini-genes that encode
homeoprotein-binding polypeptides and to
force their expression in the extracellular
milieu at the appropriate developmental periods. Such anti-homeoprotein agents are being
produced and it is our hope that their expression under the control of the appropriate regulatory sequences will allow us to prevent or
retard homeoprotein transfer without interfering with their intracrine functions.
Alain Prochiantz and Alain Joliot are both at
Centre National de la Recherche Scientifique,
Unité Mixte de Recherche 8542,
Ecole Normale Supérieure,
46 rue d’Ulm, 75230 Paris Cedex 05, France.
e-mails: prochian@wotan.ens.fr;
joliot@wotan.ens.fr
doi:10.1038/nrm1227
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Acknowledgements
We would like to thank the members of our groups for many helpful discussions and C. Goridis for his critical reading of the manuscript. Many of the experiments discussed in this article were supported by the Centre National de la Recherche Scientifique and
the Ecole Normale Supérieure, and by grants from the Association
Française de lutte contre les Myopathies, the Human Frontier
Research Program and the European Economic Community.
Online links
DATABASES
The following terms in this article are linked online to:
FlyBase: http://flybase.bio.indiana.edu/
Antennapedia | Engrailed
LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/
eIF4E | FGF1 | FGF2 | Gbx2 | Gsh2 | IL-1 | IL-1β | HMGB1 |
Otx2 | Pax6
Access to this interactive links box is free online.
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