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Introduction
Caenorhabditis elegans (C
elegans) is a 1-mm long free-living nematode that currently has
tremendous popularity as a model organism, especially regarding
questions of interest to developmental biologists. Given its ease of
culture (it is typically grown on Escherichia coli lawns), a
3-d life cycle from egg to egg, a transparent body that allows
visualization of any cell of interest, a fully described cell
lineage, and the ease with which genetic screens can be carried out
at low costs, it is no wonder that C elegans is such a
versatile and popular laboratory model organism. The 97 Mb genome of
C elegans is also completely sequenced, and computer
algorithms predict nearly 20 000 functional genes. Furthermore, the
C elegans research community has a traditional helpfulness
and openness: thousands of mutants are freely available to academic
laboratories from the C elegans Genetics Center (http://biosci.umn.edu/CGC/CGChomepage.htm),
and public databases of anatomical and molecular information are
available on the internet (http://www.wormatlas.org/;http://www.wormbase.org/).
Here, we review the developmental
genetics of the C elegans pharynx, with an emphasis on the
development of its small 20-neuron
network. However, here is one last
introductory note: several lines of evidence suggest that the C
elegans pharynx evolved from an organ that was also the common
ancestor to the vertebrate heart. This evidence consists mostly of
physiological and molecular similarities: (i) like the heart, the
pharynx is a rhythmically contracting neuromuscular pump[1];
(ii) the muscle cells of the pharynx have autonomous contractile
activity reminiscent of cardiac myocytes[2]; and (iii)
ceh-22, the C elegans homolog to the homeobox gene NK2.5
that plays an important role in heart development in vertebrates,
participates in pharyngeal development, and can partially be
replaced functionally by the zebrafish NK2.5[3]. The
evolutionary relatedness of the C elegans pharynx and the
vertebrate heart suggest that insights regarding heart development
and function may be gained by studying the simpler and
experimentally more convenient C elegans pharynx.
Pharynx form and function
The pharynx represents the foregut
of the nematode digestive tract (Figure 1). Food (typically
E coli in the labora-tory) is pumped through the mouth by the
action of the muscular pharynx, ground by specialized cuticle lining
the pharynx (the "grinder" in the posterior bulb), and transferred
to the intestine via a pharyngeal-intestinal valve. The main
anatomical features of the pharynx are, from anterior to posterior,
the procorpus, the metacorpus, the isthmus, and the posterior bulb
in which the grinder is located (Figure 1). The mature pharynx is
composed of 62 cells (for a total of 80 nuclei, since several of the
cells are binucleate as a result of cell fusion). These cells can be
categorized into 5 types: neurons (20), muscles (20 cells; 37
nuclei), marginal cells (9), epithelial cells (9), and gland cells
(4 cells; 5 nuclei). The muscle cells and marginal cells constitute
a single-cell-thick tube, continuous at its anterior end with the
tube of the hypodermis that encloses the worm. Muscle and marginal
cells are joined by tight junctions, which divide the membrane into
apical and basal surfaces. The apical surfaces face the lumen and
secrete cuticle, continuous with the cuticle made by the hypodermis.
The basal surfaces face a basal lamina that is continuous with the
basal lamina that separates the hypodermis and intestine from the
pseudo-coelom (fluid-filled body cavity) and mesoderm. Components of
this basal lamina are likely produced by body-wall muscles[4,5].
The 9 epithelial cells are arranged so as to form a narrow ring at
the anterior end of the pharynx, where it connects with the buccal
cavity. There is otherwise no epithelial sheet covering the bulk of
the pharynx. Precise knowledge of pharyngeal anatomy is available at
the ultrastructural level, thanks to detailed electron microscopy
studies[6].
Pharyngeal neurons lie deep within
folds of the basal membrane of pharyngeal muscle cells (note that
this is not a "basal lamina" or "basement membrane", but is that
part of the muscle cell membranes that is on the "basal" side),
between the muscle and basal lamina, just as the extrapha-ryngeal
nervous system is between the basal membrane of the hypodermis and
the basal lamina. No basal lamina separates pharyngeal motor neuron
presynaptic terminals from the post-synaptic muscle membrane. In
contrast, extrapha-ryngeal motor neurons are separated from the
muscle cells on which they synapse by the basal lamina that
separates the mesodermal muscle cells from the ectodermal neurons.
The role of the pharyngeal nervous
system in regulating pumping is somewhat of a mystery. Normal
feeding consists of two primary motions: pumping and isthmus
peristalsis[1]. A pump is a near-simultaneous contraction
of the muscles of the corpus, anterior isthmus, and terminal bulb,
followed by a near-simultaneous relaxation. The contractile fibers
of the pharyngeal muscles are radially oriented, so contraction
pulls the lumen open from its resting closed
Y-shape to a triangular shape. The second motion, isthmus
peristalsis, occurs after the main relaxation is complete. It is a
peristaltic wave of contraction in the posterior isthmus that
carries bacteria trapped in the anterior isthmus back to the
grinder. Typically, only every fourth pump is followed by an isthmus
peristalsis. The nervous system is not essential for pumping;
pumping continues even when the entire pharyngeal nervous system is
killed[2]. However, many neurons are important; efficient
pumping and trapping of bacteria by the pharynx requires the
presence of the neurons I5, MC, M3, M4, and NSM[2,7,8].
Development of the pharynx
In order to begin understanding how
the pharyngeal neurons develop, it is necessary first to describe
pharyngeal development itself (Figure 2). The C elegans
pharynx offers a very simple model to understand morphogenesis and
differentiation. The pharynx develops through the morphogenesis of a
primordium composed of 80 undifferentiated cells (plus many
apoptotic cells; there are 19 apoptotic cells that are sisters to
final pharyngeal cells and that die within 350- 420 min of
development[9]). Morphogenesis is accompanied by
differentiation but not by new cell divisions, so the mature pharynx
contains 80 nuclei but only 62 cells as a result of cell fusion
among some of the muscle and two gland cells; these fusions occur
around the time of hatching and seem irrelevant to the developmental
process[9], although it would be interesting to
understand how these fusions are regulated.
0-100 min: early cell divisions
and establishment of main lineages The cells that make up the
pharyngeal primordium originate from two early embryonic blastomeres:
the ABa and the MS blastomeres. This is quite remarkable: members of
two distinct lineages are recruited to form one organ. Not only
that, but cells with these two very different ancestries may end up
adopting nearly identical fates. For example, the muscle cell m3VL
has the ancestry ABalpappppp, whereas the identical cell m3DL has
the ancestry Msaaapaaa (these two cells will fuse later). Note that
even though each cells is normally specific to adopt a developmental
fate, there is some degree of developmental plasticity. For example,
Avery and Horvitz showed that the pharyngeal neuron M4 is essential
for feeding in wild-type C elegans, but that in a ced-3
mutant (in which the sister cell of M4 does not die of apoptosis),
the now viable sister of M4 can sometimes take over the function of
M4[10].
The respective contributions of the
ABa and MS lineages are more or less spatially consistent with their
initial positions within the 8-cell embryo. For example, the
anterior cell ABa contributes cells of the anterior pharynx, whereas
the more posterior MS cell contributes mostly posterior pharyngeal
cells. This observation holds true for later descendents and
narrower scopes of spatial contributions. Figure 3 shows the adult
pharyngeal contributions from the pharyngeal precursors of the
100-cell stage embryo, and emphasizes the preservation of spatial
relationships during development. Thus, ABalpa contributes mostly to
the anterior left subventral area, etc.
100-250 min: gastrulation At
100 min after first cleavage, when the egg comprises 28 cells,
gastrulation begins. During gastrulation, several cells enter deep
into the embryo through a ventral cleft. The first cells to enter
are the gut precursor cells Ea and Ep. Next are the P4 and MS
progeny at 120-200 min of development, and the AB-derived pharyngeal
precursors enter more anteriorly at 210-250 min. The ventral cleft
closes from posterior (230 min) to anterior (290 min). As
gastrulation proceeds, the E cell descendents and the pharyngeal
precursors form a central cylinder. Note that as gastrulation
proceeds, so do cell divisions. Active pre-pharyngeal cell divisions
continue until approximately 350 min of development, and some late
divisions occur until approximately 400 min.
250-400 min: compaction of
pharyngeal primordium Between 250 min and 400 min the pharyngeal
primordium becomes clearly defined. The non-pharyngeal precursor
cells are somehow excluded from the pharyngeal primordium. Perhaps
they are squeezed out in a process by which the pharyngeal cells
have more adhesive affinity to each other than to any other cell (in
line with the theory of Malcolm Steinberg; eg see Duguay et al[11]).
This aspect of primordium formation has not been investigated
experimentally.
400-430 min: extension of
pharyngeal primordium The approximately 400-min-old primordium
is insulated by a basement membrane (present at or before 400 min[9]),
such that the pharynx develops autonomously, perhaps with no
extrapharyngeal cues, or with very few. Such autonomous development
is also true of the 20-cell intestine that, together with the
pharynx, makes up the entire C elegans gut[12]. At
approximately 400 min, the pharyngeal primordium is approximately
spherical, and most of the cell nuclei appear located in spatial
relationships that are consistent with their final positions, at
least along the anterior-posterior axis, although the relative
distances between these nuclei can be very different from those of
the mature organ. For example, at approximately 430 min, the sister
cells M2 and M3 have their nuclei next to each other, whereas in the
final pharynx M2 has its nucleus in the posterior bulb and M3 in the
meta-corpus. It therefore seems that development of the pharynx is
mostly a question of cell differentiation and morpho-genesis, not of
active cell migration. However, some cells do migrate within the
developing pharynx. For example, Sulston et al observed that
the 3 g1 gland cells migrate in a reproducible way. They wrote:
"Their movements approximately follow the subsequent course of their
secretory processes, and may be responsible for laying down the
latter"[9].
Beginning at approximately 400 min,
the primordium elongates anteriorly then posteriorly. The primordium
develops into a tube connected anteriorly to the buccal cavity and
posteriorly to the midgut. The adherens junctions that connect many
pharyngeal cells with each other form simultaneously with the
process of elongation. Note that there is no evidence of any
basement membrane within the elongated pharynx during or after
elongation or at any other stage of development or adulthood[5].
Portereiko and Mango have studied the morphogenesis of the
pharyngeal primordium and divided the process into three stages: (i)
lengthening of the nascent pharyngeal lumen by reorientation of the
apicobasal polarity of anterior pharyngeal cells ("Reorientation");
(ii) formation of an epithelium by the buccal cavity cells, which
mechanically couples the buccal cavity to the pharynx and anterior
epidermis ("Epithelialization"); and (iii) a concomitant movement of
the pharynx anteriorly and the epidermis of the mouth posteriorly to
bring the pharynx, buccal cavity, and mouth into close apposition
("Contraction")[4].
430-800 min: completion of
functional pharynx Between 430 min and 490 min, as elongation
proceeds, the pharyngeal bulbs and isthmus become apparent. It is
probably at this time that the pharyngeal cells interpret their
final differentiation programs and adopt their final shapes. Between
600 min and 650 min, the pharyngeal cuticle is produced and the
lumen becomes distinct. The pharyngeal glands are active by 720 min
and the pharynx is pumping by 750 min. Hatching occurs at
approximately 800 min following first cleavage.
Genetics of pharyngeal
development
What follows is a brief overview of
some of the genes that have been shown to play a role in pharyngeal
develop-ment.
pha-4 pha-4
encodes the C elegans homolog of FoxA, a fork-head
transcription factor[13]. The pha-4 gene is
expressed in all pharyngeal cells, and also in some cells of the
rectum[13]. Expression of PHA-4 is detected in all
pharyngeal precursor cells beginning from at least 200 min of
development (and perhaps even earlier). By the comma stage
(~430 min), all the pharyngeal cells are present and express PHA-4.
PHA-4 is also expressed in the 6 cells of the pharyngeal intestinal
valve, which is not considered a part of the pharynx per se.
At 430 min, PHA-4 expression is also found in 6-8 rectal cells,
including the 2 rectal valve cells and the 3 rectal epithelial
cells. This expression pattern is therefore conserved with that of
the Drosophila forkhead gene (high levels in the
foregut/pharynx and hindgut/rectum). The pha-4 mutants
completely lack all pharyngeal cells, even though the AB and MS
lineages are otherwise completely normal[14]. It seems
that pha-4 acts as an organ identity factor. Indeed, Gaudet
and Mango have proposed that the PHA-4 protein may directly activate
most or all pharyngeal genes, with the expression timing being
regulated by the presence of binding sites of varying affinity: poor
binding sites will have delayed expression, as they will require
higher levels of PHA-4 before becoming activated[15]. The
consensus binding site for PHA-4 has been defined as: TRTTKRY
(R=A/G, K=T/G, Y=T/C). This site is present in the myo-2
gene, a pharyngeal-specific muscle myosin that is a confirmed direct
target of PHA-4[13]. Ectopic expression of PHA-4 causes
ectopic expression of myo-2, ceh-22 (a homeodomain
protein that is also a coactivator of the myo-2 gene),
pha-2 (another homeodomain protein important for pharynx
development, see below), and most likely other otherwise pharyngeal
specific genes[13,16].
pha-1 In pha-1
mutants, the pharyngeal primordium appears to form normally, with a
full complement of nuclei and surrounded by a basal membrane. In
these mutants, elongation also appears normal up to at least 420 min
of development, including the expression of an antigenic marker for
the pharyngeal muscle cell precursors detected with the monoclonal
antibody 3NB12[17,18]. After elongating and contacting
the buccal cavity, the developing organ detaches from the buccal
cavity and retracts, causing a "Pun" (pharynx unattached) phenotype.
The end result is a worm in which the incompletely formed pharynx is
slightly elongated, surrounded by the visible basement membrane and
unattached to the mouth. It is difficult to determine if all
pharyngeal cell differentiation events take place in the pha-1
mutant, but expression of MYO-2::GFP is detected and a pharyngeal
lumen forms[17]. Thus pha-1 affects pharynx
development after pharynx cells are committed to a specific cell
fate, but before terminal differentiation/morphogenesis of the
different pharyngeal cell types occurs[17,18].
Initial analysis of the PHA-1 amino
acid sequence suggested that it was a basic leucine zipper (bZIP)
transcription factor. Expression of a PHA-1::LacZ reporter also
suggested restricted expression in pharyngeal cells as well as in
body muscle cells[18]. However, a more recent evaluation
of the PHA-1 amino acid sequence indicates that pha-1
actually does not encode a bZIP transcription factor[17].
Consistent with this last analysis, a rescue-competent PHA-1::GFP
fusion protein suggests that PHA-1 is a cytosolic protein[17]
that is widely expressed (essentially in all cells by the 100-cell
stage). Because the biochemical function of PHA-1 is unknown at
present, little can be said about its actual mechanism of action.
However, genetic interaction experiments have shown that pha-1,
lin-35 (the C elegans Retinoblastoma protein homolog),
and ubc-18 (a ubiquitin-conjugating enzyme) play partially
redundant functions to control pharyngeal morphogenesis[17].
Indeed, lin-35/Rb; ubc-18 double mutants exhibit a
synthetic pharyngeal phenotype; that is, failure to undergo
pharyngeal primordium elongation, typically failing already at the
reorientation step during which the anterior epithelial cells of the
primordium should align their long axis with the dorsoventral axis
of the embryo[19]. The ubc-18 and pha-1
also both show strong synthetic pharyngeal phenotypes when combined
with class B synthetic multivulval (SynMuv) genes. The SynMuv genes
form two molecularly heterogeneous classes (class A and B) of genes
that contribute redundantly to vulva development; class B SynMuv
genes obviously also play a hitherto unknown role in pharyngeal
development that is redundant with both ubc-18 and pha-1[17,19].
pha-2 The pha-2
mutant worms exhibit a late defect in pharyngeal morphogenesis that
results in the two pharyngeal bulbs being next to each other rather
than being separated by a narrow, nucleus-free isthmus. We cloned
the pha-2 gene and found that it encodes a homeobox gene most
homologous to the vertebrate Hex gene[16]. Using a
PHA-2::GFP translational fusion reporter in which a pha-2
genomic fragment containing 2.7 kb of pha-2 5'UTR plus the entire
gene fused to GFP at its C-terminal codon, we observed expression in
several pharyngeal cells: the pm5 muscle cells that form the isthmus
but have their cell bodies within the posterior bulb; the pm4 cells
that make up the bulk of the metacorpus; and pharyngeal epithelial
cells. As this translational fusion reporter was able to rescue the
mutant pheno-type, we are relatively confident that the expression
profile of the reporter reflects normal PHA-2 expression. We
hypothesize that PHA-2 confers an isthmus cell identity to the pm5
muscle pharyngeal cells that express it and that form the isthmus.
The main characteristic of isthmus cells is that they have a long
elongated shape extending into the isthmus, but that their cell
bodies are embedded within the metacorpus or posterior bulb. This
isthmus cell shape likely results from directional growth of the
cells occurring after the comma stage (~430 min), because at this
stage there is no nuclear-free zone along the length of the
elongated primordium. As Sulston et al documented, it is
during the 430-490 min interval, as the emerging pharynx continues
its elongation, that the pharyngeal bulbs become apparent[9].
What are the genes regulated by
pha-2? Experimental evidence suggests that pha-2 acts as
a repressor of ceh-22 in the pm5 cells. In wild-type animals,
expression of a CEH-22::GFP reporter is downregulated in the isthmus
by late embryogenesis. In contrast, in pha-2 mutants the
expression of the CEH-22::GFP reporter persists and even increases
in the isthmus during late embryogenesis, and also post-embryonically.
Because of the late effects of the pha-2 mutation, we also
surmise that at some downstream level, pha-2 acts via genes
that implement the differentiation program by driving the final cell
shape changes, such as cytoskeletal genes. It is perhaps worth
noting that located just next to the pha-2 gene is the
intermediate filament 2c gene, IF-C2 (M6.1), which is expressed in
pharyngeal and intestinal desmosomes and thus likely plays a role in
cell-cell connections[20]. Given that intermediate
filaments are important in several morphogenesis processes,
including cell elongation[21,22], there is a possibility
that M6.1 contributes to pharyngeal morphogenesis. Consistent with
this line of reasoning, the expression of IF-C2 begins in the late
embryo, when final pharyngeal morphogenesis occurs.
ceh-22 Like vertebrate
cardiac and smooth muscles, the pharyngeal muscles of C elegans
do not express any of the known members of the MyoD family of
myogenic factors. In addition, like vertebrate cardiac muscle cells,
the pharyngeal muscles exhibit an intrinsic rhythmic contraction
activity that does not depend on any neuronal input. Two myosin
heavy chain genes myo-1 and myo-2 are specifically
expressed in pharyngeal muscles. In 1994, Okkema and Fire
characterized the myo-2 promoter and identified a
transcription factor that binds this promoter and regulates its
expression in pharyngeal muscles[23]. This transcription
factor was CEH-22, a homeobox protein most homologous to the
vertebrate Nkx2.5 and the Drosophila tinman, which regulate
heart development in their respective organisms. Furthermore,
expression of the zebrafish nkx2.5 gene in C elegans
can activate myo-2 and can rescue the ceh-22 mutant,
suggesting that ceh-22 and nkx2.5 share a conserved
molecular function[3]. Somewhat confusingly, the
phenotype of the ceh-22 mutant includes a slightly abnormal
pharyngeal shape (slight thickening of the isthmus), but no defect
in the expression of myo-2, suggesting that other regulatory
pathways act in parallel with ceh-22 to regulate myo-2[24].
Okkema et al showed that PHA-1 itself also directly regulates
the myo-2 gene, and that a pha-1; ceh-22 double
mutant is more severe than either mutant alone; the early pharyngeal
3NB12 antigen is not even expressed[24].
Development of the pharyngeal
neurons
Pharyngeal nervous system overview
The mature pharynx contains 20 neurons. Each establishes a unique
and predictable morphology that is reproducible from worm to worm.
The pharyngeal nervous system is organized into four general
structures: two subventral nerve cords, one dorsal nerve cord, and
one circular pharyngeal ring, which is located within the posterior
half of the metacorpus and to which 12 neurons contribute processes[6].
The synapses and gap junctions made by the pharyngeal neurons have
also been described and thus a basic wiring diagram of the
pharyngeal network exists[6]. Speculations that the
pharyngeal neural network plays an important role in regulating the
pumping activity, as has been postulated for the intrinsic cardiac
ganglia in the vertebrate heart[25], are stunted by the
above-mentioned observation that most of the pharyngeal neurons can
be laser-ablated without visible effects on pharyngeal behavior.
How does the intricate network of
axon trajectories and synapses become established within the small
cramped space of the developing pharynx? In particular, it is
important to re-emphasize that the pharyngeal neurons, in contrast
to body neurons, are not projecting between a basal lamina and an
epithelial cell. Rather, they project within muscle cell folds,
directly in contact with the muscle cell surface. Does this rather
unique substrate for the neurons involve guidance cues different
from those guiding body neurons? Also of importance is the fact that
the pharyngeal neuron cells are already present in undifferentiated
form within the spherical pharyngeal primordium. This offers
intriguing developmental possibilities. For example, the pharyngeal
neurons can take advantage of instructive interactions between cells
that are neighbors within the primordium but are widely separated in
the mature pharynx.
Establishing axon trajectories
without growth cones We have shown that the M2 pharyngeal axons
establish their trajectories via at least two independent mechanisms[26].
The straight proximal M2 trajectory (between the cell body, through
the isthmus, and reaching into the metacorpus; Figure 2) does not
depend on genes that act as axon guidance cues or that are important
for growth cone functions. Thus, this proximal straight trajectory
is established in a growth-cone-independent manner. We have
suggested that the M2 cell forms, within the primordium, a physical
connection with some neighboring cell that is ultimately located
within the metacorpus, and that these connections elongate to form
the proximal M2 axon trajectory as the primordium undergoes
morphogenesis. It is a long-standing observation that mechanical
tension exerted on neuronal cells can induce formation of a
projection that can elongate rapidly as tension is maintained, "as a
fishing line from a reel"[27,28]. Such mechanically
induced axon formation and elongation can take place even when
growth cone function is impaired[29]. This would be a
process similar to the scenario that Sulston et al described
for the pharyngeal gland cell g1 (discussed earlier), and also
similar to the immature sensilla neurons that, after contacting the
tip of the head, move posteriorly while laying down their dendritic
processes[9]. The ability of a neuronal cell body to
migrate and leave an extending axon behind has also been attributed
to vertebrate facial moto-rneurons, although that particular case
involves the movement of the cell nucleus ("nucleokinesis") into a
dendrite[30]. In Drosophila, the larval optic
nerve undergoes a period of elongation by intercalation of membrane
as the neuron cell body and a distant guidepost cell move away from
each other; later, a growth-cone-dependent process completes the
establishment of the distal trajectory[31]. Similarly,
neurons of the larval imaginal leg disc also lengthen axons in
keeping with the vast leg morphogenesis process[32].
Distal ends are established using
a growth cone Axon trajectories are usually established by
specialized structures at their growing ends, the growth cones, that
sense the molecular environment and interpret guidance cues so as to
migrate along the correct paths[33]. The distal end of
the M2 axon, which exhibits a complex trajectory within the
metacorpus (first turning outward laterally, then dorsally, before
extending towards the midline to establish a gap junction with the
contralateral M2 neuron), depends on basic growth cone function
genes (unc-73, unc-51) as well as several well-known
axon guidance cues and receptors for these cues (sax-3, slt-1,
unc-6, unc-5, unc-40)[26]. It is therefore our
conclusion that the rough trajectories of pharyngeal neurons may be
established during morphogenesis in the absence of growth cones,
relying instead on cell-cell contacts that are stretched to form
axons during cell movements, but that fine-tuning of the ends of the
trajectories depends on functional growth cones and multiple
guidance cues. A screen for mutants with abnormal M2 trajectories
allowed the isolation of several mnm (M neuron morphology
abnormal) mutants, three of which specifically affected the
formation of the distal M2 ends (Figure 2)[26]. These
mutations likely affect the function of genes important for the
function/guidance of the M2 growth cones.
An instructive developmental role
for neurons? Interestingly some of the pharyngeal neurons
themselves may act as sources of guidance cues. At least 2 of the
pharyngeal neurons likely play developmental roles. The interneuron
I5 is a source of UNC-6[34] that is likely to be
important for the guidance of other axons, notably M2, which
exhibits abnormal trajectories in a unc-6 mutant background.
The interneuron I4 expresses UNC-129[35], also a secreted
guidance molecule. Hence, it seems likely that many of the
apparently unimportant pharyngeal neurons (most neurons of the
pharynx can be ablated in the adult worm without impairing
pharyngeal function) play an important role during development. It
would be interesting to ablate the neuronal precursor in the embryo
prior to pharyngeal morphogenesis end, and thus test directly the
hypothesis that these neurons play an instructive developmental
role. It would also be interesting to determine whether this is a
function that neurons play in other developing organs and other
organisms.
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