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First published online 1 August 2007
doi: 10.1242/dev.006635
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Center for Regenerative and Developmental Biology, Forsyth Institute, and Developmental Biology Department, Harvard School of Dental Medicine, 140 The Fenway, Boston, MA 02115, USA.
* Author for correspondence (e-mail: mlevin{at}forsyth.org)
Accepted 21 June 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Planarian, Gap junctional communication, Stem cell, Regeneration
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Thus, neoblasts must effectively integrate the signals
required for self-renewal, controlled proliferation, migration,
differentiation and cell incorporation into target tissues during
physiological turnover and maintenance of complex pattern (morphostasis).
Furthermore, after injury, neoblasts proliferate and regenerate all missing
tissues in a short period of time, restoring the original form and proportion
of the organism (Reddien and
Sánchez Alvarado, 2004). All of these properties highlight
the interactions between adult stem cells and the context of their existing
differentiated neighbors (Joseph and
Morrison, 2005; Li and Xie,
2005; Scadden,
2006; Yamashita et al.,
2005). Understanding the endogenous controls of stem cell behavior
is of high priority for therapeutic applications in regenerative medicine and
cancer biology, as well as for the fundamental understanding of highly
regulative morphogenesis. However, the molecular mechanisms of the crosstalk
between stem cells and their surrounding differentiated tissues are largely
unknown.
Gap junctional (GJ) communication is increasingly revealed as an important
regulatory modality functioning alongside traditional secreted signaling
molecules. In metazoans, gap junction proteins are conserved membrane channels
involved in direct cell-cell communication via small molecule signals during
numerous physiological events (Levin,
2002; Nicholson,
2003; Phelan,
2005). Gap junction-permeable signals have been implicated in
embryonic morphogenesis and neoplasm
(Bruzzone et al., 1996;
Levin, 2002;
Mesnil et al., 2005).
Importantly, GJ communication is not just a housekeeping event necessary for
cell survival; several studies have revealed subtle alterations of embryonic
patterning, but not generalized inhibition of cell proliferation or toxicity,
following abrogation of GJ communication (GJC)-dependent signaling (reviewed
in Levin, 2007). Invertebrate
gap junctions are composed of proteins from the innexin family, whereas
vertebrate gap junctions consist of the homologous pannexins as well as
connexins. Innexins have been shown to be a crucial component for tissue
polarity, morphogenesis, locomotion, electrical cell coupling, and germ-line
cell maintenance and differentiation (Bauer
et al., 2004; Gilboa et al.,
2003; Nogi and Levin,
2005; Phelan,
2005; Tazuke et al.,
2002); however, no specific gap junction protein has yet been
functionally shown to regulate adult somatic stem cells in vivo. Moreover, no
specific gap junction gene product has been implicated in the interplay of
signals required during the regeneration of complex structures.
In recent work, we characterized the expression of a family of innexin
genes in planaria (Nogi and Levin,
2005). Crucially, however, the specific innexins involved in
patterning, and the molecular mechanisms by which innexin function could
regulate cell growth, fate specification and regeneration, remain completely
unknown. The molecular implication of a specific innexin gene in regulative
morphogenesis and stem cell behavior, as well as the characterization of the
consequences of its abrogation, are the key steps that must now be addressed.
Thus, we investigated the potential roles of GJ-mediated signals in
regeneration and homeostasis, using the planarian species Schmidtea
mediterranea as a tractable model system in which to probe novel cellular
mechanisms of communication between stem cells and their niche.
We identified 12 transcripts encoding innexin proteins (smedinx-1-12). Additional qualitative and quantitative expression studies suggested that neoblast-associated innexins are present and enriched in specific adult stem cell subpopulations. Functional analysis using RNA interference (RNAi) revealed a neoblast-associated gene, smedinx-11, that is specifically required for tissue regeneration, homeostasis and neoblast maintenance. Reduction of smedinx-11 expression resulted in a phenotype that is different in several key properties from those obtained with known genetic and radiation-based treatments. Our data show that smedinx-11 is required for the normal expression of well-conserved key stem cell regulators such as PIWI and other proteins. Taken together, our data reveal a novel role for GJC proteins and characterize a new element functioning in the regulation of adult stem cells in their natural environment.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Xenopus GJ assay
Full-length innexin gene sequences were obtained by RACE and subcloned into
the pCS2+ expression vector. Full-length smedinx-11 was entered into
Genbank under accession number DQ851133 (formerly AY067505). Ventral or dorsal
blastomeres of four-cell Xenopus embryos were injected with in
vitro-synthesized, full-length, capped mRNA encoding smedinx-11, Dugesia
japonica innexin-11 (DjInx-11; the smedinx-11 homolog)
or a truncated mutant of smedinx-11 (for additional details, see Fig.
S2 in the supplementary material). Effects were assayed as described
previously (Levin and Mercola,
1998). Injection of mRNA encoding smedinx-11 or
DjInx-11 into ventral blastomeres, in which cells are normally
gap-junctionally isolated (Levin and
Mercola, 1998), specifically randomized embryonic laterality (35%
and 26% heterotaxia, respectively, n=119, P<<0.01 by
2 test with Pearson correction); by contrast, injections into
dorsal blastomeres (expressing the innexin in a zone that is already
well-coupled by gap junctions) had no effect on embryonic asymmetry.
Similarly, a non-functional smedinx-11 mutant did not randomize in
any injection locale (P=0.3). Thus, smedinx-11 behaved in
this assay identically to all of the connexin gap junction constructs tested
previously (Levin and Mercola,
1998).
Quantitative real-time PCR (qRT-PCR)
The RNA isolation procedures and internal control used were as previously
described (Reddien et al.,
2005b). We used the iScript cDNA synthesis kit and iQ SYBR green
supermix (BioRad); PCR reactions and analyses were performed using iCycler
(BioRad).
RNAi
In vitro double-stranded (ds)RNA preparation (including the 65-1469 bp
fragment) and injection procedure was as previously described
(Reddien et al., 2005b). The
injection schedule was: a total of three injections distributed over two
consecutive days, one day of rest, followed by one more day of injections (one
injection per day with three pulses of 32 nl of respective dsRNA). To rule out
the possibility of local neoblast depletion, microinjections in some
experiments targeted either pre- or post-pharyngeal areas. In most cases,
dsRNA injections were targeted to the gastro-vascular system, to assure
effective dsRNA incorporation as well as homogeneous distribution throughout
the body. Some animals were amputated at 8 days after first injection and,
when possible (for all S. mediterranea innexin genes other than
smedinx-11), a second round of injections plus amputation was
performed. The control group in RNAi experiments was injected with water. Some
animals remained intact for more than 3 weeks, and then were fixed and
processed for immunostaining or in situ hybridization (ISH).
In situ hybridization
ISHs were performed as previously described
(Reddien et al., 2005b). Once
the ISH signal for positive controls was reproducible, experiments were
carried out with different S. mediterranea innexin riboprobe
dilutions to determine the optimal ratio of positive-signal:background. The
specificity of the ISH pattern was also confirmed by three sets of experiments
performed in parallel (Fig.
2A): (i) 
-irradiation (6000 rad), which resulted in the
selective removal of stem cells (neoblasts), significantly reduced the signal
of two S. mediterranea innexins compared to the un-irradiated worms
(see below; results were additionally confirmed by qRT-PCR); (ii) sense probe,
which resulted in no detectable signal; and (iii) specific RNAi exposure,
which reduced the signal of the target genes.
Flow cytometry experiments
Planarian dissociation and cellular labeling was performed as described
(Reddien et al., 2005b) with
only one filtration through a 35 µm Nitex filter. Propidium iodide was
added (5 µg/ml) to eliminate dead cells. Labeled cell fractions were sorted
using a MoFlo high-speed cell sorter and analyzed with Summit software (Dako).
Combinations of Hoechst and calcein stains were used in standard protocols to
separate cells on the basis of dye efflux and DNA content
(Goodell et al., 1996).
Image collection and processing
Digital images were collected using a Nikon SMZ1500 microscope connected to
a Cool Snap CF camera (Roper Scientific Photometrics), and with Openlab
software (Improvision), a Leica TCS SP2 Spectral Confocal Imaging System and
an Olympus BX61 motion-controlled compound microscope attached to a Hamamatsu
Orca AG high-resolution, monochrome, cooled CCD camera with IP Labs analysis
software.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Saló,
2006; Sánchez Alvarado,
2006; Sánchez Alvarado
and Kang, 2005). Twelve cDNAs coding for innexins were identified
in two public S. mediterranea databases
(Sánchez Alvarado et al.,
2002; Zayas et al.,
2005) and numbered smedinx-1 to smedinx-12
(Fig. 1A, and see Fig. S1 in
the supplementary material). Phylogenetic analysis and in situ hybridization
(ISH) revealed considerable similarity of sequence and expression pattern to
Dugesia japonica innexins (inter-species comparison in Fig. S1 in the
supplementary material). These transcripts were segregated into four
categories of expression: excretory, digestive, mesenchymal and nervous tissue
(Fig. 1A). Although innexin
genes are now widely known as encoding gap junction proteins, we indirectly
confirmed this function in a Xenopus assay (see Materials and methods
and Fig. S2 in the supplementary material).
The expression of mesenchymal S. mediterranea innexin genes is
widely distributed. To confirm that these patterns are correct and specific,
and to conclusively establish expression of these genes, several rounds of
optimization of the ISH protocol used were performed with negative (sense
probe) and positive (other genes with expression patterns known to involve
different tissues) (Newmark and
Sánchez Alvarado, 2002;
Sánchez Alvarado et al.,
2002) controls (data not shown). Moreover, wild-type intact
animals were sectioned (transversally and longitudinally along the
anteroposterior axis) and ISH was performed on sections
(Reddien et al., 2005b) to
ensure that probe penetration differences did not bias the results
(Fig. 1B and see Fig. S3 in the
supplementary material). The results of whole-mount and section ISH under a
variety of conditions consistently revealed expression in nervous, excretory
and digestive systems, as well as in the mesenchyme - a tissue that contains a
number of different cell types (Fig.
1 and see Fig. S3 in the supplementary material). Please see the
legend to Fig. S1 in the supplementary material for a discussion of the
comparison of innexin expression patterns among S. mediterranea and
D. japonica.
Expression of smedinx-11 and smedinx-2 was associated with neoblasts
Because neoblasts are the only proliferative cells in adult planaria,
-irradiation provides a convenient way to selectively kill them and
thus identify neoblast-associated gene expression
(Guo et al., 2006;
Hayashi et al., 2006;
Ogawa et al., 2002;
Orii et al., 2005;
Reddien et al., 2005b;
Rossi et al., 2006;
Salvetti et al., 2000;
Salvetti et al., 2005;
Shibata et al., 1999). ISH and
quantitative real-time PCR (qRT-PCR) revealed that the expression of only two
S. mediterranea innexin genes (smedinx-11 and
smedinx-2) was reduced by -irradiation
(Fig. 2A,B and data not shown),
suggesting a relationship between these S. mediterranea innexins and
neoblasts.
|
; Ogawa et al.,
2002; Orii et al.,
2005; Reddien et al.,
2005b; Rossi et al.,
2006; Salvetti et al.,
2000; Salvetti et al.,
2005). Proliferative neoblast-associated gene expression is
commonly observed throughout the mesenchyme, but not in tissue in front of the
photoreceptors or in the pharynx (Guo et
al., 2006; Ogawa et al.,
2002; Orii et al.,
2005; Reddien et al.,
2005b; Salvetti et al.,
2000; Salvetti et al.,
2005). However, in addition to the smedinx-11 signal in
mesenchymal cells, irradiation-sensitive expression of smedinx-11 was
also present in areas known to be postmitotic (i.e. cells anterior to the
photoreceptors, surrounding cephalic ganglia, and in the pharynx) (Figs
1,
2 and see Fig. S3A,B in the
supplementary material) as well as in regenerating blastemas (see Fig. S3C in
the supplementary material). Such irradiation-sensitive gene expression in
areas in front of the photoreceptors and pharynx (see Fig. S3A,B in the
supplementary material) has also been described for another stem
cell-associated transcript, vasa-related
(Shibata et al., 1999).
Moreover, although the expression of both smedinx-11 and
smedinx-2 was reduced after irradiation, a small
irradiation-insensitive component remained
(Fig. 2B).
smedinx-11 gene expression is enriched in the postmitotic X2 subpopulation
Recent work using fluorescent activated cell sorting (FACS) identified two
irradiation-sensitive subpopulations (X1 and X2) with neoblast-like morphology
(Hayashi et al., 2006;
Reddien et al., 2005b). The X1
subpopulation in S. mediterranea consists mainly of dividing
neoblasts expressing the piwi genes smedwi-1 and smedwi-2,
and smedcyclinB; however, no marker or functional characterization
are yet available for the X2 population (which consists of both
irradiation-sensitive and -insensitive cells). Gene expression experiments
using FACS-isolated cells demonstrated that smedinx-11 is mainly
expressed in the X1 and X2 compartments, and its expression levels are
specifically enriched in X2 cells (Fig.
2C).
Together, smedinx-11 quantitative and qualitative expression
analyses in FACS-isolated cells also revealed that this gene is expressed at
low levels in most (
60%) proliferative neoblasts (X1), whereas, in X2
cells, in which the total smedinx-11 levels are higher, the number of
cells expressing this gene only reach approximately 24%
(Fig. 2C,D). Because there is
more total expression of smedinx-11 in X2 cells than in X1 cells, but
fewer X2 cells express detectable smedinx-11 mRNA, we suggest that
smedinx-11 is expressed at high levels in a subset of X2 cells.
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90%)
(Reddien et al., 2005b),
suggesting that the expression in many X1 cells must overlap for S.
mediterranea piwi genes and smedinx-11. To investigate whether
smedinx-11 expression in X2 cells might be related to other neoblast
markers, we stained FACS-isolated cells with a recently characterized SMEDWI-1
antibody that recognizes postmitotic irradiation-sensitive cells
(Guo et al., 2006). In both X1
and X2 cell groups, over 90% were positive for SMEDWI-1 protein (see Fig. S4
in the supplementary material), suggesting that smedinx-11 expression
in X2 neoblasts overlaps with SMEDWI-1-positive cells. Together, these
quantitative and spatial expression data suggest that smedinx-11
represents a novel marker for irradiation-sensitive cells that provides a
molecular entry-point into understanding the differences among stem cell
subpopulations (see also the legend for Fig. S1 in the supplementary
material).
smedinx-11 is required for tissue regeneration and for proper neoblast function
To investigate whether S. mediterranea innexin proteins regulate
neoblast biology, RNAi experiments were performed
(Sánchez Alvarado and Newmark,
1999) for each S. mediterranea innexin clone, and the
effects on regenerating fragments and intact animals were evaluated
(Reddien et al., 2005a).
Although five sequences encoding S. mediterranea innexin
(smedinx-5 to smedinx-9) were previously included in an RNAi
screen, no significant phenotypes associated with regeneration or homeostasis
were described (Reddien et al.,
2005a). We used RNAi to screen all S. mediterranea
innexin genes and found that only two out of 12 (smedinx-10 and
smedinx-11) produced a detectable phenotype (100% penetrance) in both
regenerating and intact worms. smedinx-10 is expressed in the
excretory system and its abrogation produced the disruption of body
homeostasis, revealed by a conspicuous swelling followed by subsequent lysis
of the animals within 10 days (data not shown). The swelling phenotype,
together with the spatial expression of smedinx-10, might suggest
that loss of innexin function results in a failure in the excretory system to
maintain efficient osmoregulation.
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-irradiation
or RNAi for several genes, including smedwi-2
(Guo et al., 2006;
Reddien et al., 2005a;
Reddien et al., 2005b).
Although regression of the tissue in front of the photoreceptors (head
regression) was not observed even after 1 month following the first injection
of smedinx-11 dsRNA, all other phenotypes of irradiated worms
(Reddien et al., 2005b) were
recapitulated. These results suggest that smedinx-11 is required for
proper neoblast function.
smedinx-11 function is required for the proper expression of other neoblast markers
To link innexin gene function with known molecular pathways and to further
characterize the smedinx-11(RNAi) phenotype, we studied whether
smedinx-11 is required for the proper expression of neoblast markers
(Reddien et al., 2005b).
qRT-PCR analyses showed that, at 7 days after smedinx-11(RNAi),
smedwi-1 expression was reduced, whereas smedwi-2 expression was
slightly increased; but, by 1 week later, the expression of both S.
mediterranea piwi genes was severely reduced
(Fig. 4A). We characterized the
spatial expression of the S. mediterranea piwi genes,
smedcyclinB and smedbruno-like (smedbruli) in
smedinx-11(RNAi) worms. Expression of these neoblast markers
disappeared gradually (Fig.
4B,C), consistent with the quantitative assay data
(Fig. 4A). By contrast, markers
of tissues other than neoblasts (S. mediterranea innexins expressed
in excretory, nervous and digestive tissue) did not disappear in
smedinx-11(RNAi) worms at 14 days after RNAi
(Fig. 4C). Interestingly,
smedbruli expression, which is observed in neoblasts and
differentiated tissues (Guo et al.,
2006), was strongly reduced in the neoblast compartment but
remained in differentiated CNS cells (Fig.
4C). Although it is not known whether the regulation of other
neoblast genes by smedinx-11 is cell-autonomous, taken together,
these data firmly establish smedinx-11 as an endogenous regulator of
neoblast biology (it is required for the proper expression of key stem cell
genes in the piwi, cyclin B and bruno families).
The inability to regenerate within the first 7 days after smedinx-11(RNAi) demonstrates that the smedinx-11 phenotype actually begins well before external signs are visible (the early phenotype). Within the first week after smedinx-11(RNAi), animals failed to form a regeneration blastema despite a slight increase in smedwi-2 expression and the presence of mitotic activity. Thus, the early obligate role of smedinx-11 in regeneration does not function via smedwi-2 but is required to form a blastema upon amputation. By 14 days after smedinx-11(RNAi), there was a strong reduction in the expression of S. mediterranea piwi genes. By contrast, 8 days after smedwi-2(RNAi) exposure [when smedwi-2(RNAi) animals are unable to regenerate], smedinx-11 expression was not affected (see Fig. S5 in the supplementary material). This epistatic analysis strongly suggests that smedinx-11 function is required for the expression of the neoblast genes smedwi-1 and smedwi-2 during the late phenotype. It is possible that a significant component of the reduction in the expression of neoblast markers might result from a physical loss of neoblasts after smedinx-11(RNAi). However, the simple possibility that the loss of marker expression is due entirely to the disappearance of expressing cells is refuted by the upregulation of smedwi-2 following smedinx-11(RNAi); instead, this indicates the control of gene expression by SMEDINX-11 function. Taken together, our results suggest that SMEDINX-11 activity is required for the normal expression of S. mediterranea piwi genes in the animal and has additional consequences that are not dependent upon smedwi-2.
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10 days after RNAi)
(Fig. 4D). At 10 days after
smedinx-11(RNAi), the number of dividing neoblasts was reduced,
whereas the X2 subpopulation was not affected in this way
(Fig. 4E). However, as the
phenotype progressed, the X2 irradiation-sensitive subpopulation became
strongly reduced (comparable to the irradiated group), a phenomenon observed
only after -irradiation and in the late phases (18 days) of
smedwi-2 RNAi treatment (Reddien
et al., 2005b) (Fig.
5). The time-dependent way in which neoblast subpopulations
disappear in smedinx-11(RNAi) animals suggests that
smedinx-11 is crucial in order to maintain a pool of proliferative
stem cells and to support continuity of its postmitotic progeny. Thus, these
data reveal functional, molecular differences between X1 and X2 neoblast
subpopulations. Based on these expression and functional studies, we propose
that smedinx-11 is required for stem cell maintenance and that both
neoblast subpopulations require smedinx-11 in order to participate in
body homeostasis and regeneration.
smedinx-11(RNAi) reverses the endogenous anterior-posterior neoblast gradient
Strikingly, after smedinx-11(RNAi), the mitotic activity of
neoblasts disappeared in anterior-posterior (AP) progression
(Fig. 6A). We quantified three
regions (pre-pharyngeal, pharyngeal and post-pharyngeal,
Fig. 6B)
(Reddien and Sánchez Alvarado,
2004) and counted mitotic activity in each region
(Reddien et al., 2005a;
Reddien et al., 2005b). In
wild-type animals, we detected an intrinsic AP gradient of mitotic activity
(more dividing cells in anterior than in posterior areas,
Fig. 6C), consistent with
previous reports on the distribution of neoblasts and mitotic activity
(Baguñà, 1976;
Brondsted, 1969). However, we
noted a sharp reduction in mitotic activity in pre-pharyngeal and pharyngeal,
but not in post-pharyngeal, regions within the first week after
smedinx-11(RNAi). Eventually, as the phenotype progressed, the
reduction in mitotic activity also reached the post-pharyngeal region, ending
with the depletion of mitotic cells in the whole animal after 14 days
following smedinx-11(RNAi). A similar microinjection schedule
targeting only post-pharyngeal areas was performed to test whether the
depletion of mitotic activity in the anterior areas was due to a local
neoblast response to repair the wounds caused by the three successive rounds
of microinjections. In both cases (microinjections in the pre- or
post-pharyngeal area, data not shown), mitotic activity disappeared in the
same AP spatiotemporal fashion, suggesting that this is a specific response to
the functional abrogation of SMEDINX-11 and not because of localized effects
at the injection site. These data also revealed that, even though the anterior
region normally possesses more mitotic cells than the posterior region in the
worm, this is also the area with the highest propensity to lose mitotic
activity after the loss of SMEDINX-11 function. Remarkably, this pattern of
mitotic-activity disappearance has not been reported for any type of
-irradiation treatment or RNAi of genes required for stem cell
maintenance, and is unique for the reduction of SMEDINX-11.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Reddien et al., 2005b;
Salvetti et al., 2005). First,
in known genes required for homeostasis, animals usually die <1 month after
first exposure to RNAi. By contrast, smedinx-11 inhibition did not
lead to head regression; fragments of pre-existing tissue survived without
brains or other head organs for over 1 month (verified by synaptotagmin
expression, data not shown). This long survival will enable the
characterization of morphallaxis and the transitions among stem cell states.
The way in which irradiation-sensitive cells disappear after
smedinx-11(RNAi) is unique to this phenotype (i.e. first
proliferative cells, followed by the X2 population). Moreover, although
smedwi-2 is already known to be required for regeneration, abrogation
of this gene seems not to affect stem cell maintenance - a stark contrast to
smedinx-11. Lastly, the reversion of the AP gradient of proliferative
cells that we observed after smedinx-11(RNAi) has not been described
for any previous RNAi phenotype or -irradiation treatments.
One major consequence of the smedinx-11(RNAi) phenotype is the
inability to regenerate (despite the presence of cell proliferation and the
lack of early toxicity), unlike in bipolar heads previously described in
D. japonica using heptanol (Nogi
and Levin, 2005). This probably reflects the different dynamics of
GJ closure caused by the two techniques: external heptanol exposure targets
different gap junctions non-selectively, changing the open-state probability
in cells accessible to the drug; by contrast, RNAi abrogates
smedinx-11-dependent GJC. The RNAi is specific for one innexin
transcript and thus allows us to separately characterize GJ signals involved
in AP polarity versus stem cell regulation. Combinatorial analysis of
individual innexins will be necessary to understand these distinct roles and
to determine whether the lack of phenotype from RNAi of the other innexin
family members might indicate that the roles of individual innexins can be
compensated for by other innexin family members (as is evident in the connexin
literature on vertebrate gap junctions in patterning).
Because smedinx-11 and smedinx-2 expression was
irradiation-sensitive (Fig.
2A,B), these postmitotic S. mediterranea innexin
expression domains are likely to reveal committed progeny of neoblasts, in
addition to cells expressing the known smedwi-1 and smedwi-2
markers (dividing neoblasts) (Reddien et
al., 2005b). The smedinx-11 gene expression pattern in X1
and X2 cells opens an important opportunity to dissect the transition between
proliferative and postmitotic neoblasts. Moreover, expression studies for
smedinx-11 revealed its irradiation-sensitive expression in
postmitotic areas (Fig. 2 and
see Fig. S3 in the supplementary material), its presence in X1 with enrichment
in X2 cells (Fig. 2C,D) and its
overlapping expression with other neoblast markers (see Fig. S4 in the
supplementary material). Together with the way in which the X1 fraction
disappears before the X2 cells after smedinx-11(RNAi) (Figs
4,
5), these data are suggestive
of a progression between X1 and X2 cells. Therefore, one likely possibility is
that X2 represent the postmitotic progeny of the proliferative X1 compartment
[consistent with our observation that both smedinx-11 and SMEDWI-1
are observed in irradiation-sensitive postmitotic areas
(Guo et al., 2006), and that,
whereas SMEDWI-1 protein is expressed in both X1 and X2, its mRNA is expressed
mostly in X1 cells]. Future work will further elucidate the complexity and
composition of the X2 cells and the possible roles of SMEDINX-11-mediated
signaling as a component of the transition of stem cells between
sub-population states (Fig.
7D).
Analysis of smedinx-11(RNAi) also revealed unique effects on the
endogenous neoblast AP gradient (Fig.
6). Because we detected no obvious AP gradient in
smedinx-11 expression, the higher number of neoblasts in the
pre-pharyngeal area might be due to the anterior regions of planarians
possessing a higher density of complex organs and systems that are subject to
greater cellular turnover compared with posterior regions. The neoblasts
anterior to the pharynx support more-varied tissues and/or tissues with higher
cellular demand. Thus, after smedinx-11(RNAi) (which limits the
available neoblast pool), the anterior regions might deplete the neoblasts
more rapidly than posterior tissues, consistent with the requirement of
neoblast maintenance for smedinx-11. Another possibility is that the
propensity of neoblasts to respond to gap junction-mediated signal differs in
a graded manner along the AP axis. This AP progression confirms the existence
of AP gradients that were hypothesized to exist by classical workers in the
regeneration field (Brondsted,
1969), provides a molecular entry-point into the known but still
not understood relationship between position along the AP axis and
proliferation rates of neoplastic and normal cells in mammals
(Auerbach and Auerbach, 1982),
and suggests that a primary component of physiological gradients are the
neoblasts themselves. These data additionally demonstrate that the control of
neoblast proliferation by gap junction signals functionally interacts with the
morphogenetic polarity of the animal; future work must address the mechanisms
integrating neoblast behavior with large-scale axial patterning.
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Abrogating smedinx-11 function partially isolates neoblasts from
their environment; their subsequent disappearance suggests that, within the X1
population, gap junction-mediated signals might inform the neoblasts that they
are in the midst of differentiated tissue. Future work will focus on
identifying the small-molecule or electric-current signals that traverse
smedinx-11 gap junctions, but this mechanism is of general
significance to stem cell biology. Innexin homologs (pannexins) have recently
been identified in mammals (Baranova et
al., 2004; Bruzzone et al.,
2003); human pannexin 2 (PANX2) has been associated with
bone marrow stem cells (Baranova et al.,
2004), and gap junctions have been described in human embryonic
stem cells and neural progenitors (Cheng
et al., 2004; Leung et al.,
2002; Pearson et al.,
2005; Rozental et al.,
1998; Wong et al.,
2004). In vertebrates, connexin43 functions in the proper
targeting of neural crest cells (Xu et
al., 2006) - a cell population heavily involved in embryonic
morphogenesis of diverse body regions.
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; Leung et al.,
2002; Rozental et al.,
1998). Although our data implicate gap junctions in the control of
somatic, adult stem cells in a morphogenetic context for the first time, a
number of recent studies have indicated that GJC is crucial for germ cell
function in Drosophila (Gilboa et
al., 2003; Tazuke et al.,
2002), suggesting that GJ signaling is likely to be broadly
important in stem cell regulation.
Taken together, these findings suggest that similar GJ-dependent mechanisms
might be widely relevant for controlling the behavior of migratory, plastic
cells. Moreover, tantalizing mammalian data
(Gersdorff Korsgaard et al.,
2001; Goodell et al.,
1996) have suggested that ion transport is a hallmark of stem cell
state, but functional roles have not yet been probed. Likewise, exciting
recent data have linked ion and small-molecule transport to regenerative
ability in vertebrates (Adams et al.,
2007; Nuccitelli,
2003; Zhao et al.,
2006). Thus, GJC might provide an ancient and powerful mechanism
for mediating the complex interplay of regulatory signals among differentiated
and stem cells in multicellular systems
(Sánchez Alvarado and Kang,
2005). Further molecular analyses of GJ roles in planaria and
vertebrate systems will provide crucial opportunities to understand the
evolution of morphogenetic controls as well as identifying novel therapeutic
targets for manipulating stem cell behaviors in biomedical settings.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/17/3121/DC1
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ACKNOWLEDGMENTS |
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