Neuroscience Research Institute and Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, Santa Barbara, CA 93106, USA
* Author for correspondence (e-mail: rothman{at}lifesci.ucsb.edu)
Accepted 12 May 2005
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SUMMARY |
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Key words: C. elegans, Fer kinase, Morphogenesis, Adhesion, ß-catenin
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Introduction |
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Modulation of cell adhesion mechanisms governs cell shape changes and
migration during morphogenesis. Morphogenesis of the C. elegans
epidermis requires cadherin adhesion complexes at the plasma membrane.
Although C. elegans mutants defective in cadherin, -catenin or
ß-catenin function are able to undergo epidermal enclosure, they do not
elongate properly, resulting in lumpy larvae that arrest at the first larval
stage (Costa et al., 1998
).
Adhesion and migration during ventral enclosure are also mediated by the
ephrin signaling pathway. A fraction of embryos lacking either the VAB-1 Eph
receptor or the VAB-2 ephrin ligand arrest without undergoing enclosure by the
epidermis (Chin-Sang et al.,
1999
; George et al.,
1998
). The interaction of this ephrin/Eph receptor pair in
underlying neurons creates a substrate for the migration of the epidermis
during epiboly. The cell adhesion roles of the cadherin and Eph signaling
systems are each associated with regulation of tyrosine kinase activity
(Tepass et al., 2002
;
Zantek et al., 1999
). Whereas
cadherins control localization and phosphorylation of the Eph receptors by
stabilizing cell adhesion, the activated Eph receptors are capable of
autophosphorylation, which promotes their interaction with a variety of
cytoskeletal and signaling proteins through SH2 domains.
Another type of kinase shown to participate in adhesion complexes in
epithelial cells is Fer, a member of the Fes/Fps proto-oncogene family of
non-receptor tyrosine kinases (NRTKs), none of which contains a
membrane-anchoring domain. Fer interacts with cadherin complex components
including N- and E-cadherins, ß-catenin, and p120 catenin, and is thought
to stabilize the interactions within the complex via its kinase activity
(Piedra et al., 2003;
Rosato et al., 1998
;
Xu et al., 2004
). Fer has also
been shown to move between cadherin and integrin complexes while mediating
cell adhesion (Arregui et al.,
2000
). Two forms of mammalian Fer exist: a ubiquitously expressed,
full-length version (Letwin et al.,
1988
; Pawson et al.,
1989
), and a nuclear, truncated version, FerT, which has been
observed only in primary spermatocytes
(Fischman et al., 1990
). While
Fer has been implicated in cell adhesion complexes, the essential in vivo
function of fer has not been reported, with the exception that a
mouse knock-in of a kinase-inactive form of the protein showed no obvious
developmental defects (Craig et al.,
2001
). Although it was previously reported that there were
probably no Fer homologs in C. elegans
(Greer, 2002
;
Plowman et al., 1999
),
fer-like genes have been found in sponges
(Cetkovic et al., 1998
), flies
(Katzen et al., 1991
;
Paulson et al., 1997
), birds
and mammals (Fischman et al.,
1990
; Pawson et al.,
1989
; Feldman et al.,
1986
).
In this study, we demonstrate the essential in vivo role of a Fer-like protein, FRK-1, in morphogenesis of developing C. elegans embryos. We report that expression of FRK-1 in the epidermis is required for enclosure of the embryo at the onset of morphogenesis. We made the unexpected finding that the kinase activity of FRK-1 is not required for its role in morphogenesis, suggesting that FRK-1 may act to stabilize adhesion complexes independent of its enzymatic activity. This kinase-independent function of FRK-1 is also required cell-autonomously in the lateral row of epidermal cells, the seam cells, for late stages of their differentiation. FRK-1 is normally localized at the plasma membrane beginning at the onset of morphogenesis; however, it relocalizes to the nucleus in the absence of either the cadherin-associated ß-catenin, HMP-2, or the muscle-expressed ß-integrin homolog, PAT-3; the latter indicates a cell-non-autonomous action of ß-integrin on FRK-1 function. Mouse FerT can substitute for FRK-1 in worms, and expression of FRK-1 in mammalian cells causes loss of adhesion, similar to the effects of overexpressing Fer in mammalian cells. These findings suggest that the crucial role for Fer-like proteins in epithelial morphogenesis is conserved throughout animal phylogeny.
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Materials and methods |
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To analyze the tissue distribution of frk-1 expression, we created
a transcriptional fusion construct containing 1.8 kb upstream of the
frk-1 gene fused to GFP and a nuclear localization sequence using
pPD96.62 (Fire et al., 1990)
(a gift from A. Fire). Arrays containing this construct and the
rol-6(su1006) marker (pRF4)
(Mello et al., 1991
) were
analyzed.
Expression of epidermis-specific genes in frk-1(RNAi) embryos was
examined using the following strains: JR672 contains an integrated array
(wIs54) of the seam-specific SCM::GFP marker (previously described by
Terns et al. (Terns et al.,
1997) plus ajm-1::GFP (a gift from J. Hardin). JG5
contains an elt-3::GFP fusion and was a gift from J. Gilleard.
nhr-72 (PY1215) and nhr-81 (PY1282) GFP reporter lines were
gifts from P. Sengupta (Miyabayashi et
al., 1999
). JR1736 contains nhr-75::GFP (P. Sengupta) and
was made as described in Koh and Rothman
(Koh and Rothman, 2001
).
RNA-mediated interference (RNAi)
RNA interference was performed by using dsRNA made from a PCR product
containing the T7 promoter on each end fused to a genomic region of
frk-1 of approximately 1300 bp. dsRNA was synthesized and purified
using the MEGAscript kit (Ambion) according to the manufacturer's protocol.
dsRNA was either injected into young hermaphrodites
(Fire et al., 1998) or used to
soak young hermaphrodites overnight. Progeny were analyzed 8 to 20 hours after
injection.
Four-dimensional time-lapse analysis of development
Embryonic development in wild-type, mDf7 and frk-1(RNAi)
embryos was analyzed via time-lapse video microscopy as described previously
(Moskowitz et al., 1994).
Images were collected through ten focal planes, encompassing the entire
embryo. Scans were made every 5 minutes for the first 8 hours of development
(sufficient time for completion of ventral enclosure in wild-type embryos).
For each mutant embryo recorded, a wild-type embryo was placed in the field of
observation to allow for direct comparison of mutant versus wild type
throughout development. At each time point the planes of focus that included
developing epidermal cells were analyzed for cell division, shape and
migration.
In vitro kinase assays
frk-1 and frk-1(D308R) PCR products were transcribed and
translated in vitro using the TnT Quick Coupled kit (Promega) according to the
manufacturer's protocol. The translated FRK-1 was purified by
immunoprecipitation using FRK-1 antibodies and Sepharose A-agarose beads
(Zymed) and resuspended in 1x PBS buffer. Each tube of purified FRK-1
was then tested for autophosphorylation activity by adding 5x tyrosine
kinase buffer (100 mmol/l HEPES, 5 mmol/l MnCl2, 5 mmol/l DTT, 500
µmol/l Na3VO4), ATP (5 µmol/l final), 5 µCi
[32P]ATP, and 2.75 µl dH2O. Each reaction was
incubated at 30°C for 10 minutes and terminated by boiling. Unused
radioactivity was eliminated by Micro bio-spin 6 chromatography columns
(Biorad). Samples were boiled for 5 minutes and run on gradient (4-15%)
tricine gels (Jule). Gels were dried on Whatman paper and exposed to
radiographic film (Kodak).
Antibody production and immunofluorescence
Anti-FRK-1 antibodies were raised against two FRK-1-specific peptides:
FRK-1a (EKSSNNDASVTDDIRAE) and FRK-1b (QKNPEKRSTMDSIHKKLRE). The peptides were
synthesized by United Biochemical Research. The sequences of the two peptides
were derived from each end of the protein and chosen to minimize homology with
other proteins. Two rabbits, UCSB64 and UCSB65, were immunized against each
peptide by Cocalico Biologicals, Inc. Antiserum from each rabbit was
affinity-purified with the original peptide using the SulfoLink kit (Pierce)
and tested. Both antibodies gave identical staining patterns. Embryos were
fixed and stained according to Sulston and Hodgkin
(Sulston and Hodgkin, 1988)
using a 1:5 dilution of each purified FRK-1 antibody.
MH27 is a mouse monoclonal antibody that recognizes epithelial apical
junctions (Priess and Hirsh,
1986). NE2-1B4 is a mouse monoclonal antibody that recognizes an
antigen expressed only in seam cells
(Schnabel, 1991
). LIN-26
antibodies (rabbit) were a generous gift from M. Labouesse.
|
Expression of FRK-1 in cultured human cells
The mammalian ecdysone-inducible expression system (Invitrogen) was used to
express FRK-1 in cultured cells. A frk-1 cDNA was amplified using
oligonucleotides that included a 5' Kozak sequence necessary for
efficient translation in mammalian cells and ligated into the EcoRI
site of pIND-lacZ (Invitrogen). The pIND-lacZ;
frk-1 plasmid (320 ng DNA per 25 µl medium) and pVgRXR (which
expresses the ecdysone receptor) were then transiently transfected into the
human embryonic kidney cell line HEK293, using Lipofectamine 2000
(Invitrogen). Cells were plated onto 96-well plates coated with poly-D-lysine
(100 µg/ml), human placental laminin-2 (500 µg/ml; Sigma), or plasma
fibronectin (10 µg/ml; Invitrogen). FRK-1 expression was induced by
exposing cells to the ecdysone analog Ponasterone A (5 µmol/l) for 20
hours, according to the manufacturer's protocol. The cells were washed with
PBS and fixed with 4% paraformaldehyde/4% sucrose. Cells transfected with
pIND-lacZ were stained with BluOGal (5 mmol/l ferricyanide, 5 mmol/l
ferrocyanide, 2 mmol/l MgCl2) to assess transformation efficiency
(which was typically 50%). Adhesion was quantified by staining triplicate
wells with amido black (Davis and
Camarillo, 1993
).
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Results |
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The predicted product of frk-1, FRK-1, is most similar to
mammalian FerT, the truncated form of the protein that lacks the amino
terminal coiled-coil domain of full-length Fer. FRK-1 is 67% similar and 39%
identical to FerT. The similarity between the C. elegans and
mammalian proteins is more pronounced in the SH2 binding domain (86%
similarity), which contains the signature FLVR element, and the tyrosine
kinase domain (73% similarity), where the ATP binding site resides and
conserved amino acid residues (numbers 252 and 308) required for kinase
activity (Cole et al., 1999)
are located. The predicted FRK-1 protein may be the only isoform of FRK-1 in
C. elegans, as corroborated by RT-PCR and Western blotting, which
revealed a single transcript and polypeptide of approximately 1180 bp and 45
kDa, respectively (data not shown).
|
|
The discrepancy between the two mutants may be attributable to partial
rescue of mDf7 by maternally contributed FRK-1; such maternal
contribution is expected to be eliminated by RNAi. At least one other protein
involved in embryonic morphogenesis, the cadherin HMR-1, shows a similar
phenomenon: homozygous hmr-1 embryos produced by heterozygous mothers
are only partially defective in enclosure, whereas a substantial fraction of
hmr-1(RNAi) embryos arrest without any signs of enclosure
(Raich et al., 1999). We
obtained evidence that maternally provided FRK-1 is required for embryogenesis
by specifically eliminating it in frk-1(RNAi) homozygous embryos that
were rescued zygotically with the mammalian ferT gene (see below),
which is not susceptible to frk-1(RNAi) owing to the highly divergent
DNA sequences of the two genes. All embryos specifically lacking maternal
FRK-1/FerT function arrested with only 200-300 cells, far short of the number
of cells required for enclosure. Elimination of FRK-1 by RNAi in wild-type
embryos resulted in early arrest in a small fraction (
5%) of the embryos.
Thus, the maternal FRK-1 contribution in mDf7 probably functions both
in cell proliferation in early embryos and in organizing the epidermis before
enclosure in later embryos.
Analysis of frk-1 mutant embryos demonstrated that FRK-1 function is required for late stages of epidermal differentiation. Early markers of epidermal differentiation, including the apical junction protein AJM-1 and LIN-26, a transcription factor expressed in all epidermal nuclei, were both observed on the dorsal side of the embryo. Two markers specific for late stages of differentiation of the lateral rows of epidermal cells, the seam cells, failed to be expressed in terminal embryos (Fig. 3A). However, an earlier marker of seam cells, the engrailed homolog CEH-16 was always expressed in arrested embryos (not shown). Expression of four other markers that normally first appear as seam cells begin to differentiate was attenuated in arrested embryos (Fig. 3A). FRK-1 is also required for non-seam epidermal differentiation: elt-3::GFP, which is normally expressed in all epidermal cells except for seam cells, also showed attenuated expression in frk-1 mutants (Fig. 3A). These results indicate that FRK-1 is required for completion, but not initiation, of epidermal differentiation.
|
Mammalian FerT can substitute for C. elegans FRK-1
To investigate whether the function of Fer proteins in morphogenesis is
evolutionarily conserved, we tested whether mammalian FerT could substitute
for FRK-1. A mouse ferT cDNA was fused to the frk-1 promoter
and 3'-UTR and introduced into a strain carrying mDf7. We were
able to isolate stable transgenic lines carrying this construct only when it
was microinjected at very low concentrations, suggesting that high levels of
FerT are toxic in C. elegans. However, several independent arrays
containing the frk-1p::ferT transgene rescued the enclosure and
elongation defects and embryonic lethality of mDf7
(Fig. 2E,F), albeit somewhat
less efficiently than FRK-1 (Fig.
4); thus, FerT family members perform a function that is conserved
throughout metazoans.
|
Surprisingly, we found that this kinase-dead FRK-1(D308R) mutant was
capable of rescuing mDf7 homozygotes
(Fig. 5C,D) to hatching,
implying that the kinase activity of zygotic FRK-1 is not required for its
role in enclosure and elongation. Rescued, hatched larvae arrested at various
stages of larval development (L1-L4), most often with a ruptured epidermis
somewhere along the ventral midline. The only evidence we obtained that the
kinase activity of FRK-1 could perform any function in the embryo came from
observations of embryos overexpressing FRK-1. While overexpression of
wild-type FRK-1 from an extrachromosomal transgenic array in wild-type worms
often results in defective morphogenesis and lethality, this effect was
eliminated by the D308R mutation, suggesting that it may be excessive kinase
activity per se that causes the overexpression phenotype. The lack of a
phenotype in the mouse line expressing kinase-inactive Fer has been attributed
to possible redundancy with another kinase or its non-essential function in
adhesion and/or morphogenetic pathways
(Craig et al., 2001). Our
findings instead suggest that FRK-1 performs an essential structural, rather
than enzymatic, role in morphogenesis.
FRK-1 shows dynamic localization to the cell surface and nucleus
Mammalian Fer was reported to localize to nuclei during the S-phase of
mitosis (Ben-Dor et al., 1999)
and co-purifies with the chromatin fraction in nuclear extracts
(Hao et al., 1991
). However,
the in vivo localization of Fer remains a source of debate, as others reported
that it never localizes to the nucleus
(Zirngibl et al., 2001
). We
found that FRK-1 in C. elegans showed a dynamic localization pattern
during embryogenesis that could account for both observations. Using two
independent antibodies elicited to two different FRK-1 peptides (see Materials
and methods), we found that immunoreactive FRK-1 was initially present both in
nuclei and at cell-cell contact points of all cells in early embryos
(Fig. 6A,B). Expression later
became restricted to epithelial cells, body wall muscle and the germline,
including mature sperm. Staining was eliminated in frk-1(RNAi)
embryos, confirming specificity of the antibody (not shown). Epidermal
expression of FRK-1, which is apparently sufficient for morphogenesis (see
above), requires ELT-1, a transcription factor required to generate all
epidermal cells (Page et al.,
1997
): FRK-1 was seen only in body wall muscle cells in late
elt-1(zu180) mutant embryos.
Localization of FRK-1 to nuclei correlates with phases of active cell division: nuclear localization was prominent in early embryos and the adult germline (Fig. 6A,B and not shown). The protein remained in the nucleus and at the cell surface until shortly before embryonic enclosure, at which stage most cells became mitotically inactive and nuclear FRK-1 became undetectable (Fig. 6C,D). In elongated embryos, FRK-1 was present in the cytoplasm and at the membrane of epidermal cells; cell surface staining was especially prominent in the seam cells (Fig. 6E,F). While we cannot exclude a nuclear function for FRK-1 in morphogenesis, the protein was not detectable in nuclei during this phase of development, suggesting that it is the plasma membrane and cytoplasmic forms of the protein that underlie its essential morphogenetic function. Later, during larval development, FRK-1 reappeared in the nuclei of seam cells and in the nuclei of the developing germline, correlated with their active post-embryonic division. In the adult soma, FRK-1 stably localized to apical junctions of the intestine and somatic cells of the gonad (not shown).
|
|
If plasma membrane-associated FRK-1 is essential for epidermal enclosure, these findings raise a paradox: while the absence of HMP-2 resulted in elimination of FRK-1 from the cell surface and its dramatic mislocalization to the nucleus, hmp-2 mutants, unlike frk-1 mutants, were nonetheless able to undergo epidermal enclosure. It is possible that an undetectable amount of FRK-1 remaining at the cell surface is sufficient to direct epidermal enclosure in hmp-2(zu364) mutants. However, we found that FRK-1 was localized at the plasma membrane before enclosure in hmp-2(zu364) mutant embryos and that its mislocalization to the nucleus occurred only once enclosure had initiated (Fig. 7C,D); thus, it is reasonable to suggest that by the time FRK-1 was absent from the plasma membrane in hmp-2 mutants, it had already provided its essential function in epidermal enclosure. We considered the alternative possibility that FRK-1 may be required for epidermal enclosure only when ß-catenin is present; however, this appears not to be the case, as we found that frk-1; hmp-2 double mutants, like the frk-1 single mutant, failed to enclose (not shown).
The integrin adhesion system is essential for normal morphogenesis in
C. elegans (Gettner et al.,
1995), and in vitro experiments suggest that Fer participates in
cross-talk between the cadherin and integrin complexes
(Arregui et al., 2000
). We
found that normal FRK-1 localization required the ß-integrin homolog,
PAT-3: in pat-3(st564) mutant embryos, FRK-1 partially mislocalized
to the nucleus of epidermal cells (Fig.
7F). A startling aspect of this result is that PAT-3 was expressed
only in the body wall muscle of C. elegans and was not detectable in
the epidermis (Gettner et al.,
1995
). Thus, these results suggest cell non-autonomous signaling
across the basement membrane that separates the body wall muscle from the
epidermis. This effect might be attributable to general disruption of the
basement membrane adhesion system, or instead to a more specific,
cell-non-autonomous role of muscle-expressed integrin on plasma membrane
complexes containing FRK-1 in epidermal cells. We found that FRK-1
localization was not conspicuously altered in two mutants defective in
different basement membrane collagens, let-2
(Sibley et al., 1993
) and
emb-9 (Guo et al.,
1991
), suggesting that the effect of the PAT-3 ß-integrin on
FRK-1 localization in epidermal cells is not the result of a general
perturbation of the basement membrane per se. The effect of the pat-3
mutation on FRK-1 localization also raises the possibility that the elongation
defect in this mutant may result in part from disruption of FRK-1
function.
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Discussion |
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The essential role of FRK-1 in morphogenesis appears to be functionally
conserved across metazoans, as mammalian FerT could substitute for FRK-1
during enclosure and elongation in C. elegans. While FRK-1 is most
similar in sequence to FerT, its pattern of expression broadly in the embryo,
as well as in sperm, resembles that of both Fer, which is expressed
ubiquitously, and FerT, which is specific for spermatocytes, in
Drosophila and mammals. Fer contains an amino-terminal sequence not
present on FerT, which determines its phosphorylation state
(Craig et al., 1999) and which
may control its cellular functions
(Orlovsky et al., 2000
). This
sequence is apparently dispensable in C. elegans: unlike
Drosophila and mammals, which both produce a full-length and
truncated version of Fer, C. elegans appears to contain only a single
Fer-like protein that apparently performs the function of both Fer forms in
other animals.
Kinase independence of FRK-1 function
Our finding that the kinase activity of FRK-1 was not required for
embryonic morphogenesis suggests that the critical action of FRK-1 in
morphogenesis depends not on its enzymatic activity, but on its interactions
with other proteins; for example, it may be required for assembly of complexes
at the plasma membrane that mediate dynamic changes in adhesion and cell
movement. Similar to our findings with FRK-1, elimination of the kinase
activity of the integrin linked kinase (ILK) does not abolish its function in
cell adhesion: a form of Drosophila ILK that carries the same
sequence change as one in human ILK shown to drastically reduce kinase
activity rescues as efficiently as the wild-type protein
(Zervas et al., 2001). The
same mutation in the C. elegans ILK homolog, PAT-4, does not prevent
its binding to UNC-112, a cytoplasmic attachment protein
(Mackinnon et al., 2002
). Both
studies support a kinase-independent role for ILK as an adaptor protein that
stabilizes integrin complexes and facilitates muscle attachment.
Our findings do not rule out an essential kinase-dependent function for
FRK-1. Maternally contributed FRK-1 was apparently required for completion of
early embryogenesis, based on its early expression and the apparent rescue of
the early embryonic arrest by maternally, but not zygotically, provided FRK-1
product. The kinase activity of FRK-1 may well be integral to this early role
for the protein. It is during early embryogenesis that FRK-1 was
nuclear-localized and it is interesting to note that Fer has been shown to
phosphorylate nuclear localized factors such as Stat3
(Priel-Halachmi et al., 2000)
and the TATA element modulatory factor
(Schwartz et al., 1998
). The
role for nuclear FRK-1 may be of particular importance, as it has been shown
that Fer kinase is upregulated in proliferating prostate cancer cells
(Allard et al., 2000
), and that
inappropriately phosphorylated Fer prevents progression beyond the G0/G1 phase
of the cell cycle (Orlovsky et al.,
2000
). At least some of the early function of FRK-1 as a kinase
may also relate to its role in maternally directed Wnt-type signaling, as we
have found that FRK-1 acts in concert with multiple components of the Wnt
pathway (A.P.P. and J.H.R., unpublished).
It is conceivable that FRK-1 does function as a kinase later in
embryogenesis but this activity is non-essential for epidermal morphogenesis
owing to the redundant function of another kinase. Such redundancy has
recently been suggested for the mammalian non-receptor kinases Fer and Fyn,
and has been invoked as an explanation for the mild knockout phenotypes of
each of the two genes in mice (Craig et
al., 2001; Stein et al.,
1992
). While the region deleted by mDf7 included two
genes, in addition to frk-1, encoding SH2 domain-containing tyrosine
kinases, the debilitation of either gene by RNAi did not result in a
conspicuous embryonic phenotype; it remains to be determined whether another
kinase can function as a redundant partner with FRK-1 during embryonic
morphogenesis.
FRK-1 and the pathway for epidermal development and differentiation
In addition to its role in promoting epidermal morphogenesis, FRK-1 was
required for late stages of epidermal differentiation. Although early
epidermal markers, such as LIN-26, which is required for specification and
maintenance of epidermal cell fates (Page
et al., 1997; Quintin et al.,
2001
), and CEH-16, are unaffected by the loss of frk-1
function, and the appropriate number of epidermal cells appears to be
specified, later markers of both seam and non-seam cells were either absent or
severely reduced in frk-1 mutants. The requirement for FRK-1 in
epidermal differentiation is not likely to be a consequence of the enclosure
defect per se, as other mutants defective in enclosure, such as those lacking
cadherin complex components, make differentiated seam cells
(Costa et al., 1998
;
Hoier et al., 2000
). Our
results suggest that FRK-1 acts downstream of the regulators that initially
specify epidermis, i.e. ELT-1, LIN-26 and CEH-16, but upstream of factors that
confer final stages of differentiation on different subsets of the epidermis,
e.g. EGL-18 (Koh and Rothman,
2001
) in seam cells and possibly ELT-3
(Gilleard et al., 1999
) in
non-seam epidermis (Fig. 3C).
Further studies will help to elucidate whether FRK-1 acts directly on the
regulatory machinery for epidermal differentiation or instead results in
cellular rearrangements that are required for the function of such
regulators.
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ACKNOWLEDGMENTS |
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