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Expression patterns of bone morphogenetic proteins (Bmps) in the developing mouse tooth suggest roles in morphogenesis and cell differentiation

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Keratin 8 and 18 Expression in Mesenchymal Progenitor
Cells of Regenerating Limbs Is Associated With Cell
Proliferation and Differentiation
Developmental Biology Unit, Institute of Child Health, University College London, London, United Kingdom
Keratins are considered markers of epithelial differentiation. In lower vertebrates, however, immunoreactivity for keratin 8 and
18 has been reported in nonepithelial cells, particularly in mesenchymal progenitor cells of regenerating complex body structures. To confirm that
such reactivity does indeed reflect keratin expression and to investigate their possible role in
regeneration, we have isolated clones coding for
the newt homologues of keratin 8 and 18 (NvK8
and NvK18, respectively) and studied their distribution and changes in their expression following
experimental manipulations. Analysis of NvK8
and NvK18 transcripts confirms that K8 and K18
are expressed in the blastemal cells of regenerating newt limbs and that their expression is first
observed 3-5 days after amputation, when the
blastemal cells start to proliferate under the influence of the nerve, whose presence is essential for
regeneration to proceed. In contrast, no induction of these keratins is observed following amputation of a larval limb at a stage when organogenesis is proceeding in a nerve-independent manner.
To establish whether there is a causal relationship between keratin expression and cell proliferation in the adult limb blastema, we have investigated whether their expression is nerve-dependent
and whether suppression of their expression in
cultured blastemal cells affects cell division and
differentiation. Analysis of keratins in denervated limbs demonstrates that the nerve is not
necessary to induce their expression. However,
treatment of cultured blastemal cells with K8 and
K18 anti-sense oligonucleotides significantly decreases DNA synthesis and induces changes in
cell morphology, suggesting that expression of
these keratins during regeneration may be necessary for the maintenance of the undifferentiated
and proliferative state of blastemal cells. Dev.
Dyn. 1997;210:355–370. r 1997 Wiley-Liss, Inc.
Key words: differentiation; limb; keratin; regeneration; proliferation; urodele amphibians
Urodele amphibians, such as newts and axolotls, can
regenerate their limbs as adults through a process
called epimorphic regeneration. In the course of this
regenerative process, a mound of undifferentiated mesenchymal progenitors cells, the blastemal cells, accumulate under a specialized wound epidermis, which rapidly covers the stump surface following amputation.
After this accumulation phase, the blastemal cells start
to proliferate. Up to 2–3 weeks (Thornton, 1968) after
amputation, before the regeneration blastema has become palette-shaped and differentiation and morphogenesis begin, limb denervation results in the arrest of
DNA synthesis and consequent inhibition of regeneration (Singer, 1952; Thornton, 1968). However, when the
regenerating limb is denervated after reaching a critical size (from the late bud stage onward), a complete
limb, although smaller than the amputated one, will
form (Schotté and Butler, 1944; Singer, 1948). Nervedependent growth control of regeneration is established
as a consequence of innervation. In fact, whereas the
presence of the nerve is required during regeneration of
limbs that have been innervated, regeneration of a
developing limb bud can occur before it becomes innervated.
A distinction between the progenitor cells of developing and regenerating limb buds is highlighted by their
different content of intermediate filaments. Although
both populations express the mesenchymal intermediate filament vimentin, reactivity with the monoclonal
antibody (mAb) 22/18 and mAbs against human simple
epithelial keratins 8 and 18 (K8 and K18) is observed
only in regenerating limbs (Fekete and Brockes, 1987;
Ferretti et al., 1989). This apparent expression of K8
and K18 in mesenchymal cells is puzzling because
cytokeratins, obligatory heteropolymers encoded by
two large multigene families, are usually considered
markers of epithelial differentiation and are normally
expressed in glandular epithelia. In lower vertebrates,
keratin immunoreactivity has also been reported in
some nonepithelial tissues that grow throughout life
and have significant regenerative ability, such as optic
nerve and fins in fish (Giordano et al., 1989; Ferretti et
al., in preparation), and in regeneration blastemas of
Grant sponsor: Wellcome Trust.
Dr. Corcoran’s current address is Developmental Biology Research
Center, The Randall Institute, King’s College London, 26-29 Drury
Lane, London WC2B 5RL, United Kingdom.
*Correspondence to: Dr. Patrizia Ferretti, Developmental Biology
Unit, Institute of Child Health, UCL, 30 Guilford Street, London
WC1N 1EH, United Kingdom. E-mail:
Received 10 July 1997; Accepted 19 August 1997
different organs in amphibians (Ferretti et al., 1989;
Ferretti and Ghosh, 1997). Furthermore, K8 and K18
are present in the oocyte and are among the first genes
to be activated during embryogenesis when the cells are
still pluripotent and no differentiation is apparent. The
role of this keratin pair in cell function is still elusive,
but the view that these and other intermediate filaments are only needed to stabilize the cell structure has
been challenged by the fact that their assembly and
disassembly has turned out to be much more dynamic
than initially believed and that changes in their expression can be associated with changes in cell physiology.
For example, depletion of these keratins in early Xenopus embryos results in gastrulation abnormalities
(Heasman et al., 1992), activation of K8 and 18 in
certain tumors may have a role in their increased
migratory and invasive ability (Chu et al., 1993; Oshima et al., 1996), lack of K8 in transgenic mice is lethal
(Baribault et al., 1993), and mutations in human K18
appear to be associated with cryptogenic cirrhosis (Ku
et al., 1995, 1997). Therefore, the fact that simple
epithelial keratins are expressed in many instances in
nonepithelial tissues following amputation may indeed
reflect a significant role of these molecules in the
regenerative process. This possibility has yet to be
thoroughly investigated, and such studies might not
only help to elucidate the mechanisms underlying the
regenerative process, but also to reveal yet unidentified
roles of simple epithelial keratins in cell function. The
regenerating limb offers an important model for such a
study, because it is so far the best characterized of the
regenerating systems in lower vertebrates and in culture (Géraudie and Ferretti, 1997).
As mentioned above, in the newt, Notophthalmus
viridescens, reactivity with antibodies to human K8
and K18 has been observed in the blastema of the
regenerating adult limb but not in progenitor cells of
normally developing limb buds in a related species,
Pleurodeles waltl, although various embryonic tissues
were strongly stained (Ferretti et al., 1989). Therefore,
it was important to demonstrate conclusively that K8
and K18 are the proteins upregulated following amputation and that they are not expressed in the developing
limb buds in the same species. Furthermore, we wished
to establish whether expression of newt K8 and K18
could be induced following amputation of limb buds in
N. viridescens embryos or whether, like 22/18, their
expression was induced only after the transition from
the nerve-dependent to the nerve-independent mode of
regeneration. Finally, we wished to investigate whether
there is a causal relationship between expression of
these keratins in blastemal cells and the regenerative
capability of the urodele limb, particularly in relation to
nerve dependency, because of the apparent difference in
keratin expression in embryonic limb buds and regeneration blastemas.
Therefore, we have isolated cDNA clones coding for
the newt keratin homologues of mammalian K8 and
K18 and studied the distribution of these transcripts
both in developing and regenerating limbs under different experimental conditions for two purposes. First, to
confirm that the immunoreactivity detected in blastemal cells with anti-keratin mAbs was indeed due to the
expression of simple epithelium keratins. Second, to
assess a possible role for these proteins in the regenerative process. We show here that these keratins are
expressed in the blastema mesenchyme and in cultured
limb cells, but not in regenerating limb buds before they
become innervated, and that, although they are first
expressed in the regeneration blastema at the onset of
cell proliferation, their induction is not dependent on
the presence of innervation. We have also explored the
possibility that newt K8 and K18 might be permissive
for cell proliferation in the blastema by downregulating
their expression in culture using anti-sense oligonucleotides. We show that this results in inhibition of proliferation and changes in cell morphology that are consistent
with a role of simple epithelial keratins in maintaining
the blastemal phenotype.
Isolation of NvK8 and NvK18 cDNA Clones
Eight of the plaques identified by screening an expression cDNA library from cultured blastemal cell with the
mAb LP1K, which recognizes a single protein of apparent molecular weight 52 kD in these cultures (Ferretti
et al., 1989), were purified. Seven of the eight clones
isolated shared the same PstI and SacI restriction sites.
The three longest overlapping clones (1700, 1440, and
1250 bp) were selected for sequence analysis (Fig. 1A).
All these clones extended into the 38 end untranslated
region, and one of them extended toward the 58 end
region 225 bp further than the others but did not
contain a methionine initiation codon. Of the plaques
identified by hybridization with the human K18 probe,
five were isolated and they all shared the same BglII
and PstI restriction sites. Three of these clones were
used for further analysis, and the longest clone (which
contains the two shorter ones) was 2400 bp (Fig. 1B).
DNA Sequence and Deduced Amino Acid
Sequence of cDNA Clones
The basic structure of an intermediate filament protein that comprises a linear N-terminus (head), an
a-helical central portion (rod), and a linear C-terminus
(tail) is shown in Figure 1C. The nucleotide and deduced amino acid sequence of the overlapping clones
isolated by LP1K screening is shown in Figure 2A.
These clones code for a type II cytokeratin, which was
named N. viridescens keratin 8 (NvK8), since sequence
comparison with other proteins shows that the highest
percentage of NvK8 homology is with proteins of the
type II keratin gene family, particularly with the simple
epithelia keratin 8 (Fig. 2B). The percentage of amino
acid identity in the entire rod domain of NvK8 is much
higher with goldfish, Xenopus, and mouse K8 (83%,
87%, and 81%, respectively) than with the previously
cloned type II newt keratin, NvKII (69%) (Ferretti et
initiation codon, suggests that NvK8 sequence is not
complete and is probably missing 10 amino acids.
Partial sequencing of the clones isolated using the
human K18 probe shows that they encode the newt
homologue, N. viridescens keratin 18 (NvK18), of K18
(Fig. 3). The sequenced portion of the NvK18 rod
domain, which includes most helix 1B (82 of 101 amino
acids) and 11 amino acids of the linker joining helix 1B
and helix 2, contains the motif DNARPAADDFR, which
is highly conserved in type I keratins. Comparison
analysis shows that this region of NvK18 shares 70%
and 69% amino acid identity with Xenopus and mouse
K18, respectively (Singer et al., 1986; LaFlamme et al.,
1988), 47% identity with human keratin 16 (HK16;
Rosenberg et al., 1988), and 57% identity with human
keratin 17 (HK17; RayChaudhury et al., 1986). The
carboxyl tail of NvK18 has high amino acid identity
with Xenopus (72%). However, in contrast to the high
degree of conservation with mammalian type I keratins
in the rod domain, the amino acid identity of NvK18 tail
with mouse K18 is 55% and that with HK16 and HK17
only 10% and 28%, respectively. However, there is a
conserved motif at the end of the C-terminus that is
also found in the simple epithelia type I keratin HK17
and in K8s of amphibians, fish, and mammals (Figs.
2B, 3).
Tissue Distribution of the NvK8
and NvK18 Transcripts
Fig. 1. Restriction maps of newt clones isolated using the mAb LP1K
(A) and a human cDNA K18 probe (B) as well as a schematic representation (C) of the structure of keratin proteins showing the target regions of
the anti-sense oligomers used in this study. (A) Three overlapping clones
coding for the newt keratin 8 (NvK8). The 300 nucleotide NvK8 PstI-PstI
subclone used to make riboprobes is shown. (B) Three overlapping
clones coding for the newt keratin 18 (NvK18). The 290 nucleotide NvK18
BglII-EcoRI subclone used to make riboprobes is shown. The dotted lines
in A and B indicate 38 untranslated sequences. The double bar in B
indicates the beginning of the coding region. (C) Highly schematic
representation of a keratin. The hatched boxes indicate the a-helical
portions of the central rod domain (helix 1A, helix 1B, and helix 2), which
are joined by linear linkers (thin lines), L1, and L12. The straight lines
flanking the rod represent the linear N-terminus region (head) and
C-terminus region (tail). The regions against which the anti-sense oligomers to NvK8 and NvK18 are targeted and their sequences are shown.
al., 1991). In the head and tail, a high percentage of
identity with other K8s is observed in the regions
flanking the rod domain and in motifs toward the end of
both termini. The motif at the end of the C-terminus
(Fig. 2A) is found in a similar position in all K8
proteins, in several type I keratins, and in other
nonepithelial intermediate filaments such as vimentin
(Quax et al., 1983). The tail of NvK8, as that of simple
epithelia keratins from other lower vertebrates (Franz
and Franke, 1986; Giordano et al., 1989; Charlebois et
al., 1990), is longer than its mammalian counterpart
and is rich in glycine. Five repeats of the type GGS/Y
are present. The sequence of NvK8 at the N-terminus is
10 amino acids shorter than that of other K8s; this,
together with the fact that we did not find a methionine
To obtain an initial indication of the transcriptional
regulation of NvK8 and NvK18, Northern analysis was
performed using RNA from unamputated forelimbs,
forelimb blastemas, liver, intestine, and esophagus.
Both NvK8 and NvK18 probes detected single transcripts of approximately 2.1 and 1.4 Kb, respectively
(Fig. 4A,B), in liver, proximal forelimb blastemas, and
intestine. No signal was detected with either probe in
normal limbs and esophagus. Thus, it appears that
there is a single functional copy of both NvK8 and
NvK18 and that these transcripts are upregulated in
the regenerate.
Further analysis of the expression of NvK8 at different axial levels in normal and regenerating limbs and
tails was carried out by RNAase protection because of
the higher sensitivity of the method (Fig. 4C). This
analysis shows that in the unamputated limb, the
NvK8 transcript is hardly detectable in the arm but
clearly visible in the hand and fingertips. The levels of
the NvK8 transcript, however, are significantly higher
in the regenerate, but no difference between proximal
and distal blastemas is observed. A significant increase
in NvK8 mRNA is also observed in newt tail blastemas
compared with normal tail. These results further indicate that upregulation of NvK8 is associated with the
regenerative process and, as previously suggested by
immunocytochemistry, that the fingertips are blastemalike.
To establish whether NvK8 and NvK18 transcripts
are restricted to the mesenchyme and not expressed
Fig. 2. Nucleotide and amino acid sequence of NvK8. GenBank Accession Number 136454. (A) The nucleotide and predicted amino acid
sequence of the composite NvK8 cDNAs is shown. There is no initiation
methionine, suggesting that the available cDNA is not full length; the
comparison analysis in B indicates the lack of 10 amino acids. Two amino
acid motifs, TYRKLLEGE, which marks the end of the helical central rod
domain, and DGKVTSES, which is similar to a motif found in the
C-terminus of K8 in other species, are underlined. (B) Comparison of the
amino acid sequence of NvK8 with that of Xenopus (XK8), mouse (MK8),
bovine (BK8), and goldfish (gfK8) keratin 8 and with the newt type II
keratin, NvKII. Identical amino acids are in bold. The rod domain is
indicated by double arrows, and the helical regions separated by linear
linkers L1 and L2 (Fig. 1C) are indicated by single arrows.
Fig. 3. Amino acid comparison of partial amino acid sequences of
NvK18 with Xenopus K18 (XK18), mouse K18 (MK18), human K16
(HK16), and human K17 (HK17). (A) Part of helix 1B and of the linker
between helix 1 and 2. (B) Part of the C-terminus. Identical amino acids
are in bold, and an amino acid motif at the end of the C-terminus
(DGKVVS) that is similar to a motif found in the same position in other
K18, in HK17, and in K8 in newt and other species is underlined.
also in the epithelium of the limb blastema, their
pattern of expression in the regenerating limb was
examined by in situ hybridization. Figure 5A,B shows
that by 10 days after amputation (early blastema
stage), both NvK8 and NvK18 are expressed in the
majority of cells in the blastema mesenchyme but not in
the wound epidermis. The time course of expression of
the NvK8 transcript parallels that of the protein (Table
1), with the only exception being at day 3 after amputation, when the NVK8 mRNA but not the NVK8 protein
could be detected. At this stage, only a few blastemal
cells have accumulated beneath the wound epidermis,
and cell proliferation has not yet begun or is just
starting (the onset of DNA synthesis has been reported
to occur approximately 4 days after amputation; Hay
and Fishman, 1961). By 5 days after amputation,
however, both transcript and protein are clearly detectable. In normal limbs, expression of these keratins is
observed in subepidermal glands (not shown), consistent with K8 and K18 immunoreactivity observed in
newts and other species (Ferretti et al., 1989). Also,
cultured cells with the ability to differentiate into
myotubes express high levels of NvK8 (Fig. 5C) and
NvK18 (not shown) transcripts. These data demonstrate that undifferentiated newt limb mesenchymal
cells do indeed express simple epithelial keratins both
in vivo and in vitro.
observed. Expression of NvK8 and NvK18 was analysed by in situ hybridization 2–3 days after amputation, when a well-formed blastema was apparent, and
compared with that in the contralateral unamputated
limb (Fig. 5D). Expression of NvK8 and NvK18 was
detected neither in unamputated limb buds at any
stage of development (Fig. 5D,E) nor in regenerating
forelimbs amputated between stages 32 (Fig. 5E) and
39. In contrast, upregulation of NvK8 and NvK18
following amputation was observed from stage 41 onward, when the forelimb has become richly innervated
(Fekete and Brockes, 1987); the keratin-positive regenerating limb bud of a larva amputated at stage 45 is
shown in Figure 5F.
Expression of NvK8 and NvK18 in Amputated
and Normal Limb Buds
Previous immunocytochemical studies in P. waltl
embryos suggested that NvK8 and 18 are not expressed
in the developing limb bud. To confirm this observation
in N. viridescens and establish whether these transcripts are upregulated following amputation in developing and regenerating limbs, limbs from stage 32–45
embryos were amputated. In animals left to regenerate
for 2–3 weeks, regeneration of normal limbs was always
Comparison of the Expression of NvK8 in Adult
Denervated and Normal Blastemas
Since induction of NvK8 and NvK18 expression
correlates with the onset of innervation during development and because these keratins are first detected in
the adult regeneration blastema at the onset of the
nerve-dependent proliferative phase (3–5 days after
amputation, Table 1), we investigated whether the
presence of innervation was causally related to keratin
expression in adult blastemas. Limbs denervated 2
days before amputation were analysed 7 days later
(5-day regenerates) for the expression of NvK8 and
NvK18 either by immunocytochemistry using the LP1K
and RGE53 antibodies (Fig. 6) or by in situ hybridisation (not shown). We consistently found significant
Nvk8 (Fig. 6A) and NvK18 (Fig. 6B) reactivity in
blastemal cells of denervated limbs, suggesting that the
presence of the nerve is not required for their induction.
However, this experiment did not rule out the possibility that keratins have to be expressed for blastemal
cells to maintain the undifferentiated state and respond to the nerve-derived mitotic signals.
Effect of NvK8 and NvK18 Anti-Sense Oligomers
on DNA Synthesis and Phenotype of
Cultured Limb Cells
To address the issue of whether there is any correlation between keratin expression and the ability of
blastemal cells to proliferate, we have taken advantage
of two newt cell lines (Ferretti and Brockes, 1988)
originated from normal limbs (TH4B) and from limb
blastemas (BlH1) which express NvK8 and NvK18 (Fig.
5C). To downregulate keratin expression, these cells
were treated for 7 days with anti-sense oligomers
targeted against NvK8 and NvK18 (Fig. 1C), either
separately or in combination. Scrambled oligomers
were used as controls. This relatively long treatment
time was chosen because of the long half-life of intermediate filament proteins.
At the highest concentration of oligonucleotides used,
5 µM, either separately or in combination, some toxic
effect was observed. Nonetheless, as shown in the
example given in Figure 7A, when cells were treated
with 2.5 µM each of K8 and K18 oligomers, DNA
synthesis was significantly lower both in the BlH1 and
TH4B cultures treated with anti-sense oligomers than
in those treated with scrambled ones (Fig. 7A). This
suggests that anti-sense oligomers to NvK8 and NvK18
have a specific effect on cell growth. When cells were
treated with NvK18 anti-sense and scrambled oligomers at a lower concentration, 2.5 µM, 3H-thymidine
incorporation was not affected by the scrambled oligomer but was significantly decreased in anti-sense–
treated cells (Fig. 7B). A decrease in 3H-thymidine
incorporation was also observed when cells were treated
with 1 µM anti-sense oligomer, although the experimental variability was much higher, and when the same
concentrations of NvK8 rather than NvK18 anti-sense
were used (data not shown).
To confirm that keratin anti-sense oligomer treatment could indeed lead to a decrease in keratin levels,
we used an antibody specific for K18 (Ramaekers et al.,
1984; Ferretti et al., 1989) in an enzyme-linked immunoadsorbent assay (ELISA) to measure changes in NvK18
Fig. 4. Tissue distribution of NvK8 and NvK18 transcripts. (a,b)
Northern blot analysis. Lane 1, normal adult forelimb; lane 2, proximal
forelimb blastema; lane 3, liver; lane 4, intestine; lane 5, esophagus with
the 300 nucleotide PstI-PstI NvK8 probe (a) and the 290 nucleotide
BglII-EcoRI NvK18 probe (b). Five µg poly(A)1RNA per lane were used.
The size of the transcripts were estimated from the mobility of newt 28S
and 18S ribosomal RNAs. Exposure times were 2 days with intensifying
screens. Note that high levels of expression of both NvK8 and NvK18 are
found in the regenerating blastema, liver, and intestine, but no expression
is detected either in normal limb or esophagus. (c) RNase protection
analysis of NvK8 expression in limbs and tails with the PstI-PstI probe.
Normalised RNA samples (5 µg per lane) were used. Lane 1, input probe;
lane 2, fingertip; lane 3, hand; lane 4, arm; lane 5, proximal forelimb
blastema; lane 6, distal forelimb blastema; lane 7, tail blastema; lane 8,
normal tail; lane 9, tail without tip; lane 10, tRNA. The size of the protected
fragment is indicated. Note the significant increased in NvK8 expression
both in regenerating limbs and tails.
Fig. 5. Localization of NvK8 and NvK18 transcripts by in situ hybridisation in adult limb blastemas (A,B), in cultured myogenic limb cells (C), and
in normal and amputated developing limbs (D–F). (A) Expression of the
NvK8 transcript in a distal 10-day blastema. (B) Expression of the NvK18
transcript in a proximal 10-day blastema. Note that NvK8 and NvK18 are
expressed in most blastemal cells but not in the wound epidermis (WE in A
and B). (C) Expression of NvK18 transcript in undifferentiated cultured
limb cells. (D) Expression of NvK18 in the normal developing limb bud (dl)
and contralateral regenerating limb bud (rl) of a stage 32 embryo. Both
limb buds are negative; positive tissues include the area surrounding the
pharyngeal cavity, liver, and gut. (E) Expression of the NvK8 transcript in
the limb of a stage 41 larva. The developing limb is negative. High levels of
the transcript are detectable in the notochord (n); some expression is also
detectable in the gills, some of which are indicated (g). (F) Expression of
the NvK18 transcript in a regenerating limb amputated at stage 45; the
NvK18-positive regenerating limb bud is indicated by arrowheads. g, gills;
e, eye. Scale bars 5 100 µm. A,B and D,E are at the same magnification.
expression following 1 week treatment with 2.5 µM
anti-sense. As shown above, this treatment induces a
significant decrease in 3H-thymidine incorporation. Both
in TH4B and BlH1 cells treated with NvK18 anti-sense,
the levels of NvK18 protein were significantly lower
than in controls (Fig. 8). These results confirm that the
anti-sense oligomer downregulates keratin expression
and suggest a causal relationship between the decrease
in keratin expression and inhibition of DNA synthesis.
Finally, the morphology of the cells was carefully
monitored throughout the duration of each experiment
to further confirm that the changes observed were not
TABLE 1. Expression of NvK8 in Limb Blastemas
Days after amputation
aThe NvK8 protein was detected in cryostat sections by LP1K
bThe NvK8 mRNA was detected by in situ hybridization and
at 14 days also by RNAase protection.
in any way due to toxicity. We did not find any evidence
of toxic effect in cultures treated with 2.5-µM oligomers,
but we observed morphological changes in cells treated
with either NvK18 or NvK8 anti-sense oligomers that
were rather striking by 7 days of treatment (Fig. 9). In a
typical experiment, TH4B cells in five of six wells and
BlH1 cells in 4 of six wells had acquired a bipolar
morphology, and some of them had fused (Fig. 9 A,B).
Bipolar morphology in these newt cells reflects progression toward myogenic differentiation (Ferretti and
Brockes, 1988), as also confirmed in the present experiments by the occurrence of myotube formation (Fig. 9
A,B). In control cultures maintained either in normal
serum or treated with scrambled oligomers, cells with
bipolar morphology were very rare (Fig. 9C–F). Therefore, there is a significant correlation between downregulation of NvK18 and NvK8 and changes in the cell
Newt K8 and 18 are More Similar to Xenopus
and Fish Than to Mammalian K8 and 18
Cloning and expression analysis of the newt homologues of K8 and K18 (NvK8 and NvK18, respectively)
has firmly established that these simple epithelial
keratins are expressed in the mesenchymal progenitor
cells of the regenerating newt limb and in cultured
blastemal cells with myogenic potential.
Sequence comparison at the amino acid level of NvK8
with another type II newt keratin, NvKII, and with K8s
from other species has shown that sequence conservation between NvK8 and NvKII is lower than the
conservation between NvK8 and mammalian K8s. This
is consistent with our current understanding of the
evolutionary changes of keratin genes. The tail of NvK8
is longer than that of mammalian simple epithelia
keratins and rich in glycine, like that of Xenopus and
goldfish K8 (Franz and Franke, 1986; Giordano et al.,
1989). In mammalian cytokeratins, glycine-rich repeats are only found in the tail of keratins typical of
stratified epithelia (Hanukoglu and Fuchs, 1983; Jorcano et al., 1984). The presence of these glycine-rich
motifs in amphibians and fish but not in mammalian
K8 may reflect not only the likelihood that lower
vertebrate K8 is an ancestral cytokeratin, but also
different functional roles of this keratin in lower and
higher vertebrates. In fact, the tail of intermediate
filaments has been shown to have a role in filament
stabilization and cellular localization (Wilson et al.,
1992; Rogers et al., 1995).
The partial sequence of NvK18 clearly shows that
this keratin is the newt homologue of K18 proteins in
other vertebrates and that this protein has been highly
conserved through evolution.
NvK8 and Nv18 Are Expressed in the
Mesenchyme of Regenerating Adult Limbs but
not of Regenerating Embryonic Limb Buds
In situ hybridization analysis of NvK8 and NvK18
expression in regenerating and developing limbs has
demonstrated that although these keratins are indeed
expressed in the mesenchyme of regenerating adult
limbs, the developing limb does not express them either
at the early bud stage or at later stages of development.
This is consistent with previous observations in the
embryo of a different urodele species, P. waltl (Ferretti
et al., 1989), and suggests that this difference in keratin
expression in developing and regenerating limbs is a
feature common to urodeles rather than a speciesspecific phenomenon. In addition, it indicates that
co-expression of vimentin and keratin is not a normal
attribute of limb mesenchyme but is associated with
the regenerative process.
The different phenotype of progenitor cells in developing and regenerating limbs may reflect the different
origin of these cells in the two systems. Blastemal cells
are believed to originate from dedifferentiation of the
mature tissues of the stump, a process that would not
be required when an early limb bud is amputated
because it is still rather undifferentiated. Regrowth of
the missing part could therefore be achieved through a
regulative rather than regenerative process. In addition, growth of these progenitor cells is nerve-independent, unlike in adult or more developed limbs. Therefore, the transition between keratin nonexpressing and
expressing cells in regenerating limbs could be related
to the occurrence of dedifferentiation and nerve dependency, as discussed below.
Expression of NvK8 and 18 may also reflect the fact
that when dedifferentiated cells accumulate beneath
the wound epidermis to form the blastema they are
very loosely associated rather than tightly packed like
the progenitor cells of developing limb buds. Therefore,
blastemal cells might need a more rigid cytoskeleton to
maintain their shape and position, and this could be
accomplished by expressing both keratin and vimentin
filaments rather than only vimentin. Finally, because
cell motility and co-expression of the keratin pair 8-18
and vimentin have been found to be associated with
invasiveness in certain tumors (Chu et al., 1993), it is
conceivable that K8-18 expression in conjunction with
vimentin in the loose blastemal mesenchyme may have
a role in supporting cell movement and spatial organization during regeneration. These processes may require
an additional level of control given the different ‘‘struc-
Fig. 6. Keratin expression (red) in 5-day limb regenerates denervated 2 days before amputation; nuclei are in blue. (A) RGE53 immunoreactivity in a
denervated blastema; note that expression of NVK18 is clearly detectable in blastemal cells. (B) LP1K immunoreactivity in a denervated blastema;
expression of NVK8 is clearly detectable in blastemal cells and in subepidermal glands (arrows). A few positive cells (arrowheads) are also observed in
the thickened wound epidermis (WE), where LP1K reacts with another type II keratin, NvKII (Ferretti et al., 1991, 1993; Ferretti and Ghosh, 1997). Scale
bar 5 100 µm.
Fig. 8. Changes in NvK18 protein in BlH1 and TH4B cells following
7-day treatment with 2.5 µM scrambled and anti-sense NvK18 oligomers
was evaluated by ELISA immunoassay using the mAb CK18.2. The data
are expressed as percentage of controls in normal medium. Both in BlH1
and TH4B cultures, the levels of the NvK18 protein are significantly lower
in anti-sense–treated than in scrambled-treated cells or in normal medium
(*P , 0.0001 by Student’s t-test; n 5 6; bars 5 SE).
Keratin Expression in Regenerating Limbs
Appears to be Associated With Innervation
but Is not Controlled by it
Fig. 7. Effect of 1-week treatment with NvK8 and NvK18 oligomers,
either separately or in combination, on 3H-thymidine incorporation in BlH1
and TH4B cells. The data are expressed as percentage of controls in
normal medium. (A) Treatment with 2.5 µM each of NvK8 and NvK18
scrambled and anti-sense oligomers. Some toxic effect is observed at this
concentration (5 µM total) of oligomers; however, the effect of anti-sense
oligomers in reducing 3H-thymidine incorporation is significantly higher in
anti-sense than scrambled oligomer-treated cultures (*P , 0.001). (B)
Treatment with 2.5 µM NvK18 scrambled and anti-sense oligomers. Note
that at this concentration the scrambled oligomer does not affect 3Hthymidine incorporation, which in contrast is significantly reduced in
anti-sense–treated BlH1 and TH4B cells (*P , 0.0001 by Student’s t-test;
n 5 6; bars 5 SE).
ture’’ of the limb mesenchyme in developing and regenerating limbs and the greater scale, both in term of
volume and number of cells involved, of the regenerative process as compared to development (Ferretti and
Brockes, 1991).
In addition to comparing developing limb buds and
adult limb blastemas, we have examined NvK8 and 18
expression in regenerating limbs that had been amputated at different developmental stages. We have shown
that keratin expression is not induced in the regenerating mesenchyme when the developing limb is amputated before it becomes innervated, but is induced in
more mature, innervated limbs like in the adult. A
similar response to amputation in developing and adult
limbs has been previously reported in the case of the
22/18 antigen. This phenotypic change has been taken
to reflect a transition controlled by the nerve from
nerve-independent to nerve-dependent growth of blastemal cells (Fekete and Brockes, 1987, 1988). Our
studies have identified two additional molecules, the
developmentally regulated and regeneration-associated simple epithelia keratins 8 and 18, which reflect
this transition.
Notwithstanding these similarities between 22/18
and keratin expression, there is a significant difference
in their time course of expression in the adult regeneration blastema and in the type of regulation. Whereas
22/18 is detected within 24 hr after amputation (Gordon and Brockes, 1988), and this is most likely due to a
conformational change of the protein rather than to de
novo synthesis (Ferretti and Brockes, 1990), keratin
transcripts are first detected at 3 days and the proteins
slightly later, when the blastemal cells start to divide
Fig. 9. Effect of 2.5 µM anti-sense, either NvK8 or NvK18, oligomers
on the phenotype of BlH1 (A,C,E) and TH4B (B,D,F) cells. (A) NvK8
anti-sense–treated BlH1; note the presence in this culture of bipolar cells,
some of which have fused or are fusing (arrows). (B) NvK18 anti-sense–
treated TH4B; note the presence in this culture of bipolar cells, some of
which have fused or are fusing (arrows). (C) Untreated BlH1; note the
absence of bipolar cells. (D) Untreated TH4B; note the absence of bipolar
cells. (E) NvK8 scrambled-treated BlH1; note that these cells are not
significantly different from untreated controls in C. (F) NvK8 scrambledtreated TH4B; note that these cells are not significantly different from
untreated controls in D. Scale bar 5 50 µm.
under the influence of the nerve (Fig. 10A). Although
the pattern of expression of K8 and 18 during development and regeneration suggests that the nerve itself
might be required to control their expression, our
experimental results have clearly shown that this is not
the case. Although we cannot completely rule out a
decrease in K8 or 18 levels in denervated animals
because of the nonquantitative techniques used in
these experiments (immunocytochemistry and in situ
hybridization), both proteins are still clearly detectable
in 5-day denervated blastemas. This is consistent with
the fact that the initial accumulation of blastemal cells
is not nerve-dependent and does not rule out the
possibility that keratin expression is a prerequisite for
limb blastemal cells to start to divide. This possibility is
consistent not only with their pattern of expression in
embryonic and adult regenerating limbs, but also with
the observation that even in denervated limbs, a fraction of proliferating cells is present 4 days after amputation (Barger and Tassava, 1985).
Downregulation of NvK8 and NvK18 in the Newt
Limb Cells Is Associated With Inhibition
of Proliferation and Differentiation
To test the hypothesis of a causal relationship between keratin expression and the ability of blastemal
cells to divide, we have used anti-sense NVK8 or NvK18
oligonucleotides to downregulate these keratins in cultured newt cells. Because NVK8 or NvK18 are obligatory heteropolymers, downregulation of either of them
Fig. 10. Summary. (A) The onset of keratin expression in vivo
following amputation coincides with the beginning of nerve-dependent
proliferation of blastemal cells and is downregulated when cells stop
dividing and differentiate into muscle. (B) Downregulation of keratin
expression in vitro coincides with a significant inhibition of proliferation
and the onset of myogenic differentiation.
would impair filament formation, and downregulation
of either protein could be expected to produce the same
effects. This was indeed the case, and we have found a
marked decrease in cell proliferation in cultures treated
either with anti-sense NVK8 or NvK18 oligonucleotides
or with the two in combination, which is significantly
higher than that of scrambled oligonucleotides even at
a concentration at which some toxic effects occur (5
µM). The fact that we do not see complete mitotic
arrest, as in serum-deprived cultures (not shown), may
be due to the fact that treatment with anti-sense
oligomers at nontoxic concentrations does not result in
the complete disappearance of the keratin proteins.
Nonetheless, the significant decrease in protein levels
and changes in the cell phenotype (the switch to a
bipolar morphology in these cells is indicative of the
onset of myogenic differentiation; Ferretti and Brockes,
1988) in the NvK18 anti-sense–treated cultures, as
compared with controls, supports the view that there is
a causal relationship between keratin expression, proliferation, and differentiation. This is not entirely surprising because it has recently become apparent that
intermediate filaments and their associated proteins
are highly dynamic structures; it has also been proposed that in addition to their structural functions as
integrators of the intracellular space, they may be
active players at different stages of the cell cycle
(Foisner, 1997). However, it is presently difficult to
assess whether keratins can somehow play a direct role
in the control of proliferation, and it is the inhibition of
proliferation following keratin downregulation which
induces differentiation, or whether keratin downregulation induces differentiation and, as a consequence, the
cells exit the mitotic cycle (see Fig. 10B).
Notwithstanding suggestions that intermediate filaments can act as transcription factors (Traub and
Shoeman, 1994), the possibility that this may be the
way they control division in blastemal cells seems see
rather remote. What may be more likely, although at
this stage highly speculative, is the possibility that
keratins are somehow involved in the mitotic cycle
indirectly, through their ability to bind/sequester factors important in the control of the cell cycle. For
example, the fact that intermediate filaments bind
plectin, which is thought to integrate the organization
of actin and intermediate filaments in a cell-cycle and
phosphorylation-dependent fashion, has been recently
demonstrated (Foisner et al., 1995), as has the binding
between K8/18 filaments and proteins of the 14-3-3
family, which interact with several signal transduction
kinases (Liao and Omary, 1996). It has also been shown
that the Retinoblastoma (Rb) protein in the hyperphosphorylated state binds to lamins, and it has been
suggested that disassembly of such complex following
phosphorylation events may be required for cell cycle
progression (Mancini et al., 1994). Finally, intermediate filaments connect the nuclear and cell membrane,
and it has been shown that expression of keratins can
be modulated by the extracellular matrix and that their
interaction with it is associated with changes in the
motile behaviour of certain cell types (Chu et al., 1993;
Hendrix et al., 1997). This suggests that these molecules can ‘‘read,’’ either directly or indirectly, molecular cues presumably mediated by membrane receptors.
Therefore, it is not inconceivable that a decrease in
keratin filaments in blastemal cells may affect intracellular communication between the plasma membrane
and the nucleus, possibly by affecting the function of
keratin-associated molecules involved in growth control, and that consequently keratin expression is required to make blastemal cells responsive to the nerve.
As mentioned above, another possible explanation for
our results is that keratins play a role in the maintenance of the undifferentiated state, rather than in
proliferation, and that their downregulation may favour myogenic differentiation. It has been shown that
other members of the intermediate filament family,
such as desmin, can play a role in differentiation in
vitro (Li et al., 1994; Bahler, 1996). In addition, overexpression of the glial intermediate filament GFAP in an
astrocytoma cell line has been shown to induce changes
in cell shape and inhibit growth (Toda et al., 1994). How
intermediate filaments might control differentiation is
rather unclear, but arguments similar to those discussed above could be invoked to explain loss of the
dedifferentiated state/induction of differentiation. In
addition, it has been shown that K18 transcription can
be stimulated through its Ets-1 and AP-1 responsive
elements by the Ras signal transduction pathways,
suggesting that its expression may be important in the
behaviour of certain tumors (Pankov et al., 1994). For
example, expression of both K8 and 18 in conjunction
with vimentin has been associated with tumor invasiveness (Chu et al., 1993; Hendrix et al., 1997), and this
suggests that K8 and 18 are not necessarily terminal
targets of the cascade of events controlling differentiation, but are somewhere more upstream and might
exercise some ‘‘control’’ over this pathway themselves. A
parallel between certain tumor and blastemal cells is
that they are undifferentiated/poorly differentiated,
highly proliferative, motile cells found in adult organisms that express a mixed (vimentin and keratin)
phenotype; the main difference is that the growth of
blastemal cells, unlike that of tumors, is under strict
control and they eventually differentiate.
In conclusion, our results indicate a casual relationship between NvK8-18 expression in limb blastemal
cells, the undifferentiated state and proliferation, since
cells stop dividing and undergo myogenic differentiation when these keratins are downregulated. However,
it is not currently possible to say whether the primary
effect of keratin downregulation is on proliferation or
differentiation, and it will be a significant challenge to
answer this complex question.
Animals and Surgery
Adult N. viridescens supplied by Sullivan & Co.
(Nashville, TN) were maintained at 20°C and fed
shredded bovine heart on alternate days. All surgical
procedures were performed on newts anaesthetized in
0.1% tricaine (3-aminobenzoic acid ethylester methanesulphate salt; Sigma, St. Louis, MO). Following bilateral forelimb amputation at either the level of the
mid-humerus (proximal amputation) or the mid-radius/
ulna (distal amputation) and recovery in 0.5% sulfamerazine (Sigma), the regenerating newts were maintained at 25°C. Denervated blastemas were obtained by
severing the right limb brachial nerves at the midscapular level 2 days before proximal limb amputation.
Regeneration blastemas were harvested at different
times following amputation (3, 5, 10, 15 days, depending on the experiment to be performed).
N. viridescens embryos were obtained by injecting
pregnant females with human chorionic gonadotropin
(HCG; Sigma) as previously described (Ghosh et al.,
1994). Amputation of developing limbs was performed
unilaterally in embryos between stage 32 (early limb
bud) and 45 (3 digit stage). Embryos were killed by an
overdose of tricaine and processed for in situ hybridization 2–3 days after amputation, when a regeneration
blastema was always apparent. To check that regeneration could ensue normally, some animals were left to
regenerate for 2–3 weeks.
Isolation and Analysis of cDNA Clones
A cDNA library in lZapRII from cultured blastemal
cells (gift of J.P. Brockes) was screened with the mAb
LP1K to isolate cDNAs encoding the cytoskeletal protein of apparent molecular weight 52 kD recognized by
this mAb in blastemal cells. Five 3 105 pfu of the
amplified library were plated onto Escherichia coli
strain Y1090rk-; after 3–4 hr incubation at 37°C, nitrocellulose filters (previously soaked in 10 mM IPTG and
dried) were laid over the plates. The filters were
processed and reacted with LP1K as previously described (Ferretti et al., 1991). Selected LP1K-positive
plaques were purified, Bluescript SK- plasmids containing the inserts of interest were excised and propagated
in the E. coli strain XL1blue, and the cDNA clones were
analyzed for their size, restriction map, and nucleotide
composition. The same cDNA library was also screened
for the newt homologue of K18 with a human K18 DNA
probe (USB, Cleveland, OH). The dideoxy sequencing
reactions were performed using the Sequenase DNA
sequencing kit (United States Biochemical). Sequences
were compared using the software from the University
of Wisconsin Genetics Computer Group (UWGCG) and
the ‘‘Gap’’ programme used for alignment of amino acid
RNA Preparation and Analysis
Blastemas, cultured cells, and other tissues to be
used for RNA analysis were harvested, pooled, and
stored in liquid nitrogen until the extraction was
performed. RNA was extracted from newt tissues by the
guanidine isothiocyanate procedure as previously described (Brown and Brockes, 1991). A 300 bp PstI-PstI
fragment, which encodes part of the a-helix 1B of NvK8
(Fig. 1A), and a 290 bp BglII-EcoRI fragment, which
encodes part of the a-helix 1A of NvK18 (Fig. 1B), were
used to prepare DNA probes for Northern blotting, and
riboprobes for RNAase protection and in situ hybridization experiments.
Either 10 µg of total RNA or 5 µg of poly(A)1RNA
isolated by using the polyATract mRNA system IV
(Promega, Southampton, UK) were used for Northern
blot analysis, which was performed essentially as previously described (Casimir et al., 1988). Hybridization
was carried out in 50% formamide at 42°C, and the
filters were washed twice for 15 min at room temperature and once for 30 min at 42°C with 0.15 M SSC
containing 0.1% SDS.
The labelled riboprobe for RNAase protection analysis was hybridized with 5 µg of total RNA, and the
assay was essentially performed according to Casimir
et al. (1988). All the RNA samples were normalized by
using the satellite 2 DNA probe pSP6D6 (Epstein and
Gall, 1987) in conjunction with measurement of the
O.D. at 260 nm.
In situ hybridization was performed as previously
described (Ghosh et al., 1996). In brief, tissues were
fixed overnight at 4°C in 4% paraformaldehyde (PFA) in
A-PBS, paraffin wax-embedded, and 6-µm sections cut.
Dewaxed sections were fixed in 4% PFA for 20 min,
treated with 20 µg/ml of proteinase K, post-fixed,
acetylated, and hybridized overnight at 55°C with 105
cpm/ml of either the transcribed NvK8 (PstI-PstI) or
NvK18 (BglII-EcoRI) riboprobe (Fig. 1 A,B). Controls
with sense experiments and with another keratin probe,
NvKII (Ferretti et al., 1991; Ferretti and Ghosh, 1997),
were also carried out and confirmed the specificity of
the signal detected with the anti-sense NvK8 and
NvK18 probes. After high-stringency washing (65°C
with 50% formamide, 2 3 SSC, 10 mM DDT) and
RNAase A digestion for 30 min at 37°C followed by
further washing as previously described (Ghosh et al.,
1996), the slides were dehydrated and processed for
autoradiography. Slides were and exposed for 5 days at
4°C before being developed and counterstained with
toluidine blue.
effect of treatment with scrambled and anti-sense oligomers was compared by Student’s t-test.
Proliferation Assay
Immunohistochemistry was performed as previously
described using LP1K to detect NvK8 and either RGE53
or CK18.2 to detect NvK18 (Ramaekers et al., 1984;
Broers et al., 1986). The bound antibody was detected
by the nuclei stained with Hoechst dye (Ferretti et al.,
The effect of 2.5 µM anti-sense and scrambled oligomers on NvK18 and NvK8 protein levels was evaluated
by ELISA using CK18.2 and LP1K supernatants diluted 1:2. As in the thymidine incorporation assay, cells
were plated at an initial density of 750 cells/well and
grown for 1 week in the presence of different oligomers.
At the end of the treatment, the tissue culture medium
was removed and the wells washed with 70% L-15. The
cells were fixed with cold acid-alcohol for 10 min. The
fixed cells were processed as for immunocytochemistry,
except that the secondary antibody used was an affinity
purified alkaline-phosphatase-conjugated rabbit antiserum to mouse immunoglobulins (Dako) diluted in newt
culture medium. After 45 min incubation with the
secondary antibody, the wells were rinsed three times
with PBS-0.05% Tween-20 and then with 0.1 M Tris, pH
9.5. Two hundred µl of r-Nitrophenyl Phosphate substrate solution (Sigma FAST rNPP) was then added to
each well, and the plate read at 405 nm using an
automatic plate reader after a 30-min incubation. Each
experiment was repeated at least twice, and each group
consisted of at least five samples. The effect of treatment with scrambled and anti-sense oligomers was
compared by Student’s t-test.
The effect of anti-sense oligomers on DNA incorporation was studied in long-term cultures of limb cells that
express the blastemal phenotype. These long-term cultures originated from cells that had migrated from
small explants of either normal limbs (TH4B) or limb
blastemas (BlH1). Cells were grown and passaged
essentially as previously described (Ferretti and
Brockes, 1988), apart from the fact that 0.1% bovine
skin gelatin (Sigma) instead of collagen was used to
coat the dishes.
Eighteen base anti-sense oligomers targeted against
NvK8 and NvK18 modified at both ends with phosphorothioate to increase their stability and purified by
reverse-phase high-pressure liquid chromatography
(GENOSYS, Cambridge, UK) were used at 1, 2.5, and 5
µM. The relative position and sequence of the two
anti-sense oligomers used is shown in Figure 1C. The
NvK8 anti-sense oligomer is targeted against part of
helix 2 which has been shown to deplete Xenopus K8
(Heasman et al., 1992). The NvK18 anti-sense oligomer
is targeted against the border between helix 1B and the
linker between helix 1 and 2 (L12). To control for
nonspecific effects, such as toxicity, scrambled sequences of the NvK8 and NvK18 anti-sense were used
as controls.
For DNA incorporation assays, 750 cells/well were
plated in 96-well plates (Gibco, Paisley, UK). The
treatment was started the day after plating, when the
cells had attached and spread onto the substrate. Fresh
oligomers were added every 2 days; on the sixth day
from the beginning of the treatment, 3H-thymidine
(methyl-18, 28-3H-Thymidine, 124 Ci/mmol, Amersham,
Slough, UK) was added to medium at 1 mCi/ml. Cells
were collected 24 hr later on filter using an automatic
cell harvester. The filters were washed and dried, and
the incorporated radioactivity measured by liquid scintillation counting. Experiments were repeated at least
twice, and more usually three times; each experimental
group always consisted of at least five samples. The
Immunohistochemistry was essentially performed as
previously described (Ferretti et al., 1989). Reactivity of
the anti-keratin mAbs LP1K (Lane et al., 1985), RGE53,
and CK18.2 (Ramaekers et al., 1984; Broers et al.,
1986) was assayed on 8-µm cryostat sections briefly
fixed with cold acid-alcohol (95% ethanol–5% acetic
acid). Bound antibodies were detected by a rhodamineconjugated rabbit anti-mouse immunoglobulin antibody (Rh-Rb-aMIg, Dako, High Wycombe, UK), and the
nuclei stained with 1.25 µg/ml of Hoechst dye 33258
(Sigma). The results were recorded using an Axiophot
(Zeiss) fluorescent photomicroscope.
Protein Analysis
Morphological Analysis
The morphology of the cells was monitored throughout the duration of each experiment, and micrographs
of live cells in 96-well plates were taken using an IM45
inverted microscope (Zeiss).
The authors thank E.B. Lane and F. Ramaekers for
the gifts of LP1K and CK18.1, respectively; J.P. Brockes
for the gift of the cultured blastema cDNA library; R.
Oshima for the gift of human K18 DNA probe; and P.
Thorogood for reading the manuscript. This work was
supported by a Wellcome Trust grant to P.F.
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expressions, developing, mouse, toots, bones, cells, bmps, patterns, differentiation, protein, morphogenetic, role, morphogenesis, suggests
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