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First published online 29 August 2007
doi: 10.1242/dev.004556
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1 Max-Planck-Institute of Immunobiology, Stuebeweg 51, D-79108 Freiburg,
Germany.
2 Center of Cancer Research, Massachusetts Institute of Technology, Cambridge,
MA 02139, USA.
3 Children's Memorial Research Center, Northwestern University, Feinberg School
of Medicine, Chicago, IL 60614, USA.
4 Institute for Developmental Biology, University of Cologne, D-50923 Cologne,
Germany.
* Author for correspondence (e-mail: hammerschmid{at}immunbio.mpg.de)
Accepted 26 June 2007
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SUMMARY |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Hai1, Spint1, Matriptase1, St14, HGF, Met, Epidermis, Scattering, EMT, Zebrafish
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). This is
achieved either by cleaving a pro-protein to a functional form or by the
inactivation/degradation of a substrate. Strict control of protease activity
and expression is thus crucial for proper tissue ontogeny and homeostasis, and
deregulated activity of a number of proteases is associated with human disease
states.
Matriptase1 [also known as Matriptase, membrane-type serine protease 1
(MT-SP1) and Suppression of tumorigenicity 14 (St14)] is a type II
transmembrane serine protease, first identified in human breast cancer cells
(Lin et al., 1999b;
Shi et al., 1993), and is
expressed in a broad range of epithelia
(Kim et al., 1999;
Oberst et al., 2001;
Oberst et al., 2003;
Takeuchi et al., 1999).
Increasing interest in this enzyme has centered on its strong correlation with
epithelial tumor progression and the deleterious effects resulting from
altering its activity levels in vivo (reviewed in
List et al., 2006). One
crucial regulator of the proteolytic activity of Matriptase1 has been shown,
in vitro, to be the membrane-associated serine protease inhibitor Hepatocyte
growth factor activator inhibitor 1 (Hai1, also known as Spint1)
(Benaud et al., 2001;
Lin et al., 1999a), which is
almost invariably co-expressed with Matriptase1
(Oberst et al., 2001) and was
first described as an inhibitor of the circulating serine protease Hepatocyte
growth factor activator (Hgfa, also known as Hgfac)
(Shimomura et al., 1997). The
inhibitory activity of Hai1 is conferred via its extracellular Kunitz domains,
which bind directly to the protease sites
(Denda et al., 2002;
Kirchhofer et al., 2003). Via
the inhibition of both Matriptase1 and Hgfa, Hai1 is thought to be able to
attenuate cell motility via limiting serine protease-mediated activation of
the potent motogen Hepatocyte growth factor (Hgf, also known as Scatter
factor, Sf) (Parr and Jiang,
2006). In addition to Hgf, Matriptase1 has been shown to activate
other proteins and zymogens, such as urokinase plasminogen activator (uPA,
also known as Plau), protease activated receptor 2 (PAR2, also known as
F2rl1), matrix metalloprotease 3 (MMP3), insulin-like growth factor binding
protein-related protein-1 (IGFBP-rP1, also known as IGFBP7) and CUB domain
containing protein 1 (CDCP1) (Ahmed et al.,
2006; Bhatt et al.,
2005; Jin et al.,
2006; Lee et al.,
2000; Takeuchi et al.,
2000). Furthermore, Matriptase1 can degrade extracellular matrix
and structural proteins, including Laminin and Fibronectin
(Satomi et al., 2001). In
vitro models have shown that Matriptase1 induces cell scattering and invasion
via both Hgf-Met-dependent and -independent mechanisms
(Forbs et al., 2005); however,
the in vivo relevance of these downstream pathways remains largely
untested.
Knockout of matriptase1 in the mouse produced epidermal structural
and barrier defects due to insufficient processing of profilaggrin (Flg) to a
mature form in cornifying outer keratinocytes
(List et al., 2002;
List et al., 2003). The
hai1-knockout mouse dies in utero because of placental defects
underscored by a loss of epithelial integrity of chorionic trophoblasts
(Tanaka et al., 2005) and the
underlying basement membrane (Fan et al.,
2007). These placental defects are due to deregulated matriptase1
activity, as confirmed by a double-knockout strategy
(Szabo et al., 2007).
Similarly, squamous cell carcinogenesis caused by overexpressing matriptase1
in the mouse skin was rescued by the concomitant overexpression of Hai1
(List et al., 2005). However,
it remains to be demonstrated whether Hai1 repression of Matriptase1 is
necessary for normal epidermal development.
Here, we describe the roles of two Hai1 and a Matriptase1 homologues during
skin development in the zebrafish (Danio rerio), based on mutant and
antisense-mediated loss-of-function studies. During embryonic and larval
stages, the zebrafish epidermis is bilayered, consisting of a basal and an
outer layer of keratinocytes (Le Guellec
et al., 2004). As in mammals, basal keratinocytes are attached to
the basement membrane via hemi-desmosomes and to each other via desmosomes
(Sonawane et al., 2005), and
are characterized by the expression of the p53-related transcription factor
Np63 (also known as Tp73l - Zebrafish Information Network)
(Bakkers et al., 2002).
Zebrafish hai1a (also known as spint1la - Zebrafish
Information Network; GenBank accession number NM_213152), hai1b
(spint1lb; GenBank accession number EF424430) and
matriptase1a are expressed in the developing basal layer of the
epidermis. Via live imaging and marker analysis, we describe both epithelial
and inflammatory skin phenotypes caused by loss of Hai1 activity, and thus
provide direct genetic evidence for an essential role of Hai1 to maintain
epithelial integrity of the epidermis during development. Furthermore, by
inactivating both Hai1 and Matriptase1a, we demonstrate that Hai1 fulfills its
epidermal function by blocking Matriptase1a. Finally, we show that deregulated
Matriptase1a activity does not require the Hgf receptor Met or related
receptors to induce the observed scattering of hai1 mutant
keratinocytes, indicating that other Matriptase1 target proteins must be
involved.
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MATERIALS AND METHODS |
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MATERIALS AND METHODS
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DISCUSSION
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). hai1a mutant embryos were obtained from
heterozygous or homozygous parents. Embryos lacking early Smad5 function and
specification of ectodermal precursor cells were obtained by crossing mothers
heterozygous for the dominant-negative allele dtc24 with wild-type
males (Hild et al., 1999). The
Tg(bactin:hras-egfp) (allele vu119) and the
Tg(fli1a:egfp) (allele y1) transgenic lines have been
described previously (Cooper et al.,
2005; Lawson and Weinstein,
2002).
Cell transplantations
Clusters of mGFP-labeled basal keratinocytes were obtained by homotypic
(wild type
wild type; hai1a morphanthai1a
morphant; hai1a+hai1b
morphanthai1a+hai1b morphant) transplantation of
non-neural ectodermal cells from Tg(bactin:hras-egfp)
transgenic donor embryos into non-transgenic hosts. Recipients were developed
to 24 hours post fertilization (hpf), mounted in 1.5% low melting point
agarose under E3 medium and analyzed by time-lapse confocal microscopy.
RNA isolation, cDNA synthesis, RT-PCR and 5'RACE
Total RNA was isolated from embryos using Trizol-LS (Invitrogen, CA) and
cDNA synthesized with SuperscriptII reverse transcriptase (Invitrogen).
Sequences corresponding to zebrafish orthologs of hai1a, hai1b,
prgfr3 and hgfa were obtained from the zebrafish genome
(Ensembl, Sanger Center), and were amplified via reverse transcriptase
(RT)-PCR. To determine the full 5' sequence of hai1b and
prgfr3 cDNAs, 5'RACE was performed using the SMART RACE kit (BD
Biosciences, CA).
Cloning of cDNAs, probe synthesis and heat-shock treatment
Zebrafish cDNA fragments for hai1a, hai1b and prgfr3
obtained by RT-PCR were cloned into pCRII-TOPO (Invitrogen) or pGEM-T Easy
(Promega, WI). cDNA clones of matriptase1a (IMAGp998P136587),
matriptase1b (IMAGp998H0614296) and ron (IMAGp998M1514792)
were obtained from RZPD (Berlin, Germany). The matriptase1a insert
was shuttled into pCRII-TOPO. For probe synthesis, plasmids were linearized
with NcoI (hai1b, hgfa), NotI (matriptase1a,
hai1a, prgfr3), EcoRI (matriptase1b) or SmaI
(ron). Probes for hai1a, hai1b, matriptase1a, prgfr3 and
hgfa were synthesized using Sp6 RNA polymerase (Roche, Mannheim,
Germany), and probes for matriptase1b and ron with T7 RNA
polymerase.
For matriptase1a overexpression studies, the heat-inducible
construct pTol2-hse-GFP/matriptase1a was generated, cloning
the matriptase1a cDNA into the bicistronic vector pSGH2
(Bajoghli et al., 2004), which,
in addition to matriptase1a, drives expression of GFP under the
control of heat-shock elements (hse). Subsequently, the cassette was ligated
into vector pT2AL200R150G, which contains Tol2 recognition sites to allow
early genomic integration and widespread expression upon co-injection with
Tol2 transposase mRNA (Kawakami et al.,
2004; Urasaki et al.,
2006). For transgene activation, injected embryos were transferred
from 28°C to 39°C from 20-22 hpf.
In situ hybridization and immunostainings
In situ hybridizations were performed as previously described
(Hammerschmidt et al., 1996),
using probes for hai1a, hai1b, matriptase1a, myoD
(Weinberg et al., 1996),
met (Haines et al.,
2004), pu.1 (spi1)
(Lieschke et al., 2002),
lcp1 (Herbomel et al.,
1999), mpx (Lieschke
et al., 2001) and e-cadherin (cdh1)
(Babb et al., 2001).
Whole-mount antibody stainings were visualized with the Vectastain ABC kit
(Axxora) as described (Hammerschmidt et
al., 1996), or with fluorescent secondary antibodies. For combined
colorimetric in situ hybridizations and immunostainings
(Fig. 1F,G,I and
Fig. 7A), embryos underwent
standard in situ hybridization, followed by a fixation for 4-6 hours in 4%
paraformaldehyde/PBS, and a standard immunostaining
(Hammerschmidt et al., 1996).
Antibodies, dilutions used and sources were as follows: 4A4 anti-p63 (1:200,
Santa Cruz), anti-GFP (1:400, Invitrogen), anti-E-cadherin (1:200, BD
Biosciences), Alexa-Fluor-546 goat anti-mouse (1:400, Invitrogen) and
Alexa-Fluor-488 goat anti-rabbit (1:400, Invitrogen).
Western blotting
Protein extracts of embryos were separated by 8% SDS-PAGE under reducing
conditions and transferred to nitrocellulose membrane. Anti-E-cadherin
antibody was used at 1:5000 dilution, and secondary HRP-coupled goat
anti-mouse antibody (Dianova, Hamburg, Germany) at 1:10000.
Morpholino oligonucleotides (MOs)
MOs were obtained from Gene Tools (Philomath, OR) and diluted in Danieau's
buffer (Nasevicius and Ekker,
2000). MO solution (1.5 nl) was injected per embryo.
hai1a MO (5'-ACCCTGAGTAGAGCCAGAGTCATCC-3') and
matriptase1a MO (5'-AACGCATTCCTCCATCCATAGGGTC-3') were
injected at 100 µM; ron MO
(5'-AACAAGGTCTTTGGGCTGATGAACA-3') and prgfr3 MO
(5'-CTAAATGAGTGGCCCAATGGACCAT-3') at 200 µM; and hai1b
MO (sequence 5'-CACCACGAACCCATTTTTGATTGAT-3'), the met MO
(Haines et al., 2004) and
pu.1 MO (Rhodes et al.,
2005) at 500 µM.
BrdU and acridine orange stainings
Epidermal cell proliferation was assessed by BrdU incorporation followed by
combined 
-p63 and -BrdU antibody detection as described
(Lee and Kimelman, 2002).
Apoptotic cells were visualized by acridine orange staining as described
(Furutani-Seiki et al.,
1996).
Microscopy
Fluorescent images were taken with a Zeiss Confocal microscope (LSM510
META); all other microscopy was performed on a Zeiss Axiophot.
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RESULTS |
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SUMMARY
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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), staining larvae at 48 hours post fertilization (hpf)
for p63 protein, a marker of basal keratinocytes
(Lee and Kimelman, 2002). One
mutant that we identified, hi2217, displayed aggregations of
p63-positive keratinocytes (Fig.
1B) compared with the even distribution of these cells in
wild-type siblings (Fig. 1A).
Such aggregates were most prominent on the yolk sac and yolk extension, and
were already visible at 24 hpf (see below). In addition, skin cells were seen
floating in the chorion of mutant embryos from 24 hpf
(Fig. 1C).
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), we found that the mutagenic insertion had occurred in
intron 1 of the hai1a gene, which is homologous to the hepatocyte
growth factor activator inhibitor 1 (Hai1, Spint1) gene in mammals.
Because the insertion is upstream of the first coding exon
(Fig. 2A), it most probably
leaves the structure and function of the Hai1a protein intact. However, it
attenuated hai1a transcription to below detection thresholds. At 24
hpf and 48 hpf, wild-type embryos displayed strong hai1a expression
in epithelia such as the epidermis, the olfactory epithelium, the otic
vesicle, the gut, and the lateral line primordium and neuromasts
(Fig. 1D,E). Combination with
anti-p63 immunostaining revealed that, within the epidermis, hai1a
transcripts were present around the p63-positive nuclei, pointing to
expression in the basal layer (Fig.
1F,G). However, in contrast to wild-type siblings
(Fig. 2B), no hai1a
expression could be detected by in situ hybridization in hai1a
mutants between 24 and 72 hpf (Fig.
2C; and data not shown). Consistent results were obtained using
semi-quantitative reverse transcriptase (RT)-PCR of extracts from whole-mutant
and wild-type sibling embryos at 24 hpf
(Fig. 2D), indicating that the
viral insertion strongly abrogates hai1a transcription or transcript
stability. As further evidence that the viral insertion was causative of the
observed skin phenotype, we injected an antisense morpholino oligonucleotide
(MO) targeting the hai1a translation start site (hai1a MO)
into wild-type embryos, which led to a robust phenocopy of the epidermal
defects (compare Fig. 3G-I with
3A-F). In addition to the epidermis, hai1a mutants also displayed disrupted morphology of the olfactory epithelium (see Fig. S1A-C in the supplementary material), another ectodermal derivative displaying prominent hai1a expression (Fig. 1D), whereas, in the pronephric ducts and the gut, hai1a-positive epithelia (Fig. 1D,F) derived from the mesoderm or endoderm, respectively, appeared unaffected by the mutation (see Fig. S1D-G in the supplementary material).
hai1a acts in partial redundancy with its paralog, hai1b
By searching genomic and EST databases of the zebrafish, we identified a
second zebrafish hai1 gene, named hai1b. On the amino acid
level, Hai1a and Hai1b are 43% identical, and both are more similar to mouse
Hai1 (34 and 37% identity, respectively) than to mouse Hai2 (also known as
Spint2 - Mouse Genome Informatics; both 14% identity), indicating that they
are true paralogs that might have arisen from the genome duplication that has
occurred during teleost evolution
(Postlethwait et al.,
1998).
Whole-mount in situ hybridizations revealed that, as is hai1a (Fig. 1D,G), hai1b is expressed in the basal epidermis (Fig. 1H,I), raising the possibility that Hai1a and Hai1b might have partly redundant functions. Therefore, we inactivated hai1b by injecting a specific hai1b MO into wild-type or hai1a mutant or morphant embryos. hai1b morphant embryos displayed completely normal morphology and normal p63 expression (Fig. 3J-L), whereas the skin defects of hai1a, hai1b double-morphant embryos (Fig. 3M-O) were much more severe than in hai1a single mutants (Fig. 3D-F) or morphants (Fig. 3G-I). In 24 hpf hai1a single mutants, aggregates of basal keratinocytes were largely restricted to the yolk sac, the yolk extension and the ventral trunk, whereas cell shedding mainly occurred at the border between the yolk sac and yolk extension (Fig. 3D,E). By contrast, hai1a, hai1b double-morphant embryos displayed keratinocyte aggregations and cell shedding in all regions of the skin (Fig. 3M-O). At 24 hpf, many embryos had already lysed (Table 1) or were about to die, most probably due to the loss of functional skin, whereas hai1a mutants often recovered quite well, with skin aggregations and lesions only remaining in the forming body fins (data not shown). Together, these data indicate that Hai1b, although dispensable during normal skin development, can, to some extent, compensate for the loss of Hai1a.
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), which
lack basal keratinocytes, demonstrating that they are the major source of
E-cadherin at this stage (Fig.
5H; and data not shown). This suggests that E-cadherin in basal
keratinocytes of hai1 mutants has been redistributed, indicating the
loss of epithelial and the incomplete acquisition of mesenchymal
properties.
The epidermis of hai1a mutants displays enhanced apoptosis and inflammation
In mammals, diseases of the epidermis are often either due to, or invoke,
an excessive inflammatory response
(Thivolet et al., 1990). To
determine whether hai1a mutants display similar defects, we examined
leukocytes using the in situ markers leukocyte-specific-plastin
(lcp1) (Herbomel et al.,
1999; Meijer et al.,
2007) and myeloid-specific peroxidase (mpx)
(Lieschke et al., 2001). At 24
hpf, wild-type embryos showed a few leukocytes below the epidermis of the yolk
sac and the anterior trunk (Fig.
6A; and data not shown), in concordance with previous reports
(Herbomel et al., 2001).
hai1a mutant embryos at this stage, by contrast, had a strong
accumulation of leukocytes over the yolk sac and yolk extension, corresponding
to sites of epidermal aggregate formation
(Fig. 6B; and data not shown).
At later stages (48 hpf), enhanced inflammation was also seen at other sites
in hai1a mutant embryos, such as the posterior trunk and fins
(compare Fig. 6E with 6D).
In order to determine whether this inflammation is causative of the
epithelial defects described above, we genetically ablated innate immune cells
in hai1a mutants by injecting MOs against pu.1, which is
required for the specification of the myeloid lineage
(Rhodes et al., 2005). In situ
hybridizations revealed a complete loss of leukocytes in pu.1
morphant embryos (Fig. 6C).
However, epidermal defects of hai1a mutants were not ameliorated
(Fig. 6C), suggesting that
inflammation does not cause, but might instead be induced in parallel to or
by, the epidermal defects of hai1a mutants.
In several instances, inflammation is induced by dying cells to ensure the
proper clearance of apoptotic cell debris by innate immune cells (reviewed in
Henson and Hume, 2006). The
same might be true for the epidermal inflammation of hai1a mutants.
As early as 24 hpf, epidermal aggregates of hai1a mutants contained a
high number of acridine orange-positive apoptotic keratinocytes
(Fig. 6G,H), whereas
keratinocyte death was not evident in the epidermis of wild-type embryos
(Fig. 6F). Similar results were
obtained by TUNEL labeling (data not shown). Time-lapse movies of
hai1a mutants carrying a fli1:EGFP transgene
(Redd et al., 2006) further
showed that yolk sac leukocytes were strongly attracted by epidermal
aggregates with acridine orange-positive cells
(Fig. 6J), whereas, in
wild-type controls, leukocytes moved more slowly and were less directed
(Fig. 6I). In summary, this
suggests that epidermal inflammation of hai1a mutants is secondarily
caused by the death of keratinocytes.
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To test whether the epidermal defects of hai1 mutants might be due to a lack of Matriptase1a inhibition, we attempted to phenocopy and rescue the defects by Matripase1a overexpression or inactivation, respectively. Heat-treated wild-type embryos that had been injected with pTol2-hse-GTP/matriptase1a DNA (see Materials and methods) displayed severe dissociation of GFP-positive keratinocytes and skin aggregate formation both in dorsal (data not shown) and ventral (Fig. 7C; n=7/8 embryos with GFP-positive keratinocytes) positions, similar to the phenotype of hai1 mutants/morphants (Fig. 3). Defects were rescued to wild-type condition upon co-injection of pTol2-hse-GTP/matriptase1a with an MO targeting the matriptase1a translation start site (Fig. 7D; n=0/6 embryos with GFP-positive keratinocytes), indicating that they are indeed due to a gain of Matriptase1a activity. Similarly, whereas the knockdown of Matriptase1a in wild-type embryos had no effect (compare Fig. 7F with Fig. 7E), hai1a, matriptase1a double- and hai1a, hai1b, matriptase1a triple-mutant/morphant embryos displayed a robust rescue of the hai1 mutant phenotype, with epidermal morphology indistinguishable from that of wild-type siblings (compare Fig. 7G,H with Fig. 3E,N; Table 1). In particular, basal keratinocytes did not form aggregates and were not shed, but maintained their even distribution, as was seen in wild-type embryos (compare Fig. 7I with Fig. 3F,O). Similarly, in vivo imaging revealed that keratinocytes dorsal of the yolk extension of matriptase1a-MO-injected hai1a mutants maintained epithelial properties (Fig. 7K; 4/4 movies of transplanted cells), in contrast to the acquisition of mesenchymal-like characteristics in hai1a mutants at this location, as described above (Fig. 5B). Furthermore, apoptosis of keratinocytes was suppressed (compare Fig. 7J with Fig. 6G,H), as was skin inflammation (compare Fig. 7M with 7L). In conclusion, all phenotypic traits caused by the loss of Hai1 activity could be rescued by concomitant inactivation of Matriptase1a, indicating that, during normal development, Hai1 promotes skin homeostasis by blocking Matriptase1a activity.
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). To
study whether elevated Hgf signaling might account for the loss of epithelial
integrity seen in the hai1a mutant epidermis, we injected
hai1a mutants with met MO. However, in contrast to
Matriptase1a (Fig. 7G),
knockdown of Met activity did not alleviate epidermal aggregate formation
(compare Fig. 8C with 8A,B) or
inflammation (data not shown), although embryos lacked pectoral fin muscles
(compare Fig. 8F with 8D,E),
demonstrating that the MO was functional. To discount potential redundancies
with other related receptors, we also cloned zebrafish orthologs of the
closely related mammalian Ron receptor (GenBank accession number CF997695) and
a Ron-relative most similar to avian Sea and Fugu rubripes, Prgfr3
[Plasminogen related growth factor receptor 3
(Cottage et al., 1999);
GenBank accession number EF424429]. However, we were unable to detect
expression of any of these receptor genes in the zebrafish epidermis at 24 hpf
(data not shown). Furthermore, co-injection of MOs against all three members
of this receptor family (Met, Ron and Prgfr3) failed to rescue the skin
defects of hai1a mutants (see Fig. S2E-H in the supplementary
material), although all MOs efficiently suppressed the translation of their
respective mRNA upon co-injection (see Fig. S2A-D in the supplementary
material). We conclude that the defects caused by the loss of Hai1 and gain of
Matriptase1a activity are not due to enhanced signaling via the Hgf receptor
Met or any of its relatives. |
DISCUSSION |
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DISCUSSION
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).
These early defects have precluded studies on possible other essential roles
of Hai1 during later development, such as in the skin. The recent observation
that Hai1, Matriptase1 double-mutant mice develop to term
(Szabo et al., 2007) and only
show skin defects associated with the loss of Matriptase1
(List et al., 2002;
List et al., 2003) could have
two meanings. It could indicate either that Hai1 is dispensable for other
developmental processes, or that, as in the placenta, the role of Hai1 in such
other tissues is to block Matriptase1. Tissue-specific knockout of the mouse
Hai1 gene would be necessary to distinguish between these two
possibilities.
Here, we have analyzed the skin phenotype caused by loss of Hai1 function
in a non-placental vertebrate, the zebrafish. Skin defects of hai1a
mutants are most prominent around 24-26 hpf, but recover to almost wild-type
condition during further development. By contrast, combined loss of Hai1a and
its paralog, Hai1b, which, per se, is dispensable, causes a much stronger
phenotype, characterized by a dissociation of the entire epidermis and
embryonic death between 18 and 26 hpf. Both the moderate defects of
hai1a mutants and the stronger defects of hai1a mutants or
morphants injected with hai1b MO were rescued upon injection of a
matriptase1a-specific MO, indicating that Hai1a and Hai1b act in
partial redundancy and by blocking Matriptase1a activity. Our data are in line
with results obtained upon transgene-driven overexpression of Matriptase1 in
mouse keratinocytes, which leads to epidermal hyperplasia, skin inflammation
and heightened tumorigenicity in the adult epidermis, all of which can be
suppressed upon concomitant skin-specific overexpression of Hai1
(List et al., 2005). In
addition to providing the first direct proof for an indispensable role of Hai1
in the vertebrate epidermis, our data also highlight that the primary function
of Hai1 is concerned with the maintenance of epithelial integrity within the
basal epidermal layer, whereas skin inflammation and epidermal hyperplasia
appear to be secondary consequences.
Keratinocyte aggregate formation and shedding in hai1 mutants result from a loss of epithelial integrity
At 24 hpf, aggregation and shedding of keratinocytes in
hai1a-/- homozygotes was largely restricted to the ventral
side of the embryo, and most prominent on the yolk sac and the forming yolk
extension, whereas, at 36 hpf, aggregates were mainly found on the outgrowing
body fins. These are the regions of the embryo with the most-pronounced
morphological changes, suggesting that here, the epidermis is exposed to
highest mechanical stress and undergoes tissue remodeling even during normal
development. Such remodeling processes most probably involve a transient loss
of epithelial properties of keratinocytes, which might be driven by
Matriptase1a. If so, however, it remains unclear why the loss of Matriptase1a
activity does not cause developmental defects. Functional redundancy with
other serine proteases could be one explanation. In addition, Matriptase1a
might only become essential for tissue remodeling that occurs during
pathological conditions, such as wound healing. To test this notion, we
compared wound healing in wild-type and matriptase1a morphant larvae,
which, at this stage, normally involves actin cable-driven purse-string
contractions of keratinocytes, as in mammalian embryos
(Redd et al., 2004). This
process occurred normally in wounded matriptase1a morphants, (T.J.C.
and M.H., unpublished observations). Further analyses are necessary, including
wound-healing studies of matriptase1a mutants during adulthood, when
cutaneous wound closure involves EMT of keratinocytes (T.J.C., Krasimir
Slanchev and M.H., unpublished observations).
Nonetheless, our in vivo time-lapse recordings of fluorescently labeled hai1a mutant keratinocytes revealed that, during development, these cells have the potential to become mesenchymal. We observed such behavior, localized to ventral regions, in hai1a single mutants at 24 hpf, whereas the loss of both Hai1a and Hai1b led to the enhanced, earlier and more-widespread acquisition of mesenchymal-like properties of basal keratinocytes. In these movies, we also observed mutant basal keratinocytes crawling on top of each other. Furthermore, mesenchymal-like behavior was observed in keratinocytes far removed from aggregates, suggesting that the acquisition of fibroblastoid properties and motility is causative, rather than a secondary consequence, of aggregate formation.
It is important to note that hai1 mutant keratinocytes did not
become completely mesenchymal. Thus, they lacked the mesenchymal marker
Vimentin (T.J.C. and M.H., unpublished data). In addition, mRNA and protein
levels of the epithelial marker E-cadherin remained unchanged. However,
E-cadherin protein appeared to be redistributed, indicating a loss of
epithelial polarity. Acquisition of fibroblastoid shapes and motility under
such conditions is sometimes termed `scattering', in reference to its initial
discovery as an effect induced by Scatter factor (Sf, identical to Hgf), and
is opposed to complete EMTs, which require additional and/or prolonged
signaling (for a review, see Grünert
et al., 2003).
Keratinocyte apoptosis, skin inflammation and epidermal hyperplasia of hai1 mutants are secondary consequences of the loss of epithelial integrity
In addition to keratinocyte scattering, loss of Hai1 activity leads to
keratinocyte death, enhanced skin inflammation and epidermal hyperplasia.
Although the phenotype caused by the combined loss of Hai1a and Hai1b was too
severe to dissect these traits, analyses of hai1a single mutants
suggest that all are secondary consequences of the loss of epithelial
integrity. Thus, apoptosis of keratinocytes was usually observed only in the
most severely affected regions and within cell aggregates, whereas
keratinocyte scattering was also recorded in regions devoid of dying cells.
This suggests that keratinocyte death might be a secondary consequence of
their detachment from the basement membrane when they pile up on each other, a
phenomenon called anoikis, which has been well characterized in vitro
(Gilmore, 2005). This death of
keratinocytes might in turn induce skin inflammation, characterized by
enhanced numbers of leukocytes in the hai1a mutant skin. Strikingly,
the spatial pattern of inflammation resembled that of keratinocyte apoptosis
(compare Fig. 6B with 6G).
Whereas apoptosis preceded inflammation and had its peak between 24 and 28
hpf, inflammation continued beyond 48 hpf. Furthermore, time-lapse recordings
revealed that innate immune cells were strongly attracted by apoptotic
keratinocytes. Ultimate proof for a causative relationship between
keratinocyte death and inflammation would require analysis of inflammation in
hai1a mutants after the suppression of cell death. For this purpose,
we injected mutants with MOs targeting the pro-apoptotic transcription factor
p53 (Plaster et al., 2006) or
with mRNA encoding the anti-apoptotic Bcl-2 protein
(Langenau et al., 2005).
However, both treatments failed to lower the number of dying keratinocytes in
hai1a mutants (T.J.C. and M.H., unpublished data), suggesting that
keratinocyte death is accomplished independently of the intrinsic, and
possibly driven by the extrinsic, apoptosis pathway [compare with Rytomaa et
al. (Rytomaa et al., 1999)].
Although it cannot be ruled out that inflammation in hai1a is induced
by other means in parallel to dying keratinocytes, we can rule out that
inflammation is causative of the epithelial defects, because they persisted in
hai1a mutants after genetic ablation of all innate immune cells by
pu.1-MO injection.
Finally, our BrdU-incorporation studies indicate that proliferation of
keratinocytes is only secondarily affected, most probably due to the loss of
epithelial integrity and possibly contact inhibition. Thus, at 24 hpf,
keratinocyte aggregates in hai1 mutants were solely composed of
non-proliferating cells, whereas keratinocyte hyper-proliferation was only
seen later and after the onset all other phenotypic traits (48 hpf).
Similarly, transgenic mice overexpressing Matriptase1 in keratinocytes display
late epidermal hyperplasia in vivo, although isolated keratinocytes show
normal proliferative behavior when cultured in vitro
(List et al., 2005), arguing
for an indirect effect of the Hai1-Matriptase1 system on cell
proliferation.
What are the substrates of Matriptase1 accounting for the epidermal defects in hai1 mutants?
Biochemical analyses have identified multiple Matriptase1 substrate
proteins; among them is Hgf, which is involved in the regulation of several
epithelial-mesenchymal transitions during vertebrate development (for a
review, see Birchmeier and Gherardi,
1998) and which requires Matriptase1- or Hgfa-mediated proteolytic
cleavage to become biologically active. This suggests that the epithelial
defects of hai1 mutants might be due to Matriptase1-mediated
upregulation of Hgf activity. Furthermore, applied Hgf has been shown to
attract innate immune cells to skin wounds
(Bevan et al., 2004),
suggesting that elevated Hgf activity might also account for the observed skin
inflammation of hai1 mutants. Additionally, Hai1 and Matriptase1
could act via other related ligands such as Mst1 (Macrophage-stimulating
protein), shown to stimulate chemotactic migration of macrophages
(Leonard and Skeel, 1978) as
well as keratinocyte mobility (Santoro et
al., 2003). Indeed, a zebrafish mst1 homolog has been
reported to be highly expressed on the yolk sac and the yolk extension
(Bassett, 2003), the two most
strongly affected sites of hai1a mutants. However, knockdown of the
Hgf receptor Met (Haines et al.,
2004), the Mst1 receptor Ron
(Gaudino et al., 1994) and the
related Plasminogen related growth factor receptor 3
(Cottage et al., 1999) failed
to rescue the epidermal defects of hai1a mutants. This strongly
suggests that Hai1 and Matriptase1a regulate skin homeostasis and remodeling
via other or additional proteins.
A recent report on the placental phenotype of Hai1 mutant mice has
described compromised basement membrane integrity
(Fan et al., 2007), suggesting
that epithelial defects might be due to increased degradation of Laminins,
other described Matriptase1 substrate proteins (see Introduction). However,
whole-mount immunostainings and western blotting experiments of hai1a
mutant and hai1a, hai1b double-deficient embryos revealed unchanged
Laminin protein levels and cleavage patterns (T.J.C. and M.H., unpublished
data), suggesting that the epidermal defects are not due to direct alterations
in Laminin processing.
In conclusion, although we can exclude some of the most obvious candidates as the prime or sole Matriptase1 target proteins, the crucial players downstream of Matriptase1, accounting for the epidermal defects caused by the loss of Hai1 activity, remain elusive. Systematic antisense-mediated knockdowns of other candidates in hai1a zebrafish mutants, as well as genetic hai1a suppressor screens, are in preparation to hopefully reveal the nature of these proteins in the future.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/19/3461/DC1
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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K. Laue, S. Daujat, J. G. Crump, N. Plaster, H. H. Roehl, Tubingen 2000 Screen Consortium, C. B. Kimmel, R. Schneider, and M. Hammerschmidt The multidomain protein Brpf1 binds histones and is required for Hox gene expression and segmental identity Development, June 1, 2008; 135(11): 1935 - 1946. [Abstract] [Full Text] [PDF]
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