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First published online 1 November 2006
doi: 10.1242/dev.02658
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1 Okazaki Institute for Integrative Biosciences, National Institutes of Natural
Sciences, Myodaiji, Okazaki, 444-8787, Japan.
2 Graduate School of Biostudies, Kyoto University, Kitashirakawa, Sakyo-ku,
Kyoto, 606-8502, Japan.
3 Laboratory for Cellular Morphogenesis, Center for Developmental Biology,
RIKEN, Kobe, 650-0047, Japan.
4 The Graduate University for Advanced Studies (SOKENDAI), Myodaiji, Okazaki,
444-8585, Japan.
Author for correspondence (e-mail:
stakada{at}nibb.ac.jp)
Accepted 22 September 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Grainyhead, Organogenesis, Kidney, Salivary gland
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Hogan and Kolodziej,
2002). During the course of morphogenesis, the epithelial cells of
the ducts differentiate to acquire their physiological functions; for example,
the maintenance of water, mineral and acid-base homeostasis in renal tubules.
By contrast to the molecular mechanisms underlying tubule morphogenesis,
however, the mechanisms leading to the differentiation of duct epithelial
cells remain largely unknown. Exocrine glands and kidney are typical examples of tubular organs with well-developed ducts. Exocrine glands, including the salivary gland, lachrymal gland and mammary gland, consist of two major epithelial components: a proximal part called the acinus, which generally produces primary fluid by secreting serous fluid into the lumen; and a distal part, the duct, which resorbs or secretes fluids and solutes from or to the primary fluid flowing through its lumen. Thus, in spite of the characteristic and distinct appearance of each exocrine gland, they exhibit similar fundamental structures and functions.
The kidney shares the basic design of these exocrine glands. The kidney processes blood in the proximal part, the glomeruli, to form primary fluid, the primary urine, and then modifies it by absorption and/or secretion of minerals and water in the distal parts, including the ducts and tubules. Therefore, it is thought that a common molecular mechanism underlies the maturation of exocrine glands and the kidney.
Members of the grainyhead gene family of transcriptional factors, which are
evolutionarily conserved from Caenorhabditis elegans to humans
(Venkatesan et al., 2003),
have been implicated in the development of epithelial tissues. The molecules
of this family exhibit structural similarities in the DNA-binding and
oligomerization domains. Six members of this family have been identified in
mammals: CP2 (also referred to as UBP-1, LSF, LBP-1c and LBP-1d)
(Lim et al., 1992;
Yoon et al., 1994), NF2d9
(also referred to as LBP-1a and LBP-1b)
(Parekh et al., 2004;
Sueyoshi et al., 1995),
CP2-like 1 [CP2L1 (TCFCP2L1 - Mouse Genome Informatics); also referred to as
CRTR1 in mouse and LBP-9 in humans] (Huang
and Miller, 2000; Rodda et
al., 2001), mammalian grainyhead [also referred to as LBP-32 and
grainyhead-like 1 (GRHL1)], brother of mammalian grainyhead (also known as
GRHL2) (Wilanowski et al.,
2002) and sister of mammalian grainyhead (also referred to as
GET-1 or GRHL3) (Kudryavtseva et al.,
2003; Ting et al.,
2003). Intriguingly, many, but not all, of the genes of the
grainyhead family are expressed in the epithelial tissues and play roles in
epithelial morphogenesis and/or homeostasis in a wide variety of animals. For
instance, grainyhead-deficient Drosophila displays abnormalities in
epithelial development and homeostasis, including tracheal development,
epidermal cuticle formation and wound healing
(Bray and Kafatos, 1991;
Hemphala et al., 2003;
Mace et al., 2005). Likewise,
loss of sister of mammalian grainyhead in mice leads to defects in dorsal
epithelial sheet closure and failure in the formation and maintenance of the
epidermal barrier (Ting et al.,
2005). Inhibition of Grhl1 activity in Xenopus results in
defective epidermal differentiation (Tao
et al., 2005).
As in the case of these grainyhead family genes, Cp2l1 is also
expressed in epithelium in a characteristic pattern: in the developing duct
epithelium of the salivary gland and kidney in the mouse embryo
(Rodda et al., 2001;
Yamaguchi et al., 2005).
Interestingly, we found that Cp2l1 is expressed in the developing
epithelial ducts of other organs. Because the ducts of the exocrine glands and
the kidney have similar functions, we suspected that CP2L1 plays a role in the
developing ducts of these organs. To determine whether this is the case, we
examined the precise expression and roles of Cp2l1 during the
development of the salivary gland and the kidney.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
These mice were backcrossed into a C57BL/6 background for six to ten
generations and then analyzed. Genotypes were determined by PCR amplification
using the following primers: a, 5'-CCATAGTGGAGGCCACACGCCCAAGAT-3';
b, 5'-CAAAGGGACCTAAACCACCAGAAGGAGAA-3'; and c,
5'-CTCAGCCTTGAGCCTCTGGAGCTGCTC-3'
(Fig. 1L). Amplification was
performed for 35 cycles of 94°C for 30 seconds and 68°C for 30
seconds. The presence of a 725 bp fragment, a 525 bp fragment, or both,
indicated animals with tra/tra, +/+, and +/tra genotypes, respectively. Mice
carrying the CP2L1 allele in which its exon 2 was flanked by two loxP
sites were crossed with Pc36 Cre-expressing mice (provided by H.
Takebayashi) (Ding et al.,
2005) to excise exon 2, leading to a frame-shift mutation that
generates a null allele of Cp2l1 (see Fig. S1 in the supplementary
material). The complete characterization of these mice will be reported
elsewhere.
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Histology and electron microscopy
Organs were fixed in Bouin's fixatives or 4% paraformaldehyde (PFA),
dehydrated, embedded in paraffin and sliced into 6 µm sections. The
sections were stained with hematoxylin and eosin or Periodic acid/Schiff (PAS)
reaction according to standard procedures. For electron microscopic
observation, organs were cut and fixed in 2.5% glutaraldehyde and 2%
formaldehyde in 0.1 mol/l cacodylate buffer (pH 7.2) and then processed for
thin sectioning and transmission electron microscopy.
Immunofluorescence
Cryosections (10 µm) were fixed in 4% PFA and then stained with
antibodies specific to aquaporin 2 (AQP2; gift from S. Sasaki), keratin 7
(Dako), pan-cytokeratin (Sigma) or a rabbit antiserum (As2375) raised against
the N-terminal 19 amino acids of CP2L1
(NH2-MLFWHTQPEHYNQHNSGSC-COOH) conjugated to Keyhole Limpet
Hemocyanin. For the staining of keratin 7, sections were heated for 10 minutes
in a microwave in 10 mmol/l Tris-HCl (pH 9.5) containing 1 mmol/l EDTA.
Secondary antibodies conjugated to Cy3 (Jackson ImmunoResearch) or Alexa Fluor
488 (Molecular Probes) were used. Photomicrographs were obtained with a Zeiss
Axiophot2 photomicroscope or with a Zeiss LSM510.
Dissection of nephrons
To visualize the nephron segment, kidneys were dissociated and cultured for
12 hours with mesenchyme. Briefly, embryonic day (E) 14 to 16 kidneys were
isolated from embryos, incubated in 2% pancreatin (Sigma) for 2 to 5 minutes
on ice, and washed three times with Dulbecco's modified Eagle's medium (DMEM)
containing 20% fetal bovine serum. The kidneys were then dissociated by
pipetting vigorously for 50 times using a Gilson pipetman with 200 µl tip.
After this treatment, the samples were seeded on gelatin-coated dishes,
cultured for 12 hours in DMEM containing 10% fetal bovine serum and then
processed for X-gal staining. In this culture system, dissociated mesenchyme
serves as a feeder layer for the undissociated nephrons.
X-gal staining and in situ hybridization
X-gal staining was performed as described previously
(Yamaguchi et al., 2005).
Briefly, fresh cryosections were fixed in 1% formaldehyde or 2% PFA for 5 to
10 minutes, washed twice with phosphate-buffered saline, and stained for
lacZ activity. For whole-mount visualization, post-fixed stained
embryos were incubated in 1% KOH overnight and gently shaken in distilled
water until the embryos became transparent. In situ hybridization on
cryosections (10 µm thickness) was performed as previously described
(Muroyama et al., 2002).
X-gal-stained sections were fixed with 4% PFA for 10 minutes and then
processed for in situ hybridization. The following template cDNA plasmids were
used for the synthesis of probes: keratin 7 (IMAGE clones 354520);
Gk-6 (IMAGE clones 581222); keratin 19 (IMAGE clones 3164228);
Atp6b1 (IMAGE clones 4489144); Clcnkb (IMAGE clones
4975792); Umod, Nkcc2, Ncc and Ncx (gifts from S. Nakai)
(Nakai et al., 2003);
Cp2l1 (Yamaguchi et al.,
2005); Pax2 (gift from P. Gruss); and ß-NGF,
Sgn1 and Mist1 (gifts from S. Yoshida)
(Yoshida et al., 2001). The
cDNAs of Fxyd2 variant b, Fxyd2 variant c, Scnn1b, Slc34a1 and
Slc4a1 were cloned by PCR using the primers described in
Table 1. INT/BCIP (Roche)
solution was used as a substrate for signal detection of in situ hybridization
after X-gal staining. Nuclear Fast Red (Ventana) was used for
counterstaining.
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Quantitative RT-PCR
Quantitative RT-PCR was performed using a LightCycler thermal cycler
(Roche). Total RNA was extracted from the kidneys and submandibular glands
(SMGs) of embryos and newborn mice using TRIZOL reagent (Invitrogen) and then
purified using the RNeasy total RNA purification kit (Qiagen). Following
treatment with DNaseI, cDNA was generated from 2 µg of total RNA using
SuperScriptIII reverse transcriptase (Invitrogen) and a random hexamer in a
total volume of 20 µl. PCR was then carried out using SYBR Green Premix Ex
Taq (Takara) and gene-specific primers
(Table 2). At least four
samples per genotype or stage were quantified, and the expression level of
each gene was normalized with that of hypoxanthine guanine phosphoribosyl
transferase (HPRT) or cyclophilin D1 (identical results were obtained with
both). Differences were analyzed using Student's t-test.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), we
found that Cp2l1 is expressed in the epithelium of the developing
salivary (submandibular, sublingual and parotid) glands
(Fig. 1A-D) and kidney
(Fig. 4)
(Yamaguchi et al., 2005). More
extensive and sensitive analysis of expression by in situ hybridization and
lacZ staining revealed that this gene was also expressed in the ducts
of other exocrine glands, including the lachrymal glands
(Fig. 1D), nasal glands
(Fig. 1E), minor nasal glands
in the olfactory epithelium (Fig.
1F), mammary glands (Fig.
1G) and eccrine sweat glands
(Fig. 1H). However, we did not
detect expression of Cp2l1 during the development of the pancreas,
which is an exocrine gland. In addition to exocrine glands, Cp2l1 was
also expressed during development of other epithelial tissues, including the
ductus epididymis, ductus deferens and seminal vesicles
(Fig. 1I). We also detected
expression of Cp2l1 at birth in the endolymphatic sac in the inner
ear (Fig. 1J), lung
(Fig. 1K), thyroid gland and
meninx (data not shown). Thus, the expression of Cp2l1 is mainly
associated with epithelial structures in which fluids and solutes are actively
transported.
Submandibular ducts are defective in Cp2l1tra/tra mice
We previously generated mice homozygous for a gene-trap insertion in the
Cp2l1 locus (Cp2l1tra)
(Fig. 1L). These mutant mice
express little mRNA for the full-length CP2L1 protein. Instead, they express a
truncated form that contains only the N-terminal portion and lacks the
DNA-binding domain (Yamaguchi et al.,
2005). This truncated protein did not appear to be localized in
the nucleus because we detected membrane or cytosolic but not nuclear staining
for CP2L1 in the submandibular duct of Cp2l1tra/tra mice
(Fig. 2I; left panels).
Furthermore, expression of the truncated protein was below the detectable
level in the kidney of Cp2l1tra/tra mice, although the
anti-CP2L1 antibody used in this study reacted with unidentified proteins in
the cytoplasm of glomerulus cells, which do not express Cp2l1 mRNA
(Fig. 1M). Thus, we concluded
that the truncated CP2L1 produced in these mice mostly lacks the ability to
function as a transcription factor. We also found that mice homozygous for the
Cp2l1 null locus generated separately by a gene-targeting strategy
exhibit the same phenotype as the Cp2l1tra/tra mice (see
Fig. S1 in the supplementary material). We then used the trapped allele for
further analysis of CP2L1 function. Intercrossing within the heterozygotes
resulted in homozygous mice with nearly the expected Mendelian distribution
(Cp2l1+/+: Cp2l1+/tra:
Cp2l1tra/tra=49:73:45). Approximately half these mice died
before weaning and showed growth retardation during early postnatal stages,
although mutant mice that survived past weaning recovered to 80-90% of the
weight of the wild-type mice (Fig.
1N,O and data not shown).
To address whether CP2L1 is required for the development of ducts, we first
investigated the salivary glands in surviving adult
Cp2l1tra/tra mice. Mammals have three major salivary
glands, the submandibular, sublingual and parotid glands, which are formed
through similar developmental processes. The patterns of Cp2l1
expression in these glands during development and in adults were similar.
Because the submandibular gland (SMG) is the most prominent structure, we
mainly focused on it. The SMG of rodents contains several types of ducts
distinguished by their location and morphological appearance: namely,
intercalated duct, striated duct, excretory duct and granular convoluted
tubule (GCT) (Fig. 2A)
(Tandler, 1993). Maturation of
the GCTs occurs under the control of androgen and thyroxine after birth
(Gresik and Barka, 1980), and
there is sexual dimorphism in this process; in the GCT, the volume of
cytoplasm was greater in males than in females
(Fig. 2B; see Fig. S1D in the
supplementary material). By contrast to Cp2l1+/+ and
Cp2l1+/tra mice, the Cp2l1tra/tra mice
exhibited defects in the morphology of the ducts; duct structure was not well
developed, and the volume of cytoplasm in the duct epithelial cells was
reduced (Fig. 2B). In
particular, we did not observe cell shapes characteristic of striated and
excretory ducts and GCT. Consistent with this observation, we found that the
expression of ß-NGF (Ngfb - Mouse Genome Informatics)
and glandular kallikrein 6 (Gk-6; Klk1 - Mouse Genome
Informatics), which is increased according to the maturation of GCTs
(Penschow et al., 1991;
Yoshida et al., 2001), was
diminished in both male and female Cp2l1tra/tra mice
(Fig. 2C and data not shown).
In addition, we did not detect the expression of Sgn1 (also known as
Ascl3 - Mouse Genome Informatics) in basal cells
(Yoshida et al., 2001) of the
salivary duct in Cp2l1tra/tra mice
(Fig. 2D). By contrast,
morphological observation and analysis of Mist1 (Bhlhb8 -
Mouse Genome Informatics), an acinus-specific basic helix-loop-helix
transcriptional factor (Yoshida et al.,
2001), indicated that there were no obvious abnormalities in the
acini of either male or female Cp2l1tra/tra mice, a
portion of exocrine gland that does not express CP2L1 in normal mice
(Fig. 2E). These results
indicated that the duct epithelial cells did not mature properly in the SMG of
Cp2l1tra/tra mice.
|
). In
Cp2l1tra/tra embryos, the SMG did not exhibit obvious
morphological abnormalities until E16 (data not shown). At E18, however,
luminal cells in the SMG ducts of Cp2l1tra/tra embryos
were shorter in the apico-basal direction than those of
Cp2l1+/+ and Cp2l1+/tra mice
(Fig. 2F). Consistent with this
abnormal cell shape, the lumen of the SMG ducts was noticeably wider in the
mutant embryos. Formation of adherens and tight junctions in the duct appeared
to be normal, because the localization of E-cadherin and ZO-1 were normal
(data not shown). Electron microscopic analysis, however, revealed that the
apical structure of the luminal cells in normal embryos did not develop
sufficiently in the mutant embryos (Fig.
2G,H). In agreement with this observation, cytokeratin proteins
were not detected in the apical side of the luminal cells in the ducts of
mutants (shown by white arrows in the right panels of
Fig. 2I). Except for the lumens
of the ducts, Cp2l1 expression was not detected in the lobule or
primodium of acini, and the morphology and cytokeratin localization were
normal in the acini of Cp2l1tra/tra embryos (shown by
asterisks in the right panels of Fig.
2I), suggesting a specific defect in the development of salivary
gland duct cells in Cp2l1tra/tra embryos.
|
). Although
keratin 7 mRNA was detected from E14 in the duct of the SMG in normal embryos,
its expression was obviously reduced throughout development in
Cp2l1tra/tra embryos
(Fig. 3A and data not shown).
In addition, some other genes also failed to be expressed in the SMG duct of
Cp2l1tra/tra mice. The expression of Gk-6
(Penschow et al., 1991),
Fxyd2a and Fxyd2b (genes encoding
Na+,K+-ATPase 
-subunit variants a and b)
(Jones et al., 2001;
Jones et al., 2005), and
Scnn1b (gene encoding the ß-subunit of the epithelial sodium
channel) in the SMG was detected during normal development but severely
reduced in Cp2l1tra/tra embryos throughout development
(Fig. 3B-D,F and data not
shown). These results indicate that, despite formation of the duct structure,
the cells constituting the duct failed to differentiate properly in
Cp2l1tra/tra embryos. In addition, these genes were not
properly expressed in the ducts of the sublingual or the parotid gland of
Cp2l1tra/tra embryos, indicating that CP2L1 plays similar
roles in the maturation of major ducts in the salivary glands (data not
shown). In addition to the salivary glands, we observed expression of keratin
7 and Gk-6 in the duct of the nasal gland, and their expression was
also lost in Cp2l1tra/tra embryos
(Fig. 3E and data not shown).
Thus, CP2L1 appears to play similar roles in different exocrine glands.
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Expression of Cp2l1 during kidney development
Our preliminary analysis indicated that the kidney is developed in
Cp2l1tra/tra mice but with occasional hypoplasia
(Yamaguchi et al., 2005). In
the current studies, we first performed a precise analysis of Cp2l1
expression during kidney development. The kidney consists of numerous nephrons
and collecting duct (CD) systems. A secretory nephron consists of Bowman's
capsule, which filters blood to form the primary urine, and a tubule, which is
subdivided into several compartments: the proximal convoluted tubule (PCT);
the descending limb (DL); the thick ascending limb (TAL); and a distal tubule,
which is made up of the distal convoluted tubule (DCT) and the connecting
tubule (CNT), arranged in the order of primary urine flow. A secretory nephron
is connected to the CD by the CNT (Fig.
4I).
CP2L1 was expressed both in CD lineages and nephrons. During renal
development, CP2L1 is first expressed in the nephric duct and subsequently in
the branching ureteric epithelium, which differentiates into the CD
(Yamaguchi et al., 2005).
CP2L1 expression was subsequently observed in CD cells, which are
characterized by expression of cytokeratin and AQP2
(Fig. 4A and data not shown),
and later localized to the medullary and cortical, but not papillary, CDs
(Fig. 4H,I). In the nephron,
expression of CP2L1 (lacZ expression from the trapped CP2L1 allele)
was first detected in a distal region of S-shaped bodies, which differentiates
into the tubular portion of the nephron
(Fig. 4A; arrowhead)
(Yamaguchi et al., 2005).
After the S-shaped body stage, the nephron maturates through four sequential
stages characterized by development of the loop of Henle (HL, consisting of DL
and TAL): the anlage, the primitive loop, the immature loop and the mature
loop (Fig. 4I)
(Neiss, 1982;
Nakai et al., 2003), and
nephrogenesis continues repetitiously in the peripheral region of the kidney
until around P10, resulting in a large number of nephrons in a kidney. At the
anlage stage, CP2L1 expression was detected in all nephric tubule (data not
shown). Then, at the primitive loop stage, CP2L1 was expressed strongly in the
distal portion (TAL, DCT and CNT) and weakly in the proximal portion (DL and
PCT) but not in the glomerulus (Fig.
4B). At the immature loop stage, strong expression of CP2L1 was
observed in cells expressing Ncx1 (Slc8a1 - Mouse Genome Informatics)
(Na+/Ca2+ exchanger 1;
Fig. 4C), a marker for CNT.
Modest or low expression was colocalized with expression of the DCT marker,
Ncc (Slc12a3 - Mouse Genome Informatics) (Na+/Cl-
co-transporter; Fig. 4D). We
observed very weak expression in the form of a faint dot in more proximal
regions of the nephron along with expression of Nkcc2 (Slc12a1 - Mouse Genome
Informatics), Umod and Slc34a1 (Fig.
4E-G), which are specific for TAL, HL and PCT, respectively
(Grieshammer et al., 2005;
Nakai et al., 2003). At the
mature loop stage (adult kidney), CP2L1 was no longer expressed in the TAL
(Fig. 4G, lower panel), and it
was detected weakly in the CNT, and a part of the DCT (data not shown). Thus,
during the maturation of the kidney, CP2L1 expression is localized to more
distal regions in the nephron (Fig.
4I).
Defective maturation in distal nephric tubules and the collecting duct in the kidney of Cp2l1tra/tra mice
We next examined the kidney phenotype of Cp2l1tra/tra
mice. Because Cp2l1tra/tra mice often died within 2 days
after birth (Fig. 1N), we first
examined the mutant kidney around birth. Obvious structural abnormalities were
not apparent at this time in the kidney of Cp2l1tra/tra
mice (Fig. 5A,B). To examine
whether the nephron and CD were properly formed, we used in situ hybridization
and quantitative RT-PCR to examine the expression of a series of genes
specific to distinct portions of the renal tubule in
Cp2l1tra/tra mice. We found normal expression at E18 and
birth in the nephron of Cp2l1tra/tra mice for the
following marker genes: Podxl, a marker for glomeruli;
Slc34a1 for PCT; Umod for Henle's loop; Nkcc2 for
TAL; Nos1 for macula densa; Ncc for DCT; and Ncx1
for CNT (Fig. 4C-F,
Fig. 5E and data not shown)
(Grieshammer et al., 2005;
Nakai et al., 2003). The
expression of two CD markers, Aqp2 and Pax2, was also
unchanged in Cp2l1tra/tra mice
(Fig. 5C-E)
(Dressler et al., 1990;
Fushimi et al., 1993). Thus,
the expression of the markers specific for distinct regions of nephric tubules
and the CD was normally detected in Cp2l1tra/tra mice.
|
). Type A ICs
mediate proton secretion into and bicarbonate resorption from the lumen, and
type B (non-A) ICs mediate bicarbonate secretion. The expression of
Atp6b1 (Atp6v1b1 - Mouse Genome Informatics), which encodes
the B1 subunit of vacuolar H+-ATPase and mediates proton secretion
at the apical membrane of type A ICs
(Finberg et al., 2005), was
lost in Cp2l1tra/tra mice at birth
(Fig. 6A). Likewise, expression
of Slc4a1, which encodes anion exchanger 1 and is a marker gene for
type A ICs (Southgate et al.,
1996), and pendrin, which is a marker gene for type B (non-A) ICs
(Kim et al., 2002;
Royaux et al., 2001), was
absent in Cp2l1tra/tra mice
(Fig. 6B,M and data not shown).
These abnormalities were evident from E15, before a time when the expression
of these two genes is drastically increased during normal development
(Fig. 6M and
Table 3). These results showed
that differentiation of ICs was defective in the
Cp2l1tra/tra mice.
|
Differentiation of not only the CD but also the distal tubules was also
disrupted in Cp2l1tra/tra mice. The expression of
Gk-6 and Fxyd2c, which are expressed in newly formed DCT and
CNT (Fig. 6H) and encode a
serine protease and Na+,K+-ATPase -subunit
variant c, respectively, was diminished in the mutants
(Fig. 6I,J,M). Furthermore, the
expression of Clcnkb, which encodes a chloride channel and is
expressed in TAL and DCT (Kobayashi et
al., 2001), was severely reduced in the mutants
(Fig. 6K,M).
The CD and distal tubules are crucial compartments for the tight regulation
of fluid, mineral and acid-base homeostasis, and gene expression in these
tissues is regulated by physiological changes
(Reilly and Ellison, 2000).
Therefore, the observed phenotype might be an indirect consequence of possible
physiological changes occurring in the mutants, such as altered fluid flow and
composition caused by the loss of CP2L1. To see whether this is the case, we
examined the development of kidney in culture explants, wherein the lack of a
blood supply precludes the influence of physiological changes. The kidneys
were isolated from E14 embryos, cultured for 2 days and subjected to gene
expression analysis. The observed changes in gene expression were similar to
those observed in embryos. For instance, the expression of Atp6b1,
keratin 7, Scnn1b, Fxyd2c, Clcnkb and Gk-6 was significantly
lower in explants from Cp2l1tra/tra mice than in wild-type
mice, whereas the expression of Aqp2 did not differ significantly
(Fig. 6L). This strongly
suggested that the abnormal gene expression in the mutants was not a secondary
consequence of a physiological abnormality. Taken together, the results
indicate that CP2L1 is required for proper maturation of the CD and distal
tubules during development of the kidney.
Abnormal composition of saliva and urine of Cp2l1tra/tra mice
To examine whether the defects observed in perinatal and postnatal
developmental stages actually lead to functional abnormalities of these organs
in adulthood, we measured the volume and electrolyte composition of saliva and
urine collected from the surviving mutants
(Table 4). Saliva is primarily
generated in the acini and subsequently modified in the duct by resorption of
electrolytes (Young et al.,
1987). Cp2l1tra/tra mice secreted the same
volume as control Cp2l1+/+ or
Cp2l1+/tra mice. Thus, the function of the acinus in
Cp2l1tra/tra mice appeared normal, which is consistent
with our previous observations indicating normal acinus formation. Sodium and
chloride concentrations were much higher, and the potassium concentration was
significantly lower, in the saliva from both male and female mutants than in
saliva from normal mice. By contrast, the concentrations of these ions in
plasma were not significantly different in mutants and normal mice. In
consistent with the abnormal electrolyte composition of saliva in adult
Cp2l1tra/tra mice, the expression of Scnn1b was
not detected in the SMG duct of these mice (data not shown). Thus,
modification of the electrolyte composition in saliva, a physiological
function of the ducts, appeared to be abnormal in
Cp2l1tra/tra mice.
|
). In
any case, these results showed that both the salivary gland and kidney require
CP2L1 for their proper physiological function as intact organs. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cp2l1 was also required for duct maturation in the kidney. The tubule architecture and regionalization monitored by expression of marker genes specific for distinct regions of nephrons appeared normal in Cp2l1tra/tra mice, but gene expression characteristic of CD and distal tubule maturation was abnormal in the mutants. In particular, the expression of Atp6b1, Slc4a1 and pendrin, which are typical markers of differentiated ICs, was not detected during any of the embryonic stages, indicating that generation of ICs was severely defective in Cp2l1tra/tra mice (Fig. 6M and Table 3). The expression of keratin 7 and Scnn1b, which is elevated during maturation of the CD, was also reduced in Cp2l1tra/tra mice. Because the reduced expression of these genes recovered to the normal level after birth, at least some aspects of CD development appear to be delayed in the mutants. Likewise, it appeared that maturation of distal tubules (TAL, DCT and CNT) was also disrupted in Cp2l1tra/tra mice, because they did not express Clcnkb, Gk-6 or Fxyd2c. Importantly, abnormal expression of these genes was also detected in cultured mutant kidneys, indicating that defective gene expression in the mutant kidney was not due to changes in the external environment caused by the loss of Cp2l1. Rather, it appears that the defective maturation of nephric tubules leads to insufficient renal function and frequent death within a few weeks after birth.
The identification of a gene essential for the maturation of the exocrine
ducts and kidney should help to elucidate their underlying molecular
mechanisms. Duct maturation includes both duct formation and the acquisition
of physiological function. Because CP2L1 is a transcriptional regulator, genes
with expression is directly or indirectly regulated by this factor may be
involved in these processes. In Cp2l1-deficient embryos, the
expression of genes involved in the physiological function of the ducts was
reduced; for example Cp2l1-deficient mice displayed reduced
expression of Scnn1b, Fxyd2 and Atp6b1, which encode the
ß-subunit of the epithelial sodium channel
(Nakai et al., 2003;
Schmitt et al., 1999), a
possible regulatory subunit of Na+,K+-ATPase
(Arystarkhova et al., 2002;
Jones et al., 2001;
Jones et al., 2005), and the
B1-subunit of vacuolar ATPase (Finberg et
al., 2005), respectively. In addition to the genes directly
involved in physiological function, we found that the expression of keratin
genes was also abnormal in the ducts of the mutants. Keratin 7 is a type 2
intermediate filament protein distributed broadly in, and a hallmark of,
glandular ducts (Smith et al.,
2002). The apical localization of keratin 7
(Fig. 1C) may be involved in
morphological differentiation in luminal cells because a cytoskeleton-rich
structure in the apical side of the cells did not develop sufficiently in the
salivary duct of Cp2l1tra/tra mice, which lack keratin 7
expression. Given that keratin 8, a type 2 basic keratin filament like keratin
7, is essential for the proper intracellular trafficking of membrane channels
and transporter proteins in colon cells
(Toivola et al., 2004), it is
possible that keratin 7 contributes to proper glandular duct formation by
regulating intracellular trafficking. Likewise, the expression of keratin 19
(Lussier et al., 1990), which
encodes a type 1 acidic keratin filament, was not properly reduced during
development but remained at a high level in the ducts of
Cp2l1tra/tra mice. Together, these results indicate that
CP2L1 participates in establishing the function of ducts by coordinating the
expression of several genes that are involved in physiological function and
generate the appropriate cellular architecture.
Different organs developing from distinct lineages often show similarities
in their morphogenetic processes (Davies,
2002; Hogan and Kolodziej,
2002). This is also true for their physiological properties. The
exocrine glands and kidney share common basal structures and function; in the
proximal part of the glands, secretions into the lumen are generated, and in a
distal part, the duct, the primary fluid flowing through its lumen is modified
by absorption and secretion. Hence, it is thought that there is a common
molecular basis for the generation of the structural and functional
similarities in the exocrine glands and kidney. Cp2l1 was
specifically expressed during the development of the ducts in many exocrine
glands, including ducts in the salivary, nasal, lachrymal, sweat and mammary
glands as well as in the kidney. In this study, we revealed that CP2L1 is
essential for proper maturation of the salivary ducts and the kidney.
Interestingly, we found that CP2L1 is required for the expression of a set of
genes, including Fxyd2, Gk-6, Scnn1b and keratin 7, which are
expressed commonly in the developing ducts of the salivary gland and kidney
(Fig. 6N). In addition, the
expression of keratin 7 and Gk-6 was reduced in the nasal gland of
Cp2l1-deficient mice (Fig.
3E and data not shown). These lines of evidence suggest that a
Cp2l1-dependent gene expression program constitutes a molecular
mechanism that generates the analogous properties of the ducts in exocrine
glands and kidney. Combination of higher resolution analysis at a single-cell
level and exhaustive and comparative analysis of different organs using DNA
microarrays is needed to determine the validity of this hypothesis.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/23/4737/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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