Cell and Molecular Development Group, Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK
*Author for correspondence (e-mail: eoliver-jones{at}dna.bio.warwick.ac.uk)
Accepted 8 January 2002
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Annexin, Pronephros, Kidney, Xenopus, Morpholino
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We, and others, have started to establish some of the biological parameters that control pronephros formation. The temporal specification of all three components of the kidney have been established and all occur between stages 12.5 and 14 (Brennan et al., 1998; Brennan et al., 1999
). A molecular map of many of the early markers of the pronephric field and pronephric anlagen is also being built up in order to unravel the molecular control of early kidney induction and patterning. In previous studies, more than 30 genes have been shown to be expressed in the pronephros and their temporal and spatial patterns of expression established (http://golgi.ana.ed.ac.uk/kidhome.html) (Wallingford et al., 1999
; Carroll and Vize, 1999
; Carroll et al., 1999a
; Carroll et al., 1999b
; Brändli, 1999
; Sato et al., 2000
; Onuma et al., 2000
; McLaughlin et al., 2000
). Many of these genes have also been shown to be expressed in mesonephric and metanephric kidneys in higher vertebrates. Evidence from knockout mutant mice has shown that Lim1, Pax2 and Wt1 are essential for pronephros development. The role of known growth factors in the process has also been investigated (Moriya et al., 1993
; Uochi and Asashima, 1996
; Brennan et al., 1999
).
However, the initial events in kidney organogenesis are not yet fully understood and Xenopus provides an ideal vertebrate system to identify and establish the functional role of genes involved in early kidney development. It is relatively easy to establish the domains of expression of novel genes expressed in particular regions of interest, and then it is possible, by overexpression, antisense depletion or morpholino treatment to perturb normal expression pattern and get a developmental handle on function (Zhang et al., 1998; Summerton and Weller, 1997
; Heasman et al., 2000
). Furthermore, direct expression cloning is proving an invaluable tool in identifying novel genes with defined roles in developmental patterning and organogenesis (Smith and Harland, 1991
; Smith and Harland, 1992
; Smith et al., 1995
; Hsu et al., 1998
; Grammer et al., 2000
).
In order to identify novel genes that may be involved in pronephros development, we have adopted a subtractive hybridisation strategy that increases the levels of those genes expressed early in kidney development. This approach is based on the observations that animal caps treated with a combination of retinoic acid and activin develop in vitro into differentiated kidney tubules. Recent studies in our laboratory have also shown that glomus can be induced in animal caps by treatment with retinoic acid and activin or retinoic acid and FGF (Brennan et al., 1999). No combinations of RA, activin or bFGF have been found to induce pronephric duct at high frequency (E. A. J., unpublished). Evidence from histological studies indicates that these tubules have normal morphology (Moriya et al., 1993
) (H. C. Brennan and E. A. J., unpublished). Furthermore, these tubules express differentiation markers characteristic of the correct developmental stage and in the correct developmental sequence (Uochi and Asashima, 1996
). The kidney tubules formed have been reported to rescue pronephric function in tadpoles in which the pronephros has been extirpated by dissection. During the course of our experiments a similar strategy has been used by others to successfully clone the pronephros specific genes XCIRP (Uochi and Asashima, 1998
) and XSMP-30 (Sato et al., 2000
). In vitro induction followed by differential display has recently resulted in the isolation of Xsal-3 that is also expressed in the pronephros (Onuma et al., 2000
).
We describe the isolation of Xenopus annexin IV (Xanx-4) via a subtractive hybridisation strategy designed to increase the levels of tubule-specific genes, which are expressed specifically and at high levels in the pronephric tubules. We have established the temporal and spatial expression patterns both of mRNA and protein in embryos. We have established the mRNA expression pattern by northern analysis in the adult frog. Finally, we have used morpholino oligonucleotides to specifically inhibit the translation of Xanx-4 and show that a tubule phenotype results, which can be rescued by the addition of wild-type message. The tubules appear less coiled and have a diameter that is significantly greater than that seen in control embryos. These results indicate that Xanx-4 plays an important role in morphogenesis of the pronephric tubules.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tester cDNA was prepared from 2 µg of group one polyA+ RNA. The polyA+ RNA from groups two and three were pooled and Driver cDNA was prepared from 2 µg of this pool. The subtracted probe was prepared by subtracting Driver cDNA from Tester cDNA, according to manufacturers protocol using Clontech PCR-Select cDNA Subtraction Kit (K1804-1).
Library screening and clone sequencing
Whole embryo stage 13 Xenopus laevis gt 11 cDNA library (gift from I. Dawid) was plated at a density of 2.5x105 plaques on 25 cm2 plates and duplicate plaque lifts taken on Hybond N+ (Amersham) nylon filters. The subtracted probe was hybridised at 62°C (0.1%SDS, 5xSSC, 0.1% BSA, 0.1% poly vinyl-pyrollidone, 0.1% Ficoll, type 400). The first wash was performed at 62°C (2xSSC, 0.1% SDS), followed by two further washes (1xSSC/ 0.1% SDS, 0.5xSSC/0.1% SDS and 0.2xSSC/ 0.1% SDS). The 1131 bp K2 (annexin IV) cDNA insert was subcloned into pGEM-T Easy (Promega) and both strands were sequenced using an Applied Biosystems 373A instrument.
Embryo culture and dissection
Embryos were obtained by in vitro fertilisation of hormonally stimulated Xenopus laevis and staged according to Nieuwkoop and Faber (Nieuwkoop and Faber, 1994). Standard embryological procedures were used as described by Jones and Woodland (Jones and Woodland, 1986
). Embryos were dejellied in 2% cysteine hydrochloride pH 8 and cultured in 1/10 BarthX. Dissected animal caps were cultured in BarthX and staged using whole embryo controls.
Expression clones, mRNA Synthesis and micro-injection
The full-length Xanx-4 cDNA was removed by digestion with EcoRI from pGEM-T Easy plasmid and cloned into the RN3 pBluescript vector (a gift from J. B. Gurdon, Cambridge) at the EcoRI sites. Xanx-4 mRNA was synthesised from Xanx-4/RN3 plasmid template, linearised with SfiI, using the mMessage mMachine (T3 RNA polymerase, Ambion). For the Myc-tagged Xanx-4 expression construct, a PCR cloning approach was used. Primers were designed to contain BamHI restriction sites (U-gccgggatcccatggcagcactc, D-cggcggatccgtcttcccctccg) and the resulting product cloned into pCS3+MT vector (a gift from H. Benisek, Michigan). Myc-tagged Xanx-4 mRNA was synthesised by linearising the Xanx-4/MT3 construct with EcoRI and transcribed using SP6 RNA polymerase mMessage mMachine Kit. The Sox17ß mRNA and Myc-tagged constructs were a gift from D. Clements (Warwick). Approximately 0.5 ng of mRNA was injected into dejellied embryos at the one-cell stage, alone or in combination with MOs (as specified in the text), under 3% Ficoll in BarthX.
Morpholinos
Xanx-4 and Xsox17ß (gift from D. Clements) morpholinos (MO) were designed and supplied by GeneTools, LLC (Corrallis, OR):
Xanx-4: 5'-acccttagttccgagtgctgccatg-3'
XSox17ß: 5'-cctcttacctcagttacaatttata-3'
control: 5'-cctcttacctcagttacaatttata-3'.
The MOs were dissolved in double distilled H2O to a stock concentration of 10 µg/ml and were injected into one-cell stage embryos (5 ng, 10 ng or 20 ng) alone or in combination with mRNA as specified in the text.
RT-PCR
Total RNA from whole embryos was isolated and used for RT-PCR as described by Barnett et al. (Barnett et al., 1998). Primers used in this study are as follows.
Xanx-4: U-AGCAGGCACGATGAAGATG and D-TCATTCACGGTGCTGCTCTG (this work)
Xlim-1: U-GAAGGATGAGACCACTGGTGG and D-CACTGCCGTTTCGTTCATTTC (Witta and Sato, 1997)
XPax-2I: U-TCGGAAGAAGAGTGGTCTAC (this work) and D-GGTATTCATATTCCGCATTC (Accession Number, AF179300)
XPax-8: U-CCAACAGCAGCATCAGATC (this work) and D-CAATGACACCTGGCCGGATA (Accession Number, AF179301)
XWnt-4: U-GAGTGGAATGCAAGTGTC (this work) and D-TACACTGCCGACCAGTTG (Accession Number, U13183)
XWnt-11: U-GAAGTCAAGCAAGTCTGCTGG and D-GCAGTAGTCAGGGGAACTAACCAG (http://www.lifesci.ucla.edu/hhmi/derobertis/index.html)
xWT1: U-CACACGCACGGGGTCT and D-TGCATGTTGTGATGACG (Carroll and Vize, 1996)
ODC: U-GGAGCTGCAAGTTTGGAGA and D-TCAGTTGCCAGTGTGGTC (Bassez et al., 1990)
EF1: U-CAGATTGGTGCTGGATATGC and D-CACTGCCTTGATGACTCCTA (Mohun et al., 1989
)
Whole-mount in situ hybridisation
Whole-mount in situ hybridisation was carried out as described elsewhere (Harland, 1991). The embryos were fixed in MEMFA (0.5 M MOPS, pH 7.4, 100 mM EGTA, 1 mM MgSO4, 4% formaldehyde) and hybridised with RNA probes produced from cDNA clones. The Xanx-4 antisense probe was transcribed with SP6 RNA polymerase from the full-length Xanx-4 in pGEMT-Easy. The XPax-8, Xlim-1 and xWT1 were kind gifts from T. Carroll (Texas). Probes were synthesised and labelled using a DIG labelling kit (Boehringer) and visualised using anti-DIG-alkaline phosphatase secondary and NBT/BCIP for the colour reaction according to manufacturers recommendations (Boehringer).
Immunohistochemistry
Whole-mount immunohistochemistry was performed using standard methods on MEMFA fixed embryos. The primary antibodies used were pronephric tubule-specific monoclonal antibody 3G8 and pronephric duct specific monoclonal antibody 4A6 (Vize et al., 1995), and an anti-annexin IV monoclonal antibody BL7B1, a kind gift from D. Massey-Harroche, Marseille (Massey et al., 1991
). The secondary antibodies were alkaline phosphatase-conjugated goat anti-mouse (Sigma) and FITC-conjugated goat anti-mouse (Sigma). BCIP/NBT (Boehringer) or Fast Red TR/Napthol AS/MX (Sigma) was used for the colour reaction, according to manufacturers recommendations.
Nuclear staining was carried out using Hoechst stain (33258) at a concentration of 1 µg/ml on acrylamide embedded sections, mounted in glycerol and viewed on Nikon microscope and using a u.v. filter.
Acrylamide embedding and cryostat sectioning
Embryos were fixed in MEMFA, rinsed in phosphate-buffered saline (PBS) and incubated at 4°C for 5 hours in embedding acrylamide (8.4 g acrylamide, 13.4 mg bis-acrylamide, 700 µl TEMED to 100 ml in PBS). The embryos were embedded in acrylamide using 5 µl/ml 10% ammonium persulphate to polymerise overnight at 4°C. The acrylamide blocks were frozen in iso-pentane over liquid nitrogen for 5 minutes. The blocks were then allowed to warm to 20°C and sectioned on a cryostat at 12 µm. The sections were lifted onto 0.1% gelatin subbed slides (300 bloom), fixed in acetone and mounted in 50% PBS/glycerol.
In vitro and in vivo translation of Myc-tagged construct mRNA
mRNA (10 ng) was translated in vitro in the Rabbit Reticulocyte Lysate System (Promega) using the according to manufacturers protocol. Reactions (5 µl) were denatured at 95°C in 2xSDS loading buffer (Harlow and Lane, 1988) and subjected to western immunoblot analysis.
For in vivo analysis, 0.5 ng of mRNA was microinjected into one-cell stage embryos, which were then cultured to the appropriate stage. Groups of five embryos were homogenised in 50 µl of homogenisation buffer (0.1 M NaCl, 1% Triton X-100, 1 mM PMSF, 20 mM Tris-Cl pH 7.6) at 4°C and centrifuged in a bench top microcentrifuge at 4°C for 10 minutes at 10,000 g. The cytosolic layer was removed, 5 µl of each sample was denatured at 95°C in 2xSDS loading buffer and subjected to western analysis.
Western analysis
SDS-PAGE was performed on 12% (w/v) resolving gel using a vertical minigel apparatus for 1 hour at 20 mA. The proteins were transferred to nitrocellulose membrane (Amersham) according to Harlow and Lane (Harlow and Lane, 1988) for 2 hours at 350 mA or overnight at 50 mA. After transfer the nitrocellulose was incubated for 1 hour at room temperature in TBS-Tween (0.15 M NaCl, 10 mM Tris-Cl, pH 7.4, 0.1% Tween-20) containing 3% (w/v) powdered milk. The blots were then incubated in 1:1000 anti-Myc monoclonal antibody (gift from D. Stott, Warwick) in TBS-Tween overnight at room temperature. After washing, the blots were incubated in 1:2000 alkaline phosphatase-conjugated goat anti-mouse (Sigma) in TBS-Tween for 1 hour at room temperature. An Immun-star Detection Kit (BioRad) was used for detection according to manufacturers recommendations.
Preparation of adult tissue RNA samples and northern blot analysis
Total RNA was extracted from different adult tissues using Trizol (Gibco-BRL Life Technologies) following the manufacturers protocol. Samples of denatured RNA (30 µg per lane) were fractionated by electrophoresis though 1.2% agarose, 2.2 M formaldehyde gels in MOPS buffer (50 mM MOPS pH7; 1 mM EDTA; 20 mM sodium acetate), blotted for 48 hours in 10xSSC onto a Hybond-N membrane (Amersham) and fixed by baking for 2 hours at 80°C. The filters were hybridised overnight at 42°C with [32P]-labelled Xanx-4 specific probe in 0.5M phosphate buffer pH 7.2 in the presence of 7% SDS and 5mM EDTA. The probe was prepared from a 3' specific fragment, by PCR amplification (Primers U-gcataaagagcaggccagcc, D-cgattggttatgtgttcaat). After hybridisation, the filters were washed at room temperature in 2xSSC, 0.1% SDS, twice for 15 minutes at 42°C, and then for 15 minutes at 65°C in 1xSSC, 0.1%SDS. Autoradiographs were obtained by exposure of Fuji super RX films with intensifying screens at 80°C.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Sequencing revealed K2 to be a clone of 1131 nucleotides, containing an in-frame coding sequence corresponding to Xenopus laevis annexin IV (Xanx-4) of 963 bases, including a 50 base 5'UTR and a 118 base 3'UTR (Accession Number, AY039235). Conceptual translation of Xanx-4 yielded a predicted amino acid sequence of 321 amino acids that displays identity with annexin IV in other vertebrate species of between 67-74% (Fig. 1A).
|
Temporal expression of Xanx-4
The temporal expression profile of Xanx-4 transcripts was revealed by RT-PCR (Fig. 2). Maternal expression of Xanx-4 was detected in the egg but subsequently declined rapidly and was substantially reduced by the 32-cell stage. Zygotic expression was detected at a very low level between stages 9-12.5 and at a significantly increased level at stage 13. This coincides with the time of pronephric tubule specification (Brennan et al., 1998). Expression then continues at a similar level up to and beyond the stage when the pronephros becomes functional (stage 37). ODC was used as a loading control.
|
|
|
|
Xanx-4 MO depletion of the translation of Xanx-4 mRNA in vitro
Initially the ability of the Xanx-4 MO to deplete Xanx-4 translation was established in vitro. Myc-tagged Sox17ß mRNA (a kind gift from D. Clements, Warwick) was used as a control to test for the Xanx-4 MO specificity. A combination of 10 ng of Myc-tagged Sox17ß mRNA and 10 ng of Myc-tagged Xanx-4 mRNA was incubated either alone or in combination with 1 µg, 5 µg or10 µg of Xanx-4 MO in the reticulocyte lysate system. The lysates were subjected to SDS-PAGE and western blotting using anti-Myc antibody (Fig. 6A). Both Myc-tagged Sox17ß mRNA and Myc-tagged Xanx-4 mRNA were successfully translated at similar levels (lanes 1, 3 and 5) but when incubated with 1 µg, 5 µg or 10 µg Xanx-4 MO, Xanx-4 translation was blocked, whereas Sox17ß translation was not (lanes 2, 4 and 6). In a similar experiment, the Xanx-4 MO was also shown to have no effect on the translation of XEZ (Barnett et al., 2001) mRNA (data not shown). This shows that the Xanx-4 MO preferentially depletes Xanx-4 mRNA in vitro.
|
Xanx-4 depletion using Xanx-4 MO produces pronephric tubules with an enlarged diameter
In order to examine the activity of Xanx-4 in vivo, we have used overexpression and depletion to perturb endogenous expression of Xanx-4. Embryos were injected at the one-cell stage with 10 ng control MO, 10 ng Xanx-4 MO or 0.5 ng Xanx-4 mRNA. The injected embryos were cultured to stage 40 and whole-mount antibody stained with 3G8 and 4A6, which are tubule- and duct-specific monoclonal antibodies, respectively. Although no obvious effect on pronephric tubule or duct morphology was observed after overexpression of Xanx-4 mRNA or on injection of control MO (two experiments, counting each pronephros individually, total number of animals scored n=116, n=102), a clear effect on tubule morphology was observed after Xanx-4 MO injection. On whole-mount inspection, the normal coiled tubular structure of the pronephric tubules appeared completely disrupted in MO-injected embryos. This led to the formation of apparently shortened and enlarged tubules (24/56, 38/60 in two independent experiments; Fig. 7). In some embryos, the tubules were reduced in overall size and in some cases missing completely (8/56, 12/60 in the same independent experiments). In order to investigate the phenotype, a random sample of six immunostained embryos from each treatment group were acrylamide embedded, frozen and cryostat-sectioned (12 µm). The slides were counterstained stained with Hoechst 33258, mounted and compared with uninjected controls. Analysis of the sections revealed that MO-treated embryos had tubules that were substantially wider than control uninjected, control MO injected or Xanx-4 mRNA injected embryos.
|
|
|
As previously described, pronephric tubules of the embryos injected with the Xanx-4 MO appeared in general to be wider, shorter and less coiled than those of the normal control embryos. It was also observed that the most severe phenotype occurred in those embryos injected with the higher concentration of MO. Apart from the aberrant tubule phenotype, the embryos appeared to be of normal morphology. Those embryos co-injected with 5 ng Xanx-4 MO and Xanx-4 mRNA appeared almost completely rescued and had almost normal tubules (Fig. 8, compare C-F with G,H). The 10 ng and 20 ng Xanx-4 MO embryos co-injected with Xanx-4 mRNA showed significant, although incomplete, rescue. The tubules appeared somewhat less normal and displaying some larger and less coiled tubules. Seemingly the rescue was not complete at the higher concentrations of MO. Other than the pronephric tubule phenotype described above, the gross phenotype of the embryos in all groups appeared normal.
In order to quantify this phenotype, a random sample of six embryos from each group were acrylamide embedded, frozen and cryostat-sectioned as described previously. The mean number of cells in the circumference of the pronephric tubules, of the embryos co-injected with 5 ng Xanx-4 MO and 0.5 ng Xanx-4 mRNA (mean=9, range 5-21), was not significantly different (P<0.01) from control normal embryos (mean=9, range 5-15) (Fig. 9B). It appears therefore, that 0.5 ng Xanx-4 mRNA was able to rescue 5 ng Xanx-4 MO completely. Those injected with 10 ng (mean=20, range=6-52) or 20 ng (mean=21, range=6-44) of MO, were partially rescued by 0.5 ng Xanx-4 mRNA (10 ng/mRNA rescue mean=12, range 6-38, 20 ng/mRNA rescue mean=14, range 6-38). Embryos injected with 10 ng of MO were rescued to a 5 ng MO phenotype (mean=12, range=6-24) and 20 ng was rescued to an intermediate phenotype between 10 ng and 20 ng. The data collected from the serial sections of each of six randomly chosen embryos from each group is shown as a graphical representation in Fig. 9B. Similar results were obtained from a repeat experiment (data not shown).
Analysis of the expression of pronephric molecular markers in Xanx-4 over-expression and Xanx-4 MO treated embryos
In order to assess whether either the observed phenotype or other unidentified earlier events was related to other genes known to be expressed in the pronephros, the following experiments were carried out. Both semi-quantitative RT-PCR and in situ hybridisation were performed for a selection of genes whose mRNAs have been shown to play an early role in kidney development. Embryos were injected at the one-cell stage with 0.5 ng Xanx-4 mRNA or 10 ng Xanx-4 MO, cultured to stage 25. Groups of five embryos (in duplicate) were analysed by RT-PCR (Barnett et al., 1998) using primers designed against a selection of genes known to be expressed in the pronephros (see Materials and Methods). EF1
is used as a loading control. No effect on expression of any of the marker genes was observed (Fig. 10). Embryos injected with 0.5ng Xanx-4 mRNA or 10 ng Xanx-4 MO were cultured to stages 26-28 and subjected to in situ hybridisation using RNA probes prepared from cDNA clones of pronephric marker genes XPax-8, Xlim-1 and xWT1. The expression pattern of each marker gene RNA in the embryos injected with Xanx-4 mRNA and Xanx-4 MO1 were compared with uninjected control embryos (Fig. 11). No apparent change in XPax-8 or Xlim-1 expression pattern was observed in either group with perturbed Xanx-4 expression, compared with uninjected control. However, a reduced field of xWT1 expression was observed in 27% (n=12/44) of the embryos that overexpressed Xanx-4, compared with controls. This apparent reduction may have been due to a more dispersed xWT1 expression domain, as no detectable reduction in xWT1 expression was observed by RT-PCR. No altered xWT1 field of expression was seen in the embryos injected with Xanx-4 MO1. It seems perturbation of expression of Xanx-4 is not linked to any of the tubule markers analysed. We also assume from these results that the Xanx-4 depletion phenotype is not associated with more or less tissue being specified to become pronephric tubule in character.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cloning of Xanx-4
A gradient of the TGFß superfamily member activin A will induce different kinds of mesodermal tissues in dissociated cells (Green and Smith, 1990; Green and Smith, 1991
; Green et al., 1992
) and in embryonic explants in culture (Gurdon et al., 1994
; Moriya et al., 1993
). As pronephric tubules can be induced at high frequency by treatment of animal caps with a combination of activin and RA (Moriya et al., 1993
), this provides a very attractive method with which to produce large amounts of specific tissues for use in a variety of cloning strategies or screens. We have successfully used this approach to produce a high frequency of pronephric tubules in Xenopus explants as a source of mRNA enriched with pronephric transcripts.
As caps treated with RA or activin alone do not induce pronephric tubules with high frequency, our approach of preparing a subtracted probe allowed the elimination of molecules that were upregulated by RA or activin alone. Thus, we have created a probe specific for molecules upregulated only by the combination of activin and retinoic acid, which would include those expressed at the time of induction and specification of pronephric tubules. In our study, we chose to screen an early Xenopus cDNA library, stage 13, based on the work carried out in our laboratory that demonstrated that the tubules were specified at stage 12.5 (Brennan et al., 1998). In choosing to screen an early library, we were able to select for genes not only specific for pronephric tubule specification, but also genes that are involved in early events of pronephric differentiation, patterning and development.
The zygotic upregulation of Xanx-4 occurs at stage 12.5-13. RT-PCR carried out on dissected stage 13 embryos (dorsal, lateral and ventral domains) revealed that Xanx-4 is expressed in the lateral domain at this stage (data not shown). Xanx-4 expression is maintained through all the stages of pronephric tubule development to and beyond the time when the pronephros becomes functional. This implies an important and specific role for Xanx-4 not only in the development, but also perhaps in the function of the pronephros. In this work, we have described the highly restricted distribution of both Xanx-4 mRNA and protein during embryogenesis. The expression patterns of Xenopus annexin II and annexin VII are less restricted, being expressed in various neural and mesodermal tissues (Izant and Bryson, 1991; Srivastava et al., 1996
).
We have shown by northern blot analysis that the expression pattern of Xanx-4 in the adult frog is restricted to epithelial tissues. In agreement with this result, annexin IV displays polarised expression in adult epithelial tissues of other animal species: lungs (Sohma et al., 1995), intestines and pancreas (Massey et al., 1991
), liver (Boustead et al., 1993
) and kidney (Massey-Harroche et al., 1995
; Kojima et al., 1994
). It appears that the common theme for localisation of annexin IV expression in adult tissues is that of polarised epithelial tissue.
The effects of altered expression of Xanx-4
We have shown that Xanx-4 is expressed at the right time in the right place to have a functional role in pronephric tubule development. To investigate this, we have perturbed the expression of Xanx-4 in Xenopus tadpoles and identified a clear phenotype on pronephric tubule morphology in whole-mount and in section.
Depletion of Xanx-4 protein by MO causes an enlarged, shortened and uncoiled pronephric tubule phenotype. The effect of Xanx-4 MO can be rescued by co-injection of Xanx-4 mRNA. Longevity of the MO is suggested to be considerable (Summerton and Weller, 1997; Nutt et al., 2001
), and may be still acting at much later times that that of the ectopic mRNA. The rescue of the MO phenotype observed may be due to action of the Xanx-4 mRNA antagonising the action of the Xanx-4 MO on co-injection. We have shown that 0.5 ng of injected message will completely rescue the tubule phenotype seen in 5 ng Xanx-4 MO embryos, whereas at higher concentrations of Xanx-4 MO, the phenotype is only partially rescued. Interestingly Xanx-4 mRNA injected alone does not produce a phenotype. This enables a rescue experiment to be performed that will return the MO phenotype to the normal range, rather than to producing an overexpression phenotype. The lack of overexpression phenotype may be due to the relatively late requirement of Xanx-4 in kidney morphogenesis and the inability of injected mRNA to survive to this point. We are currently investigating the potential of transgenics to overcome this problem.
The pronephric tubules of the Xanx-4 MO-injected embryos, although enlarged in diameter, were constructed and organised in the normal way. The tubules consisted of a lumen, albeit enlarged, and the walls were constructed of one epithelial cell layer, though they contained more cells in their circumference. The pronephroi of Xanx-4-depleted embryos consisted of the normal complement of pronephric components, including capsule, nephrostomes, tubules and duct, as shown by the expression of marker genes (Fig. 11). It appears that the pronephroi were functional, maintaining body water and electrolyte homeostasis, as none of the components appeared cystic and no general oedema was observed. Recent work (Drummond et al., 1998) has isolated 18 independent recessive mutations that affect pronephric development from a large scale ENU mutagenesis screen (Driever et al., 1996
). A common theme for the phenotypes of these mutants was the appearance of fluid-filled cysts in the pronephric region followed by general oedema. The authors suggest that the phenotypes observed are the consequence of pronephric failure and altered osmoregulation. Histological analysis of the double bubble mutant showed that the glomerulus was loose and distorted, swelling was apparent, and the cells appeared flattened. The glomerulus overall architecture was disorganised and the basement membrane was severely distorted (Drummond et al., 1998
). No such tissue disorganisation, cell shape distortion or fluid filled cysts were observed in any of the Xanx-4-depleted embryos.
In counting all cells in serial sections, we have shown that the number of cells making up the circumference of the pronephric tubules in normal controls and Xanx-4 MO-injected embryos differs. A transverse section of the tubules does not, in general, provide perfect circles. By using cell counting as a measure of the phenotype observed, we have taken a measure of the size of tubule sections, thereby removing errors that could complicate the analysis and introduce errors.
The pronephric tubule markers tested were not affected by perturbation of Xanx-4 expression. We suggest that the phenotype observed is not due to a change in the amount of pronephric tubule tissue specified but is due to an alteration in the morphological process during tubulogenesis. Preliminary studies of the pronephric tubule morphology of Xanx-4 MO-depleted embryos at earlier stages (35-36) revealed no aberrant tubule phenotype (data not shown). It appears that the enlarged tubule phenotype, caused by depletion of Xanx-4, may only manifest itself at later stages of tubulogenesis during tubule maturation and elongation.
Previous studies have shown that co-expression of either XPax-2 or XPax-8 with Xlim-1 results in a synergistic effect producing increased pronephric tubule complexity, enlarged tubules and ectopic tubules, while expression of either XPax-2 or XPax-8 alone has a moderate effect (Carroll and Vize, 1999). We did not see complex or ectopic tubules under any of the conditions tested. We have also shown that perturbation of Xanx-4 expression does not affect the expression of either XPax-8 or Xlim-1. Ectopic expression of xWT1, which is required for metanephric development in vivo, inhibits pronephric tubule development, probably by repressing tubule specific gene expression in the region of the pronephros fated to become tubules (Wallingford et al., 1999
). Depletion of Xanx-4 does not effect the expression of xWT1. The effect of Xanx-4 overexpression on xWT1 was unexpected, as no Xanx-4 overexpression phenotype was previously observed. The fact that RT-PCR analysis gave similar levels of xWT1 expression in controls and Xanx-4 overexpressing embryos suggests that the effect of Xanx-4 overexpression results in more diffuse expression of xWT1. This is consistent with the results observed in the xWT1 in situ hybridisation of embryos overexpressing Xanx-4, where in some embryos the xWT1 expression domain was more dispersed. These results place Xanx-4 downstream of Xlim-1/XPax-2/8 and xWT1 in a molecular pathway of tubulogenesis. Further experiments will be required to confirm the relative positions of these pronephric genes.
It is thought that annexins participate in calcium homeostasis, regulation of ion channel activities and membrane traffic events. Annexins have been well characterised with regard to their binding activities. They have been shown to bind various proteins, including proteoglycans, F-actin and collagen, in addition to calcium and phospholipids (Lecat and Lafont, 1999; Seaton and Dedman, 1998
). Annexins have been shown to bind phospholipid membranes in ordered arrays, and it has been suggested that annexin complexes may function to modify membrane structure (Oling et al., 1999
). A role for annexin IV in epithelial membrane integrity, adhesion or plasticity could account for the aberrant tubule phenotype observed. Depletion of Xanx-4 in the pronephric tubules may cause a remodelling of tubule morphology during development. Annexin IV has been shown to be involved in calcium-activated cellular signal transduction events (Raynal et al., 1996
), exocytosis (Sohma et al., 2001
) and in the regulation of calcium-activated epithelial chloride ion channel activity (Kaetzel et al., 1994
; Chan et al., 1994
; Jorgensen et al., 1997
). The primary function of the pronephric tubules is the transport of ions, water and other molecules, and as the site of Xanx-4 localisation (apical) in the pronephric tubules is in agreement with a possible role in these events, further studies will be directed at ascertaining its function directly. Although, as previously discussed, a role in osmoregulation seems unlikely, the enlarged tubule phenotype observed could well be attributed to incorrect modulation of exocytosis, ion channel activity or other calcium signalling events. Direct investigations of these processes in MO-treated embryos will form the basis of future work. This study represents a study of the role of Xanx-4 in the formation of pronephric tubules in Xenopus laevis.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Barnett, M. W., Old, R. W. and Jones, E. A (1998). Neural induction and patterning by fibroblast growth factor, notochord and somite tissue in Xenopus. Dev. Growth Diff. 40, 47-57.[Medline]
Barnett, M. W., Seville, R. A., Nijjar, S., Old, R. W. and Jones, E. A. (2001). Xenopus Enhancer of Zeste (XEZ); an anteriorly restricted polycomb gene with a role in neural patterning. Mech. Dev. 102, 157-167.[Medline]
Bassez, T., Paris, J., Omilli, F., Dorel, C. and Osborne, H. B. (1990). Post transcriptional regulation of ornithine decarboxylase in Xenopus laevis oocytes. Development 110, 955-962.[Abstract]
Boustead, C. M., Brown, R. and Walker, J. (1993). Isolation, Characterisation and localisation of annexin V from chicken liver. Biochem. J. 291, 601-608.[Medline]
Brändli, A. W. (1999). Towards a molecular anatomy of the Xenopus pronephric kidney. Int. J. Dev. Biol. 43, 381-395.[Medline]
Brennan, H. C., Nijjar, S. and Jones, E. A. (1998). The specification of the pronephric tubules and duct in Xenopus laevis. Mech. Dev. 75, 127-137.[Medline]
Brennan, H. C., Nijjar, S. and Jones, E. A. (1999). Growth Factor inducibility and specification of the pronephric glomus in Xenopus laevis. Development 126, 5847-5856.
Carroll, T. J. and Vize, P. D. (1996). Wilms tumor suppressor gene is involved in the development of disparate kidney forms: evidence from expression in the Xenopus pronephros. Dev. Dyn. 206, 131-138.[Medline]
Carroll, T. and Vize, P. D. (1999). Synergism between Pax-8 and lim-1 in embryonic kidney development. Dev. Biol. 214, 46-59.[Medline]
Carroll, T., Wallingford, J., Seufert, D. and Vize, P. D. (1999a). Molecular regulation of pronephric development. Curr. Top. Dev. Biol. 44, 67-100.[Medline]
Carroll, T., Wallingford, J. and Vize, P. D. (1999b). Dynamic patterns of gene expression in the developing pronephros of Xenopus laevis. Dev. Genet. 24, 199-207.[Medline]
Chan H. C., Kaetzel, M. A., Gotter, A. L., Dedman, J. R. and Nelson, D. J. (1994). Annexin IV inhibits calmodulin-dependent protein kinase II-activated chloride conductance. A novel mechanism for ion channel regulation. J. Biol. Chem. 269, 32464-32468.
Driever, W., Solnica-Krezel, L., Schier, A. F., Neuhauss, S. C., Malicki, J., Stemple, D. L., Stainier, D. Y., Zwartkruis, F., Abdelilah, S., Rangini, Z. et al. (1996). A genetic screen for mutations affecting embryogenesis in zebrafish. Development 123, 37-46.
Drummond, I. A., Majumdar, A., Hentschel, H., Elger, M., Solnica-Krezel, L., Schier, A. F., Neuhauss, S. C., Stemple, D. L., Zwartkruis, F., Rangini, Z. et al. (1998). Early development of the zebrafish pronephros and analysis of mutations affecting pronephric function. Development 125, 4655-4667.
Grammer, T. C., Liu, K. J., Mariani, F. V and Harland, R. M. (2000). Use of large scale expression cloning screens in the Xenopus laevis tadpole to identify gene function. Dev. Biol. 228, 197-210[Medline]
Green, J. B. A. and Smith, J. C. (1990). Graded changes in dose of a Xenopus activin A homologue elicit stepwise transitions in embryonic cell fate. Nature 347, 391-394.[Medline]
Green, J. B. A. and Smith, J. C. (1991). Growth factors as morphogens. Trends Genet. 7, 245-250[Medline]
Green, J. B. A., New, H. V. and Smith, J. C. (1992). Responses of embryonic cells to activin and FGF are separated by multiple dose thresholds and correspond to distinct axes of the mesoderm. Cell 71, 731-739.[Medline]
Gurdon, J. B., Harger, P., Mitchell, A. and Lemaire, P. (1994). Activin signalling and response to a morphogen gradient. Nature 371, 487-492.[Medline]
Harland, R. (1991). In situ hybridisation: an improved wholemount method for Xenopus embryos. Methods Cell Biol. 36, 685-695.[Medline]
Harlow, E and Lane, D. (1988). Antibodies: A Laboratory Manual. New York: Cold Spring Harbour Laboratory Press.
Heasman, J., Kofron, M. and Wylie, C. (2000). Beta-catenin signalling activity dissected in the early Xenopus embryo: a novel antisense approach. Dev. Biol. 222, 124-34.[Medline]
Hsu, D. R. Economides, A. N., Wang, X., Eimon, P. M. and Harland, R. M. (1998). The Xenopus dorsalising factor Gremlin identifies a novel family of secreted proteins that antagonise BMP activities. Mol. Cell 5, 673-683.
Izant, J. G and Bryson, L. J. (1991). Xenopus Annexin II (Calpactin I) Heavy chain has a distinct amino terminus. J. Biol. Chem. 266, 18560-18566.
Jones, E. A. and Woodland, H. R. (1986). Development of the ectoderm in Xenopus: tissue specification and the role of cell association and division. Cell 44, 345-355.[Medline]
Jorgensen, A. J., Bennekou, P., Eskensen, K. and Kirstensen, B. I. (1997). Annexins from Ehrlich ascites cells inhibit the calcium-activated chloride current in Xenopus laevis oocytes. Eur. J. Physiol. 434, 261-266.[Medline]
Kaetzel, M. A., Chan, H. C., Dubinsky, W. P., Dedman, J. R. and Nelson D. J. (1994). A role for annexin IV in epithelial cell function. Inhibition of calcium-activated chloride conductance. J. Biol. Chem. 269, 5297-5302.
Kojima, K., Utsumi, H., Ogawa, H. and Matsumoto, I. (1994). Highly polarised expression of carbohydrate-binding protein p33/41 (annexin IV) on the apical plasma membrane of epithelial cells in renal proximal tubules. FEBS Lett. 342, 313-318.[Medline]
Lecat, S. and Lafont, F. (1999). Annexins and their interacting proteins in membrane traffic. Protoplasma 207, 133-140.
Massey, D., Traverso, V., Rigal, A. and Maroux, S. (1991). Cellular and subcellular localisation in rabbit intestinal epithelium, pancreas and liver. Biol. Cell 73, 151-156.[Medline]
Massey-Harroche, D., Traverso, V., Mayran, N., Francou, V., Vandewalle, A. and Maroux, S. (1995). Changes in expression and subcellular localisation of annexin IV in rabbit proximal tubule cells during primary culture. J. Cell Physiol. 165, 313-322.[Medline]
McLaughlin, K. A., Rones, M. S. and Mercola, M. (2000). Notch regulates cell fate in the developing pronephros. Dev. Biol. 227, 567-580.[Medline]
Mohun, T. J., Taylor, M. V., Garrett, N. and Gurdon, J. B. (1989). The CARG promoter sequence is necessary for muscle specific transcription of the cardiac actin gene in Xenopus embryos. EMBO J. 8, 1153-1161.[Abstract]
Moriya, N., Uchiyama, H. and Asashima, M. (1993). Induction of pronephric tubules by activin and retinoic acid in presumptive ectoderm of Xenopus laevis. Dev. Growth Diff. 35, 123-128.
Nasevicius, A. and Ekker, C. C. (2000). Effective targeted gene knockdown in zebrafish. Nat. Genet. 26, 216-220.[Medline]
Nelsestuen, G. L. and Ostrowski, B. G. (1999). Membrane association with multiple calcium ions: vitamin-K-dependent proteins, annexins and pentraxins. Curr. Opin. Struct. Biol. 9, 433-437.[Medline]
Nieuwkoop, P. D and Faber, J. (1994). Normal table of Xenopus laevis (Daudin). Amsterdam: North Holland.
Nutt, S. L., Bronchain, O. J., Hartley, K. O. and Amaya, E. (2001). Comparison of morpholino based translational inhibition during the development of Xenopus laevis and Xenopus tropicalis. Genesis 30, 110-113.[Medline]
Oling, F., Bergsma-Schutter, W. and Brisson, A. (1999). Trimers, dimers of trimers, and trimers of trimers are common building blocks of annexin a5 two-dimensional crystals. J. Struct. Biol. 133, 55-63.
Onuma, Y., Nishinakamura, R., Takahashi, S., Yokota, T. and Asashima, M. (2000). Molecular cloning of a novel Xenopus spalt gene (Xsal-3). Biochem. Biophys. Res. Commun. 264, 151-156.
Raynal, P., Kuipers, G., Rojas, E. and Pollard, H. B. (1996). A rise in nuclear calcium translocates annexins IV and V to the nuclear envelope. FEBS Lett. 392, 263-268.[Medline]
Sato, A., Asashima, M., Yokota, T. and Nishinakamura, R. (2000). Cloning and expression pattern of a Xenopus pronephros-specific gene XSMP-30. Mech. Dev. 92, 273-275.[Medline]
Saxén, L. (1987). Organogenesis of the Kidney. Cambridge, UK: Cambridge University Press.
Seaton, B. A. and Dedman, J. R. (1998). Annexins. Biometals 11, 399-404.[Medline]
Smith, W. C and Harland, R. M. (1991). Injected Xwnt-8 RNA acts early in Xenopus embryos to promote formation of a vegetal dorsalising centre. Cell 67, 753-765.[Medline]
Smith, W. C. and Harland, R. M. (1992). Expression cloning of noggin, a new dorsalising factor localised to the Spemann organiser in Xenopus embryos. Cell 70, 829-840.[Medline]
Smith, W. C., McKendry, R., Ribisi, S., Jr and Harland, R. M. (1995). A nodal-related gene defines a physical and functional domain within the Spemann organiser. Cell 82, 37-46.[Medline]
Sohma. H. M. N., Watanabe, T., Hattori, A., Kuroki, Y. and Akino, T. (1995). Ca2+-dependent binding of annexin IV to surfactant protein A and lamellar bodies in alveolar type II cells. Biochem. J. 312, 175-181.[Medline]
Sohma, H., Creutz, C. E., Gasa, S., Ohkawa, H., Akino, T. and Kuroki, Y. (2001). Differential lipid specificities of the repeated domains of annexin IV. Biochim. Biophys. Acta 1546, 205-215.[Medline]
Srivastava, M., Goping, G., Caohuy, H., McPhie, P. and Pollard, H. (1996). Detection and localization of synexin (Annexin VII) in Xenopus adult and embryonic tissues using an antibody to the Xenopus N-terminal PGQM repeat domain. Exp. Cell Res. 229, 14-19.[Medline]
Summerton, J. and Weller, D. (1997). Morpholino antisense oligomers:design, preparation and properties. Antisense Nucleic Acid Drug Dev. 7, 187-195.[Medline]
Uochi, T. and Asashima, M. (1996). Sequential gene expression during pronephric tubule formation in vitro and in Xenopus ectoderm. Dev. Growth Diff. 38, 625-634.
Uochi, T. and Asashima, M. (1998). XCIRP (Xenopus homologue of cold-inducible RNA-binding protein) is expressed transiently in developing pronephros and neural tissue. Gene 211, 245-250.[Medline]
Vize, P. D., Jones, E. A. and Pfister, R. (1995). Development of the Xenopus pronephric system. Dev. Biol. 171, 531-540.[Medline]
Vize, P. D., Seufert, D. W., Carroll, T. and Wallingford, J. B. (1997). Model systems for the study of kidney development: use of the pronephros in the analysis of organ induction and patterning. Dev. Biol. 188, 189-204.[Medline]
Wallingford, J. B., Carroll, T. J and Vize, P. D. (1999). Precocious expression of the Wilm tumor gene xWT1 inhibits embryonic kidney development in Xenopus laevis. Dev. Biol. 202, 103-112.
Witta, S. E. and Sato, S. M. (1997). XClPOU2 is a potential regulator of Spemanns organiser. Development 124, 1179-1189.
Yang, Z. Liu, N. and Liu, S. (2001). A zebrafish forebrain-specific zinc finger gene can induce ectopic dlx2 and dlx6 expression. Dev. Biol. 231, 138-148.[Medline]
Zhang, J., Houston, D. W., King, M. L., Payne, C., Wylie, C. and Heasman, J. (1998). The role of maternal VegT in establishing the primary germ layers in Xenopus embryos. Cell 94, 515-524.[Medline]