J Biol Chem, Vol. 275, Issue 17, 12363-12366, April 28, 2000

ACCELERATED PUBLICATION
ICln Is Essential for Cellular and Early Embryonic Viability^*

William T. Pu, Kevin Wickman^§, and David E. Clapham^¶

From the Cardiovascular Division, Howard Hughes Medical Institute, Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

pICln is a 26-kDa protein that is ubiquitously expressed and highly conserved from Xenopus laevis to Homo sapiens. The physiological functions of pICln remain to be established. To address this question, we disrupted the ICln gene in embryonic stem cells. We found that murine embryos lacking ICln die early in gestation (between stages E3.5 and E7.5). Furthermore, we found that ICln is essential for embryonic stem cell viability. Previously, we showed that pICln interacts directly with a homolog of a yeast protein that binds a PAK-like kinase and participates in the regulation of cell morphology and cell cycling. pICln also forms a complex with several core spliceosomal proteins, and this interaction may play a role in the regulation of spliceosomal biogenesis. Collectively, these data strongly suggest that pICln participates in critical cellular pathways, including regulation of the cell cycle and RNA processing.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

pICln is a 26-kDa protein that is ubiquitously expressed (1-3) and highly conserved from Xenopus laevis to Homo sapiens. No protein with known function has homology to the predicted amino acid sequence of pICln, and the predicted secondary structure lacks typical membrane-spanning domains. pICln is a soluble protein that is present in both the cytoplasm and the nucleus (4). A small fraction of total cellular pICln also associates with cytoskeletal elements (4, 5).

The physiological function of pICln has not been established. Although pICln was proposed initially to encode a chloride channel because overexpression of pICln in Xenopus oocytes led to the induction of a chloride conductance (6), the structure and localization of pICln suggest that it is not a channel (4, 7). Induction of the chloride conductance may be unrelated to the physiological function of pICln, because an identical endogenous chloride conductance is present in some uninjected oocytes (8) and can be induced by overexpression of structurally unrelated proteins (9).

To determine the physiological function of pICln, we purified and microsequenced several proteins that form stable, soluble complexes with pICln. We have found that pICln interacts directly with IBP72, a protein that is homologous to yeast Skb1 (10). Skb1 binds to a yeast p21-activated kinase (PAK)¹ homolog, and this interaction has been implicated in the regulation of cell morphology and cell cycling (11, 12). We have also found that pICln interacts directly with several core spliceosomal (Sm) proteins (13). Overexpression of pICln in Xenopus oocytes interfered with spliceosomal biogenesis by inhibiting the interaction of Sm proteins with spliceosomal RNAs. Binding of pICln to core spliceosomal proteins also prevented Sm proteins from interacting with SMN (10), the protein that is mutated in spinal muscular atrophy (14). SMN interaction with Sm proteins facilitates the assembly of Sm proteins on spliceosomal RNAs (13); disruption of this interaction may account, at least in part, for the inhibitory effect of pICln on spliceosomal biogenesis.

The high degree of pICln sequence conservation between distant species, the ubiquitous pattern of pICln expression, and the interaction of pICln with proteins that are active in critical cellular pathways suggest that pICln is important for fundamental cellular processes. We used gene targeting in embryonic stem cells to ask whether pICln is essential for viability.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Targeting Vectors-- Cloning and mapping of the 129Sv/J mouse genomic DNA fragment containing the two ICln coding sequence exons was described previously (3). In the targeting vector ICln^KO-TV, the first coding exon was replaced by a neomycin resistance gene driven by the mouse phosphoglycerate kinase promoter (pgk-neo). The targeting vector Flox-neo-ICln is composed of a 10-kb fragment of ICln genomic DNA that has been modified by the insertion of a loxP site upstream of the first ICln coding exon. A pgk-neo cassette flanked by directly repeated loxP sites (21) was inserted downstream of the first ICln coding exon. Both targeting vectors also included a diphtheria toxin negative selection cassette.

Embryonic Stem (ES) Cell Manipulations-- 129Sv/J ES cells (Genome Systems) were cultured on a monolayer of mitomycin-treated murine embryonic fibroblasts (MEFs), transfected with linearized targeting construct, and selected for G418 resistance using standard protocols (Genome Systems). Drug-resistant colonies were screened by a PCR-based assay to identify homologous recombinants. Clones identified as homologous recombinants by PCR were amplified and tested by Southern analysis as described previously (22) using both internal and 3' flanking probes.

Where indicated, recombination between loxP sites was performed by transiently transfecting ES cells with a Cre expression vector (23). Single colonies were picked in duplicate into 96-well dishes. Cells in one dish were grown in the presence of neomycin. Neomycin-sensitive colonies were amplified and genotyped by Southern blotting. Colonies containing the ICln² allele were subjected to a second round of purification by plating at low density and screening the resultant colonies for the proper genotype by Southern blotting.

Knockout Mice-- Chimeric mice were generated by injection of 8-20 ICln^KO (+/) ES cells into C57BL6/J blastocysts as described previously (22). Male chimeric mice were back-crossed with C57Bl6/J females to obtain germline transmission of the targeted ICln allele. Timed matings were established between mice heterozygous for the ICln null allele, and embryos were isolated 3, 7, and 9 days following detection of a vaginal plug. DNA from tail samples as well as embryos was prepared as described (24) and genotyped by PCR. DNA from isolated blastocysts was prepared by freezing in 50 µl of 0.1× phosphate-buffered saline, heating to 95 °C for 8 min, incubating at 55 °C for 30 min in the presence of 10 µg of proteinase K, and then reheating at 95 °C for 8 min to destroy the enzyme.

Reverse Transcriptase-PCR-- Total RNA was recovered from ES cells or NIH3T3 cells using TRIzol (Life Technologies, Inc.). ES cells were grown in the absence of MEF feeder cells on gelatin-coated tissue culture dishes. 1 µg of total RNA was transcribed with Superscript II reverse transcriptase (Life Technologies, Inc.) using oligo(dT) primers. Serial dilutions of the reverse transcription reaction were then used as templates for PCR amplification. Twenty cycles of amplification were performed using primers that hybridize in separate exons. Products were separated on a 1.5% agarose gel, transferred to a nylon membrane, and hybridized to a radiolabeled probe that anneals between the 5' and 3' primers.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

ICln Is Essential for Embryonic Viability-- To investigate the requirement for pICln function in the mouse, we disrupted the ICln gene in ES cells. Using a genomic DNA clone that contains a portion of the ICln gene (3), we constructed a targeting vector (ICln^KO-TV) that would eliminate the first coding exon (allele ICln^KO; Fig. 1a). ES cells were transfected with the targeting vector, and neomycin-resistant colonies were screened for homologous recombination. Of 192 neomycin resistant colonies tested, homologous recombination occurred in 16 (8%; Fig. 1b). When injected into blastocysts, two of these ES cell lines yielded highly chimeric mice that gave germline transmission of the ICln^KO allele. Although ICln (+/) ES cells show a 50% reduction in cellular pICln content as demonstrated by quantitative immunoblotting (data not shown), heterozygous animals showed no overt phenotype. No mice homozygous for the ICln null allele were born from heterozygous intercrosses from either mouse line.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Generation of ICln (+/) ES cells. a, organization of the wild-type (WT) ICln gene, the IClnKO targeting vector (IClnKO-TV), and the targeted locus (IClnKO). B, BamHI; H, HincII; Hd, HindIII; E, EcoRI; A, start codon; DTA, diphtheria toxin gene; NEO, neomycin resistance gene. Black boxes symbolize ICln coding exons, and arrows indicate primers used for PCR-based genotyping. b, genotyping of ES cell clones. Genomic DNA was digested with BamHI and analyzed by Southern blotting. Hybridization with an internal probe (probe A in panel a) revealed the expected 12- and 3.7-kb fragments from the wild-type and targeted alleles, respectively.

To determine the developmental stage at which homozygous embryos die, embryos from timed matings between ICln (+/) mice were genotyped using PCR (Table I). In embryos obtained 3.5 days post-coitus, homozygous null embryos were present in the expected Mendelian ratio. In contrast, in embryos obtained 7.5 days post-coitus, no homozygous null embryos were present (n = 47), demonstrating that ICln is necessary early in gestation for embryonic viability.

View this table: [in this window] [in a new window]   Table I Genotypes of embryos derived from timed matings between ICln (+/) mice

pICln Is Essential for Cellular Viability-- We next asked whether ICln is essential for cellular viability. We used two sequential gene targeting steps to attempt to disrupt both ICln alleles in ES cells (Fig. 2a). In the first step, we used a targeting vector in which the first coding exon and a neomycin resistance marker were flanked by loxP sites. Of 96 neomycin resistant colonies, 3 (3%) were properly targeted, as determined by Southern blotting (Fig. 2b). One properly targeted ES cell line was transiently transfected with a Cre recombinase expression plasmid. The resultant colonies were screened for the desired recombination event, which yielded neomycin-sensitive ES cell lines in which one ICln allele was disrupted (allele ICln²; Fig. 2b).

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Strategy for generating ICln (/) ES cells. a, organization of the wild-type (WT) ICln gene, the Flox-neo-ICln targeting vector, and the targeted locus after recombination mediated by Cre recombinase (ICln2). Xh, XhoI; RV, EcoRV. Solid triangles, loxP sites (see the legend for Fig. 1 for definitions of other abbreviations and symbols). b, genotypes of ES clones after targeting with the Flox-neo-ICln targeting vector and after transfection with Cre recombinase. Genomic DNA was digested with BamHI and analyzed by Southern blotting. Hybridization with a 3' external probe (probe D) shows the expected 12-, 7.5-, and 10-kb bands from the wild-type, targeted, and Cre-recombined alleles, respectively.

One such ES cell line was transfected with the targeting vector ICln^KO-TV, and neomycin-resistant colonies were selected. Of 480 neomycin resistant colonies screened by PCR, 22 (5%) were found to have undergone homologous recombination with the targeting vector. These were genotyped by Southern blotting (Fig. 3a). All homologous recombinants recovered contained the wild-type ICln allele, and none contained the ICln² allele, indicating that in every case homologous recombination occurred between the targeting vector and the ICln² allele. Assuming that the second targeting vector recombined with the ICln² and wild-type alleles with equal efficiency, the absence of homozygous null ES cells is highly significant (p < 10⁷). This result strongly suggests that the ICln gene is essential for ES cell viability.

View larger version (47K): [in this window] [in a new window]   Fig. 3.   ICln is essential for ES cell viability. a, genotypes of homologous recombinant ES cells obtained after treatment of ICln (wt/ICln2) ES cells with the IClnKO targeting vector. Genomic DNA was digested with BamHI and EcoRV, blotted onto nylon filters, and hybridized to probe C, which allows the IClnwt, ICln2, and IClnKO alleles to be distinguished. WT, wild type. b, semiquantitative reverse transcriptase-PCR analysis of ICln expression in ES cells. RNA was isolated from ES cells grown in the absence of MEF feeders or from NIH3T3 cells. 1 µg of total RNA was reverse transcribed, and 5-fold serial dilutions of reverse transcription products were used for subsequent PCR amplification.

ICln mRNA and protein are present in all of the adult tissues that we examined (1-3). We have also previously shown that ICln mRNA is present in late stage mouse embryos (3). However, the expression of pICln has not previously been demonstrated in undifferentiated cells. Consistent with our finding that a functional ICln allele is essential for ES cell viability, we found that ICln was expressed in wild-type embryonic stem cells (Fig. 3b).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

A relatively small number of genes has been shown to be essential for embryonic viability prior to embryonic day 7.5 (Table II). These genes can be divided into those that are crucial for embryonic development, but not for cellular viability, and those that are essential for fundamental cellular processes such as RNA metabolism, intracellular vesicle transport, cell cycling, and DNA repair. Despite the requirement of the latter class of genes for cellular viability, embryos deficient in these genes often survive until stage E3.5-E7.5. The survival of these embryos to this point has generally been attributed to maternal stores of RNA and/or protein or to the functional redundancy of genes early in embryonic development (15).

We have shown that embryos deficient in ICln degenerate between stages E3.5 and E7.5 and that ICln is essential for cellular viability. These findings demonstrate that pICln has an essential role in one or more of the basic cellular processes, consistent with the ubiquitous expression pattern of pICln and its high degree of conservation through evolution. By characterizing pICln interacting proteins, we have identified at least two candidate processes in which pICln may be active. pICln interacts with a homolog of yeast Skb1 (10), which binds to and up-regulates a yeast PAK-like kinase that has been implicated in the regulation of the cell cycle and cell morphology (11). pICln also interacts with core Sm proteins and plays a role in regulating spliceosomal biogenesis (13). Cell cycle control, RNA processing, and spliceosomal biogenesis are pathways that are critical for early embryonic development, as illustrated by the phenotypes of embryos that lack cyclin A2 (16), Os (17), RNA1 (18), Raly (19), or the spinal muscular atrophy disease gene, SMN (20). The early death of SMN knockout embryos is particularly interesting, because SMN and pICln both participate in the process of spliceosomal biogenesis. We have shown that pICln binding to Sm proteins inhibits Sm protein binding to spliceosomal RNA at least in part by blocking Sm interaction with SMN (13).

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

These authors contributed equally to this work.

^§ Present address: Dept. of Pharmacology, University of Minnesota, 6-120 Jackson Hall, 321 Church St., S.E., Minneapolis, MN 55455.

^¶ To whom correspondence should be addressed: Howard Hughes Medical Institute, Children's Hospital, Harvard Medical School, 1309 Enders, 320 Longwood Ave., Boston, MA 02115. Tel.: 617-355-6163; Fax: 617-355-3692; E-mail: clapham@rascal.med.harvard.edu.

	ABBREVIATIONS

The abbreviations used are: PAK, p21-activated kinase; Sm, spliceosomal; kb, kilobase pair(s); ES cell, embryonic stem cell; MEF, murine embryonic fibroblast; PCR, polymerase chain reaction; TV, targeting vector; SMN, survival of motor neuron protein.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES