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J. Biol. Chem., Vol. 276, Issue 30, 27745-27748, July 27, 2001

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ACCELERATED PUBLICATION
Islet-Brain1/JNK-interacting Protein-1 Is Required for Early Embryogenesis in Mice^*

Nancy A. Thompson^§¶, Jacques-Antoine Haefliger^§, Alfred Senn, Thomas Tawadros, Fulvio Magara, Birgit Ledermann^**, Pascal Nicod, and Gérard Waeber

From the  Department of Internal Medicine and Institute of Cellular Biology and Morphology and the  Reproductive Medicine Unit, Department of Obstetrics and Gynecology, CHUV-University Hospital, 1011 Lausanne and the ^** Labortierkunde, University of Zürich, 8057 Zürich, Switzerland

Received for publication, May 1, 2001, and in revised form, May 29, 2001

	ABSTRACT

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Islet-brain1/JNK-interacting protein-1 (IB1/JIP-1) is a scaffold protein that organizes the JNK, MKK7, and MLK1 to allow signaling specificity. Targeted disruption of the gene MAPK8IP1 encoding IB1/JIP-1 in mice led to embryonic death prior to blastocyst implantation. In culture, no IB1/JIP-1^/ embryos were identified indicating that accelerated cell death occurred during the first cell cycles. IB1/JIP-1 expression was detected in unfertilized oocytes, in spermatozoa, and in different stages of embryo development. Thus, despite the maternal and paternal transmission of the IB1/JIP-1 protein, early transcription of the MAPK8IP1 gene is required for the survival of the fertilized oocytes.

	INTRODUCTION

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Mitogen-activated protein kinases (MAPKs)¹ are key enzymes involved in diverse cellular processes in response to extracellular stimuli. In mammals, three major groups of MAPK have been identified, extracellular signal-regulated kinase, p38, and the c-Jun NH₂-terminal kinase (JNK) (1-3). The JNK pathway is activated by many forms of stress including cytokines, heat shock, or radiation. In response to these stimuli, specific MAPK kinase kinases are activated that modulate MAPK kinase such as MKK4 and MKK7, which sequentially phosphorylate the JNK kinase. Once activated, the JNK phosphorylates c-Jun, which in turn increases transcription activity of many target genes. The functions of the signal transduction mediated by the JNK group of MAPKs include the control of cell survival and apoptosis, the regulation of cell proliferation, and embryonic morphogenesis (1-3). JNK homologs have been identified in Drosophila, and this signaling pathway is required for embryonic epithelial cell sheet movement and planar polarity (4). In Xenopus laevis eggs and embryos, the JNK pathway is activated during oocyte maturation and stays constitutively activated until the early gastrula stage of embryogenesis suggesting a critical role of the kinase during oocyte maturation and embryogenesis (5).

The specificity of JNK activation and function requires the presence of the scaffold protein c-Jun NH₂-terminal kinase-interacting protein-1 (JIP-1) (6, 7). This protein was recently identified as the mammalian homolog of the yeast STE5 that functions as a scaffold protein that organizes the MAPK cascade into a specific module (8). JIP-1 binds to JNK, MKK7, and MLK1 and potentiates the JNK activation (1, 6, 7). JIP-1 contains an SH3 and PID domain in the COOH-terminal part of the protein, which interacts with p190 RhoGEF, the reelin receptor ApoER2, the low density lipoprotein receptor-related protein, megalin, and more recently, kinesin (9-12). JIP-1 was initially cloned from a mouse library using JNK as a bait in a two-hybrid system (6). The rat and human homolog of JIP-1 was independently identified and termed islet-brain1 (IB1), because it is expressed in the insulin-secreting -cells of the pancreas and in neurons (13, 14). The function of IB1/JIP-1 was partially investigated in insulin-secreting cells. In these cells, activation of the JNK cascade by interleukin-1 reduces IB1/JIP-1 content and promotes apoptosis (15). Reduction of the content of IB1/JIP-1 in these cells increased phosphorylation of c-Jun and the apoptotic rate. Conversely, overexpression of IB1/JIP-1 prevented JNK activation and apoptosis (15). The MAPK8IP1 gene encoding the human IB1/JIP-1 was mapped to chromosome 11p11.12 (14). MAPK8IP1 is a candidate gene for type 2 diabetes, because a missense mutation within the coding region of this gene was linked to human diabetes in a large pedigree (16). The mutation was shown to be associated in vitro with an accelerated apoptosis and a decreased insulin transcriptional activity (16).

Some of the physiological importance of the JNK signaling pathway has been investigated by gene targeting in mice. Mice deficient in JNK1, -2, or -3 have no obvious phenotype whereas double mutants (JNK1^/ and JNK2^/) die at mid-gestation with defective neural-tube closure because of an increased apoptosis (17, 18, 23). Mice lacking the JNK3 gene are resistant to kanaic acid-induced apoptosis in the hippocampal region of the brain (19). Thus, JNK functions are important during development and may play a permissive role for cell survival and/or for cell apoptosis. The selective disruption of MKK4 and c-Jun has a much more drastic phenotype than disruption of JNK1, -2, or -3. Both MKK4- and c-Jun-defective mice have an early embryological lethality caused by an abnormal liver development (20, 21).

Due to the pleiotrophic role of the JNK transduction signal in regulating cellular proliferation, apoptosis, and tissue morphogenesis, the physiological function of IB1/JIP-1 in vivo is unpredictable. The present study was therefore undertaken to clarify the biological role of IB1/JIP-1 using a gene-targeting strategy.

	MATERIALS AND METHODS

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Generation of IB1/JIP-1 Mutant Mice-- A rat IB1 cDNA probe was used to isolate a 13.5-kb genomic clone from a 129 SvJ mouse library. The murine gene organization was partially characterized, and a targeting vector was constructed by replacing a portion of exon 3 through exon 8 of the murine IB1/JIP-1 gene with a neomycin resistance cassette, which contained the PGK-1 promoter and the poly(A) site. A thymidine kinase cassette was also inserted in the targeting vector for negative selection. Homologous recombination was obtained in HM-1 embryonic stem cells by transfection of the XbaI-linearized vector. G418- and gancyclovir-resistant colonies were screened by Southern blot analysis following a XbaI and SalI digestion, using the indicated probe. The recombined ES cells were detected by the presence of a 7-kb digested DNA fragment, together with a 13-kb genomic fragment corresponding to the endogenous IB1/JIP-1 gene. Four clones were injected into C57BL/6 host blastocysts. Embryos were transferred into the uterus of 2.5-day post-coital pseudopregnant CD-1 females. Resulting chimeric mice were bred with C57BL/6 mice, and a single clone produced a germline transmission. Heterozygous males of the F1 offspring were repeatedly backcrossed to wild-type dams of the strains C57BL/6 and the 129SvJ for three generations, to investigate the effects of the mutation into two different genetic backgrounds. Intercrosses between N3 heterozygous were performed in the C57BL/6 line, as well as in the 129SvJ, and between the two strains, to test for hybrid vigour.

Mouse Oocytes and Zygotes-- 6 to 9-week-old IB1/JIP-1 heterozygous females were stimulated (day 1) with one peritoneal injection (10 IU/0.2ml) of follicle-stimulating hormone (Folligon; Intervet AG, Pfäffikon, Switzerland) followed on day 3 by a second injection (10 IU/0.2ml) of hCG (Pregnyl; Organon, Zürich, Switzerland) to induce ovulation (26). For the recovery of oocytes, females were killed 13 h after hCG administration by cervical dislocation. For the recovery of zygotes, females were mated after hCG injection with 8- to 16-week-old heterozygous males and killed 24 h after hCG injection.

Immunolabeling of Testis, Spermatozoa, and Zygotes-- For light microscopy studies, mouse gonads were rapidly excised and cut in fragments that were quickly frozen in 2-methylbutane precooled in liquid nitrogen. Testis fragments were frozen in Tissue-Tek medium (Miles Inc., Elkhart, IN) and cryo-sectioned at 5-µm thickness. Aliquots of freshly isolated spermatozoa were smeared on precleaned microscope slides, allowed to air dry for 2 h, fixed in methanol for 10 min, and left to air dry before being rinsed in phosphate-buffered saline. To improve the staining of zygotes, we drilled the zona pellucida by laser light (27).

Western Blot Analysis-- Whole brains were obtained from mice at different developmental stages. A total of 100 µg of the extracts were separated using a 10% SDS-polyacrylamide gel electrophoresis, and the proteins were transferred to nitrocellulose. Detection of IB1/JIP-1 was performed using the IB1/JIP-1 antiserum (13, 25) in 3/10,000 dilution.

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) Amplification-- Mouse zygotes (two-cell or blastocyst stages) were rapidly frozen in liquid nitrogen and homogenized in sterile water. The lysate containing the RNA was reverse-transcribed using oligo(dT) primers and 0.2 mM of each dNTP in 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl₂, and 0.01% gelatin. The reverse-transcribed products were used for PCR reactions in the presence of 20 ng of sense and antisense primers (Microsynth GmbH, Balgach, Switzerland). The antisense primer sequence for mouse IB1/JIP-1 located in exon 5 was 5'-CTC GAG CCG CAC ATC TGC C-3'; the sense primer, located in the exon 4, was 5'-AAG CAC AGT TGG CAG GAC CG-3'. These primers generated an amplicon of 275 bp. A second step of amplification was performed using a nested set of primers located within the first amplified fragment of 275 bp. The antisense primer located in exon 5 was 5-'TGA TAG TGG ATT CGA TCT C-3'; the sense primer, located in exon 4, was 5-'TGT GTC TCG ATC CTC C-3'. These primers generated a fragment of 236 bp. As negative controls, we submitted to PCR amplification samples of total RNA that had not been reverse-transcribed. Another control was obtained by PCR amplification of mice genomic DNA. The primers generated a fragment of 406 bp because of the presence of an intron of 170 bp located between exons 4 and 5.

	RESULTS

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The murine gene encoding IB1/JIP-1 was cloned from a 129/SvJ mouse genomic library. A targeting vector was designed to replace exon 3 through exon 8 of the MAPK8IP1 gene with the neomycin resistance gene (Fig. 1). Three embryonic stem cell clones that had undergone homologous recombination were microinjected into C57BL/6 host blastocysts. One clone produced a germline chimeric male resulting in offspring heterozygous for the disrupted allele (Fig. 1). The heterozygotes IB1/JIP-1^+/ mice were intercrossed in two different genetic background (C57BL/6 and 129 SvJ), and the progeny was genotyped. No mutant IB1/JIP-1^/ were found in live-born progenies of IB1/JIP-1^+/ intercrosses, indicating that the complete absence of IB1/JIP-1 caused embryonic lethality. Embryos were genotyped at various stages of gestation to determine when embryonic death occurred. As shown in Table I, a total of 148 pups were analyzed, and one single mutant IB1/JIP-1^/ embryo was detected at day 3.5 post-coitum. A total of 98 pups (66%) were heterozygous (IB1/JIP-1^+/) whereas the remaining 49 pups (33%) were wild-type animals (IB1/JIP-1^+/+), which is in accordance with the expected Mendelian distribution.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Gene targeting of the IB1/JIP-1 locus. Wild-type IB1/JIP-1 gene organization and the coding exons (E3 and E8; striped boxes) are schematically depicted in the upper line. A 1-kb cDNA probe (dark box) was designed 5' of the recombined sequence and used for Southern blot analysis. Middle line, targeting vector including the herpesvirus thymidine kinase gene (PGK-TK) and the neomycine gene (PGK-NEO). Lower line, disrupted IB1/JIP-1 allele with insertion of the neomycine resistance gene. Xb, XbaI; Xh, XhoI; N, NotI; B, BamHI; P, PstI; S, SalI. Inset, Southern blot analysis of genomic DNA obtained from two wild-type (IB1/JIP-1+/+) and two heterozygous (IB1/JIP-1+/)mice. The disrupted allele gives a 7-kb Xb-S-digested fragment whereas the wild-type allele is 13 kb.

To test whether IB1/JIP-1^/ embryos survive to the blastocyst stage, embryos were isolated from IB1/JIP-1^+/ intercrosses 24 h post-coitum and grown in culture up to 5 days. For genotyping the embryos, we used a PCR strategy to identify the wild-type and disrupted allele by amplifying DNA fragments located either in exons 4 and 5 of the murine MAPK8IP1 gene or in exon 3 and in the neomycin gene. As shown in Fig. 2, the genomic PCR amplification using the primers located in exons 4 and 5 identified a 406-bp fragment that includes the intronic sequence of 170 bp present between the two exons (Fig. 2, lane 1). Embryos were genotyped at two-cell, morula, and blastocyst stages. A total number of 41 embryos from 3 different litters were genotyped; 21 were heterozygotes (IB1/JIP-1^+/), and 20 were wild-type (IB1/JIP-1^+/+). The absence of null mutant embryos (IB1/JIP-1^/) in these 41 embryos was suggestive of a very early embryological death because of the absence of the MAPK8IP1 gene.

View larger version (38K): [in this window] [in a new window]   Fig. 2.   IB1/JIP-1 mRNA is detected during early embryogenesis. Reverse-transcribed RNAs, extracted from oocytes and zygotes at two different developmental stages (two-cell embryos and blastocysts), were PCR-amplified using primers specific for the MAPK8IP1 gene. This procedure resulted in the amplification of a single 236-bp product, seen after ethidium bromide staining. PCR amplification of mouse genomic DNA resulted in the amplification of a 406-bp product. Lane 1, PCR from mouse genomic DNA; lane 2, PCR from mouse genomic DNA and RT-PCR from wild-type oocytes RNA to show the size difference between genomic and spliced IB1/JIP-1 RNA.

Because lethality occurred early during embryogenesis, we then evaluated whether MAPK8IP1 gene expression could be detected in these early developmental stages. RNA from two-cell embryos and blastocysts were isolated, and RT-PCR was performed using the oligonucleotides located in exons 4 and 5. The RT-PCR strategy identified a 236-bp amplicon, which corresponds to the IB1/JIP-1 RNA lacking the intronic sequence located within the two exons (Fig. 2). The IB1/JIP-1 transcript was detected in the two-cell embryos indicating either that early transcription occurred during the process of embryonic genome activation and/or that IB1/JIP-1 transcripts were already present in the unfertilized oocyte (Fig. 2). IB1/JIP-1 mRNA was also found to be present in testis by Northern blot analysis (data not shown), and immunocytochemistry studies were then performed in unfertilized oocytes and mature spermatozoa and during various stages of embryonic development using affinity-purified antibodies raised against IB1/JIP-1 (Fig. 3, A-J). IB1/JIP-1 was immunodetected in zygote in pronucleate stage, in blastocysts, and in mature spermatozoa (Fig. 3, A-J). To explore the possibility that the disrupted allele could be associated with a loss of IB1/JIP-1 protein in a fraction of the spermatozoa, aliquots of freshly isolated spermatozoa were smeared on microscope slides. After immunostaining using the anti-IB1/JIP-1 antibodies, we counted the spermatozoa for positive and negative staining. Over 600 separate mature spermatozoa isolated form three heterozygous animals (IB1/JIP1^+/) were evaluated, and all expressed IB1/JIP-1. This result indicates that the presence of IB1/JIP-1 in all mature spermatozoa of heterozygous mice (IB1/JIP1^+/) is the consequence of a translated RNA transcribed during a dizygotic stage of spermatogenesis. Similarly, we detected the presence of IB1/JIP-1 in unfertilized oocytes, even when studying heterozygous animals (IB1/JIP1^+/) (data not shown). These results indicated that IB1/JIP-1 may play a role during fertilization, spermatogenesis, and oogenesis, possibly linked to the activated JNK pathway, which was described in X. laevis to be selectively activated during oocyte maturation (5).

View larger version (40K): [in this window] [in a new window]   Fig. 3.   A-J, distribution of IB1/JIP-1 in the testis, spermatozoa, and zygotes. IB1/JIP-1 is immunodetected in mature spermatozoa with a cellular expression restricted to the tail. A, low magnification; B, phase contrast; C, higher magnification; D, phase contrast; E, positive immunostaining for IB1/JIP-1 is restricted to the tail but not the head and intermediary piece of the mature spermatozoa; F, phase contrast. Immunodectection of IB1/JIP-1 in zygote at pronucleate stage (G and H, phase-contrast) and at expanded blastocyst stage (I and J, phase contrast) are shown. Bar = 55 µm in A, 5.5 µm in C and E, and 2.5 µm in G and I. K, IB1/JIP-1 content in brain during development. Western blot analysis of IB1/JIP-1 of brain extracts obtained at day 0 (P0), day 5 post-natal (P5), day 15 (P15), and in adult stage from wild-type and heterozygous IB1/JIP-1+/ mice. Tubulin (TUB) was subsequently immunodetected on the same blot for normalization. IB1/JIP-1 content in brain peaked within the first 15 days (25), and its normalized content was reduced by 40 to 50% in the heterozygous IB1/JIP-1+/ mice (+/+, wild-type animals; +/, heterozygous IB1/JIP-1+/).

Given the potential role of IB1/JIP-1 during spermatogenesis or oogenesis, we evaluated whether the IB1/JIP-1 haploinsufficiency was associated with an alteration of fertility. In a separate set of experiments, we therefore examined the genotypic ratios of the progeny obtained from male heterozygous mice (IB1/JIP-1^+/) with female heterozygous mice (IB1/JIP-1^+/), from male heterozygous mice (IB1/JIP-1^+/) with wild-type female (IB1/JIP-1^+/+), and conversely the progeny of female heterozygous mice (IB1/JIP-1^+/) with wild-type male (IB1/JIP-1^+/+). As shown in Table II, the genotypic ratios of these intercrosses were in accordance with the Mendelian distribution, thus excluding an impaired fertility linked to the haploinsufficiency of the IB1/JIP-1 protein.

View this table: [in this window] [in a new window]   Table II Genotypic ratios of the progeny obtained by intercrossing male or female IB1/JIP-1+/ heterozygotes

We next examined by Western blotting the IB1/JIP-1 content in wild-type and IB1/JIP-1^+/+-deficient mice. IB1/JIP-1 content was decreased by approximately 45% in the brain of heterozygous IB1/JIP-1^+/ mice. Fig. 3K depicts the IB1/JIP-1 content in brain during various development stages. Once normalized with the tubule content, the IB1/JIP-1 reduction found in the heterozygous IB1/JIP-1^+/ mice was more apparent prior to day 15 post-natal. Glucose homeostasis was also evaluated monthly on regular chow in wild-type and heterozygous mice during 6 months in two different genetic backgrounds (C57BL/6 and 129 SvJ). Fasting plasma levels of glucose and insulin were similar between wild-type and heterozygous IB1/JIP-1^+/ mice (data not shown). Lastly, pancreas histology was performed, and no obvious difference was observed in wild-type and heterozygous IB1/JIP-1^+/ animals.

	DISCUSSION

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We have found that disruption of the murine gene MAPK8IP1 encoding IB1/JIP-1 leads to an early embryological death, prior to the blastocyst implantation. In culture, no complete null mutant IB1/JIP-1^/ was identified as early as two-cell stage, suggesting the requirement of IB1/JIP-1 in the first cell cycles. In accordance with the potential importance of the scaffold protein IB1/JIP-1 in early embryogenesis, we detected mRNA encoding the protein from two-cell and blastocyst stages. IB1/JIP-1 RNA and protein were also detected in unfertilized oocytes and in spermatozoa. Despite the expression of IB1/JIP-1 in unfertilized oocytes and testis, we did not find an alteration in fertility in heterozygote IB1/JIP^+/ mice. The early death of fertilized null mutant IB1/JIP^/ oocytes indicates therefore that de novo transcription of the MAPK8IP1 gene is required for the survival of the fertilized oocyte.

IB1/JIP-1 functions as a scaffold protein that organizes the JNK signal transduction pathway (1, 2, 7). As mentioned, the JNK pathway plays several roles during embryogenesis (1-3). In X. laevis eggs, the JNK pathway is activated during oogenesis and stays activated in early embryogenesis (5). The selective disruption of genes involved in the control of the JNK activity leads, for several of them, to embryological death (17, 18, 20-22). However, the embryological death observed in IB1/JIP-1^+/ embryos is the earliest one observed in the JNK cascade indicating the critical role of early transcription of MAPK8IP1 during the first cell cycles. These early cell stages are characterized by the conversion of the two parental genomes into a single embryonic genome with the initiation of the first divisions (24). Early embryonic cells proliferate rapidly with doubling time as short as 2 h at day 6.5 post-coitum (24). The oocyte cytoplasm plays a critical role by controlling and reprogramming nuclear function. The maternal cytoplasmic functions include the repression of genes in the first 10 to 20 h post-fertilization followed by reactivation of appropriate specific genes. Because IB1/JIP-1 is detected in wild-type and heterozygote IB1/JIP-1^+/ oocytes, it is unlikely that cytoplasmic IB1/JIP-1 contributes to the reprogramming of the nuclear function. On the other hand, it has been shown that too early transcription or an inappropriate temporal transcription could induce embryonic lethality (24). A non-coordinated expression of specific genes during this process of embryonic genome activation may result in loss of normal embryogenesis. The earliest steps of embryonic genome activation involved decondensation of nuclear sperm and formation of the maternal and paternal pronuclei, followed by an alteration of chromatin structure to initiate early gene transcription. The disruption of any of these genes events may lead to inappropriate cell division and differentiation. Our data established that the MAPK8IP1 gene encoding IB1/JIP-1 is one of these critical genes for which early transcription is required to allow early embryo cleavage and survival. IB1/JIP-1 transcription in these stages may be required to allow proper JNK signaling to occur and/or to prevent an inappropriate level of JNK activation. In ex vivo experiments, it was shown that low cellular content of IB1/JIP-1 is associated with an increased apoptosis rate whereas experimentally increasing the cellular IB1/JIP-1 content could confer protection to stress-induced apoptosis (15).

In conclusion, our data indicate that despite the maternal and paternal transmission of IB1/JIP-1 in heterozygote IB1/JIP-1^+/ mice, the level of transmitted protein is unable to allow proper cell survival. The MAPK8IP1 gene encoding IB1/JIP-1 needs therefore to be transcriptionally active during the first cell cycles.

	ACKNOWLEDGEMENTS

We are grateful to D. W. Melton for providing the HM-1 embryonic stem cells and P. Wellauer for the 129/SvJ genomic library. We are also grateful for criticisms from and valuable discussions with A. Abderrahmani, F. Conquet, V. Mooser, C. Widmann, and P. Wellauer, and we thank A. Formenton and C. Mathieu for expert technical assistance.

	FOOTNOTES

^* This work was supported in part by Swiss National Science Foundation Grants 32-48916.96 and 32-54119.98 (to G. W.) and 31-56689.99 (to J. A. H.) and by the Placide Nicod and Octav and Marcella Botnar Foundation.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.

^§ Contributed equally to the work.

^¶ Present addess: ISREC 1010 Epalinges, Switzerland.

To whom correspondence should be addressed: Dept. of Internal Medicine B, CHUV-University Hospital, 1011 Lausanne, Switzerland. Tel.: 41-21-314-09-60; Fax: 41-21-314-09-68; E-mail: gwaeber@chuv.hospvd.ch.

Published, JBC Papers in Press, June 4, 2001, DOI 10.1074/jbc.C100222200

	ABBREVIATIONS

The abbreviations used are: MAPK, mitogen-activated protein kinase; JNK, c-Jun NH₂-terminal kinase; JIP-1, JNK-interacting protein-1; IB1, islet-brain1; kb, kilobase pair; hCG, human chorionic gonadotropin; RT, reverse transcriptase; PCR, polymerase chain reaction; bp, base pair; MKK, MAPK kinase kinase; MLK, mixed-lineage protein kinase.

	REFERENCES

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