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Keywords:

  • SHB;
  • knockout;
  • ovulation;
  • malformations;
  • viability in utero;
  • transmission ratio distortion

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

SHB is an Src homology 2 domain-containing adapter protein that has been found to be involved in numerous cellular responses. We have generated an Shb knockout mouse. No Shb−/− pups or embryos were obtained on the C57Bl6 background, indicating an early defect as a consequence of Shb- gene inactivation on this genetic background. Breeding heterozygotes for Shb gene inactivation (Shb+/−) on a mixed genetic background (FVB/C57Bl6/129Sv) reveals a distorted transmission ratio of the null allele with reduced numbers of Shb+/+ and Shb−/− animals, but increased number of Shb+/− animals. The Shb allele is associated with various forms of malformations, explaining the relative reduction in the number of Shb−/− offspring. Shb−/− animals that were born were viable, fertile, and showed no obvious defects. However, Shb+/− female mice ovulated preferentially Shb oocytes explaining the reduced frequency of Shb+/+ mice. Our study suggests a role of SHB during reproduction and development. Developmental Dynamics 236:2485–2492, 2007. © 2007 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

The Src homology 2 (SH2) domain-containing adapter protein B (SHB) was originally identified as a serum-induced gene expressed in beta cells (Welsh et al.,1994). Subsequent studies have revealed that SHB is ubiquitously expressed (Welsh et al.,1998) and that it contains at least four different domains responsible for protein–protein interactions. These domains are proline-rich motifs in its N-terminus (Karlsson et al.,1995), the phospho-tyrosine binding (PTB) domain (Welsh et al.,1998; Lindholm et al.,2002), potential tyrosine phosphorylation sites (Lu et al.,2000; Lindholm et al.,2002), and the C-terminal SH2 domain (Karlsson et al.,1995). SHB is a prototype for a family of adapter proteins consisting of at least five members, all containing homologous tyrosine phosphorylation sites and a C-terminal SH2 domain, but with SHB as the sole member containing the proline-rich motifs and the PTB domain. This family comprises SHB, SHD, SHE, SHF, and SHG (Welsh et al.,1994; Oda et al.,1997; Lindholm et al.,2000; Cujec et al.,2002).

The SHB SH2 domain interacts with several tyrosine kinase receptors such as the vascular endothelial growth factor-2 (VEGFR-2; Holmqvist et al.,2004), the fibroblast growth factor receptor-1 (FGFR-1; Karlsson et al.,1995; Cross et al.,2002), the T-cell receptor (Lindholm et al.,1999), the interleukin-2 receptor (Lindholm,2002), and the platelet-derived growth factor receptor (PDGFR; Karlsson et al.,1995). This interaction initiates further signaling through the assembly of additional proteins to the other domains in SHB. Previous studies have revealed the participation of SHB in several cellular processes. These processes include regulation of apoptosis in various cell types (Karlsson and Welsh,1996; Welsh et al.,1999; Dixelius et al.,2000), participation in blood vessel formation after FGF stimulation (Lu et al.,2002), and T-cell signaling after CD3 or interleukin-2 stimulation (Lindholm et al.,1999; Lindholm,2002). SHB has recently been shown to play a role for embryonic stem cell differentiation into endoderm (Kriz et al.,2003) and for blood vessel formation in differentiating embryoid bodies (Rolny et al.,2005; Kriz et al.,2006).

SHB or its homologues have been described as early in phylogeny as in primitive chordates (http://www.ensembl.org/Homo_sapiens/geneview?gene=ENSG00000107338). The murine Shb gene is localized on chromosome 4, and it is composed of at least of six exons. The recent data confirm a high degree of similarity at the protein level between human and mouse Shb (GenBank, and data not shown).

In the present work, we have generated and analyzed knockout mice in which the first exon of the Shb gene was deleted (Shb−/−). From these observations, we found out that Shb−/− mice are viable and fertile but show reproductive disorders. In addition, the Shb null allele is inherited by a transmission ratio distortion.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Shb Knockout Mice

The generation of embryonic stem cells, containing one Shb-loxP allele (Fig. 1A), has been described elsewhere (Kriz et al.,2006). Blastocyst injections of these embryonic stem cells yielded two chimeric males that showed germline transmission of the Shb-loxP allele. To delete the first Shb exon, we mated one female expressing Cre recombinase under the control of the β-actin promoter with one ShbloxP/loxP male from the second generation of offspring. Eight animals were born. Six of them (three males and three females) had the first exon (and neomycin gene) deleted in one allele (Shb+/−) that then was passed on to progeny (Fig. 1B). The Shb knockout mice do not express the 60-kDa SHB protein in liver or kidney (Fig. 1C).

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Figure 1. Analysis of targeted mutation in mice. A: Schematic picture of Shb wild-type (Shb+) and Shb knockout allele (Shb) with indicated EcoRI (E) and BamHI (B) restriction sites. Introns are indicated as lines, exons as closed boxes, arrows indicate pair of primers, gray box shows Southern blot probe, arrowhead in Shb allele indicates remaining loxP site after Cre-mediated deletion of the first exon and neomycin cassette. B: Analysis of animals by polymerase chain reaction (PCR; left panel) revealed 1,500-bp PCR product for Shb+ and 340-bp PCR product for Shb animals. Analysis of animals by Southern blot after cleavage with BamHI revealed 2,000 bp band for Shb+ and 700 bp for Shb allele. C: Western blot analysis showing SHB protein expression in the kidney and liver. Actin is used as a loading control. The star indicates a 55-kDa background band present in mouse liver but not related to SHB.

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Abnormal Transmission Ratio of Shb Allele

Shb+/− animals were mated and mice of all three categories (Shb+/+, Shb+/−, Shb−/−) were born, but the number of Shb+/+ and Shb−/− animals was lower than expected from Mendelian genetics (Fig. 2). The ratio of Shb+/+: Shb+/−: Shb−/− was 16.4%: 67.2%: 16.4% (128 animals, 12 litters), which was significantly different from the 25:50:25 ratio anticipated (P < 0.05 by χ2 test). Whereas the loss of Shb−/− mice could reflect embryonal mortality, the loss of Shb+/+ is not readily explained.

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Figure 2. Relative distribution of genotypes after mating Shb+/− parents on the mixed or C57Bl6 genetic backgrounds. Offspring were genotyped (n = 128 on the mixed background and n = 84 on the C57Bl6 background), and the percentage Shb+/+, Shb+/−, and Shb−/− animals shown. The ratios deviate significantly from the 25%: 50%: 25% distribution estimated by Mendelian inheritance by χ2 statistics.

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Breeding the Shb-null allele for five generations onto the C57Bl6 genetic background and mating Shb+/− heterozygote parents reveals no offspring carrying the Shb−/− genotype (84 embryos or pups, 10 litters). The skewed ratio Shb+/+: Shb+/− was still apparent (ratio 1: 4.3 instead of the expected 1: 2, p<0.05; see Fig. 2) with 16 Shb+/+ and 68 Shb+/− offspring, indicating nonmendelian inheritance of the Shb-null allele also on this genetic background.

Shb−/− and Shb+/− Embryos Exhibit an Increased Malformation Rate

We wanted to test the hypothesis that Shb−/− embryos show an increased malformation rate, which would explain the decreased number of Shb−/− animals per litter. Mating Shb+/+ mothers with Shb−/− fathers (both parents on the mixed genetic background) producing Shb+/− offspring did not increase the malformation rate compared with Shb+/+ control (Table 1; Fig. 3). However, when both parents were Shb−/− there was an increased malformation rate (Table 1). Such malformations included both severe malformations, such as resorptions or loss of embryo structure (Fig. 4D), and milder malformations, such as tail rotation defects, neural tube closure defects, or superficial hemorrhages (Fig. 4A–C). Based on their morphological appearance, the resorptions were classified as corresponding to day E6–E8 embryos. The diversity of the defects observed suggests that SHB operates at different stages of development in normal embryogenesis.

Table 1. Effects of the Shb Knockout on Embryonic Day 10.5 Malformationsa
Parental genotypesResorptions, severe malformationsModerate malformationsTotal malformationsNormal embryosTotal
  • a

    Genotyping of +/− or −/− was in certain cases not possible (GNP, +/− or −/−), due to lack of DNA or contamination with host mother DNA. The total malformation rate among the +/− × −/− or −/− × −/− embryos was significantly different from that of the +/+ × +/+ embryos.

  • *, **

    , ** P < 0.05 and P < 0.01 by χ2 test when compared to wild-type control.

Shb+/+ ♀ × Shb+/+ ♂1 +/+2 +/+3 +/+28 +/+31 +/+
Shb+/+ ♀ × Shb−/− ♂3 +/−2 +/−5 +/−32 +/−37 +/−
Shb−/− ♀ × Shb−/− ♂5 −/−6 −/−11 −/−*25 −/−36 −/−
Shb+/− ♀ × Shb−/− ♂3 +/−3 +/−6 +/−13 +/−19 +/−
2 −/−5 −/−7 −/−21 −/−28 −/−
 12 GNP1 GNP13 GNP1 GNP14 GNP
 17 total9 total26**35 total61 total
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Figure 3. Embryonic malformation rate in relation to parental Shb genotypes. Relative distribution of malformations per litter after mating different Shb genotypes. Percentage of malformed embryos was calculated from Table 1. Diagrams show the mean values for each group. Classified as severe malformations were all resorptions and abnormalities where the embryo's structure was unrecognizable. Classified as moderate malformation were growth retardation, tail rotation defects, neural tube defects and visible superficial hemorrhages.

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thumbnail image

Figure 4. Malformations in embryos from Shb+/− mothers. A: Tail rotation defect. Tail twist is indicated by star. B: Neural tube defect. Open neural tube is indicated by arrow. C: Superficial hemorrhages indicated by arrowhead. D: Severely malformed embryo. Heart (H) is indicated. E: Normal embryo.

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We also were interested in testing malformation rates when mating Shb+/− females with Shb−/− males. Again, an increased malformation rate was observed (Table 1; Figs. 3, 4) comprising a similar variety of malformations as the offspring from the Shb−/− matings. Genotyping the embryos with moderate malformations revealed no apparent relative increase of embryos with the −/− background relative to the +/− background. However, there were numerous early resorptions (E6–E8) that could not be genotyped due to DNA degradation and contamination with maternal DNA, and presumably this population contains a significant number of resorbed Shb−/− embryos.

When testing malformations on the C57Bl6 background by crossing Shb+/− heterozygote parents, the number of resorptions and malformations was very similar to that observed on the mixed background (Table 2). All embryos with moderate malformations were Shb+/−. Three resorptions (day E6–E8) were noted that could not be genotyped. No Shb−/− embryos were detected. This finding suggests that, on the C57Bl6 background, Shb−/− causes an early defect operating either before or shortly after implantation, preventing formation of recordable Shb−/− embryos.

Table 2. Malformations and Embryo Genotyping at Embryonic Day 10.5 After Crossing of SHB+/− Parents on the C57Bl6 or Mixed FVB/C57Bl6/129Sv Backgroundsa
Shb+/− ♀ × Shb+/− ♂ResorptionsModerate malformationsNormal embryosTotal
  • a

    GNP, genotyping not possible, due to advanced resorption.

C57Bl60 +/+0 +/+6 +/+6 +/+
 0 +/−4 +/−24 +/−28 +/−
 0 −/−0 −/−0 −/−0 −/−
 3 GNP0 GNP0 GNP3 GNP
Total3 total (8%)4 total (11%)30 total (81%)37 total
FVB/C57Bl6/129Sv0 +/+1 +/+4 +/+5 +/+
 1 +/−5 +/−17 +/−23 +/−
 1 −/−0 −/−8 −/−9 −/−
 2 GNP0 GNP0 GNP2 GNP
Total4 total (10%)6 total (15%)29 total (74%)39 total

Transfer of Shb Wild-Type or Knockout Blastocysts to Shb+/+ Mothers

The occurrence of resorptions or malformations could be an inherent feature of the early Shb knockout embryos or could result from the maternal environment of Shb+/− mothers. To address this question, blastocysts were transferred from Shb-mutant pregnant females to wild-type pseudopregnant females on day 3.5 days post coitum (dpc), and embryos were analyzed at 8 days after transfer. As seen in Table 3, the phenotypical difference between the knockout and wild-type alleles remained after transfer to wild-type females. The rate of visible implantations was lower and the percentage of malformed embryos higher among the knockout embryos than among the wild-type embryos. The relative number of normal embryos was higher for the wild-type than the knockout embryos, 63% and 23%, respectively (P < 0.01 by χ2 test). This finding suggests that the reduced rate of visible implantations and/or resorptions and malformations are mainly due to an inherent dysfunction of the early Shb+/− or Shb−/− blastocysts or embryos and not related to the function of the uterus of Shb+/− mothers subsequent to implantation.

Table 3. Characterization of Embryos That Have Undergone Blastocyst Transfer From +/+ or +/− Females to Pseudopregnant +/+ Recipientsa
Characteristics of embryosShb +/+♀ × +/+♂Shb +/−♀ × −/−♂
  • a

    Embryos were analyzed 3.5 + 8 days postcoitum.

  • **

    P < 0.01 compared with the corresponding normal embryo value of +/+ control by χ2 test.

Normal embryos126**
Total number of defective embryos or blastocysts not implanted720
Malformations among defective embryos04
Resorptions among defective embryos73
Blastocysts not implanted013
Total number of blastocysts transferred1926

Shb Knockout Allele Presents an Advantage for Early Blastocyst Formation

To determine whether there is an advantage for Shb gamete production explaining the relative reduction in Shb+/+ mice, the frequencies of newborn mice possessing Shb+ or Shb paternal alleles after mating female Shb+/+ mice with male Shb+/− mice were determined. In five litters, 31 mice were born. Of these, 17 mice were Shb+/+ (55%) and 14 Shb+/− (45%), suggesting that the distorted transmission does not result from male inheritance.

We then tested the hypothesis that altered female gamete production could explain the distorted ratio of Shb+/+ and Shb+/− mice by genotyping ovulated oocytes from Shb+/− females (Table 4). Such unfertilized eggs have completed meiosis I with subsequent degradation or extrusion of the first polar body DNA and can therefore be of the genotypes Shb +/+, −/−, or if crossing over has occurred on one of the sister chromatids, Shb +/−. The results in Table 4 show a strong and statistically significant increase in the proportion of ovulated Shb oocytes. The preferential increase of the maternally inherited Shb allele was also noted when blastocysts were retrieved from the uterus 3.5 dpc and genotyped. The results in Table 4 show a clear dominance of blastocysts carrying the maternal Shb allele. Also when examining embryos at embryonic day (E) 10.5, we observed that embryos possessing the maternal Shb allele were more common than those possessing the Shb+ maternal allele. The distortion was less pronounced than at the oocyte and blastocyst stage, suggesting a preferential loss of Shb−/− embryos between the blastocyst stage and day E10.5. The ratio of Shb+: Shb was 31%: 54% at E10.5 compared with 19%: 81% at the ovulated oocyte stage (Table 4). The data suggest that the preferential increase in Shb+/− offspring results from a relative increase in ovulation of oocytes carrying the mutated allele.

Table 4. Distribution of Shb − or + Maternal Alleles in Unfertilized Oocytes, Blastocysts, or Embryos When Mating Shb+/− Female Mice With Shb+/+ (blastocysts) or Shb−/− (Embryos) Male Micea
Presence of maternal allele
Mother no.No. of oocytesShb+ShbShb+/−DNR
  • a

    Oocytes were obtained after mating with vasectomized males. DNR, DNA not retrieved in sufficient amounts to allow genotyping; GNP, genotyping not possible due to advanced resorption.

  • *

    P < 0.05 by χ2 test when compared with 1:1 distribution.

1100901
2113602
3122910
4102521
5121812
Total558*3746
%1001567711
Litter no.Blastocysts/litterShb+ShbDNRGNP
11521030
291440
320200
491710
51541100
total508*3480
%1001668160
Litter no.Embryos/litterShb+ShbDNRGNP
162403
293601
373402
494501
582600
Total39142507
%1003664018

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

SHB knockout mice were generated to assess the role of SHB for mouse development and physiology. The mice were viable and fertile and did not show any major abnormality in comparison to their wild-type and heterozygous littermates. However, careful analysis revealed differences from normality with respect to reproduction.

SHB has previously been shown to operate downstream of several tyrosine kinase receptors, such as the VEGFR-2, FGFR-1, and the PDGFR receptors (Karlsson et al.,1995; Holmqvist et al.,2004), but the phenotypes associated with these receptor knockouts provide no clues as to the mechanisms behind the Shb knockout phenotype presently described. Thus, the signaling pathways responsible for the Shb knockout phenotype remain unresolved other than the fact that they involve SHB.

The most obvious difference was recorded in the number of Shb−/− and Shb+/+ offspring in the first filial (F1) generation from Shb+/− parents. There was a significant deviation from the expected number of 25% each for the Shb−/− and Shb+/+ genotypes, particularly on the C57Bl6 background. One possible explanation can be found in intrauterine embryonic death. Accordingly, the Shb−/− or Shb+/− embryos showed increased rates of resorptions and malformations compared with wild-type embryos. On the C57Bl6 background, no Shb−/− embryos were noted, indicating an early defect. For technical reasons, early resorptions could not be genotyped, but these are likely to be of Shb−/− origin to a significant extent. The blastocyst transfer experiments indicate that the maternal environment after implantation is not responsible for increased resorptions or malformations, but that the defect is inherent to the embryo. However, the malformations/resorptions noted were in many cases rather nonspecific, affecting development at different stages. Similar embryonic effects have been observed in many other conditions of teratogenesis, such as diabetes pregnancy, alcohol intoxication, and exposure to reactive oxygen species (Li et al.,2005; Sulik,2005; Wentzel et al.,2005,2006; Wentzel and Eriksson,2006). Superficial hemorrhages or petechiae are signs of a malformation relatively specific for the vasculature, since it has been described in Syk and Jag1 knockouts (Xue et al.,1999; Yanagi et al.,2001). Loss of SHB expression in embryoid bodies causes impaired formation of a vasculature (Kriz et al.,2006), thus this malformation could be a sign of vascular defects related to the Shb knockout. However, the hemorrhages were too infrequent (six embryos found in total) to allow further investigation or drawing any firm conclusions.

An abnormal transmission ratio of the Shb knockout allele was observed upon ovulation, blastocyst formation, and among embryos. The knockout allele was overrepresented at these stages and thus Shb deficiency causes a certain advantage for female gamete production. The best characterized example of transmission ratio distortion is the t-complex in mice (recently reviewed by Lyon,2005). This phenomenon has been known for more than seven decades but has only recently received a molecular explanation. The t-complex affects only male mice and produces an increased transmission of the t-haplotype in heterozygotes, whereas homozygotes are sterile. The increased transmission of the t-haplotype in heterozygotes is due to increased sperm motility. The t-complex has recently been found to result from a t responder (Smok1Tcr) and at least one distorter (Tagap1) that operate in concert to yield the transmission distortion (Bauer et al.,2005). We currently have no explanation to the transmission distortion in inheritance of the Shb null allele except that it involves female gamete production. Future analysis of oogenesis in Shb-null mice will reveal if SHB plays a role in this process.

Another striking effect was noted in the relative number of Shb−/− pups or embryos. These were absent on the C57Bl6 background and on the mixed background; Shb−/− embryos were also not represented in relation to the presence of the null allele among blastocysts or oocytes. This finding could reflect an early partial loss of similar nature as that observed on the C57Bl6 background. Although three resorptions were observed on the C57Bl6 background that could not be genotyped and thus could represent Shb−/− embryos, these were too few to explain the distortion of the allele inheritance. Thus, it seems that loss occurs before the stage of resorption, that is, E6–E8, and could involve reduced implantation or death shortly after implantation. This defect could also be operating on the mixed background but with incomplete penetrance. The Fgfr-2 knockout shows postimplantation mortality (Arman et al.,1998). Shb is known to operate downstream of FGFR-1 but no information is currently available as to whether Shb plays a role in FGFR-2 signaling.

We can conclude from our knockout study, that possessing an inactive maternal Shb allele is related to high female gamete production. On the other hand, later in fetal development, the Shb allele is associated with a reduced survival around implantation and a higher malformation rate than the wild-type allele. The overall conclusion is that SHB serves a role in reproduction.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Gene Targeting

The murine embryonic stem cell line GSI-1 derived from 129/SvJ mice was transfected with targeting vector containing 3 loxP sites (Kriz et al.,2006). The first and the second loxP sites flank the neomycin gene, and the second and third loxP sites border the coding sequences of the first exon, where the translation initiator codon is situated. The clone in which homologous recombination had taken place and all three loxP sites were present was expanded. Embryonic stem cells were then injected into blastocysts originating from C57BL/6 mice. To generate chimeric animals, these were implanted into pseudopregnant CD1 females. Chimeras were mated with C57BL/6 females, and heterozygous (Shb+/loxP) and homozygous (ShbloxP/loxP) mice for the loxP allele were obtained. The first exon and neomycin gene were deleted in all tissue by mating ShbloxP/loxP mouse with a transgenic mouse (strain FVB/N) carrying the Cre recombinase under the control of the β-actin promoter (Lewandoski et al.,1997). Heterozygotes (Shb+/−) and homozygotes (Shb−/−) for deletion of the Shb first exon were obtained and verified by polymerase chain reaction (PCR) or Southern blot analysis. Using PCR, the knockout (Shb) and wild-type (Shb+) alleles were identified with the primer pair: 5′-CCGGCCGCCTCCCTCATTCGTTCGC-3′ and: 5′-TAGACCTCAATTCCCATCTG-3′, which generated fragments of 340 bp and 1,500 bp, respectively. By Southern blot analysis, Shb and Shb+ alleles were identified as 700 bp and 2,000 bp bands, respectively (Fig. 1). The level of liver or kidney SHB protein was determined by Western blot analysis and found to be absent in the knockout (Fig. 1).

Animals

All animal experimentation had been approved by the Uppsala animal ethics committee. The weight of the mice was measured from the second week after birth when the mice were ear marked. Mice were weaned at 3 weeks after birth. The Shb knockout allele was maintained on a mix of three different wild-type strains (mix between FVB/N, C57BL/6, and 129/SvJ) or bred onto the C57Bl/6 background for five generations for experimentation.

Analysis of Blastocysts and Oocytes

Shb+/− females were mated with C57BL/6 Shb+/+ males. Females were killed 3 days after appearance of vaginal plugs (3.5 dpc). The uterus was removed and put into a dish with M2 medium. All blastocysts were washed out from the uterus by M2 medium into the Petri dish. Blastocysts were transferred into a new Petri dish with phosphate buffered saline (PBS) and then transferred in a small volume (less than 0.5 μl) into PCR tubes using a micropipette, one blastocyst per tube. Blastocysts were scored for size before PCR. PBS from the dish was used as a negative control for PCR. PCR was run for the knockout allele in a volume of 20 μl under these condition: 98°C 2 min; (98°C 20 sec, 60°C 1 min, 72°C 1 min) 45–50 cycles, 72°C 10 min. The PCR product was analyzed on a 1.5% agarose gel. The same procedure was used for analysis of oocytes except that oocytes were collected from the oviduct the same morning as when the vaginal plug was identified. In this situation, the females were mated with infertile (after vasectomy) male mice. The oocytes were cultured for 24 hr in M16 medium before genotyping. If the first PCR reaction gave ambiguous results, PCR was repeated by analyzing for the knockout or wild-type alleles.

Blastocyst Transfer

Female wild-type mice were made pseudopregnant by mating with sterile (after vasectomy) male wild-type mice. Shb+/+ or Shb+/− female mice were mated with Shb+/+ or Shb−/− male mice, respectively. On 3.5 dpc, the female mice were killed, and the blastocysts were collected and transferred to the pseudopregnant female recipients. These were maintained for 8 days further after which they were killed and embryo resorptions or malformations scored.

Analysis of Embryos

Females were killed at 10.5 dpc. If possible, the position in the uterus, the crown-rump length, the somite number and the presence of heart beat was indicated for each embryo. The number of corpora lutea and the number of resorptions were indicated for each litter. One limb, yolk sac, or all embryonic tissue (in case of resorptions) was used for genotyping by PCR or by Southern blot.

Statistical Analysis

Statistical analysis was performed using PractiStat software. Distribution of maternal and paternal allele and the malformation rate of the embryos were evaluated by the χ2 test.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

We thank the Uppsala University Transgenic Facility (UUTF) and Anne-Mari Olofsson for blastocyst microinjections and blastocyst isolation. We also thank Dr. Rolf Ohlsson for providing the Cre transgenic mouse. V.K. was supported by a grant from Henning and Gösta Ankarstrand and by a grant from Erland Wessler.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  • Arman E, Haffner-Krausz R, Chen Y, Heath JK, Lonai P. 1998. Targeted disruption of fibroblast growth factor (FGF) receptor 2 suggests a role for FGF signaling in pregastrulation mammalian development. Proc Natl Acad Sci U S A 95: 50825087.
  • Bauer H, Willert J, Koschorz B, Herrmann BG. 2005. The t complex-encoded GTPase-activating protein Tagap1 acts as a transmission ratio distorter in mice. Nat Genet 37: 969973.
  • Cross MJ, Lu L, Magnusson P, Nyqvist D, Holmqvist K, Welsh M, Claesson-Welsh L. 2002. The Shb adaptor protein binds to tyrosine 766 in the FGFR-1 and regulates the Ras/MEK/MAPK pathway via FRS2 phosphorylation in endothelial cells. Mol Biol Cell 13: 28812893.
  • Cujec TP, Medeiros PF, Hammond P, Rise C, Kreider BL. 2002. Selection of v-abl tyrosine kinase substrate sequences from randomized peptide and cellular proteomic libraries using mRNA display. Chem Biol 9: 253264.
  • Dixelius J, Larsson H, Sasaki T, Holmqvist K, Lu L, Engstrom A, Timpl R, Welsh M, Claesson-Welsh L. 2000. Endostatin-induced tyrosine kinase signaling through the Shb adaptor protein regulates endothelial cell apoptosis. Blood 95: 34033411.
  • Holmqvist K, Cross MJ, Rolny C, Hagerkvist R, Rahimi N, Matsumoto T, Claesson-Welsh L, Welsh M. 2004. The adaptor protein shb binds to tyrosine 1175 in vascular endothelial growth factor (VEGF) receptor-2 and regulates VEGF-dependent cellular migration. J Biol Chem 279: 2226722275.
  • Karlsson T, Welsh M. 1996. Apoptosis of NIH3T3 cells overexpressing the Src homology 2 domain protein Shb. Oncogene 13: 955961.
  • Karlsson T, Songyang Z, Landgren E, Lavergne C, Di Fiore PP, Anafi M, Pawson T, Cantley LC, Claesson-Welsh L, Welsh M. 1995. Molecular interactions of the Src homology 2 domain protein Shb with phosphotyrosine residues, tyrosine kinase receptors and Src homology 3 domain proteins. Oncogene 10: 14751483.
  • Kriz V, Anneren C, Lai C, Karlsson J, Mares J, Welsh M. 2003. The SHB adapter protein is required for efficient multilineage differentiation of mouse embryonic stem cells. Exp Cell Res 286: 4056.
  • Kriz V, Agren N, Lindholm CK, Lenell S, Saldeen J, Mares J, Welsh M. 2006. The SHB adapter protein is required for normal maturation of mesoderm during in vitro differentiation of embryonic stem cells. J Biol Chem 281: 3448434491.
  • Lewandoski M, Wassarman KM, Martin GR. 1997. Zp3-cre, a transgenic mouse line for the activation or inactivation of loxP-flanked target genes specifically in the female germ line. Curr Biol 7: 148151.
  • Li R, Chase M, Jung SK, Smith PJ, Loeken MR. 2005. Hypoxic stress in diabetic pregnancy contributes to impaired embryo gene expression and defective development by inducing oxidative stress. Am J Physiol Endocrinol Metab 289: E591E599.
  • Lindholm CK. 2002. IL-2 receptor signaling through the Shb adapter protein in T and NK cells. Biochem Biophys Res Commun 296: 929936.
  • Lindholm CK, Gylfe E, Zhang W, Samelson LE, Welsh M. 1999. Requirement of the Src homology 2 domain protein Shb for T cell receptor-dependent activation of the interleukin-2 gene nuclear factor for activation of T cells element in Jurkat T cells. J Biol Chem 274: 2805028057.
  • Lindholm CK, Frantz JD, Shoelson SE, Welsh M. 2000. Shf, a Shb-like adapter protein, is involved in PDGF-alpha-receptor regulation of apoptosis. Biochem Biophys Res Commun 278: 537543.
  • Lindholm CK, Henriksson ML, Hallberg B, Welsh M. 2002. Shb links SLP-76 and Vav with the CD3 complex in Jurkat T cells. Eur J Biochem 269: 32793288.
  • Lu L, Anneren C, Reedquist KA, Bos JL, Welsh M. 2000. NGF-Dependent neurite outgrowth in PC12 cells overexpressing the Src homology 2-domain protein shb requires activation of the Rap1 pathway. Exp Cell Res 259: 370377.
  • Lu L, Holmqvist K, Cross M, Welsh M. 2002. Role of the Src homology 2 domain-containing protein Shb in murine brain endothelial cell proliferation and differentiation. Cell Growth Differ 13: 141148.
  • Lyon MF. 2005. Elucidating mouse transmission ratio distortion. Nat Genet 37: 924925.
  • Oda T, Kujovich J, Reis M, Newman B, Druker BJ. 1997. Identification and characterization of two novel SH2 domain-containing proteins from a yeast two hybrid screen with the ABL tyrosine kinase. Oncogene 15: 12551262.
  • Rolny C, Lu L, Agren N, Nilsson I, Roe C, Webb GC, Welsh M. 2005. Shb promotes blood vessel formation in embryoid bodies by augmenting vascular endothelial growth factor receptor-2 and platelet-derived growth factor receptor-beta signaling. Exp Cell Res 308: 381393.
  • Sulik KK. 2005. Genesis of alcohol-induced craniofacial dysmorphism. Exp Biol Med (Maywood) 230: 366375.
  • Welsh M, Mares J, Karlsson T, Lavergne C, Breant B, Claesson-Welsh L. 1994. Shb is a ubiquitously expressed Src homology 2 protein. Oncogene 9: 1927.
  • Welsh M, Songyang Z, Frantz JD, Trub T, Reedquist KA, Karlsson T, Miyazaki M, Cantley LC, Band H, Shoelson SE. 1998. Stimulation through the T cell receptor leads to interactions between SHB and several signaling proteins. Oncogene 16: 891901.
  • Welsh M, Christmansson L, Karlsson T, Sandler S, Welsh N. 1999. Transgenic mice expressing Shb adaptor protein under the control of rat insulin promoter exhibit altered viability of pancreatic islet cells. Mol Med 5: 169180.
  • Wentzel P, Eriksson UJ. 2006. Ethanol-induced fetal dysmorphogenesis in the mouse is diminished by high antioxidative capacity of the mother. Toxicol Sci 92: 416422.
  • Wentzel P, Gareskog M, Eriksson UJ. 2005. Folic acid supplementation diminishes diabetes- and glucose-induced dysmorphogenesis in rat embryos in vivo and in vitro. Diabetes 54: 546553.
  • Wentzel P, Rydberg U, Eriksson UJ. 2006. Antioxidative treatment diminishes ethanol-induced congenital malformations in the rat. Alcohol Clin Exp Res 30: 17521760.
  • Xue Y, Gao X, Lindsell CE, Norton CR, Chang B, Hicks C, Gendron-Maguire M, Rand EB, Weinmaster G, Gridley T. 1999. Embryonic lethality and vascular defects in mice lacking the Notch ligand Jagged1. Hum Mol Genet 8: 723730.
  • Yanagi S, Inatome R, Ding J, Kitaguchi H, Tybulewicz VL, Yamamura H. 2001. Syk expression in endothelial cells and their morphologic defects in embryonic Syk-deficient mice. Blood 98: 28692871.