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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online May 16, 2005 as doi:10.1096/fj.04-3407fje. |
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Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration Bethesda, Maryland, USA
1Correspondence: E-mail: mccright{at}cber.fda.gov
SPECIFIC AIMS
Notch1 and Notch2 are structurally conserved essential genes that are critical for the development of multiple organ systems in mammals. We hypothesized that amino acid differences in their intracellular domains (ICDs) could be responsible for exerting the functional differences that have been observed between the Notch1 and Notch2 receptors in vitro. To analyze the function of the Notch ICD in vivo, we created mice that contained a targeted mutation that replaces the nonconserved region of the Notch2 ICD with the homologous region of Notch1.
PRINCIPAL FINDINGS
1. Creation of mice expressing a Notch2-Notch1 fusion protein
A targeting construct was made to replace the C-terminal 426 amino acids of the Notch2 ICD with the homologous region of Notch 1 (Fig. 1
). The genomic structure of both genes is highly conserved in the C-terminus of the ICD region even though the peptide sequence is not (37% identical; 137/426). In both genes, genomic DNA encoding the region from the sixth ankyrin repeat to the C-terminus is contained in one large exon (Fig. 1)
. This feature enabled the strategy of exchanging the nonconserved regions of the activation domains. The resulting fusion peptide consists of the first 2044 amino acids of Notch2 fused to the last 445 amino acids and 3' UTR of Notch1. We named this new allele of Notch2, Notch2N1in.
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Mice heterozygous for the Notch2N1in allele did not display any observable phenotypes, implying there is no dominant gain-of-function associated with this allele. Southern blot analysis of DNA obtained from 3-wk-old mice show the presence of a band specific to the Notch2N1in allele indicating that the targeting construct had integrated properly (Fig. 1)
. Germ line transmission of the Notch2N1in allele was obtained from two separate lines and mice from either cell line were indistinguishable from each other.
2. Notch2N1in mice express Notch2-Notch1 fusion mRNA
To demonstrate that the Notch2-Notch1 fusion message was being made, we performed RT-PCR on total RNA obtained from neonatal kidneys since both Notch1 and Notch2 are expressed in developing kidneys. Kidneys were obtained from the 3-day-old progeny of intercrossed heterozygous mice. As expected, we observed Notch2N1in-specific message only in the heterozygous and homozygous Notch2N1in mice and not in the wild-type mice (Fig. 2
). Similarly, we did not see any Notch2 message in the Notch2N1in homozygous mice but did see the wild-type Notch2 message in Notch2 heterozygotes and wild-type mice (Fig. 2)
. Notch1 message was present in all samples (Fig. 2)
. The level of expression of the chimeric message was approximately equivalent to that of the native Notch1 message as evidenced by three primer PCR that amplified both Notch2N1in and Notch1 messages in the same reaction (Fig. 2)
.
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3. Mice homozygous for the Notch2N1in allele are phenotypically normal
Surprisingly, we observed that mice homozygous for the Notch2N1in allele did not die during embryonic development and had an outwardly normal appearance. Notch2 has been shown to be required for the development of kidney glomeruli and intrahepatic bile ducts in mice. Therefore, we hypothesized that these tissues would be most sensitive to altered Notch2 function. We did not observe any differences in the histology of the Notch2N1in homozygotes relative to that of the wild-type mice in 3-day-old livers or kidneys. Specifically, the intrahepatic bile ducts and the kidney glomeruli of the homozygous Notch2N1in mice develop normally.
Whole blood samples from Notch2N1in homozygotes were analyzed to determine whether there were any disturbances in hematopoietic cell differentiation. We found total white blood cell counts and the white blood differential counts of these mice were similar to wild-type mice and published values with the exception that the number of Notch2N1in monocytes was increased relative to that of wild-type mice (5.7 to 2.0%).
To further evaluate the effects of the mutation on viability, we weighed mice obtained from heterozygote intercrosses and tabulated the genotypes of the mice at weaning. We did not observe any weight decreases or deviation from Mendelian distribution supporting the conclusion the Notch2N1in allele has no adverse effect on the health of mice. Homozygous mice derived from two independent ES cell lines were able to have normal size litters of healthy mice that have not displayed any health defects (4+ months).
CONCLUSIONS AND SIGNIFICANCE
This study was designed to identify differences in the signaling capabilities of the Notch1 and Notch2 ICDs. We hypothesized that the nonconserved region of the ICD could impart different functional capabilities to these essential Notch receptors. To test this hypothesis, we replaced the last 426 amino acids of Notch2 with the corresponding region of Notch1. Surprisingly, mice homozygous for the Notch2N1in targeted mutation, expressing no native Notch2 message, appeared phenotypically wild-type. While we did not exhaustively test for subtle defects, the kidney glomeruli and intrahepatic bile ducts, two structures that require Notch2 for their development, formed normally. Therefore, this region of the ICD, or activation domain, is functionally conserved between Notch1 and Notch2. It is unknown whether the same result would have been obtained if the entire Notch2 ICD was replaced, but because of the very high amount of conservation in the ankyrin repeat region, we hypothesize that the result would have been the same.
Most of the C-terminal half of the Notch ICD is not evolutionarily conserved at the amino acid level, but Notch receptors from Drosophila, Xenopus, and mammals have 
400 amino acids downstream of the ankyrin repeats. This region is essential for Drosophila viability and has been shown to physically interact with Disheveled. These results support our conclusion that the C-terminus of the Notch1 ICD is replacing the function of Notch2 ICD in our experiments rather than the possibility that this region of Notch2 has no essential function.
In contrast to the rest of the C-terminal half of the Notch ICD, the PEST sequences at the carboxyl terminus are not only conserved between mNotch1 and mNotch2 (Fig. 1)
but are also evolutionarily conserved. The main biological function of the C-terminal half of the Notch ICD may be to regulate the turnover rate of the Notch signal by regulating the access of the ubiquitin ligases. The ability to rapidly turn off a signal that regulates cell fate determination would be essential for the correct development of any morphologically complex structure.
Our results are consistent with the study that demonstrated that the C-terminus of both Notch1 and Notch2 could activate transcription in vitro. Other groups, however, have identified transcriptional activating differences between the different Notch ICDs and have identified a region C-terminal of the ankyrin repeats that conferred differential cytokine response to the Notch1 and Notch2 ICDs in myeloid progenitor cells undergoing differentiation. Our analysis on the hematopoietic cell populations present in Notch2N1in mice did not uncover any striking lineage imbalances other than an increase in the numbers of monocytes relative to expected values. This increase in monocyte number is not of the magnitude normally associated with monocytosis, but it could be related to differences in cytokine response as reported by Bigas et al. Further analysis of these mice could uncover additional hematopoietic lineage differences and possibly link these differences to functional differences in the Notch1 and Notch2 ICDs.
Our results are consistent with a signaling model where Notch1 and Notch2 ICDs are functionally redundant, but their activation is dependent on differential interaction with ligands or the effect of extracellular domain modifiers such as Fringe. Similarly, if Notch1 and Notch2 ICDs exert the same downstream effects, then they may be used interchangeably in scenarios where the goal is to exogenously activate the Notch signaling pathway for the purpose of controlling cell fate.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3407fje;
This article has been cited by other articles:
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  H.-T. Cheng, M. Kim, M. T. Valerius, K. Surendran, K. Schuster-Gossler, A. Gossler, A. P. McMahon, and R. Kopan Notch2, but not Notch1, is required for proximal fate acquisition in the mammalian nephron Development, February 15, 2007; 134(4): 801 - 811. [Abstract] [Full Text] [PDF]
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