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Title
High-throughput discovery of novel developmental phenotypes.
Permalink
https://escholarship.org/uc/item/06t772m3
Journal
Nature, 537(7621)
ISSN
0028-0836
Authors
Dickinson, Mary E
Flenniken, Ann M
Ji, Xiao
et al.
Publication Date
2016-09-01
DOI
10.1038/nature19356
Peer reviewed
eScholarship.org Powered by the California Digital Library
University of California
High-throughput discovery of novel developmental phenotypes
A full list of authors and affiliations appears at the end of the article.
Abstract
Approximately one third of all mammalian genes are essential for life. Phenotypes resulting from
mouse knockouts of these genes have provided tremendous insight into gene function and
congenital disorders. As part of the International Mouse Phenotyping Consortium effort to
generate and phenotypically characterize 5000 knockout mouse lines, we have identified 410
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#
Corresponding author: steve.murray@jax.org.
19
Additional contributors listed in the supplement
*
Equal contribution
Code accessibility
Analysis code for bioinformatics data presented in Figures 1 and 5 can be accessed at
https://github.com/IMPC2015/code.
Additional Contributors
19
The International Mouse Phenotyping Consortium
The Jackson Laboratory: Matthew McKay, Barbara Urban, Caroline Lund, Erin Froeter, Taylor LaCasse, Adrienne Mehalow, Emily
Gordon, Leah Rae Donahue, Robert Taft, Peter Kutney, Stephanie Dion, Leslie Goodwin, Susan Kales, Rachel Urban, Kristina Palmer
Infrastructure Nationale PHENOMIN, Institut Clinique de la Souris (ICS): Fabien Pertuy, Deborah Bitz, Bruno Weber, Patrice Goetz-
Reiner, Hughes Jacobs, Elise Le Marchand, Amal El Amri, Leila El Fertak, Hamid Ennah, Dalila Ali-Hadji, Abdel Ayadi, Marie
Wattenhofer-Donze, Sylvie Jacquot, Philippe André, Marie-Christine Birling, Guillaume Pavlovic, Tania Sorg.
Charles River Laboratories: Iva Morse, Frank Benso
MRC Harwell: Michelle E Stewart, Carol Copley, Jackie Harrison, Samantha Joynson
The Toronto Centre for Phenogenomics: Ruolin Guo, Dawei Qu, Shoshana Spring, Lisa Yu, Jacob Ellegood, Lily Morikawa, Xueyuan
Shang, Pat Feugas, Amie Creighton, Patricia Castellanos Penton, Ozge Danisment
The Wellcome Trust Sanger Institute: Nicola Griggs, Catherine L. Tudor, Angela L. Green, Cecilia Icoresi Mazzeo, Emma Siragher,
Charlotte Lillistone, Elizabeth Tuck, Diane Gleeson, Debarati Sethi, Tanya Bayzetinova, Jonathan Burvill, Bishoy Habib, Lauren
Weavers, Ryea Maswood, Evelina Miklejewska, Michael Woods, Evelyn Grau, Stuart Newman, Caroline Sinclair, Ellen Brown
RIKEN BioResource Center: Shinya Ayabe, Mizuho Iwama, Ayumi Murakami
Contributions
M.E.D, A.M.F., X.J, L.T., M.D.W., J.K.W, T.F.M, W.J.W., H.W., D.J.A., M.B., and S.A.M. contributed to the data analysis and writing
of the paper, A.Y., A.B., L.B., L.B.C., F.C., B.D., H.F., A. Galli, A.G., V. G-D., S.G., S.M., S.A.M., L.M.J.N., E.R., J.R.S., M.S.,
W.C.S., R.R.S., L.T., S.W., J.K.W., generated animal models and identified lethal genes, M.E.D, A.M.F., X.J., H.W., L.T., J.M.B.,
N.R.H., T.F.M., M.E.Dolan, S.A.M. contributed to gene list analysis, H.A., L.B, L.B.C., C.N.B., J.C., J.M.D., M.E.D, S.M.E., A.M.F.
A. Galli, C-W.H., S.J.J., S.K., L.C.K., L.L., M.M., M.L.M., T.M., S.A.M., S.N., L.M.J.N., K.A.P., D.R., E.R., Z. S-K., M.T., L.T.,
A.T., O.W., W.J.W., J.K.W., L.W., contributed to the secondary lethal screen and data analysis, J.M.B., D.C., J.G., N.R.H, T.N.L.,
J.M., I.T., and J.W. provided informatics support, M.D.W. and R.M.H. performed the automated 3D analysis, J.M.B, N.R.H, I.T., J.W.,
and H.W. developed and implemented the IMPC portal, X.J, M.J.D., S.A.M., M.L., K.E.S., D.G.M., D.J.A., and M.B. contributed to
the essential gene and human disease analysis, M.E.D, A.M.F., X.J, L.T., M.D.W., J.K.W, T.F.M, W.J.W., H.W., S.W., R.R-S., J.M.D.,
D.G.M., D.B.W., G.P.T-V, X.G., P.F., W.C.S., A.B, M.J.J., H.E.P., M.M, S.W., R.E.B., K.S., M.H.d.A, Y.H., T.M., A.-M.M., R.M.H.,
S.D.M.B., D.J.A., K.C.K.L., C.M., A.L.B., M.B., and S.A.M. contributed to the design, management, execution of the work and
review of the manuscript.
Competing Financial Interests
The authors declare no competing financial interests.
Data accessibility
All data is freely available from the IMPC database hosted at EMBL-EBI via a web portal (
mousephenotype.org), ftp (ftp://
ftp.ebi.ac.uk/pub/databases/impc
) and automatic programmatic interfaces. An archived version of the database will be maintained after
cessation of funding (exp. 2021) for an additional 5 years. Allele and phenotype summaries are additionally archived with Mouse
Genome Informatics at the Jackson Laboratory via direct data submissions (J:136110, J:148605, J:157064, J:157065, J:188991, J:
211773).
HHS Public Access
Author manuscript
Nature
. Author manuscript; available in PMC 2017 March 14.
Published in final edited form as:
Nature
. 2016 September 22; 537(7621): 508–514. doi:10.1038/nature19356.
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lethal genes during the production of the first 1751 unique gene knockouts. Using a standardised
phenotyping platform that incorporates high-resolution 3D imaging, we identified novel
phenotypes at multiple time points for previously uncharacterized genes and additional phenotypes
for genes with previously reported mutant phenotypes. Unexpectedly, our analysis reveals that
incomplete penetrance and variable expressivity are common even on a defined genetic
background. In addition, we show that human disease genes are enriched for essential genes
identified in our screen, thus providing a novel dataset that facilitates prioritization and validation
of mutations identified in clinical sequencing efforts.
Keywords
mouse; embryonic lethal; knockout; KOMP; EUCOMM; IMPC
Introduction
Our understanding of the genetic mechanisms required for normal embryonic growth and
development has been advanced by the analysis of single mutations generated in individual
labs or the identification of mutants through focused mutagenesis screens
1–4
. Systematic,
standardized approaches to mouse phenotypic analysis complement these data, capitalizing
on the efficiency provided by scale and reducing the potential for ascertainment bias,
ultimately providing a means to achieve genome-wide functional annotation. Moreover,
recent challenges in reproducibility of animal model experimentation
5,6
emphasize the need
for careful standardization of allele design, genetic background, and phenotyping protocols.
Building on these principles, the goal of the International Mouse Phenotyping Consortium
(IMPC) is to generate a catalogue of gene function through systematic generation and
phenotyping of a genome-wide collection of gene knockouts (KO) in the mouse. To date,
nearly 5000 new knockout lines have been created by IMPC from the International
Knockout Mouse Consortium (IKMC) resource
7–12
. Here we report the results of the first
international, systematic effort to identify and characterize the phenotypes of embryonic
lethal mutations using a standardised
13
, high-throughput pipeline. These findings provide
novel insights into gene function, provide new models for inherited disorders, and shed new
light on the role of essential genes in a variety of monogenic and complex human disorders.
Results
Intercrosses of 1,751 germ-line transmitted (GLT) heterozygous lines from IMPC
production colonies
1
identified 410 lines that displayed lethality (Fig. 1a), defined as the
absence of homozygous mice after screening of at least 28 pups (p<0.001 Fisher’s exact test)
prior to weaning. We also identified 198 “subviable” lines, defined as fewer than 12.5% (half
of expected) homozygous preweaning pups (full list of genes available in Supplementary
Table 1). The vast majority of the alleles employed in this study were of “tm1.1” or “tm1b”
IKMC variants, which disrupt the coding sequence (1704 of 1804 unique alleles; see
Extended Data Fig.1 for schematics of each allele and Supplementary Table 2 for all other
alleles employed). Centre-to-centre variability in the proportion of essential genes is
observed ranging from 4.8%–52.7%, which likely reflects the different biases in gene
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selection criteria between centres and specific consortium arrangements for lethal gene
characterization (TCP and UCD) (Extended Data Fig. 2A,B). No significant bias is observed
in the distribution of lethal genes across mouse chromosomes (Extended Data Fig. 2C,D).
Overall, however, the lethal proportion (23.4%) is consistent with published observations of
null alleles
7,9,12,13
, particularly when combined with subviable lines (11.3%), resulting in
65.3% viability for IMPC KO lines overall. A main goal of this project is to provide
phenotype data for unknown or novel genes, i.e. those with no prior report of a targeted null
allele in the mouse (curated in Mouse Genome Informatics). The primary viability data
indicated that such unannotated genes displayed an overall viability rate of 66.5%, compared
to the 62% viability rate among previously reported null alleles (Extended Data Fig. 2E;
novel versus prior gene lists in Supplementary Table 3; list of all first publications or reports
of gene knockouts in Supplementary Table 4). These data reveal consistent identification of
essential genes in our program, and further support that approximately 35% (24% lethal and
11% subviable) of null mutations across the genome are essential for survival at normal
Mendelian ratios.
Functional data from mouse knockouts are highly informative, and thus would be predicted
to have a strong impact on Gene Ontology (GO) Consortium
14
annotations. For the 1,751
IMPC mouse lines phenotyped to date, IMPC phenotyping provides the only experimental
evidence for over 40% of the genes in our dataset. Using the GO Slim tool, which clusters
terms associated with each gene into a set of broad categories, we observed enrichment in
lethal and subviable genes within several categories (Extended Data Fig. 3). Compared to
novel genes, the number of annotations for a majority Process and Function categories was
greater for published alleles, highlighting the value of our analysis in assigning function to
novel, previously uncharacterized genes.
We used data from three recent publications on genome-wide screens for cell-essential genes
in human cells to address the overlap between essential genes in the human and mouse
genome
15–17
. We selected core essential genes from each study and compared to human
orthologs of mouse essential genes on the consensus list of curated IMPC-MGI genes. We
found that approximately 35% of core essential genes in each study are associated with
lethality or subviability in the mouse, with 61–62% of genes currently unknown (Fig. 1b).
Of the 19 human essential genes common to all three studies that were nonessential in the
mouse, only three (
Rbmx, Dkc1, and Sod1
) could be reliably confirmed as a targeted
knockout of a nonessential gene, highlighting the remarkable concordance between mouse
and human in their core essential genes.
To expand the depth of our analysis of essential genes, we developed a comprehensive
phenotyping pipeline designed to identify the time of lethality, assign phenotypes, and
document LacZ expression patterns at discrete time points (Extended Data Fig. 4)
13
. A key
aspect of the pipeline is the incorporation of optical projection tomography (OPT)
18
, micro-
computed tomography (micro-CT)
19,20,21
and high-resolution episcopic microscopy
(HREM)
22
, which provide cost-effective, high-throughput approaches to the collection of
phenotype data, including quantitative volumetric analysis (see below). The catalogue of KO
lines and all phenotype data are available to the community via the IMPC portal
(www.mousephenotype.org), with an embryo phenotyping-specific portal at
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www.mousephenotype.org/data/embryo (a guide to accessing, viewing and using these data
is available on the IMPC portal at http://www.mousephenotype.org/data/documentation/doc-
explore)
Using a tiered strategy, we established clear viable vs. lethal (defined if homozygous
embryos were absent or lacked a heartbeat) calls at up to four different time points for a total
of 283 lethal lines (A comprehensive progress table for all 1861 alleles is provided in
Supplementary Table 5), the total number varying by progress through the pipeline. From
these data, we established windows of lethality for 242 genes with complete data to more
precisely define the timing of embryo death. Figure 1c shows that a majority of lines
(147/242; 60.7%) died prior to E12.5 and a majority of these (107/147; 72.8%),
development ceased prior to E9.5, the earliest time point examined. Remarkably only 9 total
lines die in the E12.5–E15.5 or E15.5–E18.5 windows, while most lines that were viable at
E12.5 were also viable at the latest time point examined (E15.5 or E18.5). Although viable,
many of these lines show phenotypes at E15.5 and E18.5 (see below), and ultimately
succumbed in the perinatal or early postnatal period.
Taking advantage of the LacZ cassette present in most IMPC alleles
10,11
, gene expression
was evaluated in heterozygous embryos at E12.5 in the lethal/subviable lines. Expression
patterns fell into three broad categories as shown in Figure 1d (bottom): restricted (e.g.,
Clcf1, Cgn
and
Kif26b
); ubiquitous (e.g.,
Psen1
); or undetectable expression (not shown).
All images and annotations of the expression atlas are available at the IMPC portal,
providing a rich and growing in situ expression atlas for the scientific community.
Identification of novel lethal phenotypes
At each time point, gross morphological phenotypes were recorded using a structured set of
Mammalian Phenotype (MP) terms (Supplementary Table 6). An analysis of phenotype
areas revealed that the most common phenotype overall was growth/developmental delay
(Fig. 2a–c) affecting 23.5%, 44.1% and 39.3% of lines at E12.5, E14.5/E15.5 and E18.5,
respectively. Abnormalities in cardiovascular development were also common, frequently
observed at both E12.5 and E15.5 (Fig. 2a,b), along with craniofacial malformations and
defects in development of the limbs and/or tail. At E18.5, a number of mutants exhibited
respiratory and/or body wall abnormalities (captured as “other”), in addition to the growth
abnormalities seen at other stages.
Our pipeline has identified a number of novel phenotypes for previously unreported
knockouts. In all cases, 3D imaging revealed additional phenotypes that might have been
missed by gross inspection. For example,
Tmem132a
E15.5 homozygous embryos were
smaller than littermates, displayed an obvious spina bifida, and narrow, club-shaped limbs
(Fig. 2d,f). Sagittal cross-sections through the micro-CT data showed the abnormal
curvature in the spinal column adjacent to the open neural tube, and abnormal head structure
in mutants (Fig. 2e,g). Kidney defects were also observed in E15.5 mutant embryos (n=3)
and bladder defects were also evident by E18.5 (n=4) (not shown).
Svep1
homozygous
mutant embryos display multiple defects at both E15.5 and E18.5, severe edema and
discolouration (Fig. 2h,k), and die in the perinatal period. Additionally, transverse sections
of micro-CT data from E18.5 embryos revealed abnormal development of the kidney pelvis
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