Phenotyping
Primary phenotyping: At weaning or about 21 days of age, all litters were observed for
the segregation of coat colour, sex, and visible anomalies. Lethal mutations were
designated if no test class animals were observed in a total of at least 24 carrier class
animals. If no lethal or visible phenotype was observed at this stage, four test class
animals were saved to the age of three months. During that time, they were observed for
later onset visible phenotypes. In addition, 517 of the pedigrees were bled and analyzed
by complete blood count with differential. These same 517 pedigrees were analyzed for
high urine glucose and albumin using Chemstrip 9 (Roche, Basel, Switzerland). Briefly,
urine was obtained by allowing the animal to excrete urine onto a petri dish covered with
plastic wrap. The urine was pipetted into a tube, and placed onto the ChemStrip. High
urine glucose over 500 mg/dL in more than two animals in a pedigree was an indication
of a mutant. Mice have normal urinary proteins, so high albumin was designated if total
protein in the urine was above 100 mg/dL.
The presence of albumin was confirmed by
ELISA. One-hundred eighty-four of the pedigrees were tested for infertility by setting
up matings between G3 siblings. If no pregnancy was observed after eight weeks, the
individuals were mated with wild type animals. If no pregnancy resulted, the pedigree
was rescued from a carrier sibling, and test class animals were examined for signs of
infertility. Ovary appearance and histology were assessed at different ages on females,
and sperm motility and numbers as well as testis histology were assessed on males. One
hundred-eighteen of the pedigrees were assessed by Tandem Mass Spectrometry (TMS)
for amino acids, acylcarnitine-conjugated fatty acids, neutral cholesterols, and bile acids,
which revealed no recessive mutations. X-rays on 218 of the pedigrees did not reveal
additional phenotypes that we could not observe visibly. At the end of the three month
period, necropsies were performed on 218 of the pedigrees, but revealed no phenotypes
that segregated to chromosome 11.
Phenotype Assessment
Number Number of
Number of
of
mutants on
mutants
pedigrees chromosome 11 genome-wide
assessed
Lethal
Visible
CBC
Urine Glucose/Protein
Aging to 3 months
X-rays
Necropsy
Infertile
TMS
735
735
517
517
517
218
218
184
118
55
20
3
0
2*
0
0
8
0
5 (fitness)
127
2
5
4*
0
0**
0
0**
* Two on chromosome11 had late onset neurological phenotypes. One genome-wide
developed obesity, two developed a late onset neurological phenotype, and one develops
a nonsyndromic deafness by three months of age.
** No heritable recessive mutations without other visible phenotypes were observed.
Two dominant mutations in neutral cholesterols were isolated.
Secondary phenotyping: If a lethal mutation was designated, or if a phenotype was
observed in at least two animals from the pedigree, the pedigree was saved as a stock, and
additional information was obtained on the phenotype, while the penetrance of the
mutation after an outcross to C3H.Inv/Rex (if segregating on chromosome 11) or
129S6/SvEvTAC (if genome-wide) was assessed. All mutant stocks that produced
animals living at least to weaning were bled for CBC analysis and TMS. In addition to
the blood assays, body weight curves were obtained on all small mutants. All small
mutants were also assessed for infertility. Deafness was assessed on all pedigrees with
circling, headtossing, or weaving phenotypes using a Clickbox (University of
Nottingham, UK) to assess the Preyer reflex.
All skeletal mutants undergo X-rays and
Alizarin red skeleton preparation to assess the penetrance and basis for the defect.
Embryo dissections: Timed matings between male and female carriers were set up for
each line. The presence of a vaginal plug was designated as day E0.5. Initial dissections
were performed at E12.5. Each embryo was genotyped and photographed. If embryos
that appeared abnormal were genotyped as mutants (by the absence of the HPRT
cassette), further dissections were performed to confirm the mutant phenotype and stage
of death. If embryos that appeared normal were genotyped as mutant at E12.5, then
subsequent dissections were performed at later stages until an abnormal phenotype was
found. If no mutant embryos were observed, dissections were performed at early stages
until mutant embryos were isolated. Dissections for several lines were performed with
lethal/Rex females, which allow for recombination between the mutation and the Rex
chromosome. Mutant embryos recovered from these dissections were indistinguishable
from mutants recovered from dissections with lethal/inversion females, indicating that the
lethal mutations are likely to be caused by mutations in single genes, rather than
mutations in two genes co-operating to cause the lethal phenotype.
Supplementary Figure legends
Figure 1 a, Some l11Jus51 homozygotes survive to weaning, but have
developmental delay. The homozygous mutant animal is shown in front of a
number of littermates. The K14-Agouti coat colour is penetrant on both a black
and an agouti background. Since in all photos, agouti and non-agouti are
segregating, some animals will be black and others agouti. b, l11Jus52
homozygotes are very small at weaning, and have craniofacial anomalies. c,
Skeleton preparations on l11Jus53 homozygotes (right) show the short snout
compared with a normal littermate (left) d, A l11Jus54 homozygote that survived
to weaning age (right) next to a wild type littermate (left).
Figure 2 a-f, Embryos from several lines have neural tube defects. a,
Heterozygous l11Jus8 embryo at 12.5dpc. b, l11Jus8 mutant embryos at 12.5
dpc have kinked neural tubes and abnormal somites. c, Heterozygous l11Jus15
embryo at 11.5dpc. d, l11Jus15 mutant embryo with open neural tube in spinal
cord region at 11.5dpc. Neural tube defects are observed in 10% of l11Jus15
mutants. e, Heterozygous l11Jus39 embryo at 12.5dpc. f, l11Jus39 mutant
embryo with kinked neural tube at 12.5dpc. g-l, l11Jus3 and l11Jus27 mutant
embryos have developmental defects. g, l11Jus3 mutant embryo at 7.5dpc does
not show any clear embryonic structures. h, Sagittal section of wild type 7.5dpc
embryo stained for haematoxylin and eosin. i, Sagittal section of l11Jus3 mutant
at 7.5dpc. j, Wild-type embryo at 9.5dpc. k, l11Jus27 mutant embryo at 12.5
dpc shows heart defects and developmental delay (appears arrested at 9.5dpc).
l, l11Jus27 mutant embryo at 12.5dpc shows distended heart in enlarged
pericardial sac.
Figure 3 Male infertile mutants have low sperm counts a, Wild type carrier male
testis (shown) with an average sperm count of 14.6 + 7.0 x 106 sperm/ml. Tails
of mature spermatozoa in the seminiferous tubule (*). b, inf9 homozygote
revealing an arrest at the round spermatid stage. Note the absence of mature
sperm, and the prominence of round spermatids (arrows). Released sperm
were non-motile and sperm count was significantly reduced (8.7 + 1.2 x 105; p <
0.001). c, inf4 homozygotes show seminiferous tubules with an incomplete block
at the late differentiating spermatid stage. Again, released sperm were nonmotile and sperm count was reduced (8.0 + 2.8 X 104; p <0.001). Note the
presence of round spermatids (arrows), as well as differentiating sperm,
indicated by the presence of tails in the lumen (*). d, Infertile mutant class with
normal testis histology, normal sperm morphology and motility. Sperm count was
significantly reduced (1.9 + 1.9 x 106; inf6; p <0.001). Macrocysts (cy) are
prominent throughout the kidney of inf6 homozygotes. G; glomerulus. Scale
bars represent 50 microns.
Movie: l11Jus57 homozygous animal that survived to weaning. The majority of
mutant animals of this strain die at birth. Note the severe dystonia.