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Gene: 683	Name: Ppard	Family: Nuclear Hormone Recept...	Subfamily: Peroxisome Proliferato...	Accession: U10375	GI: 507778

Gene 683
Summary of Phenotypic Analysis

Changes related to genotype:

Lethality: The majority of homozygous mutant embryos die before birth, however a few survive to weaning
Heterozygous mutant mice displayed a significant decrease in the mean response latency to thermal pain
Histopathology

Brain, cerebrum, ventricle, dilation in 49 day cohort homozygous females may be related to genotype

There were no significant differences detected in the homozygous mutant mice which survived post weaning when compared with age- and gender-matched wild-type control mice. However, the observed genotypic ratio deviates significantly from the expected ratio of 1:2:1, approaching a ratio of 1:2:0. Data suggest that the majority of homozygous mutant mice die at multiple time points between implantation and weaning.

ES cells derived from the 129/OlaHsd mouse substrain were used to generate chimeric mice. F1 mice were generated by breeding with C57BL/6 females. The resultant F1N0 heterozygotes were backcrossed to C57BL/6 mice to generate F1N1 heterozygotes. F2N1 homozygous mutant mice were produced by intercrossing F1N1 heterozygous males and females.

Wild-type control mice, as well as heterozygous and homozygous mutant mice were evaluated by the following examinations or tests:

· Physical examinations.
· Necropsy including body length, body weight, and organ weight measurements.
· Histological examination of tissues and organs.
· Bone marrow section evaluations.
· Complete blood counts and differentials.
· Clinical chemistry panels.
· Embryonic development.
· Behavioral Tests.
· Aging Studies.

When compared to age- and gender-matched wild-type control mice, heterozygous mutant mice displayed a decreased mean response latency during hot plate testing.

When compared to age- and gender-matched wild-type mice, there is moderate dilation of ventricles in the cerebrum of the brain in both homozygous mutant females. This finding may be related to genotype.

Gene 683
Behavior

Changes related to genotype:

There was a significant difference detected during hot plate testing in the heterozygous mutant animals when compared with age- and gender-matched wild-type control mice.

Heterozygous mutant and wild-type control mice were evaluated for phenotypic changes by testing on six behavioral tasks: Open field test, Tail suspension test, Rotarod test, Hot plate test, Startle/PPI, and Metrazol test.

Mouse ID numbers are as follows:

10 heterozygous mutant males (113166, 113170, 114657, 114658, 114659, 116789, 119478, 121041, 121863, 123048)
10 wild-type control males (113163, 113164, 113165, 114656, 114661, 116786, 119481, 121040, 121864, 123047)

ES cells derived from 129/OlaHsd; mouse substrain were used to generate chimeric mice. F1 mice were generated by breeding with C57BL/6 females. The resultant F1N0 heterozygotes were backcrossed to C57BL/6 mice to generate F1N1 heterozygotes. F2N1 heterozygous mutant mice were produced by intercrossing F1N1 heterozygous males and females.

Behavior Findings:

When compared to age- and gender-matched wild-type control mice, heterozygous mutant mice displayed a decreased mean response latency during hot plate testing. There were no other genotype-related differences noted between heterozygous mutant and wild-type control mice for any other parameters evaluated during behavior testing.

Gene 683
Expression Summary

RT-PCR Summary:
RNA transcripts are detectable in all tissues examined: brain, cortex, subcortical region, cerebellum, brainstem, olfactory bulb, eye, heart, lung, liver, pancreas, kidneys, spleen, thymus, lymph nodes, bone marrow, skin, gall bladder, urinary bladder, pituitary gland, adrenal gland, salivary gland, skeletal muscle, tongue, stomach, small intestine, large intestine, cecum, testis, epididymis, seminal vesicle, coagulating gland, prostate gland, ovary and uterus.

LacZ Summary:
LacZ (beta-galactosidase) expression is detectable in brain, spinal cord, eye, pancreas, kidneys, trachea, larynx, esophagus, pituitary gland, parathyroid gland, tongue, skin, male and female reproductive systems.

Expression:

Brain
In wholemount staining X-Gal staining is detectable in olfactory bulb, cortex, thalamus and cerebellum.
Frozen sections reveal expression in cortex, caudate putamen, corpus callosum, hippocampus, thalamus and ventricles. In cerebellum lacZ expression is detectable in the ventricles, granular layer, molecular layer, Purkinje cell layer and white matter.

Spinal Cord
Very weak lacZ expression is detectable in few cells dorsal of the central canal.

Eyes
Cells in the inner nuclear layer of the retina express lacZ.

Pancreas
Many acinar cells show lacZ expression.

Kidney
X-Gal staining is detectable in tubules of the cortex and medulla and in glomeruli.

Trachea
X-Gal staining is detectable in epithelial cells of the mucosal epithelium.

Larynx
Few epithelial cells of the laryngeal epithelium express lacZ faintly.

Esophagus
Many epithelial cells express lacZ moderate to strongly.

Pituitary Gland
Weak lacZ expression is detectable in pars nervosa.

Parathyroid gland
Very weak lacZ expression is detectable in few cells in the parathyroid gland.

Tongue
Many epithelial cells express lacZ.

Skin of the Ear.
X-Gal staining is detectable in chondrocytes, myocytes, and epithelial cells. Furthermore X-Gal signals are observed in hair follicles, glands and blood vessel walls.

Male Reproductive Systems
Testis
X-Gal staining is detectable in many interstitial cells; faint signals are detectable in myofibroblasts and in tunica albuginea. LacZ expression is also observed in blood vessel walls.

Penis
Many epithelial cells display moderate to strong lacZ expression.

Seminal Vesicles
Myocytes in the capsule show faint lacZ expression.

Coagulating Glands
Some epithelial cells of the coagulating glands and myocytes in the capsule express lacZ.

Prostate Glands
Myocytes surrounding the glands express lacZ.

Female Reproductive Systems
Oviduct/Uterus
Epithelial cells show moderate to strong lacZ expression in the uterine tubules.

Vagina/Cervix
Epithelial cells display moderate lacZ expression.

No Expression:
LacZ expression is not detected in: spinal cord, sciatic nerve, Harderian glands, thymus, spleen, lymph nodes, bone marrow, aorta, heart, lung, liver, gallbladder, urinary bladder, thyroid gland, salivary glands, adrenal glands, skeletal muscle and ovary.

Gene 683
Densitometry

There were no significant differences detected in the homozygous or heterozygous mutant mice when compared with age- and gender-matched wild-type control mice.

The following mice were evaluated by dual-energy x-ray absorptiometry.

49 Day Cohort Mouse ID numbers are as follows:
1 homozygous mutant female (187418)
2 heterozygous mutant females (119480, 119482)

300 Day Cohort Mouse ID numbers are as follows:
1 heterozygous mutant female (136867)
2 heterozygous mutant males (187414, 187417)
1 wild-type control female (136868)
2 wild-type control males (187415, 187416)

Bone Mineral Density (BMD in g/cm² ), fat % (fat percentage expressed as a percentage of body soft tissue compartment), and R-value of soft tissue were calculated from Bone Mineral Content (BMC in g), bone and tissue areas (cm² ), and total tissue mass (g) generated by a PIXImus densitometer.

Densitometric Findings:

Certain densitometric differences between mice, including fat %, are present that occasionally occur spontaneously in this age group. In this target, such differences are present in one 300 day heterozygous knockout female. We have not reported this finding as a phenotypic change, but we have presented it here for your consideration.

Other incidental densitometric differences were present between some mice. These findings were considered to represent background differences occasionally seen in this strain of mice, differences due to spontaneous disease, age-related differences, and/or differences of a nonspecific etiology. They were not considered to be genotype related.

Gene 683
Histopathology

Changes that may be related to genotype:

Brain, cerebrum, ventricle, dilation in 49 day cohort homozygous females

There were no significant differences detected in the heterozygous mutant mice when compared with age- and gender-matched wild-type control mice.

The following mice were evaluated were evaluated histologically.

Mouse ID numbers were as follows:

49 Day Cohort Mouse ID numbers were as follows:
2 homozygous mutant females (102477, 187418)
1 homozygous mutant male (102474)
3 heterozygous mutant females (102478, 119480, 119482)
5 heterozygous mutant males (102473, 111961, 114660, 117999, 118004)
2 wild-type control females (102475, 102479)
2 wild-type control males (101558, 101559)

300 Day Cohort Mouse ID numbers were as follows:
2 heterozygous mutant females (121865, 136867)
2 heterozygous mutant males (187414, 187417)
2 wild-type control females (121866, 136868)
2 wild-type control males (187415, 187416)

Histopathology Findings:
When comparedto age- and gender-matched wild-type mice, there is moderate dilation of ventricles in the cerebrum of the brain in both homozygous mutant females. This finding may be related to genotype.

No Significant Abnormalities:
Tissues examined and considered to have no genotypically-significant abnormality: pituitary gland, ears, nasal cavity, salivary glands, oral cavity, lymph nodes, aorta, lungs, gallbladder, pancreas, spleen, kidneys, urinary bladder, stomach, small and large intestines, larynx, esophagus, trachea, thyroid gland, thymus gland, tongue, skeletal muscle, sciatic nerve, mammary glands, vertebrae, spinal cord, bone (skull, sternum, femur, tibia, and stifle joint), reproductive tract including gonads, eyes, Harderian glands, integumentary system (skin and either clitoral or preputial glands), and bone marrow.

Bone marrow was examined in sections of sternum, vertebrae, and/or femur and tibia. Marrow cellularity, myeloid:erythroid (M:E) ratio, myeloid and erythroid maturation sequences, and numbers of megakaryocytes were evaluated.

Incidental lesions may have been present in some tissues. These findings were considered to represent background lesions occasionally seen in this strain of mice, lesions due to spontaneous disease, age-related lesions, lesions due to procedural artifacts and/or lesions of nonspecific etiology. They were not considered to be genotype related.

Gene 683
Necropsy

There were no significant differences detected in the homozygous or heterozygous mutant mice when compared with age- and gender-matched wild-type control mice.

The following mice were necropsied. Body weight, body length, and organ weights were obtained, and gross pathological findings were recorded.

49 Day Cohort Mouse ID numbers were as follows:
2 homozygous mutant females (102477, 187418)
1 homozygous mutant male (102474)
3 heterozygous mutant females (102478, 119480, 119482)
5 heterozygous mutant males (102473, 111961, 114660, 117999, 118004)
2 wild-type control females (102475, 102479)
2 wild-type control males (101558, 101559)

300 Day Cohort Mouse ID numbers were as follows:
2 heterozygous mutant females (121865, 136867)
2 heterozygous mutant males (187414, 187417)
2 wild-type control females (121866, 136868)
2 wild-type control males (187415, 187416)

Mice were examined for the following observables: adrenal glands, body length, body weight, bone marrow, bone-cranium, bone-femur, bone-sternum, bone-stifle joint, bone-vertebral column, brain, cecum, colon, duodenum, epididymis-seminal vesicle, esophagus, eyes, gallbladder, general appearance, Harderian glands, heart, heart weight, ileum, jejunum, kidney weight, kidneys, liver, liver weight, lungs, lymph nodes, mesentery, ovaries, pancreas, penis, salivary glands, sciatic nerve, scrotum, skeletal muscle, skin, skinned mouse, spleen, spleen weight, stomach, testes, testes-epididymis weight, thymus, thymus weight, tongue, trachea, urinary bladder, urine, uterus and vagina (gender specific observables apply to appropriate gender).

Necropsy Findings:

Certain lesions were present that occasionally occur spontaneously in mice of this sex and age group. In this target, a missing adrenal is present in 1 female 49day heterozygote mouse (102477). Also, both heterozygote male 300 day mice (187414,187417) have discolored, abnormal appearing livers, and one heterozygote 300 day male has a significantly enlarged spleen (187414). We were not reporting these findings as phenotypic changes, but we present them here for your consideration.

There were no other genotype-related or biologically significant differences noted between mutant and wild-type control mice for any of the parameters evaluated at necropsy. Incidental lesions may have been present in some tissues. These findings were considered to represent background lesions occasionally seen in this strain of mice, lesions due to spontaneous disease, age-related lesions, lesions due to procedural artifacts, and/or lesions of nonspecific etiology. They were not considered to be genotype related.

Body and Organ Weight Findings:

Certain findings were present that occasionally occur spontaneously in mice of this sex and age group. Also, both heterozygote male 300 day mice (187414,187417) have increased liver weights and liver weight to body weight ratios with one significantly increased, and one heterozygote 300 day male (187414) has a significantly increased spleen weight. We have not reported these findings as phenotypic changes, but we have presented them here for your consideration

Other differences in body length, body weight, organ weights, and/or organ weight to body weight ratios were present between individual mice. The variability between mice usually fell within our historical reference ranges and was not correlated with genotype.

Gene 683
Clinical Chemistry

There were no significant differences in the heterozygous mutant mice when compared with age- and gender-matched wild-type control mice.

Serum samples from the following mice were evaluated by a clinical chemistry panel.

49 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (102477, 187418)
1 homozygous mutant male (102474)
2 heterozygous mutant females (102478, 119480)
4 heterozygous mutant males (102473, 111961, 114660, 118004)
2 wild-type control females (102475, 102479)
2 wild-type control males (101558, 108965)

90 Day Cohort Mouse ID numbers are as follows:
4 heterozygous mutant females (121865, 123051, 123052, 136867)
4 heterozygous mutant males (187259, 187414, 187417, 191257)
4 wild-type control females (101553, 101554, 121866, 121867)
4 wild-type control males (187415, 187416, 189598, 204048)

180 Day Cohort Mouse ID numbers are as follows:
4 heterozygous mutant females (121862, 121865, 123051, 123052)
4 heterozygous mutant males (187414, 187417, 191257, 193039)
4 wild-type control females (101553, 101554, 121866, 121867)
4 wild-type control males (187415, 187416, 189598, 204048)

300 Day Cohort Mouse ID numbers are as follows:
2 heterozygous mutant females (121865, 187263)
2 heterozygous mutant males (187417, 195056)
2 wild-type control females (121866, 189599)
2 wild-type control males (187415, 187416)

Values for the various analytes evaluated were generally similar between heterozygous mutant and wild-type control mice. Variations in clinical chemistry values, if present, were not consistent with genotype and thus were not considered phenotypically relevant.

Gene 683
Hematology

There were no significant differences in the heterozygous mutant mice when compared with age- and gender-matched wild-type control mice.

Blood samples from the following mice were evaluated by a complete blood count and differential cell count.

49 Day Cohort Mouse ID numbers are as follows:
1 homozygous mutant female (187418)
1 homozygous mutant male (102474)
3 heterozygous mutant females (108966, 119482, 136870)
3 heterozygous mutant males (102473, 111961, 114660)
2 wild-type control females (102479, 109938)
2 wild-type control males (101558, 104971)

90 Day Cohort Mouse ID numbers are as follows:
4 heterozygous mutant females (121862, 121865, 123051, 136867)
4 heterozygous mutant males (187259, 187414, 187417, 191257)
5 wild-type control females (101554, 121866, 121867, 123053, 136868)
3 wild-type control males (187415, 187416, 189598)

180 Day Cohort Mouse ID numbers are as follows:
4 heterozygous mutant females (121862, 121865, 123051, 136867)
4 heterozygous mutant males (187414, 187417, 191257, 193039)
4 wild-type control females (101553, 101554, 121866, 136868)
4 wild-type control males (187415, 187416, 189598, 204048)

300 Day Cohort Mouse ID numbers are as follows:
2 heterozygous mutant females (121865, 136867)
2 heterozygous mutant males (187414, 187417)
2 wild-type control females (121866, 136868)
2 wild-type control males (187415, 195058)

Although minor variations of hematological values were present in some mice, these changes were not consistent with genotype and thus were not considered phenotypically relevant.

Gene 683
Physical Examination

There were no significant differences detected in the homozygous or heterozygous mutant mice when compared with age- and gender-matched wild-type control mice.

The following mice were evaluated by physical examination.

Mice were examined in detail as follows: anus, behavior, body shape, claws, coat - fur, coat color - back, coat color - belly, ear - left, ear - right, eye - left, eye - right, eye color - left, eye color - right, feces, forelimb - left, forelimb - right, forelimb number of amputated digits - left, forelimb number of amputated digits - right, forelimb number of digits - left, forelimb number of digits - right, general appearance, genitals - female, genitals - male, hair type, head shape, hindlimb - left, hindlimb - right, hindlimb number of amputated digits - left, hindlimb number of amputated digits - right, hindlimb number of digits - left, hindlimb number of digits - right, injuries, lesions, limb shape, locomotor, lumps - masses, mammary glands, mice in cage, respiration, skin appearance, snout, swelling - joints, tail, teeth color, teeth length, urine, and whiskers. (Gender-specific observables apply to the appropriate gender.)

Individual homozygous and heterozygous mutant mice had only occasional minor differences in observed physical features comparedto wild-type control mice. These findings were considered to represent individual variability, background features occasionally seen in this strain of mice, findings due to spontaneous disease, age-related findings, and/or findings of a nonspecific etiology. However, none of these differences was regarded as biologically significant or genotype related.

Gene 683
Aging Metrics

There were no significant differences detected in the heterozygous mutant mice when compared with age- and gender-matched wild-type control mice.

Body weights and body lengths were measured for mice at 49, 90, 180, and 300 days of age.

49 Day Cohort Mouse ID numbers are as follows:
5 heterozygous mutant females (121862, 121865, 123051, 123052, 136867)
4 heterozygous mutant males (187259, 187414, 187417, 191257)
5 wild-type control females (101553, 101554, 121866, 121867, 136868)
4 wild-type control males (187415, 187416, 189597, 189598)

90 Day Cohort Mouse ID numbers are as follows:
5 heterozygous mutant females (121862, 121865, 123051, 123052, 136867)
4 heterozygous mutant males (187259, 187414, 187417, 191257)
5 wild-type control females (101553, 101554, 121866, 121867, 136868)
4 wild-type control males (187415, 187416, 189597, 189598)

180 Day Cohort Mouse ID numbers are as follows:
4 heterozygous mutant females (121862, 121865, 123051, 123052)
4 heterozygous mutant males (187414, 187417, 191257, 193039)
4 wild-type control females (101553, 101554, 121866, 121867)
4 wild-type control males (187415, 187416, 189598, 204048)

300 Day Cohort Mouse ID numbers are as follows:
4 heterozygous mutant females (121862, 121865, 123051, 123052)
4 wild-type control females (101553, 101554, 121866, 121867)
1 wild-type control male (189598)

Body Weight and Length Findings:

Differences in body length and body weight were present between individual mice. The variability between mice usually fell within our historical reference ranges and was not correlated with genotype.

Gene 683
Summary of embryonic Development

The majority of homozygous mutant embryos die before birth

Embryos were isolated at E8.4 to E16.5 days post coitum. Homozygous offspring were detected by PCR from E8.5 to E16.5. Data suggests that the majority of homozygous embryos die (resorptions and/or retarded) between E8.5 to E10.5. However, a few viable animals are found at later stages, including at weaning.

Embryos were isolated at E8.4 to E16.5
Six litters were examined comprising of 45 embryos, resorptions and partial resorptions, of which 35 were successfully genotyped.

Litter	Embryonic stage	+/+	+/-	-/-	complete resorption or unknown
1	8.5	3	1	1	1
2	9.5	2	5	2	0
3	10.5	3	2	0	3
4	10.5	1	5	2	0
5	12.5	2	7	0	0
6	16.5	1	2	1	6