Gene: 801	Name: Grm1	Family: GPCR	Subfamily: Metabotropic Glutamate	Accession: AF320126	GI: 11935151

Gene 801
Summary of Phenotypic Analysis

Changes related to genotype:

• Home cage observations: ataxia/lack of coordination, stumbling, paddling movements, hypoactivity, thin, and/or hunched posture, 20 and 49 day cohorts
• Physical examination: ataxia, incoordination, runted, thinness and weakness in homozygous mutant mice, 49 day cohort 
• Necropsy examination: decreased body weights and body lengths in homozygous mutant mice at 49 days
• Mouse metrics: decreased body weight, body length, and body weight/body length ratio in a homozygous mutant mouse, 49 day cohort 
• Juvenile lethality: homozygous mutant mice do not survive to 90 day cohort

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

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. F2 homozygous mutant mice were produced by intercrossing F1 heterozygous males and females.

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

• Physical examination.
• Necropsy, including body length, body weight, and organ weight measurements.
• Histological examination of tissues and organs.
• Bone marrow section evaluation.
• Complete blood counts and differentials.
• Clinical chemistry panels.
• Densitometry.
• Expression analysis.
• Aging studies.
• Behavioral tests.

When compared to age- and gender-matched wild-type control mice and heterozygous mutant mice, findings in 20 and  49 day cohort homozygous mutant mice at home cage observations and/or physical examination included the following: ataxia, incoordination, weak, hunched posture, thin and/or runted. The 49 day cohort homozygous mutant mice also had decreased body weights and body lengths at necropsy, and a homozygous mutant mouse had a decreased body weight, body length, and body weight/body length ratio at mouse metrics. Homozygous mutant mice do not survive to reach the 90 day cohort (see the mouse ID numbers for home cage observations, mouse metrics, clinical chemistry and hematology summaries), consistent with juvenile lethality.

Gene 801
Behavior

There were no significant differences detected 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 seven behavioral tasks: Open field test, Tail suspension test, Rotarod test, Startle response/PPI test, Tail flick test, Hot plate test, and Metrazol test.

Mouse ID numbers are as follows for the N1 generation:
10 heterozygous mutant males (195140, 195142, 195143, 202014, 202016, 203874, 203876, 211286, 212729, 212733)
6 wild-type control males (170024, 195141, 202015, 203880, 211284, 211285)

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 heterozygous mutant mice were produced by intercrossing F1N1 heterozygous males and females.

Behavior Findings:
There were no genotype-related or biologically significant differences noted between heterozygous mutant and wild-type control mice for any of the parameters evaluated during behavior testing.

Gene 801
Expression Summary

RT-PCR Summary:
RNA transcripts are detectable brain, cortex, subcortical region, cerebellum, brainstem, olfactory bulb, spinal cord, eye, heart, skin, tongue and testis.

No RNA transcripts are detectable in Harderian gland, lung, liver, pancreas, kidney, spleen, thymus, lymph nodes, bone marrow,  gallbladder, urinary bladder, pituitary gland, adrenal gland, salivary gland, skeletal muscle, stomach, small intestine, large intestine, cecum, epididymis, seminal vesicle, coagulating gland, prostate gland, ovary, uterus and white fat.

LacZ Summary:
LacZ (beta-galactosidase) expression is detectable in brain, spinal cord and eye.

Expression:
Brain
In wholemount staining strong lacZ expression is detectable in olfactory bulb, cortex, thalamus, superior colliculus and cerebellum. On coronal sections strongest lacZ expression is detectable in thalamus and Purkinje cells. Further in cerebrum, expression is detectable in cortex, amygdala, hippocampus, caudate putamen, lateral septal nuclei and nuclei of the horizontal and vertical limb of the diagonal band. In cerebellum weak to moderate lacZ expression is detectable in many nuclei of the molecular layer. In brainstem some nuclei express lacZ strongly. 

Spinal cord
Weak lacZ expression is detectable in dorsal horns.

Eyes
LacZ expression is detectable in the inner nuclear and ganglion cell layer of the retina.

No Expression: 
LacZ expression is not detected in sciatic nerve, Harderian glands, thymus, spleen, lymph nodes, bone marrow, aorta, heart, lung, liver, gallbladder, pancreas, kidney, urinary bladder, trachea, larynx, esophagus, thyroid gland, parathyroid gland, pituitary gland, adrenal glands, salivary glands, tongue, skeletal muscle, skin, male and female reproductive systems.

Gene 801
Densitometry

Changes related to genotype:  

• Decreased tissue area and total tissue mass in female and male homozygous mice
• Decreased bone mineral density and bone mineral content in female homozygous mice

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

49 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (144361, 155585, 155594)
3 homozygous mutant males (150805, 154594, 155593)
3 heterozygous mutant females (165579, 165580, 165581)
3 heterozygous mutant males (165589, 165591, 165592)
2 wild-type control females (144358, 144359)
2 wild-type control males (150806, 150807)

300 Day Cohort Mouse ID numbers are as follows:
2 heterozygous mutant females (187376, 189486)
2 heterozygous mutant males (189474, 189476)
2 wild-type control females (187375, 194107)
2 wild-type control males (187372, 187373)

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:

When compared with age- and gender-matched wild-type control mice, in the 49 day cohort three homozygous mutant female mice (144361, 155585, 155594) had lower BMC, bone area, tissue area, total tissue mass.  Three 49 day cohort heterozygous mutant male mice (150805, 154594, 155593) had lower tissue area and total tissue mass.

Certain densitometric differences between mice, including fat %, are present that occasionally occur spontaneously in this age group. In this target, two 49 day cohort homozygous mutant females (155585, 155594) and one 49 day cohort heterozygous mutant female (165581) had lower % fat, one 49 day cohort heterozygous mutant male (154594) had higher % fat, and two 300 day cohort heterozygous mutant males had higher fat %. We are not reporting these findings as phenotypic changes, but we present them 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 801
Histopathology

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

Tissues from the following mice were evaluated histologically.

49 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (144361, 155585, 155594)
3 homozygous mutant males (150805, 154594, 155593)
3 heterozygous mutant females (165579, 165580, 165581)
3 heterozygous mutant males (165589, 165591, 165592)
2 wild-type control females (144358, 144359)
2 wild-type control males (150806, 150807)

300 Day Cohort Mouse ID numbers are as follows:
2 heterozygous mutant females (187376, 189486)
2 heterozygous mutant males (189474, 189476)
2 wild-type control females (187375, 194107)
2 wild-type control males (187372, 187373)

No Significant Abnormalities:
Tissues examined and considered to have no genotypically significant abnormality: brain, 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. For example, a 300 day cohort heterozygous mutant male (189474) has lymph node involvement by lymphoma. A 300 day cohort heterozygous mutant male (189476) has a bronchiolo-alveolar adenoma of the lung. These findings are 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 a nonspecific etiology. They are not considered to be genotype related.

Gene 801
Necropsy

Changes related to genotype:

• Decreased body weights and decreased body lengths in 49 day cohort homozygous mutant 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 are as follows:
3 homozygous mutant females (144361, 155585, 155594)
3 homozygous mutant males (150805, 154594, 155593)
3 heterozygous mutant females (165579, 165580, 165581)
3 heterozygous mutant males (165589, 165591, 165592)
2 wild-type control females (144358, 144359)
2 wild-type control males (150806, 150807)

300 Day Cohort Mouse ID numbers are as follows:
2 heterozygous mutant females (187376, 189486)
2 heterozygous mutant males (189474, 189476)
2 wild-type control females (187375, 194107)
2 wild-type control males (187372, 187373)

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, feces, 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:

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, except as specifically noted above. 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 a nonspecific etiology. A 49 day cohort wild-type control male (150807) had only one kidney; the right kidney was absent. A 300 day cohort wild-type female (187375) was reported to have a nodule in the renal fat for which there was no histopathological correlate. These findings were not considered to be genotype related.

Body and Organ Weight Findings:
The body lengths and body weights were lower in the 49 day cohort homozygous mutant mice compared to age- and gender-matched heterozygous mutant mice and wild-type control mice.  While organ weights were also lower in the homozygous mutant mice, there were no significant differences organ weight/body weight ratios in the homozygous mutant mice compared to the ratios noted in heterozygous mutant mice and wild-type control mice.

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 801
Clinical Chemistry

There were no significant differences detected in the homozygous mutant and heterozygous mutant mice when compared with age- and gender-matched wild-type control mice.The data are compiled from the N0F2 and N1F2 generations.

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

49 Day Cohort Mouse ID numbers are as follows:
4 homozygous mutant females (155594, 173750, 182060, 195138)
4 homozygous mutant males (150805, 163286, 170037, 170038)
3 heterozygous mutant females (165579, 165580, 165581)
3 heterozygous mutant males (165589, 165591, 165592)
2 wild-type control females (144358, 144359)
2 wild-type control males (150806, 150807)

90 Day Cohort Mouse ID numbers are as follows:
4 heterozygous mutant females (187376, 189486, 194114, 195136)
4 heterozygous mutant males (189474, 189475, 189476, 189477)
4 wild-type control females (187375, 194116, 195139, 195773)
4 wild-type control males (187372, 187373, 194110, 217726)

180 Day Cohort Mouse ID numbers are as follows:
4 heterozygous mutant females (187376, 194114, 194115, 195136)
3 heterozygous mutant males (189474, 189476, 195770)
3 wild-type control females (187375, 194116, 195139)
4 wild-type control males (187372, 187373, 194110, 217726)

300 Day Cohort Mouse ID numbers are as follows:
2 heterozygous mutant females (187376, 189486)
2 heterozygous mutant males (189474, 189476)
2 wild-type control females (187375, 194109)
2 wild-type control males (187372, 187373)

Values for the various analytes evaluated were generally similar between homozygous mutant and 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 801
Hematology


There were no significant differences detected in the homozygous mutant and 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. The data are compiled from the N0F2 and N1F2 generations.

49 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (173751, 195138, 213283)
5 homozygous mutant males (155593, 165584, 165585, 170037, 189479)
3 heterozygous mutant females (165579, 165580, 165581)
3 heterozygous mutant males (165591, 182063, 182064)
2 wild-type control females (144359, 154592)
2 wild-type control males (150806, 150807)

90 Day Cohort Mouse ID numbers are as follows:
3 heterozygous mutant females (189486, 194114, 194115)
4 heterozygous mutant males (189474, 189475, 189476, 189477)
4 wild-type control females (187375, 194116, 195139, 195773)
5 wild-type control males (187372, 187373, 187374, 194110, 217726)

180 Day Cohort Mouse ID numbers are as follows:
5 heterozygous mutant females (187376, 189486, 194114, 194115, 195136)
4 heterozygous mutant males (189474, 189475, 189476, 189477)
3 wild-type control females (187375, 195139, 195773)
4 wild-type control males (187372, 187373, 194110, 217726)

300 Day Cohort Mouse ID numbers are as follows:
2 heterozygous mutant females (187376, 189486)
2 heterozygous mutant males (189474, 189476)
2 wild-type control females (187375, 194107)
2 wild-type control males (187372, 187373)

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 801
Physical Examination

Changes related to genotype:

• 49 day cohort:
• Locomotion: Ataxia and incoordination
• Runted
• Thin body habitus

The following mice were evaluated by physical examination.

49 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (144361, 155585, 155594)
3 homozygous mutant males (150805, 154594, 155593)
3 heterozygous mutant females (165579, 165580, 165581)
3 heterozygous mutant males (165589, 165591, 165592)
2 wild-type control females (144358, 144359)
2 wild-type control males (150806, 150807)

300 Day Cohort Mouse ID numbers are as follows:
2 heterozygous mutant females (187376, 189486)
2 heterozygous mutant males (189474, 189476)
2 wild-type control females (187375, 194107)
2 wild-type control males (187372, 187373)

Mice were examined for the following observables: 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 appropriate gender.)

When compared to age- and gender-matched wild-type control mice, in the 49 day cohort three homozygous mutant female mice (144361, 155585, 155594) and three homozygous mutant male mice (150805, 154594, 155593) exhibited ataxic locomotion and lack of coordination. Additionally, one homozygous mutant female mouse (144361) and two homozygous mutant male mice (150805, 154594) were weak. The general appearance of a homozygous mutant male mouse (154594) was runted with a hunched posture, and a homozygous mutant female mouse (155594) was thin.  These findings correlate with those reported during home cage observations.

Individual homozygous mutant and heterozygous mutant mice had only occasional other minor differences in observed physical features compared to wild-type control mice, other than those findings noted above. For example, a wild-type control male (150806) had a deformity of the left forelimb, resulting in lameness.  These findings are considered to represent individual variability, background features occasionally seen in this strain of mice, findings due to spontaneous disease, age-related findings, procedural artifacts, and/or findings of a nonspecific etiology. However, none of these differences was regarded as biologically significant or genotype related.

Gene 801
Aging Metrics

Changes related to genotype:

• Decreased body weight, body length, and body weight/body length ratio in a 49 day cohort homozygous mutant male. 

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:
1 homozygous mutant male (211279)
5 heterozygous mutant females (187376, 189486, 194114, 194115, 195136)
4 heterozygous mutant males (189474, 189475, 189476, 189477)
4 wild-type control females (187375, 194116, 195139, 195773)
5 wild-type control males (187372, 187373, 187374, 194110, 217726)

90 Day Cohort Mouse ID numbers are as follows:
5 heterozygous mutant females (187376, 189486, 194114, 194115, 195136)
4 heterozygous mutant males (189474, 189475, 189476, 189477)
4 wild-type control females (187375, 194116, 195139, 195773)
5 wild-type control males (187372, 187373, 187374, 194110, 217726)

180 Day Cohort Mouse ID numbers are as follows:
5 heterozygous mutant females (187376, 189486, 194114, 194115, 195136)
4 heterozygous mutant males (189474, 189475, 189476, 189477)
4 wild-type control females (187375, 194116, 195139, 195773)
4 wild-type control males (187372, 187373, 194110, 217726)

300 Day Cohort Mouse ID numbers are as follows:
3 heterozygous mutant females (194114, 194115, 195136)
1 heterozygous mutant male (189477)
1 wild-type control female (195139)
1 wild-type control male (194110)

Body Weight and Length Findings:

When compared to age- and gender-matched wild-type control mice, body weight, body length, and body weight/body length ratio were lower in a 49 day cohort homozygous mutant male (211279). These findings correlate with the findings at necropsy.

Other 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 801
Home Cage Observations

Changes related to genotype:

• Movement
• Ataxia/lack of coordination, stumbling, paddling movements, and/or hypoactivity, 20 and 49 day cohorts.
• General
• Hunched posture and thin, 20 day cohort homozygous mutant male.

Home cage observations are collected by animal care personnel over the lifetime of the animals.  The observables are similar to those found in the physical examination.

The following mice have home cage observations:

20 Day Cohort Mouse ID numbers are as follows:
11 homozygous mutant females (155585, 155594, 165583, 173750, 173751, 182060, 195138, 196706, 196707, 211283, 213283)
18 homozygous mutant males (150805, 154594, 155593, 163286, 165576, 165584, 165585, 170025, 170026, 170037, 170038, 179569, 180429, 196700, 196701, 211279, 213284, 213357)

49 Day Cohort Mouse ID number is as follows:
1 homozygous mutant female (144361)

Twenty day cohort homozygous mutant mice and a 49 day cohort homozygous mutant female exhibited movement disorders, including the following findings: ataxia/lack of coordination, stumbling, paddling movements, and/or hypoactivity. A 20 day cohort homozygous mutant male (179569) was also reported to be thin and have a hunched posture. These findings correlate with observations reported at physical examination.