Gene: 600	Name: Htr4	Family: GPCR	Subfamily: Serotonin	Accession: NM_008313	GI: 6680324

Gene 600
Summary of Phenotypic Analysis

Change related to genotype:

• Serum Chemistry: Increased blood urea nitrogen (BUN)

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 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 differential cell counts
• Clinical chemistry panels
• Densitometry 
• Behavioral tests
• Fertility
• Aging studies

When compared to age- and gender-matched wild-type control mice and the historical reference ranges, most homozygous mutant males and females had an increased level of blood urea nitrogen (BUN) at 49, 90, 180 and 300 Days of age.

Gene 600
Behavior

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

Homozygous 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 homozygous mutant males (155195, 146556, 146533, 151435, 143751, 146542, 146554, 151430, 168212, 151459)
11 wild-type control males (151433, 143748, 146553, 151456, 168189, 146530, 146551, 151458, 151432, 151436, 168188)

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.

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

Gene 600

Fertility

Both homozygous mutant males and females were fertile.  Their progeny were viable until weaning.

Three homozygous mutant mice of each gender were set up in a fertility mating one on one with each other at seven to ten weeks of age.  The number of pups born from three litters was recorded.  Three weeks later, the live pups were counted and weaned.

Mouse ID numbers are as follows:

3 homozygous mutant males (142442, 142443, 142444)

3 homozygous mutant females (146547, 146548, 151412)

Gene 600
Expression Summary

RT-PCR Summary:
RNA transcripts are detectable in brain, cortex, subcortical region, cerebellum, brainstem, spinal cord, gallbladder, urinary bladder, stomach, small intestine, large intestine, cecum and seminal vesicle.

No RNA transcripts are detectable in: olfactory bulb, eye, Harderian gland, heart, lung, liver, pancreas, kidney, spleen, thymus, lymph nodes, bone marrow, skin, pituitary gland, adrenal gland, salivary gland, skeletal muscle, tongue, testis, epididymis, coagulating gland, prostate gland, ovary, uterus and white fat

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

Expression:
Brain
In wholemount staining strong lacZ expression is detectable in choroid plexus. Weaker X-Gal signals are detectable in brainstem. On coronal sections distinct cells in brainstem express lacZ.

Spinal cord
Faint to moderate lacZ expression is detectable in gray matter.

Male Reproductive System
Testis
A few spermatogenic cells in seminiferous tubules express lacZ weakly.

Female Reproductive System
Ovary
Scattered, faint X-Gal staining is detectable in the ovary.

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

Gene 600
Densitometry

There were no significant differences detected in the homozygous 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:
2 homozygous mutant females (142435, 143741)
3 homozygous mutant males (142396, 142397, 142431)
2 wild-type control females (142418, 142425)
2 wild-type control males (143745, 143746)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (151411, 151428)
2 homozygous mutant males (143743, 163656)
2 wild-type control females (151408, 151425)
2 wild-type control males (143747, 163665)

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

Densitometric Findings:

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

 

Gene 600
Histopathology

There were no significant differences detected in the homozygous 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 (142404, 142435, 143741)
3 homozygous mutant males (142396, 142397, 142431)
2 wild-type control females (142418, 142425)
4 wild-type control males (143745, 143746, 175373, 175386)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (151411, 151428)
2 homozygous mutant males (143743, 163656)
2 wild-type control females (151408, 151425)
2 wild-type control males (143747, 163665)

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. These findings are considered to represent background lesions occasionally seen in this strain of mice, lesions due to spontaneous disease, age-related lesions, and/or lesions of a nonspecific etiology. They are not considered to be genotype related.

Gene 600
Necropsy

There were no significant differences detected in the homozygous 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 are as follows:
3 homozygous mutant females (142404, 142435, 143741)
3 homozygous mutant males (142396, 142397, 142431)
2 wild-type control females (142418, 142425)
4 wild-type control males (143745, 143746, 175373, 175386)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (151411, 151428)
2 homozygous mutant males (143743, 163656)
2 wild-type control females (151408, 151425)
2 wild-type control males (143747, 163665)

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:

There were no 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, and/or lesions of a nonspecific etiology. They were not considered to be genotype related.

Body and Organ Weight Findings:

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 600
Clinical Chemistry


Change related to genotype:

• Increased BUN

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

49 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (142435, 143741, 163755)
3 homozygous mutant males (142396, 142397, 142431)
2 wild-type control females (142418, 143738)
4 wild-type control males (143745, 143746, 175373, 175386)

90 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (151448, 151449, 155187)
4 homozygous mutant males (163656, 163666, 163772, 165362)
2 wild-type control females (151443, 151446)
4 wild-type control males (143747, 163665, 173839, 173840)

180 Day Cohort Mouse ID numbers are as follows:
5 homozygous mutant females (151411, 151428, 151441, 151448, 151449)
5 homozygous mutant males (143743, 163656, 163772, 169949, 169951)
4 wild-type control females (151408, 151425, 151443, 151446)
4 wild-type control males (143747, 163665, 163750, 163771)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (151411, 151428)
2 homozygous mutant males (143743, 163656)
2 wild-type control females (151408, 151425)
2 wild-type control males (143747, 163665)

When compared to historical reference ranges, several homozygous mutant females and  homozygous mutant males had an increased level of BUN (blood urea nitrogen) in 49 Day, 90 Day ,180 Day and 300 Day as well. 

Values for the other various analytes evaluated were generally similar between homozygous mutant and wild-type control mice.

Gene 600
Hematology


There were no significant differences detected in the homozygous 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:
3 homozygous mutant females (142404, 142435, 143741)
3 homozygous mutant males (142396, 142397, 142431)
2 wild-type control females (142418, 143739)
4 wild-type control males (143746, 151415, 175373, 175386)

90 Day Cohort Mouse ID numbers are as follows:
5 homozygous mutant females (151411, 151428, 151441, 151448, 151449)
4 homozygous mutant males (143743, 163656, 163772, 165362)
3 wild-type control females (151408, 151425, 151443)
4 wild-type control males (143747, 163665, 163771, 165364)

180 Day Cohort Mouse ID numbers are as follows:
4 homozygous mutant females (151411, 151428, 151441, 151448)
4 homozygous mutant males (143743, 163656, 163772, 165363)
4 wild-type control females (151408, 151425, 151443, 151446)
4 wild-type control males (143747, 163665, 163750, 163771)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (151411, 151428)
2 homozygous mutant males (143743, 163656)
2 wild-type control females (151408, 151425)
2 wild-type control males (143747, 163665)

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 600
Physical Examination

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

The following mice were evaluated by physical examination.

49 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (142404, 142435, 143741)
3 homozygous mutant males (142396, 142397, 142431)
2 wild-type control females (142418, 142425)
2 wild-type control males (143745, 143746)

300 Day Cohort Mouse ID numbers are as follows:
2 homozygous mutant females (151411, 151428)
2 homozygous mutant males (143743, 163656)
2 wild-type control females (151408, 151425)
2 wild-type control males (143747, 163665)

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, feces color, feces exam, 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 exam, mice in cage, respiration, skin appearance, snout, swelling - joints, tail, teeth color, teeth length, urine, urine color, urine exam and whiskers (gender specific observables apply to appropriate gender).

Individual homozygous mutant mice had only occasional minor differences in observed physical features compared to wild-type control mice. 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, and/or findings of a nonspecific etiology. However, none of these differences was regarded as biologically significant or genotype related.

Gene 600
Aging Metrics

There were no significant differences detected in the homozygous 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 homozygous mutant females (151411, 151428, 151441, 151448, 151449)
3 homozygous mutant males (143743, 163772, 165362)
4 wild-type control females (151408, 151425, 151443, 151446)
4 wild-type control males (143747, 163665, 163771, 165364)

90 Day Cohort Mouse ID numbers are as follows:
5 homozygous mutant females (151411, 151428, 151441, 151448, 151449)
4 homozygous mutant males (143743, 163656, 163772, 165362)
4 wild-type control females (151408, 151425, 151443, 151446)
4 wild-type control males (143747, 163665, 163771, 165364)

180 Day Cohort Mouse ID numbers are as follows:
5 homozygous mutant females (151411, 151428, 151441, 151448, 151449)
4 homozygous mutant males (143743, 163656, 163772, 165363)
4 wild-type control females (151408, 151425, 151443, 151446)
4 wild-type control males (143747, 163665, 163750, 163771)

300 Day Cohort Mouse ID numbers are as follows:
3 homozygous mutant females (151441, 151448, 151449)
3 homozygous mutant males (143743, 163772, 165363)
2 wild-type control females (151443, 151446)
3 wild-type control males (143747, 163750, 163771)

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.