Gene: 1443	Name: Acvrl1	Family: Kinase	Subfamily: Serine/Threonine Kinas...	Accession: L48015	GI: 1408066

Gene 1443
Summary of Phenotypic Analysis

Changes related to genotype:

• Lethality: Homozygous embryos identified at E10.5 were resorping

Weaned progeny from the heterozygous matings were genotyped, however no homozygous mice were detected by PCR. Data derived from examination of embryonic development suggest that the mutation causes embryonic death at E9.5 to E10.5.

There were no significant differences 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 heterozygous mutant mice were produced by intercrossing F1 heterozygous males and females.

Wild-type control mice and heterozygous 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
• Embryonic development
• Densitometry
• Behavioral tests 
• Aging studies

Gene 1443
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 (242221, 242222, 247863, 250791, 256890, 256893, 265366, 265383, 265386, 274155)
12 wild-type control males (242219, 242220, 247858, 247864, 250792, 256891, 256892, 256894, 265397, 265399, 274158, 274159)

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 1443
Expression Summary

RT-PCR Summary:
The highest levels of RNA transcripts are detectable in lung.

Lower levels of RNA transcripts are also detectable in all other tissues analyzed: brain, cortex, subcortical region, cerebellum, brainstem, olfactory bulb, spinal cord, eye, Harderian glands, heart, liver, pancreas, kidney, spleen, thymus, lymph nodes, bone marrow, skin, gallbladder, 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, ovaries, uterus and white fat.

LacZ Summary:
In many tissues, expression is detectable in blood vessels.

LacZ (beta-galactosidase) expression is detectable in all tissues examined: brain, spinal cord, sciatic nerve, eyes, 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, skeletal muscle, skin, male and female reproductive systems.  

Expression:
Brain
In wholemount staining strong lacZ expression is detectable throughout the brain and in blood vessels and choroids plexus.

On coronal sections of cerebrum, cerebellum and brainstem very strong lacZ expression is detectable in chorid plexus, blood vessels and cells scattered throughout the sections.

Spinal cord
Very strong lacZ expression is detectable in cells scattered throughout the spinal cord and in blood vessels.

Sciatic Nerve
Moderate lacZ expression is detectable in a few cells.

Eyes
Very strong lacZ expression is detectable in cells scattered throughout the retina and pigment layer and in blood vessels.  Strong lacZ expression is detectable in ciliary body, iris and extraocular muscles.

Thymus
Strong lacZ expression is detectable in cells scattered throughout the thymus and surrounding adipose tissue.  Many of the cells appear to be blood vessels.

Spleen
Strong to very strong lacZ expression is detectable in cells scattered throughout the spleen.  Very strong lacZ expression is also detectable in capsule and blood vessels.  Further, weak lacZ expression is detectable in many cells of trabeculae.

Lymph Nodes
Very strong lacZ expression is detectable in cells scattered throughout the lymph node and perinodal fat.  Very strong lacZ expression is also detectable in blood vessels.

Bone Marrow Smear
Strong lacZ expression is detectable in some cells on the bone marrow smear from the female.

Aorta
Strong lacZ expression is detectable in both smooth muscle cells and endothelial cells as well as in some cells of the periaortic adipose tissue.

Heart
Very strong lacZ expression is detectable in many cells of the ventricles, some cells in the atria and valves, and in blood vessels.

Lung
Very strong lacZ expression is detectable in practically all cells of the alveoli and blood vessels.

Liver
Moderate lacZ expression is detectable in cells scattered throughout the liver.  Very strong lacZ expression is detectable in many cells of blood vessels.

Gallbladder
Very strong lacZ expression is detectable in some cells scattered throughout the gallbladder and in blood vessels.

Pancreas
Very strong lacZ expression is detectable in cells scattered throughout the pancreas including some cells in the Islets of Langerhans.  Further, very strong lacZ expression is detectable in blood vessels.

Kidney
Very strong lacZ expression is detectable in many cells of the glomeruli and blood vessels and in some cells of the medulla.  Strong lacZ expression is detectable in some cells of papilla and capsule.

Urinary Bladder
Strong lacZ expression is detectable in muscularis, lamina propria and blood vessels.

Trachea
Very strong lacZ expression is detectable in the lamina propria.

Larynx
Very strong lacZ expression is detectable some cells of the lamina propria, submucosal glands and surrounding musculature.

Esophagus
Very strong lacZ expression is detectable in lamina propria and and some cells of the muscularis.

Thyroid Gland
Very strong lacZ expression is detectable in many cells.

Pituitary Gland
Strong lacZ expression is detectable some cells of pars nervosa and in a few cells of pars intermedia.  Weak to moderate lacZ expression is detectable in some cells of pars distalis.

Adrenal Glands
Moderate to strong lacZ expression is detectable in cells scattered throughout the cortex and a few cells in the medulla.  Very strong lacZ expression is detectable in endothelial cells of blood vessels and in some cells of the pericapsular adipose tissue.

Salivary Glands
Very strong lacZ expression is detectable in cells scattered throughout the salivary gland and the surrounding adipose tissue and in endothelial cells of blood vessels.

Skeletal Muscle
Strong lacZ expression is detectable in some cells and in blood vessels.

Skin
Very strong lacZ expression is detectable in cells scattered throughout the dermis and in blood vessels.

Ear, external
Very strong lacZ expression is detectable in cells scattered throughout the external ear and in blood vessels.

Male Reproductive Systems
Testis
Strong lacZ expression is detectable in some interstitial cells and blood vessels.  Weak lacZ expression is detectable in some spermatogenic and peritubular cells of the seminiferous tubules.

Prostate Gland
Strong lacZ expression is detectable in some connective tissue cells and in blood vessels.

Female Reproductive Systems
Ovary
Strong lacZ expression is detectable in cells scattered throughout the ovaries.  Very strong lacZ expression is detectable in blood vessels.

Uterus
Strong lacZ expression is detectable in cells scattered throughout the endometrial stroma and myometrium and in blood vessels.

 

Gene 1443
Densitometry
 
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 by dual-energy x-ray absorptiometry.

49 Day Cohort Mouse ID numbers are as follows:
3 heterozygous mutant females (209561, 209562, 215228)
3 heterozygous mutant males (209557, 215227, 217304)
2 wild-type control females (209560, 217301)
2 wild-type control males (209559, 218937)

300 Day Cohort Mouse ID numbers are as follows:
2 heterozygous mutant females (237298, 242224)
2 heterozygous mutant males (237286, 237288)
2 wild-type control females (237299, 247867)
2 wild-type control males (237287, 237297)

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 were considered to represent background differences occasionally seen in this strain of mice, differences due to spontaneous disease, age-related changes, differences due to procedural artifacts, and/or differences of a nonspecific etiology. They were not considered to be genotype related.

Gene 1443
Histopathology

There were no significant differences detected in the 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 heterozygous mutant females (209561, 209562, 215228)
3 heterozygous mutant males (209557, 215227, 217304)
2 wild-type control females (209560, 217301)
2 wild-type control males (209559, 218937)

300 Day Cohort Mouse ID numbers are as follows:
1 heterozygous mutant female (237298)
1 heterozygous mutant male (237286)

No Significant Abnormalities:

The following tissues were examined and considered to have no genotype-related 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 were present in some tissues. For example, one 300 day heterozygous mutant male (237286) had bone marrow involvement by histiocytic lymphoma. 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.

Gene 1443
Necropsy

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 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 heterozygous mutant females (209561, 209562, 215228)
3 heterozygous mutant males (209557, 215227, 217304)
2 wild-type control females (209560, 217301)
2 wild-type control males (209559, 218937)

300 Day Cohort Mouse ID numbers are as follows:
2 heterozygous mutant females (237298, 242224)
2 heterozygous mutant males (237286, 237288)

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 the 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 related to genotype.

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 1443
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:
3 heterozygous mutant females (209561, 215228, 223005)
3 heterozygous mutant males (209557, 215227, 217306)
2 wild-type control females (209560, 217303)
2 wild-type control males (209559, 218937)

90 Day Cohort Mouse ID numbers are as follows:
4 heterozygous mutant females (237298, 242223, 242224, 242225)
4 heterozygous mutant males (237286, 237288, 237294, 237295)
4 wild-type control females (237299, 247867, 247869, 253797)
3 wild-type control males (237287, 237296, 237297)

180 Day Cohort Mouse ID numbers are as follows:
3 heterozygous mutant females (237298, 242224, 242225)
1 heterozygous mutant male (237288)
1 wild-type control female (237299)
4 wild-type control males (237287, 237297, 263332, 298990)

300 Day Cohort Mouse ID numbers are as follows:
2 heterozygous mutant females (237298, 242224)
2 heterozygous mutant males (237286, 237288)
2 wild-type control females (237299, 247867)
2 wild-type control males (237287, 237297)

Values for the various analytes evaluated were generally similar between heterozygous mutant and wild-type control mice. Although variations in clinical chemistry values were present in some mice, they were not related to genotype and, thus, were not considered phenotypically relevant.

Gene 1443
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:
3 heterozygous mutant females (209561, 215230, 220519)
3 heterozygous mutant males (209557, 215227, 217305)
2 wild-type control females (209560, 217310)
2 wild-type control males (209559, 218937)

90 Day Cohort Mouse ID numbers are as follows:
4 heterozygous mutant females (237298, 242223, 242224, 242225)
3 heterozygous mutant males (237286, 237288, 237294)
4 wild-type control females (237299, 247867, 247869, 253797)
4 wild-type control males (237287, 237296, 237297, 247845)

180 Day Cohort Mouse ID numbers are as follows:
4 heterozygous mutant females (237298, 242223, 242224, 242225)
3 heterozygous mutant males (237286, 237288, 237294)
4 wild-type control females (237299, 247867, 247869, 253797)
4 wild-type control males (237287, 237297, 247845, 298990)

300 Day Cohort Mouse ID numbers are as follows:
2 heterozygous mutant females (237298, 253800)
2 heterozygous mutant males (237286, 237288)
2 wild-type control females (237299, 247867)
2 wild-type control males (237287, 237297)

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

 

Gene 1443
Physical Examination

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 by physical examination.

49 Day Cohort Mouse ID numbers are as follows:
3 heterozygous mutant females (209561, 209562, 215228)
3 heterozygous mutant males (209557, 215227, 217304)
2 wild-type control females (209560, 217301)
2 wild-type control males (209559, 218937)

300 Day Cohort Mouse ID numbers are as follows:
2 heterozygous mutant females (237298, 242224)
2 heterozygous mutant males (237286, 237288)
2 wild-type control females (237299, 247867)
2 wild-type control males (237287, 237297)

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 heterozygous mutant mice had only occasional minor differences in observed physical features compared to 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 1443
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:
4 heterozygous mutant females (237298, 242223, 242224, 242225)
4 heterozygous mutant males (237286, 237288, 237294, 237295)
4 wild-type control females (237299, 247867, 247869, 253797)
5 wild-type control males (237287, 237296, 237297, 247845, 298990)

90 Day Cohort Mouse ID numbers are as follows:
4 heterozygous mutant females (237298, 242223, 242224, 242225)
4 heterozygous mutant males (237286, 237288, 237294, 237295)
4 wild-type control females (237299, 247867, 247869, 253797)
4 wild-type control males (237287, 237296, 237297, 247845)

180 Day Cohort Mouse ID numbers are as follows:
4 heterozygous mutant females (237298, 242223, 242224, 242225)
4 heterozygous mutant males (237286, 237288, 237294, 237295)
4 wild-type control females (237299, 247867, 247869, 253797)
4 wild-type control males (237287, 237297, 247845, 298990)

300 Day Cohort Mouse ID numbers are as follows:
3 heterozygous mutant females (237298, 242225, 253800)
4 heterozygous mutant males (237286, 237288, 247828, 247829)
4 wild-type control females (237299, 247867, 247869, 253797)
4 wild-type control males (237287, 237297, 247845, 263332)

Body Weight and Length Findings:

When compared to age- and gender-matched wild-type control mice, body weights were low in two 49 day heterozygous mutant males (237288, 237294), one 90 day heterozygous mutant male (237295) and two 180 day heterozygous mutant males (237294, 237295). We have not reported these findings as phenotypic changes but we have presented them here for your consideration.

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  1443
Summary of Embryonic Development

Resorping homozygous mutant embryos were identified at E10.5.

Embryos were isolated at 8.5 to 14.5  days post coitum.  Grossly normal homozygous offspring were detected by PCR at E8.5.   However by E10.5 all homozygous embryos isolated were resorping.  These data suggest that the mutation causes embryonic death at E9.5 to E10.5.

Embryos were isolated at E8.5 to E14.5
Four litters was examined comprising of 29 embryos, resorptions and partial resorptions, of which 19 were successfully genotyped.

Litter	Embryonic stage	+/+	+/-	-/-	complete resorption or unknown
1	8.5	0	3	3	0
2	10.5	1	1	1	4
3	10.5	3	3	3	1
4	14.5	1	0	0	5