Protein recoding by ADAR1-mediated RNA editing is not essential for normal development and homeostasis

Background Adenosine-to-inosine (A-to-I) editing of dsRNA by ADAR proteins is a pervasive epitranscriptome feature. Tens of thousands of A-to-I editing events are defined in the mouse, yet the functional impact of most is unknown. Editing causing protein recoding is the essential function of ADAR2, but an essential role for recoding by ADAR1 has not been demonstrated. ADAR1 has been proposed to have editing-dependent and editing-independent functions. The relative contribution of these in vivo has not been clearly defined. A critical function of ADAR1 is editing of endogenous RNA to prevent activation of the dsRNA sensor MDA5 (Ifih1). Outside of this, how ADAR1 editing contributes to normal development and homeostasis is uncertain. Results We describe the consequences of ADAR1 editing deficiency on murine homeostasis. Adar1 E861A/E861A Ifih1 -/- mice are strikingly normal, including their lifespan. There is a mild, non-pathogenic innate immune activation signature in the Adar1 E861A/E861A Ifih1 -/- mice. Assessing A-to-I editing across adult tissues demonstrates that outside of the brain, ADAR1 performs the majority of editing and that ADAR2 cannot compensate in its absence. Direct comparison of the Adar1 -/- and Adar1 E861A/E861A alleles demonstrates a high degree of concordance on both Ifih1 +/+ and Ifih1 -/- backgrounds, suggesting no substantial contribution from ADAR1 editing-independent functions. Conclusions These analyses demonstrate that the lifetime absence of ADAR1-editing is well tolerated in the absence of MDA5. We conclude that protein recoding arising from ADAR1-mediated editing is not essential for organismal homeostasis. Additionally, the phenotypes associated with loss of ADAR1 are the result of RNA editing and MDA5-dependent functions. Electronic supplementary material The online version of this article (doi:10.1186/s13059-017-1301-4) contains supplementary material, which is available to authorized users.

Note: (i) Reactive lymph nodes are defined as mild follicular hyperplasia, germinal centre formation and occasional sinus histiocytosis-a common finding in mice (ii) Mild extramedullary hematopoiesis (EMH) identified in the red pulp of all the spleens, a common finding in the mouse. EMH consists of erythroid precursors, myeloid precursors, megakaryocytes or all three. While some degree of extramedullary hematopoiesis is present in normal rodents, especially in mice, increased extramedullary hematopoiesis can result from hematotoxic insult, systemic anemia, and infections elsewhere in the body.
(iii) Sexual dimorphism, under the influence of testosterone exists in the submandibular glands of the mouse. The male glands are larger and weigh almost twice as much as those of the female. In the male, the convoluted (granular) ducts are larger and more prominent than the female, and are lined by large, tall columnar cells, containg abundant eosinophilic intracytoplasmic granules. In the female , these ducts are lined by shorter columnar cells with centrally located nuclei and fewer intra-cytoplasmic granules.

Microscopic Observations
Blood counts: Most readings are within the normal mouse reference intervals. The Hematocrit was elevated (this is likely to be a result of mild dehydration as the RBC count was normal). The platelet count for most samples including the controls was low, worth noting as the MPV was elevated in some cases indicating the presence of large, immature platelets. Please note that mouse platelets readily aggregate and instrument generated platelet counts will underestimate true platelet counts in the presence of platelet clumping. In addition, mouse platelet clumps are counted as eosinophils by some automated hematology analyser. Note that the eosinophil count for all samples was elevated.

Macro Observations
Testes: 5x4x3mm, symmetrical Spleen: 14x4x3mm Kidneys: 12x7x5mm, symmetrical Thymus: 10x9x2mm Heart: 10x7x6mm Brain: 15x11x6mm, symmetrical Pituitary gland identified, macroscopically normal Tail 80mm (straight) Teeth, tongue and oral mucosa unremarkable -Head harvested for evaluation of auditory and vestibular structures Tail suspension test for neurological defects-negative Left hind leg-Bone marrow smear Ears harvested and frozen Left tibia collected and fixed overnight in 10% NBF at room temperature then transferred to 70% ethanol Micro Observations Peripheral blood smear: Examination of the smear showed red blood cells (majority of cells shown), occasional white blood cells including segmented neutrophils and platelets (clumps). No discernible morphological changes or detectable parasites (52853) Marrow smear: Cells observed were readily identified as those from the erythroid and myeloid series. Conspicuous cells from the lymphoid series. Discernible and unremarkable megakaryoblasts (52854).

#380 was used as a male histological control
Testes-Occasional seminiferous tubule with mild testicular degeneration Cecum-Numerous intraluminal protozoa

Macro Observations
Spleen: 12x4x2mm Kidneys: 10x6x5, symmetrical Thymus: 5x7x2mm Heart: 8x6x5mm Brain: 15x10x5mm, symmetrical Pituitary gland identified, macroscopically normal Tail 80mm (straight) Teeth, tongue and oral mucosa unremarkable -Head harvested for evaluation of auditory and vestibular structures Tail suspension test for neurological defects-negative Left hind leg-Bone marrow smear A single ear was harvested and frozen Left tibia & femur collected and fixed overnight in 10% NBF at room temperature then transferred to 70% ethanol

Micro Observations
Peripheral blood smear: Examination of the smear showed red blood cells (majority of cells shown), occasional white blood cells including segmented neutrophils and platelets (clumps). No discernible morphological changes or detectable parasites (52864) Marrow smear: Cells observed were readily identified as those from the erythroid and myeloid series. Conspicuous cells from the lymphoid series. Discernible and unremarkable megakaryoblasts (52863).
#388 was used as a female histological control Peripheral blood smear: Examination of the smear showed red blood cells (majority of cells shown), occasional white blood cells including segmented neutrophils and platelets (clumps). No discernible morphological changes or detectable parasites (52851) Marrow smear: Cells observed were readily identified as those from the erythroid and myeloid series. Conspicuous cells from the lymphoid series. Discernible and unremarkable megakaryoblasts (52852).

Testes/Epididymes
Section shows typical convoluted seminiferous tubules at various stages of cycle surrounded by the tunica albuginea. Within the tubules, unremarkable spermatogenic cells including, Sertoli cells, spermatogonia, developing spermatocytes and spermatids. Section also shows unremarkable vas deferens with typical intraluminal sperm. The architecture of the epididymis is typical, with numerous intraluminal elongated spermatozoa. Section shows unremarkable ventral/coagulating prostate glands with typical intraluminal secretions. Section shows concretions in the prostatic urethra, commonly seen as an incidental finding No lesions of significance (52691) Penis/Preputial gland Section shows typical penile structures including prepuce, glans, corpus cavernosum and urethra. Unremarkable preputial glands including basal and secretory cells. Section shows small portion of mesenteric lymph node with typical nodal histology including cortex, medullary cords and sinuses.
No lesions of significance (52695) Spleen Unremarkable follicular pattern identified with typical red and white pulp micromorphology. Mild extramedullary hematopoiesis identified in the red pulp of the spleen, a common finding in the mouse.
No lesions of significance (52692) Pancreas Section shows representative exocrine tissue (serous acini) and endocrine tissue (islets of Langerhans). (52692) Kidney Section shows a cortex, medulla, and papilla. There is a uniform distribution of Glomeruli and accompanying nephron components and the micromorphology of the convoluted and straight segment tubules was unremarkable. Small clusters of tubular casts were observed in the cortex. These micromorphological changes were considered mild.

Pathology to comment
Adrenal glands Section shows adrenal glands with typical cortex/medulla micromorphology.
No lesions of significance (52701) Salivary glands and Regional lymph nodes Section shows submandibular, sublingual and parotid glands. The regional lymph nodes displayed mild follicular hyperplasia with germinal centre formation, indicating a reactive state.
No lesions of significance (52695)

Comments:
Observation: In the submandibular glands, there appears to be a paucity of serous acini compared to the control male Thyroids Normal lateral lobes of the thyroid gland with typical follicles lined by cuboidal epithelium. Section also includes representative parathyroid gland.

Head
Multiple levels through the head demonstrate dermal appendages, nasal cavity,oral cavity, teeth and tongue including muscle bundles (52876-77). Sections also show unremarkable pituitary gland including pars intermedia, pars distalis and pars nervosa as well and the trigeminal nerve/ganglia (52875).The outer and middle regions of the ear are discernible. The tympanic membrane is intact and the ossicles are unremarkable and include the stapedial annular ligaments (52876-77). Typical components of the inner ear including bony labyrinth, organ of corti, stria vascularis and scala cavities are discernible. Based on multiple levels, the organ of corti is unremarkable with no discernible loss of inner/outer hair cells and typical tectorial membrane (52876-77). The cochlear nerve and spiral ganglion is also demonstrated and based on several levels, there is no reduction in the density of the spiral ganglion cells. Examples of otolith organs can be seen with typical features such as the hair cells and mineral otoliths. The ampulla including the crista ridge with hair cells is discernible (

Tail
Section shows typical tail components including keratinized squamous epithelium, dense regular connective tissue, tendons, bone, intervertebral disc, bone marrow, skeletal muscle, nerves and blood vessels.

No lesions of significance (52781)
Eyes/Harderian glands Section shows eyes with unremarkable retina, cornea, iris, ciliary body, lens, sclera and choroid. Typical branched tubuloalveolar formation of the Harderian gland.
No lesions of significance (52774) Brain Sections were prepared from the standard levels of the brain: Level I: including cortex, corpus callosum, the lateral ventricles,anterior commissure and caudate putamen (Bregma -0.98) Level II: includes the hippocampus, thalamus, amygdala,hypothalamus, lateral and third ventricles (Bregma -1.94) Level III: includes the cerebellum, pons and fourth ventricle  Sections of brain stained with Haematoxylin and Eosin, Luxol Fast Blue appear symmetrical with no ventricular dilation observed, unremarkable meninges and typical lamination. The cerebellum appears symmetrical with typical architecture and Purkinje cells. At this level, there was no evidence of neuronal loss and the myelination appears normal.

Spinal cord
Representative thoracic and lumbar region of spinal cord, vertebral bone, striated muscle, peripheral nerves and bone marrow.

No lesions of significance (52780)
Comments: Neuropathology to comment (Hind leg) Long bone/Bone marrow/Synovial joint/Skeletal muscle Levels through the hind leg show unremarkable long bone, striated muscle, synovial joint and bone marrow including conspicuous megakaryoblasts. The skeletal muscle shows consistent fiber size with peripheral nuclei.
No lesions of significance (52776, 54808) Head Multiple levels through the head demonstrate dermal appendages, nasal cavity,oral cavity, teeth and tongue including muscle bundles (52898). Sections also show unremarkable pituitary gland including pars intermedia, pars distalis and pars nervosa as well and the trigeminal nerve/ganglia (52895).The outer and middle regions of the ear are discernible. The tympanic membrane is intact and the ossicles are unremarkable and include the stapedial annular ligaments (52897, 52898). Typical components of the inner ear including bony labyrinth, organ of corti, stria vascularis and scala cavities are discernible. Based on multiple levels, the organ of corti is unremarkable with no discernible loss of inner/outer hair cells and typical tectorial membrane (52897). The cochlear nerve and spiral ganglion is also demonstrated and based on several levels, there is no reduction in the density of the spiral ganglion cells. Examples of otolith organs can be seen with typical features such as the hair cells and mineral otoliths. The ampulla including the crista ridge with hair cells is discernible (52898) Peripheral blood smear: Examination of the smear showed red blood cells (majority of cells shown), numerous white blood cells including segmented neutrophils and platelets (clumps). No discernible morphological changes or detectable parasites (52856) Marrow smear: Cells observed were readily identified as those from the erythroid and myeloid series. Conspicuous cells from the lymphoid series. Discernible and unremarkable megakaryoblasts (52855).

Testes/Epididymes
Section shows typical convoluted seminiferous tubules at various stages of cycle surrounded by the tunica albuginea. Within the tubules, unremarkable spermatogenic cells including, Sertoli cells, spermatogonia, developing spermatocytes and spermatids. The architecture of the epididymis is typical, with numerous intraluminal elongated spermatozoa. Prostate glands Section shows unremarkable ventral and dorsal lateral prostate glands with typical intraluminal secretions, typical vas deferens and a portion of ureter. Section shows concretions in the prostatic urethra, commonly seen as an incidental finding No lesions of significance (52738) Penis/Preputial gland Section shows typical urethra and unremarkable preputial glands including basal and secretory cells.

No lesions of significance (52740) Urinary Bladder
Unremarkable distended bladder with typical urothelium and detrusor muscle. Comments:

Pathology to comment
Mesenteric lymph node Section shows small portion of mesenteric lymph node with typical nodal histology including cortex, medullary cords and sinuses.
No lesions of significance (52733) Spleen Unremarkable follicular pattern identified with typical red and white pulp micromorphology. Moderate extramedullary hematopoiesis identified in the red pulp of the spleen.

No lesions of significance (52737)
Comments: Pathology to comment Pancreas Section shows representative exocrine tissue (serous acini) and endocrine tissue (islets of Langerhans). (52737) Kidney Section shows a cortex, medulla with a uniform distribution of Glomeruli and accompanying nephron components. The micromorphology of the tubules is unremarkable. (52735) Adrenal glands Section shows one adrenal gland with typical cortex/medulla micromorphology.

No lesions of significance (52735)
Salivary glands and Regional lymph nodes Section shows submandibular, sublingual and parotid glands. The regional lymph nodes show typical nodal histology including cortex, medullary cords and sinuses.
No lesions of significance (52733)

Comments:
Observation: In the submandibular glands, there is a paucity of serous acini compared to the control male Thyroids Unremarkable colloid secreting follicles identified. Section also shows a small sheet-like mass of polygonal cells, characteristic of the parathyroid gland.
No lesions of significance (52746) Trachea/Lungs Section shows typical lung micromorphology demonstrating parenchyma/alveoli, bronchioles, blood vessels and parabronchial lymph node. Trachea with unremarkable mucosal epithelial lining and hyaline cartilage. Oesophagus with typical features including stratified squamous epithelium and discernible intraluminal protozoan aggregates.

No lesions of significance (52746)
Comments: Pathology to comment Thymus Section shows typical medulla/cortex distribution and micromorphology. Tail Section shows typical tail components including keratinized squamous epithelium, dense regular connective tissue, tendons, bone, bone marrow, skeletal muscle, nerves and blood vessels.

No lesions of significance (52743)
Eyes/Harderian glands Section shows eyes with unremarkable retina, cornea, iris, ciliary body, lens, sclera and choroid. Typical branched tubuloalveolar formation of the Harderian gland.
No lesions of significance (52731) Brain Sections were prepared from the standard levels of the brain: Level I: including cortex, corpus callosum, the third and lateral ventricles, choroid plexus and caudate putamen (Bregma -0.58) Level II: includes the hippocampus, thalamus, amygdala,hypothalamus, lateral and third ventricles (Bregma -1.94) Level III: includes the cerebellum, pons and fourth ventricle (Bregma -6.36) Sections of brain stained with Haematoxylin and Eosin, Luxol Fast Blue appear symmetrical with no ventricular dilation observed, unremarkable meninges and typical lamination. The cerebellum appears symmetrical with typical architecture and Purkinje cells. At this level, there was no evidence of neuronal loss and the myelination appeared normal.

Head
Multiple levels through the head demonstrate dermal appendages, nasal cavity,oral cavity, teeth and tongue including muscle bundles (52885-52889). Sections also show unremarkable pituitary gland including pars intermedia, pars distalis and pars nervosa as well and the trigeminal nerve/ganglia (52885).The outer and middle regions of the ear are discernible. The tympanic membrane is intact and the ossicles are unremarkable and include the stapedial annular ligaments (52887-88). Typical components of the inner ear including bony labyrinth, organ of corti, stria vascularis and scala cavities are discernible. Based on multiple levels, the organ of corti is unremarkable with no discernible loss of inner/outer hair cells and typical tectorial membrane (52887). The cochlear nerve and spiral ganglion is also demonstrated and based on several levels, there is no reduction in the density of the spiral ganglion cells. Examples of otolith organs can be seen with typical features such as the hair cells and mineral otoliths (52887-88).  (52770) Penis/Preputial gland Section shows typical penile structures including prepuce, glans, corpus cavernosum and urethra. Unremarkable preputial glands including basal and secretory cells. Comments:

Pathology to comment
Mesenteric lymph node Section shows mesenteric lymph node with typical reactive micromorphology including mild follicular hyperplasia with germinal centre formation, indicating a reactive state.
No lesions of significance (52767) Spleen Unremarkable follicular pattern identified with typical red and white pulp micromorphology. Moderate extramedullary hematopoiesis identified in the red pulp of the spleen.

No lesions of significance (52751)
Comments: Pathology to comment Pancreas Section shows representative exocrine tissue (serous acini) and endocrine tissue (islets of Langerhans). (52751) Kidney Section shows a cortex, medulla, and papilla. There is a uniform distribution of Glomeruli and accompanying nephron components and the micromorphology of the convoluted and straight segment tubules was unremarkable. Mild processing/fixation artefact No lesions of significance (52765) Comments:

Pathology to comment
Adrenal glands Section shows one adrenal gland with typical cortex/medulla micromorphology.
No lesions of significance (52765) Salivary glands and Regional lymph nodes Section shows submandibular, sublingual and parotid glands. The regional lymph nodes displayed mild follicular hyperplasia with germinal centre formation, indicating a reactive state.
No lesions of significance (52767)

Comments:
Observation: Unremarkable serous acini numbers in the submandibular glands compared to the control male.

Thyroids
Unremarkable colloid secreting follicles identified. Section also shows a small sheet-like mass of polygonal cells, characteristic of the parathyroid gland.

Head
Multiple levels through the head demonstrate dermal appendages, nasal cavity,oral cavity, teeth and tongue including muscle bundles (52891-94). Sections also show unremarkable pituitary gland including pars intermedia, pars distalis and pars nervosa as well and the trigeminal nerve/ganglia (52890).The outer and middle regions of the ear are discernible. The tympanic membrane is intact and the ossicles are unremarkable and include the stapedial annular ligaments (52891-92). Typical components of the inner ear including bony labyrinth, organ of corti, stria vascularis and scala cavities are discernible. Based on multiple levels, the organ of corti is unremarkable with no discernible loss of inner/outer hair cells and typical tectorial membrane (52892). The cochlear nerve and spiral ganglion is also demonstrated and based on several levels, there is no reduction in the density of the spiral ganglion cells. Examples of otolith organs can be seen with typical features such as the hair cells and mineral otoliths. The ampulla including the crista ridge with hair cells is discernible (52892 (52862) Marrow smear: Cells observed were readily identified as those from the erythroid and myeloid series. Conspicuous cells from the lymphoid series. Discernible and unremarkable megakaryoblasts (52861).

Mammary glands
Section shows typical mammary fat pad with developing lactiferous ducts (including primary duct), blood vessels and nerve bundles. Unremarkable dermal appendages.
No lesions of significance (52793) Ovaries/Oviducts Section shows unremarkable ovaries containing follicles at various stages of development and one corpus luteum. Unremarkable oviduct micromorphology with typical columnar epithelium and mucosal folds.

Pancreas
Section shows representative exocrine tissue (serous acini) and endocrine tissue (islets of Langerhans).

No lesions of significance (52804)
Kidney Section shows typical cortex with a uniform distribution of Glomeruli and accompanying nephron components. The micromorphology of the tubules was unremarkable.

No lesions of significance (52795)
Adrenal glands Section shows one adrenal gland with typical cortex/medulla micromorphology.
No lesions of significance (52795) Salivary glands and Regional lymph nodes Section shows submandibular, sublingual and parotid glands. The regional lymph nodes displayed mild follicular hyperplasia with germinal centre formation, indicating a reactive state.
Tail Section shows typical tail components including keratinized squamous epithelium, dense regular connective tissue, tendons, bone, intervertebral disc, bone marrow, skeletal muscle, nerves and blood vessels.

No lesions of significance (52802)
Eyes/Harderian glands Section shows eyes with unremarkable retina, optic nerve, cornea, iris, ciliary body, lens, sclera and choroid. Typical branched tubuloalveolar formation of the Harderian gland.
No lesions of significance (52807) Brain Sections were prepared from the standard levels of the brain: Level I: including cortex, corpus callosum, the lateral ventricles,anterior commissure and caudate putamen (Bregma -0.98) Level II: includes the hippocampus, thalamus, amygdala,hypothalamus, lateral and third ventricles (Bregma -2.54) Level III: includes the cerebellum, pons and fourth ventricle (Bregma -5.68) Sections of brain stained with Haematoxylin and Eosin, Luxol Fast Blue appear symmetrical with no ventricular dilation observed, unremarkable meninges and typical lamination. The cerebellum appears symmetrical with typical architecture and Purkinje cells. At this level, there was no evidence of neuronal loss and the myelination appears normal. Multiple levels through the head demonstrate dermal appendages, nasal cavity,oral cavity, teeth and tongue including muscle bundles (52901). Sections also show unremarkable pituitary gland including pars intermedia, pars distalis and pars nervosa as well and the trigeminal nerve/ganglia (52900).The outer and middle regions of the ear are discernible. The tympanic membrane is intact and the ossicles are unremarkable and include the stapedial annular ligaments (52901-52903).
Typical components of the inner ear including bony labyrinth, organ of corti, stria vascularis and scala cavities are discernible. Based on multiple levels, the organ of corti is unremarkable with no discernible loss of inner/outer hair cells and typical tectorial membrane (52901-52903). The cochlear nerve and spiral ganglion is also demonstrated and based on several levels, there is no reduction in the density of the spiral ganglion cells. Examples of otolith organs can be seen with typical features such as the hair cells and mineral otoliths. The ampulla including the crista ridge with hair cells is discernible (52903 Peripheral blood smear: Examination of the smear showed red blood cells (majority of cells shown), occasional white blood cells including segmented neutrophils and platelets (clumps). No discernible morphological changes or detectable parasites (52866) Marrow smear: Cells observed were readily identified as those from the erythroid and myeloid series. Conspicuous cells from the lymphoid series. Discernible and unremarkable megakaryoblasts (52865).

Mammary glands
Section shows typical mammary fat pad with developing lactiferous ducts, blood vessels, nerve bundles and a large reactive lymph node. Some scattered mast cells identified; a common finding in mouse mammary tissue.

No lesions of significance (52836)
Ovaries/Oviducts Section shows unremarkable ovaries containing follicles at various stages of development and several corpora lutea. Unremarkable oviduct micromorphology with typical columnar epithelium and mucosal folds.
No lesions of significance (52843) Uterus/Cervix/Vagina/Clitoral gland Unremarkable architecture of the endometrium/ endometrial glands, myometrium and adventitia. Some neutrophils and low numbers of lymphocytes identified in the endometrial glands; a common finding within a non-inflamed endometrium during the phases of estrus. The micromorphology of the uterus and vagina places the animal at metestrus. Comments:

Pathology to comment
Mesenteric lymph node Section shows typical reactive mesenteric lymph node with representative cortex including the occasional follicle, an expansive paracortical area and mild sinus histiocytosis.
No lesions of significance (52840)

Spleen
Unremarkable follicular pattern identified with typical red and white pulp micromorphology. Mild extramedullary hematopoiesis identified in the red pulp of the spleen, a common finding in the mouse.
No lesions of significance (52837) Pancreas Section shows representative exocrine tissue (serous acini) and endocrine tissue (islets of Langerhans). No lesions of significance (52837) Kidney Section shows a cortex and papilla. There is a uniform distribution of Glomeruli and accompanying nephron components and the micromorphology of the convoluted and straight segment tubules was unremarkable.
No lesions of significance (52848) Adrenal glands Section shows one adrenal gland with typical cortex/medulla micromorphology.
No lesions of significance (52848) Salivary glands and Regional lymph nodes Section shows submandibular, sublingual and parotid glands. The regional lymph nodes displayed mild follicular hyperplasia with germinal centre formation, indicating a reactive state.

Head
Multiple levels through the head demonstrate dermal appendages, nasal cavity,oral cavity, teeth and tongue including muscle bundles (52910-11). Sections also show unremarkable pituitary gland including pars intermedia, pars distalis and pars nervosa as well and the trigeminal nerve/ganglia (52910).The outer and middle regions of the ear are discernible. The tympanic membrane is intact and the ossicles are unremarkable and include the stapedial annular ligaments (52911-12). Typical components of the inner ear including bony labyrinth, organ of corti, stria vascularis and scala cavities are discernible. Based on multiple levels, the organ of corti is unremarkable with no discernible loss of inner/outer hair cells and typical tectorial membrane (52912). The cochlear nerve and spiral ganglion is also demonstrated and based on several levels, there is no reduction in the density of the spiral ganglion cells. Examples of otolith organs can be seen with typical features such as the hair cells and mineral otoliths. The ampulla including the crista ridge with hair cells is discernible (52913) No lesions of significance (52910-14)

Comment / Plan
Case APN16/010 will be referred to Professor Rolfe Howlett R&A Pathology Services NSW for a supplementary pathology report and Dr. John Finnie-SA Pathology for a neuropathology report. 19th April, 2016 Supplementary Pathology Report

(control) 378
Peripheral blood smear: The smear displayed neutrophils and lymphocytes being the main white cell types; the most numerous cell type being the erythrocyte Marrow smear: Myeloid and erythroid cells were readily identified throughout which were conspicuous darkly stained small cells. The latter cells were presumed to be a mixture of normocytes and small lymphoid cells. A heavy sprinkling of large elongated microbiological bacterial like micro-organisms occurred through out the marrow cells. These microorganisms were cigar shaped and their length measured 1 to 2 the diameter of a red blood cell. As none of the other mice displayed these micro-organism in their marrow smears their presence is judged to be a contaminant.

Testes and Epididymes
There are no abnormalities in the testes, epididymes and vas deferens. Intraluminal storage of spermatozoa was observed in the tail of epididymis and the vas deferens. No lesions of significance Secondary sex glands (e.g. seminal vesicles; prostate, coagulating gland) Unremarkable seminal vesicles, prostate-and coagulating-glands A large concretion in the prosthetic urethra appeared to have caused prominent dilation of the urethral segment; such concretions are often observed as co-incidental findings. No lesions of significance Prepuce ,Penis, Preputial gland, Urethra The sections display these structures clearly and no lesions of significance were observed.

Urinary Bladder
The bladder was sectioned in slide No.52691 and the features displayed a distended bladder wall lined with urothelium; there were no lesions of significance The sections display well formed epithelial lining with well formed villi enclosed by normal musculature coats. In a few local regions with epithelial tips of the villi being unstained and ballooning, this is judged to be artefact. The epithelium could be considered as mildly hyperplastic but without appropriately prepared sections this apparent hyperplasia is probably within normal limits. The Peyer's patches showed mild reactivity; a constant observed feature in the small bowel. No lesions of significance Large Intestine /Caecum Sections from the caecum and the colon displayed numerous intraluminal protozoa These micro-organism had not entered and parasitized the colonic epithelial lining. Hence they are best considered non-pathogenic. No lesions of significance

Mesenteric Lymph Node
Mild follicular hyperplasia and mild sinus histiocytosis The mesenteric lymph node frequently shows these changes; as would be expected because these nodes drain the small intestine. No lesions of significance Spleen The microscopic pattern of relatively discreet white cell islands scattered throughout the splenic red cell parenchyma failed to show any lesion of significance. A common finding extramedullary haematopoiesis was observed; such is almost considered normal. No lesions of significance

Pancreas
The parenchyma of this organ displayed endocrine (Islets of Langerhans) and exocrine tissue. In a normal and expected pattern No lesions of significance

Kidneys
In the distal convoluted tubular region in both kidneys small amounts of protein appear to have collected within the lumens to form hyaline casts. In a few instances this protein appears about the size of an epithelial cell. The glomerulae were scattered evenly throughout the cortex. No abnormal features were observed within the glomeruli . In the lumens of these organs were protozoan micro-organisms; such supports coprophagia. No lesions of significance

Large Intestine /Caecum
Intraluminal protozoan micro-organisms were packed into the lumen. There was no evidence of the protozoan invading or living in the mucosa. Coprophagy would enhance the proliferation of these non-parasitic micro-organisms.

Small Intestine
The issue of mild epithelial hyperplasia is difficult to assess. In this particular intestine there are areas where the villous tips are oedematous and in some regions the villi are necrotic (artefact). Additionally this mouse was recorded as having mildly distended bowel.

Mesenteric Lymph Node
A small amount of node displayed minimal changes No lesions of significance

Spleen
The parenchyma of the spleen has a pattern that displays no lesions of significance Extramedullary haematopoiesis is commonly found in normal mice. See the additional notes provided from the preliminary report There are no features of significance in the sections prepared from the stomach. The limiting ridge divides the lining of this organ into glandular and non-glandular regions. Whether areas of the glandular segment is mildly hyperplastic is debatable.

Small Intestine
No lesions of significance The villi of the mucosa do appear slightly longer but seem to be within expected parameters. Indeed the features of the mucosa of this animal seem similar to the control female mouse.

Large Intestine /Caecum
Marked protozoan intraluminal accumulation in the caecum; note the protozoan organism are not within the mucosa. Common non pathogenic protozoa listed are Tritrichomonas muris and others Intra-lumenal protozoan organisms are observed in small numbers in the colon.

Mesenteric Lymph Node
Moderate follicular hyperplasia and sinus histiocytosis The mesenteric lymph node is a perpetual state of reactivity. The medulla and cortex of the thymus are without significant lesions. There were several small cysts and these occur regularly in mice. No lesions of significance The comment re thymic cysts in the preliminary report from Maronpot is appropriate.
Heart / major vessels / valves I concur with the details in the preliminary report to which I would add the following: There is a mild myxomatous thickening of 2 leaflets of the left atria-ventricular valve. The latter changes are probably of minimum significance Skin The epidermis, dermis upper hypodermis and Panniculus carnosus together with Dermal appendages do display any features of significance.
Eyes / Harderian glands One eye appeared smaller than the other but this would appear to be due to plane of section (towards the ocular margins); particularly as all components normally found in the eye were present. No features of significance Spinal Cord /vertebrae (3 sections of the spinal cord from the thoracic and lumbar regions) I concur with the preliminary report findings. No lesions of significance Skeletal Tissue(appendicular skeleton)/ synovial joint/ skeletal muscle The section was prepared from the hind limb. No lesions occur in the bones, styfle joint and striated muscle. The bone marrow had conspicuous megakaryocytes but other wise seemed to have cells from myeloid and erythroid series Tail I concur with details presented in the preliminary report. No lesions of significance Head I concur with the detailed preliminary report indicating that the nasal and oral cavities had no significant lesions. Furthermore, microscopic features of auditory and balance were identified and seemed not to display any abnormal features. No significant lesions.

389
Blood smear: Occasional white blood cell, (often small with little in the way of cytoplasm) in a dominant background of red blood cells.
Marrow smear: Erythroid and myeloid cells were readily identified. A commonly identified cell type was small densely stained nucleated cell; the most likely types being a normocytes and lymphocytes.
The marrow smear and sections cut from the trabeculated bone do suggest that there is a possible increase in lymphocytes in the marrow.
Ovary/ Fallopian tubes One ovary displaying follicles at varying stages together with several corpora lutea. The fallopian tubules were lined by intact columnar epithelium No lesion of significance

Uterus / Vagina
The microscopic features of uterus ,myometrium, endometrial glands with mild numbers of migrating neutrophils in the uterine walls suggests that this animal was in metoestrus. No lesions of significance.

Summary
RENAL OBSERVATION / COMMENT: In the following kidneys small numbers of tubular casts were observed in the distal convoluted tubules in at least one kidney from each of the following mice; Mouse Numbers 378; 385 as well as the control mice 380 and 388. The presence of tubular proteinaceous casts should be further explored. Such an undertaking is worth while if this is a new strain of mouse, because there is a possibility that this strain of mouse is carrying a renal defect. BONE MARROW SMEAR OBSERVATION/COMMENT: The preliminary reports for bone marrow identified conspicuous lymphoid series in all the mice submitted. Indeed, considerable numbers of small deeply staining cells were present in all samples I examined. I presumed that these cells were either normoblasts/normocytes or small lymphocytes.
2nd May, 2016 385 H&E and LFB stained brain sections were examined. Representative H&E stained sections of spinal cord were examined. Sections of brain and spinal cord show no significant neuropathology.

386
H&E and LFB stained brain sections were examined. Representative H&E stained sections of spinal cord were examined. Sections of brain and spinal cord show no significant neuropathology.

389
H&E and LFB stained brain sections were examined. Representative H&E stained sections of spinal cord were examined. Sections of brain and spinal cord show no significant neuropathology.

Summary
Sections of brain and spinal cord show no significant neuropathology.
6th May, 2016 The Australian Phenomics Network advises all research groups that images or results obtained through the services offered by the APN are to be acknowledged in resultant publications. Example acknowledgement: "This study utilised the Australian Phenomics Network Histopathology and Organ Pathology Service, University of Melbourne."