Developmental expression of the mouse mottled and toxic milk genes suggests distinct functions for the Menkes and Wilson disease copper transporters
Developmental expression of the mouse mottled and toxic milk genes suggests distinct functions for the Menkes and Wilson disease copper transporters Yien-Ming Kuo, Jane Gitschier1 and Seymour Packman*
Departments of Medicine and Pediatrics and 1Howard Hughes Medical Institute, University of California, 3rd and Parnassus Ave, San Francisco, CA 94143, USA
Received March 6, 1997;Revised and Accepted April 28, 1997
Menkes disease and Wilson disease are human disorders of copper transport caused by mutations in distinct genes encoding similar copper-transporting P-type ATPases. These genes are expressed in different adult tissues in patterns reflecting disease manifestations. The mouse homologues for the Menkes (MNK) and Wilson (WND) disease genes are the mottled (Atp7a) and toxic milk (Atp7b) genes, respectively. Using RNA in situ hybridization we describe the distribution of mottled and toxic milk transcripts during mouse embryonic development. The mottled gene is expressed in all tissues throughout embryogenesis and is particularly strong in the choroid plexuses of the brain. Mottled expression in the liver is in contrast to the prior observation of absent or very low expression in the adult liver. Expression of the toxic milk gene is significantly more delimited, with early expression in the central nervous system, heart and liver. Later in gestation, toxic milk transcript is clearly seen in the liver, intestine, thymus and respiratory epithelium including nasopharynx, trachea and bronchi. In lung, toxic milk expression is restricted to bronchi, while mottled expression is diffuse. Hepatic expression of both toxic milk and mottled is in the parenchyma, as opposed to blood cells. These results suggest that the mottled gene product functions primarily in the homeostatic maintenance of cell copper levels, while the toxic milk gene product may be specifically involved in the biosynthesis of distinct cuproproteins in different tissues.
All living organisms require copper as a cofactor for numerous cuproproteins, for normal growth and development (1 -3 ). However, copper in excess of cellular needs mediates free radical production and direct oxidation of cellular components with detrimental effects (1 ). Intracellular copper content is therefore regulated and maintained by specialized cellular transport systems (1 ), which are conserved through evolution (4 ).
Two human genetic disorders, X-linked Menkes disease and autosomal recessive Wilson disease, highlight the importance of intact cellular copper transport mechanisms (3 ). These two diseases are caused by mutations in distinct genes encoding copper-transporting P-type ATPases (5 -9 ). The protein encoded by the Wilson disease gene (WND; ATP7B) (8 ,10 ) has 56% overall identity to that of the Menkes disease gene (MNK; ATP7A) (5 -7 ). The mottled (Atp7a) and toxic milk (Atp7b) genes have been confirmed as mouse homologues for the Menkes and Wilson disease genes, respectively (11 -13 ).
The patterns of MNK and WND gene expression in the adult are markedly different and correlate with the distinct clinical manifestations of the disorders. The Menkes gene is ubiquitously expressed in adult tissues, with little or no expression in liver (5 ). Mutations in this gene lead to defective cellular export of copper. In Menkes disease and the mottled mouse, copper significantly accumulates in some tissues (intestinal mucosa and kidney), leading to failure of copper delivery to other tissues, resulting in systemic copper insufficiency (3 ). Clinical manifestations include progressive neurologic degeneration, seizures, growth failure, hypopigmentation, pili torti, arterial aneurysms and skeletal defects (14 ). Deficiencies in cuproenzyme activity in multiple tissues, e.g. lysyl oxidase (cross-linking of elastin and collagen), cytochrome c oxidase (mitochondrial electron transport), superoxide dismutase (free radical detoxification), tyrosinase (pigmentation) and dopamine [beta]-hydroxylase (catecholamine production), are possibly responsible for many of the clinical features.
In contrast to MNK, WND is expressed in a few cell types: liver cells and possibly in kidney and certain neuronal cells (8 -10 ). Wilson disease is characterized by progressive and distinct neurologic findings, chronic liver disease with cirrhosis, renal tubular failure, and pigmented corneal rings (3 ,15 ). The copper content of the liver, brain, kidney and cornea is increased, and the organ failures of Wilson disease and of the animal models reflecting the toxicity of copper excess (16 ,17 ).
Such correlations represent an incomplete understanding of these disorders. Menkes disease has a prenatal onset (3 ,18 ), and the central nervous system (CNS) manifestations include not only diffuse and unspecific neurodegenerative changes, but also selective defects which may be of developmental origin (14 ). Indeed several of the mottled alleles, tortoiseshell and dappled, lead to death in utero (19 -21 ). The pattern and timing of prenatal expression of mottled and MNK, the effects of mutations in these genes on organogenesis, and the prenatal developmental origin of somatic and central nervous system (CNS) manifestations of the human and mouse disorders, are not known.
The prenatal and adult spectrum and timing of expression of the Wilson disease gene are not known. Wilson disease is of postnatal onset and affects only a few distinct organs. The functions of the Wilson disease ATPase in extrahepatic cell types (16 ) have not been elucidated, and it is reasonable to consider that such functions might be different from that of the liver protein which is thought to serve by delivering copper into the bile and for incorporation into ceruloplasmin (22 ).
Here we describe the prenatal expression of the mottled (Atp7a) and toxic milk (Atp7b) genes by RNA in situ hybridization to begin to address such questions. (We shall use the terms mottled and toxic milk when we refer to the Atp7a and Atp7b genes respectively.) This is the first comprehensive examination and report of the prenatal developmental expression of the mottled and toxic milk genes. We compared the expression of mottled with that of the Wilson disease homologue, toxic milk. We show that mottled is expressed ubiquitously throughout the embryo during gestation, and that toxic milk is expressed in a limited set of tissues. Our findings are broadly consistent with the notion that the mottled gene product functions in the individual homeostatic maintenance of cellular copper levels in all cell types. Our findings suggest that the toxic milk gene product is more likely involved in a physiologic or biochemical role that is specific to, and possibly quite different for, each of the several distinct cell types in which it is expressed and in a way which was previously unsuspected.
Before embarking on in situ hybridization studies, we first asked whether the mottled and toxic milk genes are expressed during murine embryonic development and tested probe specificity by Northern analysis. A fragment of the mottled gene was hybridized to RNA isolated from mouse forebrains at different stages of development and to organs (including brain, heart, liver, lung and limbs) isolated from E13.5 embryos. A single band of ~8.3 kb was detected from E9 to E17, PO and P8 in all the organs, indicating that the mottled gene is expressed during embryonic development (data not shown). When the forebrain blot was reprobed with a toxic milk probe, a single ~7.5 kb band was detected from E9 to E17, PO and P8. These results, combined with those below, demonstrate that our probes are hybridizing specifically.
Expression of the toxic milk gene is more delimited and at E9.5 expression is seen in the heart and liver primordia (Fig. 1 .1, B). By E11.5, toxic milk expression can be clearly seen in the ventricle of the heart, nasal epithelia, intestine, lung primordia and especially in the liver (Fig. 1 .1, E). At E15.5 through to E18.5 (Fig. 1 .2) there is expression in the lung, thymus and abundant expression in the liver and intestine. We note that toxic milk expression in the intestine is more luminal compared with mottled expression (Fig. 1 .2, H, K). At E18.5, we note that there is expression throughout the lining of respiratory tract, including nasopharynx, trachea and pulmonary bronchiae. We confirmed the in situ results by performing additional hybridizations using probes transcribed from the 5' and 3' UTR from both genes; therefore we are confident that our results are not due to cross hybridization.
From higher power magnifications specific regions within tissues and organs that express the mottled and toxic milk genes can be discerned. Figure 2 shows regions of expression in selected tissues of E18.5 embryos. We observe mottled gene expression throughout the brain, but most strongly in the choroid plexuses (Fig. 2 and Fig. 1 .2). This pattern of expression has also been seen in adult brain (Kuo, unpublished observations; 23 ,24 ). The toxic milk gene does not appear to be expressed in the brain (Fig. 1 ) or the choroid plexus (Fig. 2 ) in the embryo nor in the adult mouse (data not shown). In the lung, the mottled gene is expressed evenly throughout the lung parenchyma, whereas toxic milk is expressed most highly in the bronchial epithelium. In the liver, toxic milk expression is somewhat higher than that of mottled, and both genes are expressed in hepatic parenchyma, not in blood cells. In the intestine, mottled and toxic milk are both expressed in the villous epithelium. In skin, mottled is expressed in a diffuse pattern, but toxic milk expression is absent (Fig. 2 ).
Prototypical Menkes disease is a lethal multisystem neurologic and connective tissue disorder of both prenatal onset and severe progressive postnatal disability (3 ,14 ,18 ). Given the complexity of the Menkes/mottled phenotype and its pathogenesis, we undertook this study to help understand those aspects of the mottled phenotype that are ofprenatal onset. We note that the mottled gene is expressed diffusely throughout all embryonic tissues, from gestation day 9.5 (E9.5) through E18.5. Expression in the placenta and in the liver at E17.5 has been previously observed (11 ,12 ,25 ).
It could be argued that failure of placental transport of copper to the fetus is the primary pathogenic event, producing an intrauterine state of copper insufficiency and resulting cellular damage during critical periods of differentiation. However, the striking mottled expression throughout the embryo and throughout gestation also argues that the mottled gene may be required for the maintenance of cellular homeostasis and the extracellular microenvironment of multiple cell types during development. Such a `housekeeping' role for the mottled gene would have to be taken into consideration in the design of prenatal copper therapies based on the bypassing of a placental transport block.
The high expression of mottled in the embryonic liver parenchyma, is in contrast to the absent or low level of expression in adult human and mouse liver (11 ,12 ). The difference in mottled expression between adult and fetal liver may be viewed in the context of the anatomic and functional development of fetal liver and hepatocytes. In the fetus, biliary secretion is at exceedingly low levels, exchange by enterohepatic circulation is negligible, and ceruloplasmin levels are very low (26 -28 ). Accordingly, the fetal hepatocyte is not the bile-secreting or ceruloplasmin-synthesizing cell as it is in the postnatal animal, and these avenues of copper export are not available to the fetal hepatocyte. It is therefore reasonable to consider that the fetal liver cell requires the same `housekeeping' copper export system as any other cell type, and that such copper transport function in fetal liver is supplied by the mottled/Menkes protein. Conversely, as the postnatal liver cell develops the capacity to deliver lysosomal copper to the bile canaliculi, and to rid itself of a significant fraction of cell copper by incorporation into ceruloplasmin, the copper excretory capacity provided by mottled is no longer required, and the expression of mottled declines.
The possible association between age of onset of therapy and response to therapy (29 ) and the effects of acquired copper deficiency on neuronal histology (30 ,31 ) suggest that postnatal events contribute to CNS manifestations of Menkes disease. Nevertheless, the early onset of the disease in humans and in mice and the observation that a number of the pathologic manifestations are not corrected by postnatal copper therapy in macular mice (32 ,33 ) suggest that certain of the CNS changes reflect prenatal events (14 ). Defects which may be of prenatal origin include heterotypic innervations, abnormal dendritic arborizations of pyramidal neurons and of Purkinje cells, primary cellular degeneration in the thalamus, reduced number of Purkinje cells, and abnormalities in number and size of Purkinje cell mitochondria (14 ,32 ,34 ,35 ).
In treatment studies on brindled and macular mutants, a window period of postnatal age 7-10 days was established for amelioration of clinical neurologic manifestations (36 ,37 ,39 -41 ). It was proposed (42 ,43 ) that astrogliogenesis is not yet completed at this stage, and that parenterally administered copper can therefore directly reach neurons in mutants, without being sequestered in astrocytes. Since expression of the mottled gene has also been documented in neuronal cells in postnatal mice (23 ,24 ), the Menkes/mottled gene product may play a role in copper transport in neuronal cells themselves, and copper homeostasis in neurons may therefore not be entirely governed by transport mechanisms in astroglial cells.
Our data on prenatal expression of the mottled gene in brain point to an additional redundancy in brain copper transport. In prenatal brain sections, we observe strong expression in the choroid plexuses, which function by capillary filtration and active epithelial secretion to form the cerebrospinal fluid in equilibrium with brain extracellular fluid. This component of CNS copper transport likely functions to maintain a homeostatic extracellular copper concentration for cells of the CNS. We hypothesize that copper transport regulated by the choroid plexuses would function as an adjunct to neuronal copper transport and to copper transport mediated by astroglial cells.
In contrast to the widespread expression of the mottled gene, toxic milk expression is limited to certain tissues. Toxic milk expression in liver is certainly not unexpected, as liver disease is a cardinal feature of Wilson disease in humans and of toxicity in the toxic milk mouse and LEC rat (16 ,44 ,45 ). Expression in the heart during early gestation may be related to the cardiomyopathy of the Wilson disease patients (3 ). Given that brain and renal tubular involvement are observed in Wilson disease, we must propose that expression of toxic milk in brain and kidney is either at a low level and not detectable prenatally, or that significant expression in those tissues begins postnatally.
It is most intriguing that toxic milk is expressed prenatally in cell types which appear to be unrelated to clinical manifestations in Wilson disease. Most striking are the detection of toxic milk transcript in thymus, and in respiratory epithelium and intestine (present paper; 13 ). In order to speculate on the role of the toxic milk gene in these tissues, we first consider the possible nature of the cellular function of the toxic milk gene. Recent studies of the MNK gene have suggested that the Menkes and mottled gene products function to transport copper from the cytosol into a secretory compartment derived from the trans Golgi network (46 ,47 ). This notion of MNK gene product function has been extended to postulate that the WND gene product specifically functions in the hepatocyte to transport copper into a trans Golgi vesicle for incorporation of that copper into ceruloplasmin (4 ,18 ). Given such a putative role of the Wilson/LEC/toxic milk gene product in the hepatocyte, one can speculate that the transporter might serve the same role in other cell types. Alternatively, the transporter has a novel function that can interact with ceruloplasmin or is required in higher amounts in specific stages of development.
We note that ceruloplasmin gene is expressed in a number of extrahepatic cells, including uterus, placenta, yolk sac, lactating mammary gland, testis, lymphocytes and splenic macrophages (28 ,48 -51 ). Fetal rat lung is a major site of extrahepatic ceruloplasmin mRNA during development, and the quantity of lung ceruloplasmin mRNA increases in parallel with that of liver throughout gestation (28 ). We therefore propose that the toxic milk gene in the fetal lung and potentially other respiratory tissues may serve to transport copper into an intracellular compartment for incorporation into ceruloplasmin. Given the expression of ceruloplasmin in lymphocytes and other immune cells (50 ), and the elevations in ceruloplasmin mRNA observed in response to inflammation (48 ), we also postulate that the toxic milk gene plays a similar role in the formation of holoceruloplasmin in cells of the thymus.
There is no direct evidence of ceruloplasmin expression in fetal heart and intestine and in fact, preliminary analyses of such expression were negative (28 ). However, the reasoning which led to the above hypotheses can engender speculations on the synthesis of other cuproproteins during development. In a given tissue, the toxic milk gene might specifically mediate the synthesis of a holocuproenzyme other than ceruloplasmin. This contention is supported by the documentation of instances in which the pattern of expression of superoxide dismutase I (SOD) differs from that of mottled. It was noted that SOD is not ubiquitous, and limited to specific embryonic tissues (52 ). Further, SOD is expressed at a high level in adult liver (25 ), in which mottled is not expressed. Such discordances lead to the consideration that the Wilson/toxic milk gene product, rather than the mottled protein, might mediate SOD biosynthesis in selected tissues.
In sum, the prenatal expression patterns of mottled and toxic milk lead us to infer that copper transport mediated by the mottled gene product is required for cellular homeostasis and integrity in a multiplicity of tissues. In contrast, the toxic milk gene appears to function in a distinct subset of cell types, leading to speculations on physiologic roles for toxic milk in the biosynthesis of distinct cuproenzymes. In a few tissues, notably the fetal liver parenchyma and intestinal epithelium, both genes are expressed. Interestingly, these cell types exhibit polarity in transport functions. Therefore, it is possible that the two different copper transporters might be performing different functions and be situated in different cellular structures, membranes or membrane regions. The capacity to exploit mutations of such copper transport genes in inbred mouse strains will aid in the testing of the validity of the cellular and physiologic mechanisms that we herein propose. The data of the present work also form the basis for future tests of rational prenatal therapies to prevent the devastating multisystem involvement of Menkes disease, as well as to more completely understand the phenotype and treatment of Wilson disease.
To eliminate the possibility of cross-gene hybridization, mottled riboprobes were generated from a portion of the 5'UTR (nt 1-277, 11). Primers 5'-atgtgccgtctgtcatgaac-3' and 5'-AGTAAGTTGGGCTTCTGGAG-3' were used to amplify by polymerase chain reaction a 288 nt fragment, containing nt 497-785 (13 ) of the mouse toxic milk cDNA from reverse transcribed mouse liver RNA (according to Invitrogen protocol). The toxic milk cDNA was subsequently cloned into the Srf vector (Stratagene). From these plasmids, 33P-labeled sense and anti-sense riboprobes were made according to published protocols (53 ).
Embryos were isolated from randomly bred Swiss Webster mice of at least 6-8 weeks of age (Simenson Laboratories, Gilroy, CA), with noon on the day of finding a vaginal plug designated to be 0.5 days of gestation (E0.5). Embryos were dissected free of maternal decidual tissue, fixed overnight in 4% paraformaldehyde in phosphate-buffered saline (PBS), dehydrated and embedded in paraffin. Sections (8 [mu]m) were mounted on Superfrost Plus slides (Fisher) and processed for in situ hybridization according to published protocols (53 ), with the following modifications. After wax removal and rehydration in water, slides were fixed with 4% paraformaldehyde in PBS prior to incubation with proteinase K (20 [mu]g/ml) for 7.5 min at room temperature. Digestion was stopped by washing twice in PBS and once in 4% paraformaldehyde in PBS. Following a further wash in PBS, slides were treated in 0.25% acetic anhydride 0.1 M triethanolamine for 10 min, washed twice in 2* standard sodium citrate (SSC) and once in water. The slides were incubated for 16 h with 2 * 106 c.p.m. of probe in 100 [mu]l of hybridization buffer (50% formamide, 5* SSC, 10 mM [beta]-mercaptoethanol, 10% dextran sulphate, 2* Denhart's, 250 [mu]g yeast tRNA, 500 [mu]g/ml Salmon Sperm DNA) at 65oC in a moist chamber. Slides were washed as described (53 ) except that sections were digested with RNase A (50 [mu]g/ml) in 0.3 M NaCl/10 mM Tris-HCl, pH 8.0/5 mM EDTA. Samples were dehydrated and exposed on Bio-Max film (Kodak) for 2 days. Slides were dipped in emulsion (Kodak NTB-2), exposed for ~14 days, developed in Kodak D-19 and counterstained with toluidine blue. Silver grains were visualized with dark field microscopy.
We are grateful to Drs John Rubenstein, Yuh N. Jan and Lily Y. Jan for allowing us to use equipment in their laboratories. We especially want to thank Ming Ming Jiang and Dr Weimin Zhong for their helpful technical advice. We thank Drs Christopher Vulpe, Weimin Zhong and Barbara Levinson for their critical readings of the manuscript and thank Dr Philip Rosenthal for helpful discussions on fetal liver development. Finally, we express our sincere appreciation for the advice and support given by our laboratory colleagues. This work was supported by grants from the March of Dimes Birth Defects Foundation and the National Institutes of Health. JG is an Associate Investigator of the Howard Hughes Medical Institute.
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*To whom correspondence should be addressed. Tel: +1 415 476 2871; Fax: +1 415 476 9976; Email: pack{at}itsa.ucsf.edu
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