ARTICLE |
Correspondence to: Betty A. Eipper, Dept. of Neuroscience, MC3401, U. of Connecticut Health Center, 263 Farmington Ave., Farmington, CT 06030-3401. E-mail: eipper@uchc.edu
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Summary |
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Kalirin, a homologue of trio and UNC-73, has been previously demonstrated to cause cytoskeletal rearrangements, enhanced outgrowth of neuritic processes, and altered secretion. In the adult rat, kalirin is specifically localized to the central nervous system, with the main adult isoform, kalirin-7, concentrated in neuronal postsynaptic densities. In this study we examined the expression of kalirin in rat tissue from embryonic Day 10 (E10) through E18, using an antibody that detects all known kalirin isoforms. Kalirin expression in the embryo was more widespread than in the adult, with localization of kalirin protein to both neuronal and non-neuronal tissue, such as muscle, lung, intestinal epithelium, and pancreas. In neurons, kalirin was localized both in cell bodies and axon processes; in muscle tissue, kalirin was highly localized to migrating myogenic cells and at muscle attachment sites. Western blotting analysis indicated that kalirin-7, the major adult isoform, was a minor component of embryonic kalirin; the main isoform expressed in the embryo was kalirin-9. This is the first identification of kalirin expression in embryonic tissue and the first demonstration of non-neuronal expression of kalirin. (J Histochem Cytochem 49:833844, 2001)
Key Words: GEF, P-CIP10, duo, trio, UNC-73, spectrin, PAM
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Introduction |
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PROTEINS OF THE Dbl family of GDP/GTP exchange factors (GEFs) activate small GTP-binding proteins of the Rho subfamily, which then affect diverse functions ranging from cytoskeletal organization to axon guidance and gene expression (
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Kalirin was identified through a screen for interactors with the cytosolic domain of peptidylglycine -amidating mono-oxygenase (PAM), a membrane enzyme essential to the synthesis of many bioactive peptides. PAM functions in large dense-core vesicles, and mutations in its cytosolic domain cause its mislocalization without altering enzyme activity (
The kalirin proteins are heterogeneous (Fig 1; kalirin-7 is generated through use of an internal translational start site. Kalirin-7 contains a PDZ-binding motif, which enables this isoform to localize to the postsynaptic density fraction (
Overexpression of kalirin-7 in hippocampal neurons leads to the formation of more dendritic spine-like structures and abnormal spine morphology (
The functions of kalirin in development and adulthood are not yet clear, although the unique subcellular localization of different isoforms suggests form-specific functions (
To investigate the potential roles of kalirin during embryonic development, we evaluated kalirin protein expression in the embryonic rat using immunohistochemistry and Western blotting analysis. We found that kalirin expression in the rat embryo is predominantly restricted to kalirin-9, which is not the major form of kalirin in the adult cortex. Kalirin-12 expression is readily detected, whereas kalirin-7 expression is not detectable during embryonic development. In contrast to the adult, kalirin expression during embryonic development is not restricted to the nervous system. On the bassis of its sites of expression, kalirin may play a role in axon outgrowth, myoblast migration and fusion, and endocrine development.
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Materials and Methods |
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Animal Preparation
All experimental protocols were approved by the Johns Hopkins University Institutional Animal Care and Use Committee, and all applicable guidelines from the National Institutes of Health Guide for the Care and Use of Laboratory Animals were followed. Timed pregnant SpragueDawley rats were obtained from Harlan (Indianapolis, IN). Embryonic Day 0 is defined as the date of conception. For preparation of tissue extracts, timed pregnant female rats were anesthetized with 400 mg/kg chloral hydrate, the embryos removed, washed in PBS, placed in MEM-AIR, and dissected using a dissecting microscope. Tissue was extracted with 20 mM PIPES, pH 6.8, 2 mM Na2EDTA, 50 mM NaF, 10 mM Na4P2O7, 1 mM Na3VO4, 1% Triton X-100, 300 µg/ml phenylmethanesulfonyl fluoride, 2 µg/ml leupeptin, 16 µg/ml benzamidine, 10 µg/ml lima bean trypsin inhibitor, and 2 µg/ml pepstatin, homogenized, and centrifuged for 15 min in a microfuge at 4C. Supernatant was collected and assayed for protein concentration using the bicinchoninic acid protein reagent kit (Pierce Chemical; Rockford, IL).
Antibodies and Antibody Purification
Rabbit polyclonal antibodies to the COOH-terminus of kalirin-12 (Ab3225 and Ab3226) were affinity-purified (
Immunohistochemistry
Embryos fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 12 µm were obtained from Novagen (Madison, WI). Slides were washed in xylene to remove paraffin, then rehydrated through a series of graded ethanol solutions from 100% to 50%. Slides were microwaved for 15 min, cooled, and postfixed in Bouin's fixative for 15 min. Tissue was washed several times in PBS to remove fixative, incubated in PBS for 15 min, and permeabilized in 0.1% Triton X-100 in PBS for 30 min. Slides were washed twice in PBS for 5 min each, blocked with 4% normal goat serum (Vector Laboratories; Burlingame, CA) for 1 hr at RT, and incubated with primary antibody diluted in PBS at 4C overnight. The following day, slides were washed twice in PBS for 5 min each, incubated in Vectastain biotinylated antibody (1:1000; Vector Laboratories) for 30 min, washed twice in PBS, quenched with 0.5% H2O2 in PBS for 10 min, washed twice in PBS, and incubated in the BC reagent for 30 min. Finally, slides were rinsed in PBS and developed using 3,3'-diaminobenzidine tetrahydrochloride (Gibco BRL; Gaithersburg, MD) in 50 mM Tris, pH 7.4. Slides were viewed using a Zeiss Axioskop. Photography was performed using the Spot Camera (Diagnostic Instruments; Sterling Heights, MI).
Western Blotting
Samples from tissue extracts were prepared for electrophoresis by making them 2% in SDS and 5% in 2-mercaptoethanol and boiling for 5 min. Proteins were fractionated on SDS-polyacrylamide gels containing 6% acrylamide, 0.19% bis-acrylamide. Proteins were transferred to Immobilon-P membranes (Millipore; Bedford, MA) in 25 mM Tris-HCl (pH 8.5), 200 mM glycine, 20% methanol for 2 hr at 500 mA. Blots were blocked with 5% skim milk diluted in 50 mM Tris-HCl (pH 7.5) and 150 mM NaCl containing 0.05% Tween-20 (TTBS), incubated in a 1:1000 dilution of rabbit antiserum (Ab2581 or Ab3225) for 2 hr at RT, and rinsed. Blots were then incubated for 1 hr with HRP-conjugated donkey anti-rabbit IgG antibody (1:10,000; Amersham, Piscataway, NJ) and visualized using the Enhanced Chemiluminescence Kit (Amersham). Exposure times ranged from 30 sec to 10 min.
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Results |
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Kalirin-9 Is the Major Embryonic Isoform of Kalirin
Kalirin protein expression in E18 embryonic cortex was examined by Western blotting analysis to determine the major kalirin isoforms expressed (Fig 2A). Antibodies against spectrin-like repeats 4 through 7 were used because they recognize all forms of the kalirin protein except Duet, and adult cortex was analyzed for comparison (Fig 2A). The E18 cortex demonstrates predominant expression of kalirin-9, with readily detectable amounts of kalirin-12; the minor band at approximately 200 kD is thought to represent kalirin-8 (Fig 2A). Essentially identical patterns were obtained with two different kalirinspectrin antisera (Ab2581 and Ab2582). A protein the size of kalirin-7, the major isoform in adult cortex, was not detected in E18 cortex (Fig 2A). Western blotting analysis of E18 and adult cortex using a kalirin-7-specific antibody verified kalirin-7 protein expression only in the adult CNS (Fig 2B, Ab2959).
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We next examined extracts prepared from other E18 tissues, including the heart, lung, kidney/adrenal, intestine, tongue, and liver, for kalirin expression. Kalirin-9 represents the major isoform of kalirin in these tissues (Fig 2C). Although proteins the size of kalirin-8 and kalirin-12 are detectable, levels are generally lower than levels of kalirin-9. Kalirin expression was detectable only at very low levels in the embryonic liver. The amounts of kalirin-9 and kalirin-12 detected in the other tissues examined were quite similar. A very similar pattern was observed using both kalirinspectrin antisera. Preincubation of Ab2581 with excess antigen demonstrated the specificity of the signal observed.
Kalirin Is Expressed Early in Embryonic Development (E10)
Using an antibody to the spectrin-like regions of kalirin, which identifies the major kalirin isoforms, we next performed immunohistochemistry on developing rat embryos to determine the major sites of kalirin expression. By E10 in the rat, the neural tube and rudimentary heart have formed (
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Kalirin Becomes Widely Expressed in Developing Neural and Muscle Tissue by E12
By E12, kalirin protein expression expands to include not only the heart but also multiple nervous system tissues and newly formed, migrating myogenic cells (Fig 4A). Kalirin expression remains elevated in the cardiac region (H), similar to the pattern observed at E10, with the atrium, ventricle, and bulbus cordis demonstrating immunoreactivity. In addition to the heart, at E12 kalirin is uniformly expressed in the newly formed myogenic cells of the tongue (Tn; Fig 4A and Fig 4B). These myogenic cells are first identifiable as an outgrowth from the first five or six somites of the murine embryo and migrate along the hypoglossal cord before eventually taking their place in the tongue, which develops from the mandibular component (Fig 4A, Fig M;
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At E12, many regions of the nervous system begin to differentiate (
Interestingly, kalirin expression in not detectable in the E12 olfactory placode (OP), the earliest structure formed during development of the olfactory epithelium (
Kalirin Demonstrates Distinct Cortical, Muscle, and Olfactory Neuron Expression at E15 and E16
In the rat nervous system, differentiation continues through E15 and E16, with expansion of cortical volume and increased differentiation of the olfactory lobes and cerebellar region (
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Kalirin immunoreactivity becomes apparent at this age in the developing olfactory epithelium. At this stage in olfactory neuron development, proliferation has slowed and a majority of neurons present begin to differentiate (
By E15/E16, many of the rudimentary muscle structures have formed after myogenic cell migration (
To evaluate the specificity of the staining observed using the kalirinspectrin domain antibody, blocking experiments were performed. Antigen-blocked antibody was unable to recognize kalirin protein in neural tissue (Fig 5F) or in muscle (Fig 5G), although nonspecific staining was evident in the overlying epidermis, which was evident with all antibodies tested. This nonspecific staining may reflect the increased friability of this tissue with processing.
Differential Expression of Kalirin at E18
A dramatic shift in kalirin expression is apparent between E15/E16 and E17/E18. Although the cortex is undergoing continued differentiation at E17, kalirin immunoreactivity in the outer layer of the cortex is decreased, with only a subset of neurons containing detectable levels of kalirin (Fig 6A and Fig 6A'). In addition, there is an increased level of kalirin protein in axon bundles (Fig 6A''). Kalirin expression in a variety of muscles, including the glossopharyngeal and intercostal muscles, has decreased at E17/E18 in comparison to E15/E16 (data not shown).
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Kalirin immunoreactivity becomes evident at this age in a number of peripheral tissues, including the intestine (Fig 6B and Fig 6B'), the pancreas (Fig 6C), and the vibrissae (Fig 6D and Fig 6D'). These tissues undergo increased expansion and differentiation at this developmental stage (
Kalirin-12 Exhibits a Distinct Localization Pattern in the Developing Rat Embryo
We examined the expression of one of the embryonic isoforms of kalirin, kalirin-12, in the developing embryo using antiserum specific for the C-terminus of kalirin-12 (Fig 1). At E10, kalirin-12 expression was demonstrated in the embryonic heart, in both the common atrium and the ventricle, and staining was eliminated by immunodepleting the antiserum (data not shown). At later stages of development, kalirin-12 expression was detected in the DRG (Fig 7A and Fig 7B), olfactory neurons (Fig 7C), adrenal medulla (Fig 7D), and developing cortex (Fig 7E). Antibody specificity was established by peptide blocking experiments, which completely eliminated binding to tissue (Fig 7E).
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The tissue expression pattern of kalirin-12 parallels that demonstrated previously with the antibody that recognizes all forms of kalirin and agrees with the results of Western blotting analysis. Kalirin-12, however, exhibits a unique localization within cells that express it, occupying only a subset of the sites visualized with the kalirinspectrin antibody. In neurons, kalirin-12 is specifically localized to cell bodies rather than to axon processes (compare Fig 7A with Fig 7B, and Fig 7C with Fig 4B). The axonal immunostaining demonstrated with the kalirinspectrin domain antibody presumably reflects kalirin-9, which is the most prevalent isoform, in the processes. Kalirin-9, with only five unique amino acids at its COOH-terminus, is difficult to visualize specifically.
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Discussion |
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Kalirin Expression Correlates with Early Neuronal Development, Including Differentiation and Axon Outgrowth
Kalirin is widely expressed in the developing rat nervous system, with high levels of protein in the embryonic cortex, olfactory neurons, and dorsal root ganglia (Fig 8). Interestingly, kalirin expression in the nervous system is first detectable at E12 and appears to parallel neuronal differentiation rather than neuronal proliferation. Olfactory neurons, for example, begin to proliferate at E12, differentiate by E15/E16, and form connections to the developing olfactory bulb by E18 (
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In differentiating neurons, kalirin expression may be important in promoting axon outgrowth, because expression of kalirin in AtT-20 corticotrope tumor cells alters cytoskeletal organization and produces spike-like protrusions of actin from the cell surface (
The kalirin isoforms have divergent C-terminal domains. Kalirin-7, the major adult isoform, contains a PDZ-binding motif, while kalirin-8 contains an SH3 domain. Kalirins-9 and -12 both contain this SH3 domain and in addition contain a second DH/PH domain. Kalirin-12 is distinguished by its protein-kinase like domain (Fig 1B). Using antibody against kalirinspectrin-like repeats 4-7, we identified kalirin protein expression in both cell bodies and axons. However, using an antibody specific for the C-terminal of kalirin-12, we identified kalirin-12 protein specifically in neuronal cell bodies. These results suggest that kalirin-12 may be selectively retained in neuronal cell bodies, or that kalirin-9, which lacks the C-terminal kinase domain, may be targeted to axons. Similarly distinct localizations of endogenous kalirin-9 and -12 were observed in primary neuronal cultures (
The isoform-specific localizations observed for different kalirin proteins, which derive from alternative splicing, suggest that kalirin is important in regulating multiple physiological processes. Although kalirin expression in the adult mammal appears to be specific for the nervous system, kalirin is broadly expressed during development. In neurons of the rat embryo, kalirin may regulate growth cone motility and axon growth. In non-neuronal cells, such as endocrine cells and muscle cells, kalirin may regulate cell migration and attachment. Rescue of Drosophila mutant for dTrio requires expression of trio in both neurons and non-neuronal cells (
Expression of Kalirin in Migrating Muscle Cells and at Muscle Attachment Sites
In the adult rat, kalirin expression is restricted to the CNS. In the embryonic rat, however, kalirin expression is present in a number of muscle tissues, as well as other non-neuronal tissues. Earliest expression of kalirin in muscle tissue begins in the E10 heart, where it remains elevated throughout development. The fact that heart muscle exhibits normal morphology in trio loss-of-function mice also suggests a major role for kalirin in this tissue (
In muscle cells and tissues, kalirin demonstrates onset of expression during muscle cell migration. This is most dramatically seen with cells that will eventually form the embryonic tongue (Fig 4B). In rats and mice, these cells originate from the first five somites, migrate to the level of the diaphragm as the hypoglossal cord, and move anteriorly to form the embryonic tongue muscles (
The kalirin homologue unc-73 is broadly expressed in a number of migrating cells in the developing C. elegans, including the sex myoblasts, and mutations in the unc-73 gene lead to defects in cell migration (
The functional domains of kalirin suggest pathways through which development of nerve, muscle, and endocrine tissue could be affected. Kalirin is a close homologue of a number of proteins demonstrated to function in axon guidance and cell migration, including UNC-73 and Trio (
During embryonic rat development, kalirin, predominantly the kalirin-9 isoform, may function to promote proper formation and interaction of a number of tissues, including muscle and nervous tissue. Kalirin most likely can transduce these effects in a similar manner to UNC-73 and Trio, by activating Rac1, a member of the Rho GTPase family of proteins (
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Footnotes |
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1 These authors contributed equally to the work.
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Acknowledgments |
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Supported by DK-32948, DA-00266, DC-2979, and the Medical Scientist Training Program.
We would like to thank Marie Bell for technical support, Dick Mains for assistance with imaging, and Dr Christian Hansel for invaluable constructive comments on the manuscript.
Received for publication November 14, 2000; accepted January 31, 2001.
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