The Journal of Neuroscience, December 1999, 8(12): 4503-4512 The Sequential Appearance of Low- and High-Molecular-Weight Forms of MAP2 in the Developing Cerebellum R. P. Tucker,’ L. I. Binder,’ C. Viereck,’ B. A. Hemmings,’ and A. I. Matus’ ‘Friedrich Miescher Institute, CH-4002 Basel, Switzerland, and *Department of Cell Biology and Anatomy, School of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, Alabama 35294 Mammalian microtubule-associated protein 2 (MAPS) exists shown that a number of MAPS promote the assemblyof tubulin in high-molecular-weight (M, -280,000) and low-molecular- into microtubulesand stabilize the resulting microtubulesagainst weight (M, -70,000) forms, with the latter protein being more the depolymerizing effects of cold and various pharmacological abundant in embryonic brain homogenates than in prepa- agents(Olmsted, 1986; Matus, 1988). The most abundant MAP rations from mature brain (Riederer and Matus, 1985). In the in the mammalian CNS, MAP2, displays each of these char- current study, we have shown that avian MAP2 also exists acteristics: (1) There are 2 closely related high-molecular-weight as both high- (M, -260,000) and low-molecular-weight (M, (HMW) forms of MAP2, MAP2a, and MAP2b, which change -65,000) forms whose relative abundance changes during in relative abundanceduring development (Binder et al., 1984; brain maturation, indicating a conserved function for these Burgoyne and Cummings, 1984);(2) MAP2 is found in dendrites proteins during vertebrate neuronal morphogenesis. Using and only rarely in axons (e.g., Cacereset al., 1984; DeCamilli indirect immunohistochemistry, we have determined the cel- et al., 1984; Huber and Matus, 1984b); and (3) MAP2 stabilizes lular distribution of the high- and low-molecular-weight forms and promotes the assembly of microtubules in vitro (Murphy of MAP2 in the developing avian cerebellum. In the embry- and Borisy, 1975; Sloboda et al., 1976; Herzog and Weber, onic cerebellum, low-molecular-weight MAP2 is found in the 1978). external granular layer and in epithelial cells. High-molecu- Recently, a low-molecular-weight (LMW) form of MAP2, lar-weight MAP2 is found only in neurons that have com- MAP2c, has been described in the rat (Riederer and Matus, menced dendrogenesis, i.e., Purkinje cells and neurons with- 1985; Garner et al., 1988). Initially shownto be related to HMW in the internal granular layer. Thus, low-molecular-weight MAP2 by a common monoclonal antibody epitope, the LMW MAP2 is not only more abundant in embryonic nervous tissue form of MAP2 is probably encodedby the samegene as HMW than in the adult, but it also appears in glia and in differen- MAP2 (Garner and Matus, 1988). In contrast to MAP2a, MAP2c tiating neurons before the high-molecular-weight form. We is plentiful in embryonic and neonatal rat brain homogenates, have also shown that in the mature cerebellum high-molec- and decreasesdramatically in abundanceduring the secondweek ular-weight MAP2 cannot be detected with monoclonal an- of postnatal development. tibodies or polyclonal antisera in Purkinje cell dendrites. Using MAP2 monoclonal antibodies and polyclonal antisera, Polyclonal antisera against the regulatory subunit of the we have studied the distribution and developmental regulation CAMP-dependent protein kinase, which is associated with of HMW and LMW MAP2 forms during the morphogenesisof MAP2 in the Purkinje cell dendrites of the rat, also fail to the avian cerebellum.By using an avian model system, we have stain Purkinje cell dendrites in the mature quail cerebellum. been able to determine which aspectsof the intracellular com- This suggests that high-molecular-weight MAP2 may be nec- partmentalization and changesin MAP2 form are conserved, essary for the establishment of dendrites but is not neces- implying a fundamental function, in 2 classesof vertebrates. sary for the maintenance of dendritic form. Furthermore, by comparing the onset of expression and local- ization of the HMW and LMW forms of MAP2 in differentiating Microtubule-associated proteins (MAPS) have been implicated tissue, it is now possible to envisage the function of MAP2 as key regulators of the morphogenesis,function, and mainte- during neuronal morphogenesismore clearly. nance of the nervous system. For example, brain MAPS have been shown to be developmentally regulated, with their molec- Materials and Methods ular form and abundanceundergoing substantial changesduring Experimental animals. Quail (Coturnix coturnix) eggs were kept at 37°C development (Nunez, 1986; Matus, 1988). Moreover, immu- in a humidifiedincubator until embryonicday 10 (ElO), E12, or El4 nohistochemicalstudies with monoclonal antibodieshave shown (a day beforehatching). Juvenile quail (9 d afterhatching, P9) were used as a sourceof mature(no or greatlyreduced external granular layer) that many MAPS are compartmentalized within neurons, being cerebellums. enriched in either axons or dendrites, and in vitro studieshave Monoclonal antibodies and polyclonal antisera. The productionand characterizationof severalof the monoclonalantibodies to microtubule Received Oct. 26, 1987; revised Apr. 1, 1988; accepted Apr. 4, 1988. proteinsused in this study have been describedin detail elsewhere This work was supported in part by U.S. Public Health Service Grant AGO6969 (AP14: Binder et al., 1986; MAb/C: Huber and Matus, 1984b; Tu27b: to L.I.B. We wish to thank W. Halfter, G. Huber, and E. Mackie for constructive Binder et al., 1986). Monoclonal antibody AP14 recognizes only the criticism of the manuscript. HMW forms of MAP2 on Western blots of mammalian brain micro- Correspondence should be addressed to Richard P. Tucker, Friedrich Miescher tubule protein (Binder et al., 1986), whereas monoclonal antibody C Institute, P.O. Box 2543, CH-4002 Basel, Switzerland. (MAb/C) recognizes both ofthe HMW forms of MAP2, as well as LMW Copyright 0 1988 Society for Neuroscience 0270-6474/88/124503-10$02.00/O MAP2 (Riederer and Matus, 1985). Tu27b is specific for fl-tubulin. The 4504 Tucker et al. - MAP2 Forms in Developing Cerebellum ;=: . API4 C API8 PX2 CIP- CIP+ Figure 1. Immunoblots of P9 quail and adult rat brain microtubule Figure 2. Immunoblots of embryonic and mature quail brain super- proteins stained with monoclonal antibodies and polyclonal antisera to natant proteins stained with monoclonal antibody C. Molecular weights MAPZ. Molecular weights are indicated by dashes at the far right (from are indicated by dashes at the far right (from top to bottom: M, 200,000, top to bottom: M, 200,000, 116,000, 95,000, 66,000, and 45,000). 116,000, 95,000, 66,000, and 45,000). The relative abundance of the Monoclonal antibody AP14 stains the HMW MAP2 doublet (MAP2a LMW form of MAP2 decreases as the brain matures (A). When the and MAP2b) in the rat preparation and a single band with an apparent same proteins are incubated with calf intestinal alkaline phosphatase molecular weight of -260,000 on quail microtubule immunoblots. (CIP), the multiple LMW MAP2 bands resolve into a single M, 65,000 Monoclonal antibodies C and API 8, as well as polyclonal antisera PX2, band (B), indicating that the heterogeneity of LMW MAP2 forms is due stain the same HMW MAP2 bands as AP14, as well as a LMW (M, to phosphorylation. -65,000) doublet on the quail immunoblot. LMW MAP2 can be detected immunohistochemically by staining adjacent sections with AP14 and an antibody that recognizes both HMW and LMW MAP2 (e.g., C); Nitrocellulose filters with transferred brain proteins were blocked with the differences between the staining patterns will represent the LMW 5% skim milk in PBS, incubated in the MAP2 monoclonal antibodies form of MAPZ. (diluted hybridoma supematants, 1:20 in PBS) or polyclonal antisera (diluted 1:400 in PBS) for 2 hr, rinsed in PBS, and incubated for 1 hr with HRP-conjugated rabbit anti-mouse IgG (1:500 in PBS; Dakopatts). specificity of MAP2 monoclonal antibody AP18, which was raised ac- The antibodies were visualized using chloronaphthol as chromogen. cording to the methods described in Binder et al. (1986), was determined Immunohistochemistry. Whole embryonic quail heads or freshly dis- by Western blotting (see below). Polyclonal antisera against MAP2 (PX2) sected P9 cerebellums were fixed by immersion in cold 4% parafor- was raised using a HMW MAP from adult Xenopus laevis brain as an maldehyde in potassium phosphate buffer (0.1 M, pH 7.5) for 4 hr. The antigen. In brief, a HMW band with an apparent molecular weight tissue was then rinsed in PBS (0.1 M, pH 7.5) and cryoprotected in 25Oh corresponding to mammalian MAP2 was cut from an SDS-polyacryl- sucrose/PBS overnight. Heads or cerebellums were embedded in O.C.T. amide gel, homogenized in PBS with Freund’s complete adjuvant, and compound (Miles), frozen, and sectioned sagittally at 20 pm in a Rei- injected into the peritoneum of a Balb/c mouse. After 3 immunizations chert-Jung model 2700 cryostat. Serial sections were collected on gel- with the antigen mixed in Freund’s incomplete adjuvant, the serum was atin-coated slides, air-dried for 2-3 hr, rinsed in PBS, blocked in 0.5% collected and screened by immunoblotting (see below). The affinity- BSA, and incubated overnight with monoclonal antibodies (hybridoma purified rabbit antisera to bovine heart R,, has been described elsewhere supematants) or polyclonal antisera diluted in 0.5% BSA/PBS. Control (Hemmings et al., 1986). sections were treated identically except for incubation in 0.5% BSA/ Protein preparations and immunoblotting. Adult rat brain and P9 PBS without antibodies. Since MAP epitopes can be masked in situ by quail brain cold-warm cycled microtubules were made essentially as phosphorylation (Papasozomenos and Binder, 1987) some sections were described by Karr et al. (1979). Brain supematant proteins were made treated with CIP before incubation with the MAP2 monoclonal anti- from ElO, E14, and P9 quail by centrifuging brain homogenates at bodies (Stemberger and Stemberger, 1983; Tucker et al., 1988a). After 90,000 x g for 45 min at 4°C in MES buffer (0.1 M morpholinoeth- monoclonal antibody incubation, sections were rinsed in PBS and in- anesulfonic acid, 1 mM EGTA, 0.5 mM MgCl,; pH 6.6).