Phylogenetic conservation of brain microtubule-associated proteins MAP2 and TAU

NeuroscienceVol. 26, No. 3, pp. 893-904, 1988 Printed

in Great

0306-4522/88

Britain

$3.00 + 0.00

Pergamon Press plc Q 1988 IBRO

PHYLOGENETIC CONSERVATION OF BRAIN MICROTUBULE-ASSOCIATED PROTEINS MAP2 AND TAU C. VIERECK, R. P. TUCKER, L. I. BINDER* and A. MATUS Friedrich Miescher-Institut, P.O. Box 2543, CH-4002 Basel, Switzerland, and *Department of Cell Biology and Anatomy, School of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, AL 35294, U.S.A. Abstract-The major rat brain microtubule-associated proteins, MAP2 and tau, exhibit various properties that implicate them in the mechanisms underlying the growth of axons and dendrites during neuronal development. To determine if these properties represent fundamental morphogenetic mechanisms, we have examined the phylogenetic conservation of these proteins in Xenopus luevis, quail and rat with respect to their molecular form, cytological distribution and developmental expression. In all three species, the high-molecular weight form of MAP2 migrates as a pair of polypeptides (MAP2a and MAP2b); this doublet as well as the low-molecular weight form of MAP2 (MAP2c) and the tau proteins are markedly similar in size in the different classes of vertebrates. lmmunohistochemical staining of the Xenopus and quail cerebellum showed that MAP2 is highly concentrated in dendrites whereas the tau proteins are predominantly confined to axons, exactly as they are in rat. The developmental regulation of these proteins in Xenopus and rat is also conserved. Between the larva and the adult (i.e. during metamorphosis) MAP2c undergoes a marked decrease while MAP2a undergoes a large increase. Thus, in both classes of vertebrates the timing of changes in MAP2 expression coincides with the maturation of neuronal morphology. Taken together, these conserved properties of MAP2 and tau in three phylogenetically divergent classes of vertebrates suggest that these proteins serve fundamental functions during neuronal morphogenesis.

The microtubule-associated proteins (MAPS) of brain exhibit various properties that suggest they are important in the growth and stabilization of axons and dendrites during neuronal morphogenesis.26.3’ For two major MAPS from mammalian brain, MAP2 and the tau proteins, the evidence is as follows: First, both proteins promote tubulin polymerization in vitro8.29.46 and may thus stimulate the formation of microtubules, which is known to be essential for the elongation and stabilization of neurites.5.‘7.36 Second, these proteins are primarily associated with different domains of the neuronal structure. High-molecular weight MAP2 (HMW-MAP2) is, with very few exceptions, limited to dendrites of neurons throughout the mammalian CNS,‘,7’0.20 whereas low-molecular weight MAP2 (i.e. MAP2c) is found in axons,42.43 and the tau-MAPS are more abundant in axons than dendrites.4.42 Third, a variety of MAPS, including MAP2 and tau, undergo changes in expression that in rat brain coincide with the time at which axonal and dendritic outgrowth are completed.2~6,‘3~28 The developmental regulation of MAPS involves both the amount of the particular MAP species that

Abbreviations: EDTA, ethylene diaminetetra-acetate HMW-MAP2, high-molecular weight MAP2; MAPS, microtubule-associated proteins; PBS, phosphatebuffered saline; PMSF, phenylmethyl sulfonylfluoride; SDS-PAGE, sodium dodecylsulfate-polyacrylamide gel electrophoresis; TBS, Tris-buffered saline. 893

is expressed and the form that predominates at different times during neuronal morphogenesis. Rat brain HMW-MAP2 is represented by a pair of 280,000 mol. wt components: MAP2a and MAP2b;29 the recently described MAP2c has a molecular weight of 70,000.‘5~‘6~28~35 In the neonatal rat brain the levels of MAP2b and MAP2c are high’5,28.35 whereas MAP2a is absent.3,6 Between postnatal days 10 and 20, MAP2c undergoes a large decrease simultaneously with the appearance of MAP2a.28,35 Nunez and colleagues have shown that in the juvenile rat brain the tau MAPS are represented by a single component of 48 kDa whereas in the adult rat brain there are several tau components in the size range 52,00&68,000 mol. wt.9’3.25 In the developing rat brain, changes in the form of MAP2 and tau coincide with the transition of neurons from the juvenile phase, during which axons and dendrites are extending and branching, to the adult phase during which mature branching patterns have been achieved and are maintained. This implies a chemo-mechanical link between the form of MAP expression and the state of plasticity of neuronal processes.26 If the compartmentalization and developmental changes of brain MAPS represent chemical signs of fundamental mechanisms of axonal and dendritic growth and differentiation, then they should be present in non-mammalian vertebrates as well. We have used broadly cross-reactive monoclonal

C.

894

VIERECK et al.

antibodies to MAP2 and tau to compare the molecular form, cytological distribution and developmental regulation of these proteins in Xeno~~us iueuis, the quail and rat brain to determine how fundamental these patterns are at the phylogenetic level.

EXPERIMENTAL

PROCEDURES

Experimental animais Adult female Xenopus fueois, postnatal day 15 Japanese quails (Carurn& corurnix japonica) and Sprague-Dawley rats were obtained from Ciba Geigy Ltd, Basei. For developmental studies, Xenopus laeois embryos were obtained from hormone-induced natural matings of animals maintained in a colony at Ciba-Geigy, Basel. The larvae were staged using the developmental tables of Nieuwkoop and Faber.= Four stages were compared to the adult: pre-metamorphosis (stages 4648; no hind limb buds), early metamorphosis (stages 5ft54, hind limb buds), midmetamorphosis (stages 5460; extended hind limb buds) and late metamorphosis (stages 6@62; fore limbs).

Immunohistochemisrry Xenopus, quail and rat nervous tissue were fixed, cryoprotected and sectioned using methods described in Tucker and Matus.4Z To expose anti-tau epitopes masked by phosphorylation,‘2 alternate sections were treated with 40 LJ-of Ealf intestinal alkaline phosphatase (Boehringer) in TBSiEDTA buffer ff BS. 0.1 mM EDTA. DH 8.0) with 10 mM’PMSF for 120 min at 37°C or in TBS/EDTA/PMSF alone under the same conditions.‘2,37 The immunohistochemical techniques for anti-MAP?, anti-tau and anti-tubuiin staining are described in detail elsewhere.4’ The hybridoma supernatants were diluted at i:lO (v/v) in phosphate-b~ered saline (PBS) and stained with rhodamine-labeled rabbit anti-mouse antibody (DAKOPATTS). Sections stained for immunofluorescence were rinsed in PBS, counter-stained for 5 min in 1fig/ml bisbenzimide H 33258 (Hoechst dye; Riedei-de Haen) in PBS and rinsed again in PBS alone before mounting in 50% (w/v) glycerol and 1% (w/v) azide in PBS. RESULTS

Molecularfbrms

of MAP2

and tau in

Xenopus, quail

and rat brains

MAP2 and tau were characterized by gel electrophoresis and Western blotting. Figure 1 shows Western blots of the supernatant (lanes l), thermostable supernatant (lanes 2) and microtubule (lanes 3) fractions from the brains of adult Xenopus (marked “X”), postnatal day 15 quail (marked “Q”) and adult rat (marked “R”) stained with anti-MAP2 (left panel) and anti-tau (right panel). MAP2. The molecular form of HMW-MAP2 is highly conserved in the brains of the three classes of vertebrates studied (Fig. 1). The molecular weight of HMW-MAP2 is approximately 260,000 in quail and 270,000 in .Y~IKI~U.S and is thus very similar in size to the corresponding form of MAP2 in the rat brain (2g0,000).29 In all three species HMW-MAP2 is Monoc~o~a~antibodies the~o-stable (Fig. 1, lanes 2) and binds efficiently to All monocional antibodies used in this study were raised in mice against mammalian brain proteins. The production microtubules (Fig. 1, lanes 3). In both Xenopus and and characterization of the monoclonal antibodies against rat HMW-MAP2 appears as a pair of polypeptides MAP2 (API4 and API@, tau (TAU-I) and tubuiin (Tu27b) corresponding to MAP2a and MAP2b. To resolve have been described in detail eisewhere.4,7 On immunoblots MAP2a and MAP2b from quail brain, it was necesof rat brain microtubules API8 recognizes high-molecular sary to use urea-containing polyacrylamide_SDS gels weight (HMW-MAP2) and low-molecular weight (LMWMAPZ) forms of MAP2 whereas API4 recognizes only (Fig. 2). Quail HMW-MAP2 then also appears as a HMW-MAP2; TAU-I stains approximately 46 polypeppair of closely migrating proteins but there appears to tides and anti-tubuiin recognizes fi-tubuiin. be proportionately less MAP2a than MAP2b (Fig. 2, Gel elecrrophoresis and immunoblotting lanes 2 and 4). It should be noted that, in the presence Sodium dodecyisulfatepolyacryiamide gel eiectrophoreof urea (Fig. 2) quail MAP2a and MAP2b undergo sis (SDS-PAGE) was performed using 1.5 mm slab gels with a change in electrophoretic mobility and co-migrate or ‘without 5 M urea and a 415% linear gradient of with the corresponding rat poly~ptides. polyacryiamide.z4 Proteins were either stained with MAP2c was identified in the adult rat and quail Coomassie Brilliant Blue or electrophoreticaiiy transferred brains and exhibited similar electrophoretic mobilito nitroceliulose sheets (Miliipore) for immunobiotting.@’ All monocional antibodies were diluted at I: 10 (v/v) in ties but was not detectable in the adult Xenopus brain 0.05 M Tris-MCI (pH 7.6) and 0.2 M NaCi (TBS) contain(Fig. 1, left). MAP2c appeared as a group of polying 1% (w/v) non-fat dried milk. Immunobiots of Xenr1pu.r peptides of approximately 70,000 mol. wt in the rat brain supernatants corresponding to different stages of brain in agreement with previous reports’5.28.‘5comdevelopment were scanned using a reflectance densitometer (Camag) using the methods previously described by pared to 65,OOOmol. wt in the quail brain. Like Riederer and Matus.j’ Equai amounts of brain supernatant MAP2a and MAP2b, MAP2c was also thermo-stable protein (40 pug)were loaded onto the gel. Two series of brain and bound efficiently to microtubules (Fig. 1, left, supernatants from the different stages were scanned. Protein lanes 2 and 3). MAP2c was more abundant in the concentration was determined using BioRad reagents and quail brain than in the rat brain. bovine serum albumin as a standard. Brain ,fiactionation

Freshly dissected brain tissue was homogenized in icecold assembly b&e?’ without GTP containing 0.5 M sucrose, 1mM phenylmethyl sulfonylfluoride (PMSF; Serva) as well as pepstatin (Sigma) and antipain (Sigma), both at 0.01 mg/ml. The homogenate was centrifuged for 60min at 150,OOOgand 4°C. Aliquots of the supernatant and the fractionated thermo-stable supernatant18 were stored at -80°C before gel eiectrophoresis and immunoblotting. Taxol-stabilized microtubules were isolated from the remaining supernatant4j and were resoiubilized in assembly buffer** minus GTP. An aliquot of the microtubules was used to fractionate the thermo-stable MAPS.‘” The microtubules and thermo-stable MAPS were either used immediately for gel electrophoresis and immunobiotting or stored at -80°C until use.

Phylogenetic conservation of MAPS

X

Q

R

123123123

x

895

Q

123123

R 123

116929 66459

MAP2

TAU

Fig. 1. Molecular form of brain MAP2 and tau in adult Xenopus, quail and rat. SDS-PAGE immunoblots of supernatants (lanes I), thermo-stable supernatants (lanes 2) and microtubules (lanes 3) from Xenopus (“X”), quail (“Q”) and rat (“R”) brains were stained with monoclonal antibodies against MAP2 (AP18; left panel) and tau (TAU-I; right panel). Anti-MAP2 recognizes both HMW-MAP2 and MAP2c in the adult rat and quail brains; in the adult Xenopus brain only HMW-MAP2 is stained. The electrophoretic mobility of ~MW-MAP2 is closely similar in the three classes of vertebrates: approximately 260,O~ mol. wt in the quail. 270,090 in Xenoptis and 280,000 in the rat; the mol. wt of MAP2c is also similar in the rat and quail. Both forms of MAP2 are thermo-stable (lanes 2) and bind to microtubules (lanes 3). Anti-tau stains a group of brain proteins from the three classes of vertebrates between approximately 50,000 and 70,000 mol. wt as well as a higher mol. wt component (_ l~,~O), the latter tau is most abundant in the quail and least abundant in the rat. A number of less abundant lower mol. wt tau potypeptides (30,0~~0,~) can also be detected. The majority of the tau proteins were also found to be thermo-stable (lanes 2) and to bind to microtubules (lanes 3). Molecular weight markers (x 10e3) are indicated on the left.

Tuu. In agreement with previous reports,4’* antitau stains a group of rat brain proteins between approximately 50,000 and 70,~Omol. wt (Fig. 1, right). Although differing in their relative number and molecular weight, tau polypeptides within this range were also identified in the quail and Xenopus brains. In the adult Xenopus brain, for example, two major tau polypeptides of 55,000 and 60,000 mol. wt as well as a number of polypeptides below 40,000 kDa were identified in addition to a tau species of approximately 100,000 mol. wt. The latter was most abundant in the Xenopus and quail brains but was also present in the rat brain. This high-molecular weight tau as well as most of the lower molecular weight tau polypeptides were thermo-stable and

bound efficiently to microtubules 2 and 3).

(Fig. 1, right, lanes

Cellular d~~tri~utio~ofhigh-~~Ie~uI~r weight MAP2 and tau

To compare the cytological distribution of HMWMAP2 and tau proteins, adult Xe~~pus, postnatal day 1.5quail and adult rat cerebellums were stained with monoclonal antibodies against MAP2 and tau (Figs 3 and 4). sigh-~#Ie~uIar weight-~~~2. In agreement with the previously reported distribution of HMW-MAP2 in the rat cerebellum (Fig. 3a, b),1,2,7,‘o.20. anti-MAP2 specifically stains dendrites in the quail (Fig. 3c, d) and Xenopus cerebellums as well (Fig. 3e, f). In the

C. WRECK et al.

116926645

12

3

4

12341

XENOPUS

234

QUAIL

RAT

Fig. 2. Electrophoretic pattern of supetnatants and microtubules in adult Xenopus, quail and rat brains. SDS-PAGE in the presence of 5 M urea, of supernatant (lanes I), thermo-stable supernatant (lanes 2), microtubule (lanes 3) and thermo-stable microtubule (lanes 4) fractions from XURIJNU, quail and rat. HMW-MAP2 (arrowhead) can be identified in the thermo-stable fractions of all three species (see lanes 2 and 4) and appears as a pair of closely migrating proteins corresponding to MAP2a and MAPZb, which vary in relative proportion in the different species. The electrophoretic mobilities of HMW-MAP2 in the presence of urea are very similar: * 270,000 mot. wt in ~e~o~~s fractions and 280,ooO mol. wt in the quail and rat brains, Molecwlar weight standards ( x IO-“) are indicated on left.

cerebellums of all three classes of vertebrates, antiMAP2 stains the granule cell layer intensely. Within the ,molecular layer, anti-MAP2 stains stellate cell bodies and dendrites; in the quail and rat, verticallycoursing processes in the molecular layer are also stained. The Purkinje cell bodies and dendrites are stained by anti-MAP2 in the rat and Xeno/~u.s cerebellums but are unstained in the quaiLe No staining was found in the white matter or in the parallel fibers in the cerebellums of the three species. Tau. In contrast to the dendritic staining pattern obtained with anti-MAP2, the anti-tau monoclonaf antibody staining pattern is axonal (Fig. 4) as previously reported in the rat.4 No differences in the staining pattern or staining intensity were observed after sections were treated with alkaline phosphatase (results not shown). Within the molecular layer, parallel fibers are stained in the cerebellums of all three species. The granule cell layer is also stained but less intensely than the molecular layer. Purkinje cell bodies are faintly stained in the rat (Fig. 4a, b) and intensely stained in Xenliptts (Fig. 4e, f) but are unstained in the quail (Fig. 4c, d). The white matter of the rat cerebellum is stained by anti-&u as

previously described by Binder rf aL4 and is also stained in the adult Xenopus and quai cerebellums. MAP2

and fau in the developing Xenopus

brain

Supernatants isolated from Xenopus brains at different stages of development were subjected to immunoblotting using monoctonal antibodies against MAP2 and tau (Fig. 5). To determine whether there were changes in the relative amount of individual MAPS during brain development, equal amounts of supernatant protein from each developmental stage were loaded onto the gel and the immunoblots scanned using a reflectance densitometer. The results are expressed as relative amount of protein versus mean developmental stage (Fig. 6). Two series of brain supernatants corresponding to the different developmental stages were scanned and an identical pattern of regulation was found in each case. MAP2. Figure 5 demonstrates that the form of MAP2 differs in larval and adult Xeplopus brains; there are distinct larval. and adult MAP2 proteins. Before metamorphosis, there is a higher proportion of MAP2c (65 kDa) and proportionately less MAP2a. Quant~tation of the relative amounts of

Fig. 3. Low (a,c,e) and high (b,d,f) magnification micrographs of adult rat. quail and Xenopus cerebellums stained with monoclonal’antibodies to MAP2 (API4 and API@. Anti-MAP2 stains dendrites and not axons in the cerebellums of all three classes of vertebrates examined. In the rat cerebellum (a,b), hlAP2 is found in the granule cell layer (g) and in cell processes within the molecular layer (m). The white matter (a, arrows) is unstained. Within the molecular layer, the cell bodies and dendrites of steilate cells (b, arrows) and Purkinje cells (arrowheads) are stained. In the quail cerebellum (c,d). the overall staining pattern is very similar to the rat, with intense staining within the granule cell layer (g) and the staining of processes in the molecular layer (m), and the absence of anti-MAP2 staining in the white matter (c, arrows). In the quail cerebellar molecular layer. stellate cells and their dendrites (arrows), as well as vertically-toursing beaded processes are stained, but Purkinje cells (arrowheads) are unstained. The concentration of MAP2 in dendrites and cell bodies is also seen in Xennpu.~ (e,f) where granule cells (g) are stained intensely, and bundles of white matter (e. arrows; f,w) are unstained. Purkinje cells and their dendrites are stained intensely in the Xenopus cerebellum. as are stellate cells (f, arrows). In all three classes of vertebrates, parallel fibers are unstained hy anri-MAPZ. 897

Fig. 4. Low (a,c,e) and high (b,d,f) magnification micrographs of adult rat, quail and ,%‘wIo~u.\cerebellums stained with monoclonal antibodies against tau (TAU-I). All sections were pretreated with alkaline phosphatase. Anti-tau stains axons and not dendrites in the cerehellar cortex of the rat (a.b). quail (cd) and Xenopus(e,f). Parallel tibers within the molecular layer (m). and white matter (a,c.e, arrows; f.w,) arc stained by anti-tau. The granule cell layer (g) is stained less intensely than the molecular layer. The dendrites of Purkinje cells (arrowhead) stand out in negative relief in the rat cerebellar molecular layer (b); Purkinje cell bodies are unstained in the quail (d, arrowhead) and are anti-tau positive in ,Y’WZO,VN,F (f, arrowheads). 89X

12345

12345 cue .

*=

zoo-

11692-

45-

TAU

MAP2

Fig. 5. ~velopmental time course of MAP2 and tau in Xenopur brain. Immunoblots of brain su~rnatants from larval stages 46-48, N-54, N-60,60-62 and adults (lanes I-5, respectively) were stained with MAP:! (AP18) and tau (TAU-I) monoclonal antibodies (left and right panels, respectively). Equal amounts of protein (40 pg) from each developmental point were loaded onto the gel. During the identical period of brain development in Xenopus, expression of MAP2c and the 30,000mol. wt tau protein decreases significantly while expression of MAP2a and the 100,000 mol. wt tau protein increases. The changes were quantitated by reflectance densitometry and are summarized in Fig. 6. 2.0-

2.0MAP2

TAU

z ii8 5 ISiz c:

z ? 0 if c:

1.5-

l.O-

s

l.O-

9 E = 0.5Bz

g

$

ii ;: a

I 47

I 51

I 57

1 A

I 61

MEAN DEVELOPMENTAL

0.5-

I 47

I 51

1 57

MEAN DEVELOPMENTAL

STAGE

I 61

-l A STAGE

Fig. 6. Changes in the relative amounts of MAP2 and tau in the Xenopm brain at different developmental stages. Equal amounts of brain supernatant protein (40 pg) from each developmental stage were loaded onto the gel. Immunoblots stained with MAP2 (AP18; left panel) or tau (TAU-I; right panel) monoclonal antibodies were scanned by reflectance densitometry and the changes expressed as relative amount of protein versus the mean developmental stage. MAP2 protein values are given relative to the MAP2b (stage 47) level; tau poly~ptide values are given relative to the ~,~rnol. wt tau protein (stage 47) level. Expression of MAP2b (270.0~ mol. wt) remains unchanged during this developmental period while the relative amounts of MAP2a (270,~mol. wt) and MAP2c (65,OOOmol. wt) increase and decrease, respectively, with the largest change occurring during the same developmental interval (between stage 57 and the adult). While expression of the 55,000 and 60,000 mol. wt tau proteins remains unchanged during this developmental period, the relative amounts of the 30,000 and 100,000 mol. tau decrease and increase, respectively. MAP2a (270,OOOmol. wt; -O-O-), MAP2b (270,000 mol. wt; -e-e-), MAP2c (65,OOOmol. wt; -A-A-). Tau: 100,000mo1. wt (-@-a-), 60,000 mol. wt (-O-O-), 55,000 mol. wt (-A-A-), 30,000 mol. wt (-A-A-). 899

C. VIERECK et al.

900

MAP forms (Fig. 6) revealed that during the metamorphic climax, there was a significant decrease in MAP2c concomitant with the increase in MAP2a. The level of MAP2c drops to almost zero in the adult brain. The decrease in the Xenopus MAP2c and increase in MAP2a can also be seen in taxol-stabilized microtubule fractions from larval and adult Xenopus brains (results not shown). Tuu. As reported for the developmental time course of MAP2 polypeptides, a number of changes in the relative amount of individual tau proteins were also observed during the development of the Xenopus brain (Figs 5 and 6). During the developmental period corresponding to the metamorphic climax, there was an increase in the relative amount of the 100,000 mol. wt tau and a decrease in the expression of the 30,000 mol. wt tau. The developmental period during which these changes in tau polypeptides were measured is identical to the period that encompasses the changes in MAP2 proteins. The changes in tau were also observed in the corresponding microtubule fractions (results not shown). Cellular distribution qfMAP2 ing Xenopus spinal cord

root cells) in the stage 49/50 lumbar spinal cord (Fig. 7b). Similarly, tau is abundant in myelinated fibers running longitudinally through the white matter of the adult Xenopus spinal cord (Fig. 8b). Tau is abundant in adult spinal cord gray matter as well. Although no changes in the staining pattern or staining intensity of TAU-I were observed following alkaline phosphatase treatment of adult spinal cord sections, the staining of neuronal cell bodies in the larval spinal cord was only observed after this enzymatic treatment; treatment of immunoblots with alkaline phosphatase had no effect on the TAU-I staining intensity or staining pattern (results not shown). Tubulin. In the stage 49/50 spinal cord, the staining pattern with our monoclonal antibody to b-tubulin resembles a superimposition of the anti-MAP2 and anti-tau patterns with the ependymal layer staining as well (Fig. 7d): the white matter is stained intensely and both cell bodies and processes are stained in the gray matter. Similarly, in the adult (Fig. 8c), antitubulin stains processes in the gray matter as well as longitudinally-coursing axons in the white matter.

and tau in the develop-

To determine whether the intracellular compartmentalization of MAP2 and tau were conserved in the developing Xenopus CNS, spinal cords from larval and adult stages were stained with anti-MAP2 and anti-tau monoclonal antibodies; these staining patterns were then compared to the anti-tubulin staining pattern. MAP2. Anti-MAP2 stains cell bodies and dendrites in the gray matter of the lumbar spinal cord at stage 49/50 (Fig. 7b). There is no staining with anti-MAP2 in the ependymal layer or white matter. In the adult, anti-MAP2 stains long, beaded processes within the gray matter as well as thin processes coursing laterally through the lateral white matter (Fig. Sa). Myelinated fibers in the white matter and peripheral nerves are unstained. Tuu. In contrast to MAP2, tau is found in white matter and neuronal cell bodies (including dorsal

DISCUSSION

Evidence that MAP2 and tau ure present in Xenopus und quail brains Xenopus and quail brain tissue contain proteins that cross-react with monoclonal antibodies against mammalian brain MAP2 and tau. These proteins also possess other properties indicating that they are authentic Xenopus and quail forms of these previously characterized microtubule components. A principle indication is that both the putative Xenopus and quail MAP2 and tau occur as multiple components whose relative abundance and molecular weights closely match those of the same proteins in rat brain. Rat brain MAP2 occurs as two high molecular forms, MAP2a and MAP2b, of apparent molecular weight 280,000’y and a low-molecular weight form, MAP2c, of apparent molecular weight 70,000.‘5~‘6~~8~‘5 The anti-MAP2 reactive Xenopus brain proteins include a pair of proteins with an apparent molecular weight of

Fig. 7. MAP2, tau and tubulin distribution in the pre-metamorphic Xenopus spinal cord. Sections from a stage 49/50 lumbar spinal cord were stained with monoclonal antibodies to MAP2 (b, AP18), tau (c, TAU-I) and tubulin (d, Tu27b). Control sections were completely unstained. To expose TAU-I epitopes masked by phosphorylation, sections were pretreated with alkaline phosphatase. The Hoechst nuclear dve (a) shows the organization of the larval spinal cord. Few cells are found in the gray matter (g). The nuclei of the lateral-motor neurons are relatively large (arrow). The ependymal layer (e), white matter (w) and dorsal root ganglia (dr) are clearly seen. The monoclonal antibody to MAP2 (b) stains dendrites and cell bodies within the gray’matter exchtsively. The perikaryon of a large lateral motor neuron and its dendritic tree are indicated by the arrow. Anti-tau (c) stains white matter and cell bodies (but no processes) within the gray matter. The staining pattern with anti-tubulin (d) resembles a superimposition of the anti-MAP2 pattern onto the anti-tau pattern, with the ependymal layer stained as well. Fig. 8. MAPZ, tau and tubulin distribution in the dorsal horn of the adult Xenopus spinal cord. Sections from the dorsal horn of an adult spinal cord were stained with monoclonal antibodies to MAP2 (a, APl8), tau (b, TAU-I) and tubulin (c, Tu27b). Control sections were completely unstained. For TAU-I immunohistochemistry, sections were pretreated with alkaline phosphatase. Anti-MAP2 (a) stains beaded processes in the lateral white matter and gray matter; dorsal white matter is completely unstained. Anti-tau (b) stains the gray matter and myelinated fibers in the white matter. The anti-tubulin (c) staining pattern resembles the anti-tau pattern though it is less intense.

Phylogenetic conservation of MAPS

Figs 7 and 8.

901

902

C. VIERECK

- 270,000 corresponding to MAP2a and MAP2b and MAP2c of - 65,000 mol. wt. The electrophoretic mobility of quail MAP2a and MAP2b was shown to differ in the presence or absence of urea and have apparent molecular weights between - 260,000 and 280,000 while quail MAP2c migrates with an apparent molecular weight of 65,000. Unlike the case for Xenopus and rat MAP2a and MAP2b, where approximately equal amounts of both polypeptides are present, there was relatively less MAP2a compared to MAP2b in the quail brain. This may be related to the relatively early developmental stage of the quail brains that were used in this study. Rat tau is represented by a set of bands between 52,000 and 68,000,4,‘3 as well as an anti-tau reactive component of 100,000 mol. wt.33,34A similar pattern is found in Xenopus and quail brain fractions. Several anti-tau reactive components are present in the molecular weight range 50,000-60,000 and while their detailed patterns are slightly different from that of rat brain tau, they very closely resemble those of the tau components in other mammalian brainsi There are also Xenopus and quail anti-tau reactive bands at 100,000mol. wt. A tau protein of this approximate size has also been recently identified by Gard and Kirschner.14 We observe a very faint anti-tau reactive band at approximately the same molecular weight in rat brain supernatants and microtubules. In addition Drubin et al. have found a 110,000 mol. wt anti-tau reactive microtubule component in rat pheochromocytoma PC12 cells that differentiate in the presence of nerve growth factor to form neurites” and Peng et al. have found tau components of this approximate size in cultured rat sympathetic neurons.33,34 In the larval Xenopus brain there is an additional 30,000 tau component that is similar to a tau protein reported in rodent brain by Nunez and coworkers.‘3 We conclude that Xenopus and quail brains contain MAPS that in both antigenicity and molecular form are similar to their counterparts in mammalian brain. Conservation of cytological distribution of‘ MA P2 and tau In the rat brain, MAP2 and tau are concentrated in dendrites and axons, respectively.‘,4.7.‘0.20.42.43 In this study we found that Xenopus and quail MAP2 and tau are also associated with the same neuronal domains as the corresponding proteins in the rat brain. In the cerebellum and spinal cord the contrast is particularly obvious with anti-MAP2 staining only neuronal cell bodies and dendrites and anti-tau labeling cell bodies and axons. Although white matter staining with anti-tau was observed without pretreatment of the spinal cord sections, the staining of cell bodies with anti-tau in the larval Xenopus spinal cord was observed only after alkaline phosphatase treatment. The staining of processes in the gray matter (i.e. dendrites) was never seen. Thus, the unmasking of the anti-tau (TAU- 1) epitope by phos-

et ul

phatase treament in neuronal perikarya is another conserved feature of tau between Xenopus and the rat.32 The conservation of intracellular compartmentalization of MAP2 and tau between these three phylogenetically divergent classes of vertebrates suggests that the functions of MAP2 and tau are closely involved with dendrites and axons, respectively. Various possibilities exist for the nature of these functions. Both MAP2 and tau are known to promote the assembly of tubulin into microtubules.s~29~46 Recently Drubin and Kirschner have demonstrated such effects inside living cells by showing that both the rate of tubulin polymerization and microtubule stability increase following the microinjection of adult tau protein into fibroblast cells.12 It seems probable that both proteins would have the same stabilizing influence on microtubules in neuronal processes. MAP2 is a rod-like molecule that forms side arms on repolymerized brain microtubules in vitro,” and is believed to cross-link microtubules in the dendritic cytoplasm.26 Developmental regulation of MAP2 and tau in XenoPus Perhaps the most striking feature of the conservation of these proteins between rat and Xenopus is their developmental regulation. In both species the number and relative abundance of the MAP2 and tau-MAPS changes markedly between the juvenile and the mature brain. A significant decrease in expression of MAP2c and an increase in the expression of MAP2a during neuronal maturation have been shown in rat”,” and we observed the same changes in Xenopus. In Xenopus brain the decrease in MAP2c and the increase in MAP2a occur between mid-metamorphosis (stages 54-60) and the adult. Similarly, in the rat brain, a ten-fold decrease in MAP2c and an increase in MAP2a occur between postnatal days IO and 20.28.35Thus, in both rat and Xenopus the changes in MAP2 coincide with the end of axon and dendrite growth, the timing of the change corresponding to the maturation of neuronal morpho]ogy,15.17.21.23.3R.39

The Xenopus tau-MAPS are also developmentally regulated and the changes in tau expression occur during the same developmental period, between midmetamorphosis (stages 54-60) and adult, as the changes in MAP2 expression. In the rat, distinct tau-MAPS are expressed in the juvenile and adult brain: the juvenile brain contains only a single 48,000 mol. wt tau component’,” whereas there are several characteristic bands in the adult4,” The situation differs slightly in Xenopus. Although adultspecific tau proteins are not expressed, there is a juvenile-specific tau component. A selective drop in the 30,000 mol. wt tau is observed between metamorphosis and adult. A similar tau species has previously been identified in some rodent brains.” There is also an increase in the expression of the

Phylogenetic conservation of MAPS 100,000 mol. wt tau protein in Xenopus. The timing of the changes in tau expression, like the changes in MAP2 expression, also coincides with the transition of neurons from the phase in which there is high growth activity to the one in the adult where the branching patterns have been achieved and are maintained.2’ The juvenile and adult set of MAPS may be exerting different functions within the neuronal cytoskeleton. Mareck et al. 25 found that the rate of tubulin assembly in uitro was significantly lower when stimulated by juvenile rat brain thermo-stable MAPS (which contain predominantly MAP2b, MAP2c and juvenile tau proteins) compared to thermo-stable adult rat brain MAPS (which contain MAP2a and MAP2b, little MAP2c and a different complement of tau proteins). Juvenile and adult forms of Xenopus MAP2 and tau may similarly serve different functions in the neuronal cytoskeleton. The differential expression of MAP2 and tau proteins in Xenopus may reflect differences in the degree to which MAPS stabilize microtubules or cross-link

903

them with other components skeleton.

of the neuronal

cyto-

CONCLUSION

We have demonstrated that the molecular form, cytological distribution of MAP2 and tau in Xenopus and quail are markedly similar to those reported for the corresponding proteins in mammalian nervous systems. Furthermore, we have shown that the pattern of developmental regulation of these MAPS in Xenopus and rat is also conserved. This suggests that these proteins serve conserved functions and that the changes in MAP2 and tau expression reflect fundamental mechanisms of axonal and dendritic growth in vertebrates. Acknow[edgemenls-L.I.B. was supported by U.S. Public Health Service Grant AG06969. Taxol was generously donated by M. Suffness, National Cancer Institute, Bethesda, MD. We thank Lynne Farmer for technical assistance with the hybridoma cultures, Josef Jiricny for use of the reflectance densitometer and lnge Obergfiill for photographic services.

REFERENCES

I. Bernhardt R. and Matus A. (1984) Light and electron microscopic studies of the distribution of microtubule-associated protein 2 in rat brain: a difference between dendritic and axonal cytoskeletons. J. camp. Neurol. 226, 203-221. 2. Bernhardt R., Huber G. and Matus A. (1985) Differences in the developmental patterns of three microtubule-associated proteins in the rat cerebellum. J. Neurosci. 5, 9777991. A.. Kim H.. Caceres A.. Pavne M. R. and Rebhun L. I. (1984) Heterogeneity of 3. Binder L. I.. Frankfurter microtubule-associated protein’ 2 during rat brain development. Proc. nafn. Acad. Sci. U.S.A. 81; 5613-5617. _ 4. Binder L. I., Frankfurter A. and Rebhun L. I. (1985) The distribution of tau in the mammalian central nervous system. J. Cell Biol. 101, 1371-1378. in cultures of sensory neurons. Proc. natn. Acad. 5. Bray D., Thomas C. and Shaw G. (1978) Growth cone formation Sci. U.S.A. 81, 5613-5629. 6. Burgoyne R. D. and Cumming R. (1984) Ontogeny of microtubule-associated protein 2 in rat cerebellum: differential expression of the doublet polypeptides. Neuroscience 11, 1577167. 7. Caceres A., Binder L. I., Payne M. R., Bender P., Rebhun L. and Steward 0. (1984) Differential subcellular localization of tubulin and the microtubule-associated protein MAP2 in brain tissue as revealed by immunohistochemistry with monoclonal hybridoma antibodies. J. Neurosci. 4, 394410. 8. Cleveland D. W., Hwo S. Y. and Kirschner M. W. (1977) Physical and chemical properties of tau factor and the role of tau in microtubule assembly. J. molec. Bid. 116, 227-247. characterization of microtubule-associated proteins specific for the 9. Couchie D. and Nunez J. (1985) Immunological immature brain. Fedn Eur. biochem. Sots Lett. 188, 331-335. of microtubule-associated 10. De Camilli P., Miller P. E., Navone F., Theurkauf W. E. and Vallee R. B. (1984) Distribution protein 2 in the nervous system of the rat studied by immunofluorescence. Neuroscience 11, 819-846. M. W. (1984) Studies on the expression of the microtubule-associated II. Drubin D. G., Caput D. and Kirschner protein, tau. during mouse brain development, with newly isolated complementary DNA probes. J. Cell Biol. 98, 109t%lO97. 12. Drubin D. G. and Kirschner M. W. (1986) Tau protein function in living cells. J. Cell Biol. 103, 2739-2746. of microtubule13. Francon J., Lennon A. M., Fellous A., Mareck A., Pierre M. and Nunez J. (1982) Heterogeneity associated proteins and brain development, Eur. J. Biochem. 129, 465471. 14. Gard D. L. and Kirschner M. W. (1987) A microtubule-associated protein from Xenopus eggs that specifically promotes assembly at the plus-end. J. Cell Biol. 105, 2203-2216. 15. Garner C. C., Brugg B. and Matus A. I. (1988) A kiloDalton microtubule-associated protein 2 (MAP2c), related protein. J. Neurochem. 50, 609-615. 16 Garner C. C. and Matus A. I. (1988) Different forms of microtubule-associated protein 2 (MAPZ) are encoded by separate mRNA transcripts. J. Cell Biol. 106, 7799783. 17 Gona A. G. (1972) Morphogenesis of the cerebellum in the frog tadpole during spontaneous metamorphosis. J. camp. Neural. 146, 1333142. 18 Herzog W. and Weber K. (1978) Fractionation of brain microtubule-associated proteins, Isolation of two different proteins which stimulate tubulin polymerization in vitro. Eur. J. Biochem. 92, 1-8. 19 Hirokawa N. (1986) 270 K microtubule-associated protein cross-reacting with anti-MAP2 IgG in the crayfish peripheral nerve axon. J. Ceil Biol. 103, 33-39. of two microtubule-associated proteins, MAP1 20 Huber G. and Matus A. I. (1984) Differences in the cellular distributions and MAPZ, in rat brain. J. Neurosci. 4, 151-160. 31 Hughes A. (1981) Metamorphic changes in the brain and spinal cord. In Frog Neurobiology (eds Llinas R. and Precht W.). pp. 8566868. Springer, New York. A..

904

C. VIERECK rt al.

of brain microtubule proteins prepared by selective 22. Karr L. T., White H. D. and Purich D. L. (1979) Characterization removal of mitochondrial and synaptosomal components. J. biol. Chem. 254, 6107~6111. 23. Kollros J. J. (1981) Transitions in the nervous system during amphibian metamorphosis. In Mefamorphosis: A Problem in Derelopmenfal Biology (eds Gilbert L. I. and Frieden E.), pp. 445460. Plenum, New York. 24. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 68&685. 25. Mareck A., Fellous A., Francon J. and Nunez J. (1980) Changes in composition and activity of microtubule-associated protein during brain development. Nature 284, 3533355. 26. Matus A. (1988) Microtubule-associated proteins: Their potential role in determining neuronal morphology. A. Rev. Neurosci. II, 294. R., Bodmer R. and Alaimo D. (1986) Microtubule-associated protein 2 and tubulin are 21. Matus A., Bernhardt differentially distributed in the dendrites of developing neurons. Neuroscience 17, 371-389. 28. Matus A. and Riederer B. (1986) Microtubule-associated proteins in the developing brain. Ann. N. Y. Acad. Sci. 466, 167- 179. 29. Murphy D. B. and Borisy G. G. (1975) Association of high-molecular weight proteins with microtubules and their role in microtubule assembly in Grro. Proc. natn. Acad. Sci. U.S.A. 72, 269G-2700. 30. Nieuwkoop P. D. and Faber J. (1975) Normal Tables ofXenopus laevis (Daudin). North-Holland, Amsterdam. 31. Nunez J. (1986) Differential expression of microtubule components during brain development. Deal Neurosci. 8, 1255141. 32. Papasozomenos S. Ch. and Binder L. I. (1987) Phosphorylation determines two distinct species of tau in the central nervous system. Cell Motil. Cytoskel. 8, 21&226. analyses of cytoskeletal domains of 33. Peng I., Binder L. I. and Black M. M. (1985) Biochemical and immunological neurons. J. Cell Biol. 102, 252-262. 34. Peng 1.. Binder L. I. and Black M. M. (1985) Cultured neurons contain a variety of microtubule-associated proteins. Brain Res. 361, 200-2 1 I. 35. Riederer B. and Matus A. (1985) Differential expression of distinct microtubule-associated proteins during brain development. Proc. natn. Acad. Sri. U.S.A. 82, 60066009. 36. Seeds N. W., Gilman A. G.. Amano T. and Nirenberg M. W. (1970) Regulation of axon formation by clonal cell lines of a neural tumor. Proc. natn. Acad. Sci. U.S.A. 66, 160-167. 31. Sternberger L. A. and Sternberger N. H. (1983) Monoclonal antibodies distinguish phosphorylated and nonphosphorylated forms of neurofilaments in situ. Proc. natn. Acad. Sci. L/.S.A. 80, 6126~6130. 38. Thors F., De Kort E. J. M. and Nieuwenhuys R. (1982) On the development of the spinal cord of the clawed frog Xenopus laeois. 1. Morphogenesis and histogenesis. Anat. Embryo/. 164, 4277441. 39. Thors F., De Kort E. J. M. and Nieuwenhuys R. (1982) On the development of the spinal cord of the clawed frog Xenopus laeeis. II. Experimental analysis of differentiation and migration. Anur. Embryol. 164, 443454. transfer of protein from polyacrylamide gels to 40. Towbin H., Staehelin T. and Gordon J. (1979) Electrophoretic nitrocellulose sheets: procedure and some application. Proc. nam. Acad. Sci. U.S.A. 76, 435&4354. 41. Tucker R. P. and Matus A. (1987) Developmental regulation of two microtubule-associated proteins (MAP2 and MAPS) in the embryonic avian retina. Development 101, 535 546. 42. Tucker R. P., Binder L. I. and Matus A. (1988) Neuronal microtubule-associated proteins in the embryonic avian spinal cord. J. camp. Neural. 271, 44-55. 43. Tucker R. P., Binder L. I. and Matus A. (1988) Differential localization of the high- and low-molecular weight variants of MAP2 in the developing retina. Decl Brain Res. 38, 313-318. 44. Tucker R. P., Binder L. I.. Viereck C., Hemmings B. A. and Matus A. (1988) The sequential appearance of low- and high-molecular weight forms of MAP2 in the developing cerebellum. J. Neurosci. (in press). 45. Vallee R. B. (1982) A taxol-dependent procedure for the isolation of microtubule-associated proteins (MAPS). J. Cell Biol. 92, 435442. 46. Weingarten M. D., Lockwood A. H.. Hwo S. Y. and Kirschner M. W. (1975) A protein factor essential for microtubule assembly. Proc. nam. Acad. Sri U.S.A. 72, 185% 1862. (Accepted 8 March 1988)