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Continuous conversion of neuron phenotype in hydra
[]~12VIEWS 5 Hillman, N., Sherman, M.I. and Graham, C.F. (1972) J. Embryol. Exp. Morphol. 28, 263-278 6 Ziomek, C.A., Johnson, M.H. and Handyside, A.H. (1982) J. Exp. Zool. 221,345-355 7 Johnson, M.H. (1985) in DevelopmentalBiology: A Comprehensive Synthesis (Vol. 4) (Gwatkin, R.B.L., ed.), pp. 279-296, Plenum Press 8 Fleming, T.P. (1987) Dev. Biol. 119, 520-531 9 Hyafil, F., Morello, D., Babinet, C. and Jacob, F. (1980) Cell 21,927-934 10 Johnson, M.H., Maro, B. and Takeichi, M. (1986) J. Emb~. oL Exp. Morphol. 93, 239-255 11 Vestweber, D., Gossler, A., Boller, K. and Kemler, R. . (1987) Dev. Biol. 124, 451-456 12 Fleming, T.P. and Johnson, M.H. (1988)dnnu. Rev. Cell Biol. 4, 459-485 13 Houliston, E., Pickering, S.J. and Maro, B. (1987)J. Cell Biol. 104, 1299-1308 14 Houliston, E. and Maro, B. (1989)J. CellBiol. 108, 543-551 15 Wiley, L.M., Lever, J.E., Pape, C. and Kidder, G.M. (1991) Dev. Biol. 143, 149-161 16 Reeve, W.J.D. and Ziomek, C.A. (1981)J. Emh~.ol. Exp. Morphol. 62, 339-350 : 17 Maro, B., Gueth-Hallonet, C., Aghion, J. and Antony, C. (1991) Development Suppl. 1, 17-25 18 Levy, J.B., Johnson, M.H., Goodall, H. and Maro, B. (1986) J. Embryol. Exp. Morphol. 95, 213-237 19 Bloom, T. and McConnell, J. (1990) Mol. Reprod. Dev. 26, 199-210 20 Winkel, G.K., Ferguson, J.E., Takeichi, M. and Nucitelli, M. (1990) Dev. Biol. 138, 1-15 21 Bloom, T.L. (1989) Development 106, 159-171 22 Jones, J. and Schultz, R.M. (1990) Dev. Biol. 139, 250-262
B o t h the adult nervous system and its development have many c o m m o n features in most animals. Some time after the initial cleavage divisions of early embryogenesis, neuron precursors arise and undergo limited proliferation. The resulting progeny differentiate into neurons and organize into a coherent nervous system. Once formed, the individual neurons are considered to be terminally differentiated, as they maintain the same phenotype for the lifetime of the organism. Similarly, the resulting nervous system, in terms of the organization of its neuron populations, is generally static. The nervous system of hydra, a freshwater coelenterate, provides a striking contrast in several respects. (1) Instead of neurons being generated only during embryogenesis, they are produced continuously in the adult. Since neurons are also continually being lost, the nervous system of the adult is not static, but is in a steady state. (2) Almost all neurons are constantly changing their axial or regional location in the animal. (3) Many neurons change phenotype as they are displaced from one region to another. (4) Despite this dynamic situation, the overall organization of the nervous system remains constant. In this review, I discuss these unusual features, some of the problems that arise in maintaining the nervous system, and how these problems have been solved.
23 Ozawa, M., Ringwald, M. and Kemler, R. (1990) Proc. Natl Acad. Sci. USA 87, 4246--4250 24 Ozawa, M., Baribault, H. and Kemler, R. (1989) EMBOJ.
8, 1711-1717 25 McCrea, P.D. and Gumbiner, B.M. (1991)./'i Biol. Chem. 266, 4514-4520 26 McNeill, H., Ozawa, M., Kemler, R. and Nelson, wo. (1990) Cell 62, 309-316 27 Fleming, T.P., Pickering, S.J., Qasim, F. and Maro, B. (1986) J. EmbryoL F,x'p.Morphol. 95, 169-191 28 Johnson, M.H. and Maro, B. (1985)J. Embryol. Exp. Morphol. 90, 311-334 29 Maro, B. and Pickering, S.J. (1984)J. Embo,ol. F~cp. Morphol. 84, 217-232 30 Houliston, E., Pickering, S0. and Maro, B. (1989) Dev. Biol. 134, 342-350 3 I Johnson, M.H. el al. (1988) Development 102, 143-158 32 Johnson, M.H. and Ziomek, C.A. (1981) Cell 24, 71-80 33 Pickering, S.J., Maro, B., Johnson, M.H. and Skepper, J. (1988) Development 103, 353-363 34 Goodall, H. and Maro, B. (1986)J. Cell Biol. 102, 568-575 35 Ziomek, C.A. and Johnson, M.H. (1981) Wilhelm Roux's Arch. Dev. Biol. 190, 287-296 36 Surani, M.A.H. and Barton, S.C. (1984) Dev. Biol. 102, 335-343 37 Johnson, M.H. and Ziomek, C.A. (1983) Dev. Biol. 95, 211-218
C. GUETH-HALLONET AND ~ MARO ARE IN THE LABORATOIRE DE PHYSIOLOG1E DU DI~VELOPPEMENT~ INSTITUT JACQUES
Moxoz~ CNRS, UmvERSrr£ PARDS VII, TOUR 43 -- 2 PLACE JUSSIEU~ 75005 PARIS~FRANCE. ]
Continuous conversion of neuron phen0type in
hydra HANS R. BODE All neurons in adult hydra are constantly changing their location. This poses interesting problems for the maintenance of the organization of the hydra nervous system, The solutions provide a different perspective on the development of nervous systems.
The structure of the nervous system is simple The body plan of hydra, whose ancestors appeared very early in metazoan evolution, is quite simple (Fig. 1). It is basically a tube with, at the apical end, a head consisting of the hypostome, the mouth region and a ring of tentacles. The basal disk, or foot, is at the other end. Throughout the animal, the body wall consists of two epithelial layers, the ectoderm and endoderm, separated by the mesoglea, a basement m e m b r a n e (Fig. 2). The nervous system is equally simple, consisting of a nerve net that extends throughout the animal ~ (part
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Overall structure of a hydra and the distribution of the FLI÷ subset of neurons. Arrows running along the body column indicate the direction of tissue displacement. The locations of the FLI+ neurons and their processes are indicated by the network of dots and connecting lines. Reproduced, with permission, from Ref. 1. of the net is shown schematically in Fig. 1). The cell bodies of neurons are found mostly near the basal sides of both tissue layers, while their processes weave among the epithelial cells (Fig. 2). The density of neurons varies, being sixfold higher in the head, and threefold higher in the foot than in the body column. The only neurons that are not part of the net form a nerve ring at the base of the hypostome in the ectoderm 2. The ring is connected to the net, as processes of neurons of the ring synapse with those of the net. The net consists of a mosaic of subsets of neurons, some defined by morphology, and others by staining with antisera against neuropeptides, or with monoclonal antibodies against u n k n o w n antigens 1. Many subsets, such as the four subsets of sensory neurons, have specific regional locations, while other subsets are found in several regions. Steady-state production and loss o f neurons The tissue dynamics .of adult hydra are unusual, and play an important role ,in many aspects of the development of the animal. The epithelial cells of the body column are constantly in the mitotic cycle3, and although the epithelial tissue is continuously growing,
the adult animal does not change in size because tissue is lost at the same rate as it is produced 4. About 80-90% of the loss is due to displacement of tissue into developing buds, which eventually detach 4. (Budding is hydra's form of asexual reproduction.) The remainder is sloughed at the ends of the tentacles, hypostome and basal disk. The production and loss of cells is similar to that seen in mammalian epithelia, such as the epidermis or the intestinal epithelium. What is unusual here is that the entire animal exhibits these tissue dynamics; there are no static tissues in hydra. A consequence of this steady state is that almost all epithelial cells are constantly changing their axial location 5. Those in the upper part of the b o d y column are displaced in an apical direction into the head, and are eventually sloughed (see Fig. 1). The remainder are displaced basally either into developing buds or into the foot, where they are also sloughed. Hence there must be a stationary zone where cells are not displaced in either direction. This zone does not correspond to any particular region or set of cells, and its location can be shifted by altering the feeding regime of the animal. The tissue dynamics of the epithelia raise a problem for the nervous system. With epithelial cells of the body column continuously dividing, the 'mesh size' of the associated nerve net should continuously increase, and neuron density should decrease. Further, when epithelial tissue is displaced into an extremity or a developing bud, the nerve net is displaced too, so neurons are lost continuously from the animal. Yet the number of neurons, and the regional densities of the nerve net, remain constant. How are these parameters maintained? Neurons differentiate continuously from the multipotent stem cells among the interstitial cells along the entire length of the animal 6. Thus, new neurons are constantly intercalated into the net at a rate appropriate to the rate of epithelial cell division, thereby mainraining the mesh size as well as neuron number. How these processes are coordinated is not known. The other problem is the maintenance of the higher densities of neurons in the head and foot compared to the body column. Higher rates of neuron differentiation take place in the extremities because greater numbers of stem cells enter the neuron pathways 6, and because neuron differentiation intermediates immigrate from the body column 7. Specification o f neuron p h e n o t y p e An even more intriguing problem concerns the mosaic of subsets of neurons of the nerve net. The regional location of individual subsets of neurons is maintained despite constant expansion of the tissue, and the constant displacement of all neurons of the net. For example, a subset defined by an antiserum against the neuropeptide FMRFamide, termed neurons with FMRFamide-like immunoreactivity, or FLI+ neurons, are found in the head, and in a narrow ring directly above the foot 8 (see Fig. 1). As a result of tissue displacement, the basal border of the subset of FLI÷ neurons in the head might be expected to move towards the apical ends of the tentacles and hypostome. Yet this does not
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h a p p e n . Instead, the location of the basal b o r d e r remains constant. To achieve this, n e w FLI+ neurons must be continuously ,~ a d d e d at the base of the tentacles and hypostome, either by new differentiation, or by conversion of FLI- into FLI+ neurons as sensory mesoglea they are displaced up the b o d y column into cells the base of the head. The location of the basal border remained u n c h a n g e d in animals in which all neuron endodermal precursors were r e m o v e d 9. Similarly, decapi.,sensory tation of animals without neuron precursors ~I¢ cell resulted in the regeneration of animals with an FLI+ subset in the head 9. Both results indicate that FLI+ cells can form without n e w differentiation, and thus that FLI- neurons convert to FLI+. The only other cells in the ectoderm of these animals were epithelial cells, which are k n o w n not to be converted ganglion cell j into neurons. Another subset of neurons in the head, defined by an antiserum against endoderm -~lzI-/~- ectoderm vasopressin, is also maintained b y p h e n o " type c o n v e r s i o n ° . Although these examples of p h e n o t y p e conversion may involve only a single peptide, another e x a m p l e clearly involves more extensive changes. During head regeneration, ganglion cells of the b o d y column b e c o m e part of the regenerating head, and some are converted into epidermal sensory cells, which are found only in the head. Two antibodies, one specific for each cell type, detected cells that stained with both antibodies and had intermediate FIGS] morphologies as part of the transition of Longitudinal section of a hydra showing the two tissue layers and the ganglion to sensory cell ~1. As the sensory location of the nerve net. The two enlargements of the regions indicated by cell has a c o m p l e x stereociliary apparatus boxes illustrate the location of the nerve cell bodies and their connecting processes within the tissue layers. All cell types except nerve cells and distinct from the cilia of ganglion cells 1, epithelial cells have been omitted. Reproduced, with permission, from Ref. 1. this e x a m p l e of p h e n o t y p e conversion required the synthesis of a large n u m b e r of subset from the base towards the apical tip of the tenmacromolecules as well as a complete alteration in tacles. After 10 days the subset has vanished con> shape. pletely. An analysis of the differentiation of this subset If the p h e n o t y p e of a neuron can change as the of neurons indicates they are formed at the base of the neuron is displaced from one region to another, it is tentacles. Hence, this subset is maintained by conplausible that more than one such conversion might tinuous differentiation ofJD1 + cells at the basal border occur as it moves along the b o d y axis. In the p e d of the subset. uncle, w h e r e a ring of FLI÷ neurons is located, the Thus, each subset is as dynamic as the whole implication is that some neurons displaced into this nerve net. Neurons are continuously lost from a subset region b e c o m e FLI+, and later switch to FLI w h e n either as they are displaced out of the territo W of that they are displaced out of the p e d u n c l e and into the subset or as they are sloughed. To maintain the subset, foot. By grafting tissue of the lower p e d u n c l e into the neurons are continuously a d d e d either by new differmiddle of the b o d y column it has been shown that entiation or by p h e n o t y p e conversion of displaced FLI÷ neurons remain FLI+ if the grafted tissue maintains neurons. For some subsets, both mechanisms p r o b a b l y the character of the lower peduncle9. However, w h e n operate, as p h e n o t y p e conversion would be insufficient the tissue takes on the character of the b o d y column in regions w h e r e the neuron density increases substanor the foot, all the neurons b e c o m e FLI- (Ref. 9). tially; however, this has not yet been demonstrated. These results are consistent with two sequential The critical factor in maintaining the subset is the conversions. addition of new neurons at the subset border facing Not all subsets are maintained by p h e n o t y p e conthe oncoming tissue. This implies that neurons a n d / o r version. In another species, Hydra oligactis, a subset neuron precursors respond to quite localized environof neurons found in the tentacles, and defined by the mental cues for specification of phenotype. If all submonoclonal antibody JD1, is maintained by differensets b e h a v e like the four examined so far. positional tiation ~e. Removal of the neuron precursors results in the displacement of the basal border of the JD1 + cues are an important factor in determining neuron TIG AUGUST1992 VOL. 8 NO. 8
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FIG[] Change in the regional distribution of FLI+ neurons as a result of daily treatment of animals with diacylglycerol (DAG) for six days. The solid, roughly triangular figure to the right of each hydra represents the head activation gradient in normal animals. The dotted extension in the second and third hydra represents the increase in head activation due to DAG treatment. The arrow indicates an assumed threshold concentration of head activation that elicits FLI expression. An antibody against the neuropeptide RFamide was used for these experiments. The regional distribution of the subset defined by this antibody is identical to that defined by the antibody against FMRFamide, and is probably the same subset. For purposes here, it is assumed to be the same subset. Reproduced, with permission, from Ref. 25. phenotype. Finally, the fact that neurons of three out of the four subsets examined can arise by phenotype conversion indicates that the phenotype of many neurons in hydra is only metastable, and not an irreversibly determined property of the cell. However, the phenotype cannot be solely a response to local positional cues. The FLI÷ and VLI+ (vasopressin-like immunoreactivity) subsets are separate subsets of neurons that are both found in the head. When a FLDVLI neuron is displaced into the base of the tentacle, for example, the position could influence this neuron to express either phenotype, or possibly yet a third one. H o w is the choice made? Plausibly, a FLI-VLI- neuron could be committed to one or other phenotype before reaching the head. This seems unlikely. Migratory neuron precursors or neurons moved to different regions of the body column express phenotypes that they would not normally express if left in their original locations 7,9. These results complement those already described, and suggest neurons are not determined to undergo specific differentiation before arriving in a particular location. It is more likely that a second mechanism determines the choice between FLI and VLI. A positiondependent influence could activate both possibilities, FLI or VLI, and perhaps lateral inhibition from existing neighboring FLI÷ or VLI÷ cells could dictate the choice of one or the other. Lateral inhibition can explain the spatial pattern of neuroblasts that appear in ventral ectoderm in the developing Drosophila embryo 13, as well as the spacing of the dopaminergic amacrine neurons of the developing amphibian retina TM.
Head activation gradient and positional information If the axial position or regional location plays a major role in determining the phenotype of the neuron, what is the basis of this position dependence? For the FLI÷ subset, evidence implicates the head activation gradient, a process governing the formation of the hydra head. Head activation is the ability of a piece of the body column to induce the formation of a head, w h e n trans-
planted into the b o d y wall of a host animal 15. This property, which is associated with epithelial cells 16, is graded down the column, with a higher fraction of transplants from the apical than the basal end of the column forming heads 15. Current evidence suggests that the head activation gradient involves the following elements. A signal produced and secreted by cells of the head 17 diffuses down the column, forming a concentration gradient. The signal is transduced by epithelial cells via the diacylglycerol/protein kinase C branch of the inositol phospholipid pathway, resulting in an increase of the head activation property of these cells TM. The level of head activation would correspond to the concentration of the signal, thereby generating the observed gradient. Head activation could plausibly be the synthesis or activation of a transcription factor for head-specific genes. For the FLI+ subset in the head there is evidence that a threshold concentration of head activation provides the local environmental cue establishing the basal border of the subset. Periodic treatment of animals with diacylglycerol for six days raises the head activation concentration proportionately along the body column 18. The treatment also results in the appearance of FLI+ neurons in the body column, thereby increasing the population size of this subset 19. The additional FLI÷ neurons appear in a specific pattern 19. After three days of treatment new FLI+ neurons are found in the upper quarter of the body column, thereby effectively displacing the basal border of this subset from the base of the head to the upper part of the body column. By 6 days, FLI+ neurons are found in the upper half of the b o d y column. One way to explain the progressive basal displacement of this border is shown in Fig. 3. If neurons respond to a threshold level of head activation, and that level normally exists in the head, then FLI÷ neurons would appear in the head. If the head activation levels rise with diacylglycerol treatment, the threshold concentration would shift down the animal, and neurons, or neuron precursors, further down
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would respond by forming FLI+ neurons. With time, the head activation levels would continue to rise, and the FLI+ border would move in a basal direction, as observed. It seems likely that the effects of the altered head activation gradient on neurons or neuron precursors in the b o d y column are mediated by epithelial cells whose head activation levels have been raised. The epithelial cells are exposed to the diacylglycerol, but it is unlikely that the neurons or neuron precursors are, as they lie in the interior of the tissue.
depending on feeding conditions 4, thereby resulting in quite different possible patterns of neuron conversion. Thus, although some transitions may indeed be unidirectional, the critical element in the type of phenotype conversion a neuron undergoes in hydra is the new location or local environment in which it resides. In hydra, the metastability of the differentiated state is not restricted to neurons. Other cell types undergo changes in their differentiated state due to displacement into new regions. Ectodermal epithelial cells displaced into the tentacles or the foot differentiate into battery cells and foot gland cells, respectively 23. Gland cells of the body column are probably converted to mucous cells upon displacement into the head, though this is not as well established 23. This behavior indicates that many cell types in hydra are not 'irreversibly committed' or 'terminally differentiated'. Instead, it suggests that the differentiated state of these cells is continuously regulated in an 'active' manner, as proposed recently by Blau and Baltimore 24. Thus, changing the local environment of any of these cell types by displacement would change the change the regulatory influences they receive, leading to an alteration in their differentiated state. As these authors suggest, the differentiated state of cells in general may be metastable and regulated in this manner. This metastability is more obvious in hydra than in other animals simply because of the unusual displacement behavior of all the cells in this organism.
Conclusions At first glance the nervous system of an adult hydra appears highly unusual, in that there is a steady state of production and loss of neurons, and every neuron is constantly changing its location. Yet the processes that generate and maintain this nervous system are not so different from those of more c o m m o n static nervous systems. The neuron progenitors in hydra are stem cells with extensive, probably unlimited, selfrenewal capacity, compared to progenitors with clearly circumscribed proliferation capacity in most animals. As in most nervous systems, location or position plays an important role in specifying neuron phenotype. Perhaps the most striking difference in hydra is the obvious plasticity of the differentiated state of neurons as they are displaced from region to region. This difference may be more apparent than real, as neurons of other nervous systems experimentally moved to other locations undergo changes in phenotype. Comparison of the plasticity of the differentiated state of neurons in hydra with that in other animals reveals c o m m o n features, and a notable difference. Neuron conversion occurs without the need for DNA replication or cell division, as also happens, for example, in the conversion among amphibian chromatophores 2°. And, instead of involving dedifferentiation to an earlier c o m m o n progenitor, conversion or transdifferentiation occurs directly from one type of neuron to another (e.g. ganglion cell -+ epidermal sensory cell), as also occurs, for example, in the transition of some mammalian endocrine cells into neurons2L Transdifferentiation, or phenotype conversion, is commonly considered to be unidirectional; a cell might shift from A--+B or A--+B--+C, but does not move in the reverse direction 20,21. It is not clear that this is true for neuron phenotype conversion in hydra. Neurons arising in the body column have no particular fixed fate, and do not follow a fixed sequence of differentiated states. Conversion can be reversible, since some ganglion cells displaced d o w n the column are converted from FLI- to FLI+, and later back to FLI- after further basal displacement 9. The same can be inferred from the distribution of neurons expressing the neuropeptide neurotensin 22. In another example, ganglion cells displaced d o w n the body column towards the foot will remain ganglion cells or may form sensory cells of the foot, but would never form epidermal sensory cells of the head. But when the animal is bisected, some of these ganglion cells will be incorporated into the regenerating head, and thereafter form epidermal sensory cells 9. Similarly, a particular ganglion cell of the body column will be displaced apically or basally
Acknowledgements My work is supported by the National Institute for Child Health and Human Development.
References 1 Bode, H.R, et al. (1988) Am. Zool. 28, 1053-1063 2 Grimmelikhuizjen, C.J.P. (1985) Cell Tissue Res. 241, 171-182 3 David, C.N. and Campbell, R.D. (1972) L Cell&i. 11. 557-568 4 Otto, J.J. and Campbell, R.D. (1977) J. Cell Sci. 28, 117-132 5 Campbell, R.D. (1967)J. MorphoL 121, 19-28 6 Bode, H.R. and David, C.N. (1978) Prog. Biophy~. Mol. Biol. 33, 189-206 7 Teragawa, C.K. and Bode, H.R. (1990) Dev. Biol. 138, 63-81 8 Grimmelikhuijzen, C0.P., Dockray, G.J. and Schot, L.P.C. (1982) Histochemistry 73, 499-508 9 Koizumi, O. and Bode, H.R. (1986) Dev. Biol. 116, 407-421 10 Koizumi, O. and Bode, H.R. (1991)./. Neurosci. 11, 2011-2020 11 Koizumi, O., Heimfeld, S. and Bode, H.R. (1988) Dev. Biol. 129, 358-371 12 Yaross, M.S., Westerfield, J., Javois, L.C. and Bode, tI.R. (1986) Dev. Biol. 114, 225-237 1.3 Doe, C.A. and Goodman, C.S. (1985) Dev. Biol. 111, 206-219 14 Reh, T.A. and Tully, T. (1986) Dev. Biol. 114, 463-469 15 MacWilliams, H.K. (1983) Dev. Biol. 96, 239-272 16 Nishimiya, C., Wanek, N. and Sugiyama, T. (1986) 1)ev. Biol. 115, 469-478 17 Wilby, O.K. and Webster, G. (1970)J. Emb~.,ol. Exp. Morphol. 24, 583-593 18 Muller, W.A. (1990) Differentiation 42, 131-143
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19 Muller, W.A. (1991) Dev. Biol. 147, 460-463 20 Okada, T.S. (1991) Transdifferentiation: Flexibility in Cell Differentiation, Oxford Scientific Publications 21 Anderson, D.J. (1989) Trends Genet. 5, 174-178 22 Grimmelikhuijzen, c.J.P., Carraway, R., Rokaeus, A. and Sundler, F. (1981) Histochemistry 72, 199-209 23 Bode, H. et al. (1986) Curr. Top. Dev. Biol. 20, 257-279 24 Blau, H.M. and Baltimore, D. (1991) J. CellBiol. 112, 781-783
T h e r e are three eukaryotic nuclear RNA polymerases, each with a distinct biological function: RNA polymerase I (pol I) synthesizes ribosomal RNA, RNA polymerase II (pol II) synthesizes messenger RNA and several small nuclear RNAs, and RNA polymerase III (pol Ill) synthesizes transfer RNA, 5S rRNA, and various other small cellular and viral RNAs (reviewed in Ref. 1). Although transcription is extremely specific within the cell, a purified RNA polymerase transcribes DNA essentially randomly. Specificity can be restored by the addition of a cell-free protein extract. This reflects the presence in such an extract of transcription initiation factors, which serve to recruit RNA polymerases to the correct start sites of the appropriate sets of genes. Until recently, it was believed that the genes transcribed by pols I, II and III (referred to as class I, II and III genes, respectively) utilized distinct and nonoverlapping sets of initiation factors. However, it now appears that one factor is involved in all cases.
25 Bode, H.R. in Determinants of Neural Identity (Shankland, S.M. and Macagno, E.R., eds), Academic Press (in press)
H.R. BODE IS IN THE DEVELOPMENTAL BIOLOGY CENTER AND I THE DEPARTMENT OF DEVELOPMENTAL AND CELL BIOLOGY, UNIVERSITY OF CALIFORNIA, IgRVINE, CA 9 2 7 1 7 , USA.
The TATA-binding protein: a central r01e in transcription by RNA p01ymerases I, II and Ill ROBERTJ. WHITEAND STEPHENP. JACKSON The TATA-box-binding protein, first notedfor its association with the general transcription factor T¥IID, has recently been shown to be required for t r a n s c r i p t i o n by all three classes of nuclear RNA polymerase found in eukaryotes. As such, it plays a unique and pivotal role in gene expression in higher organisms.
were required to direct specific initiation by purified pols II and III. In this way it was hoped to determine whether there are multiple initiation factors or just one, and whether the different polymerases use the same or different factors. The scheme began with chromatography on phosphocellulose (PC), to generate four HUMAN CELL EXTRACT fractions, A-D (Fig. 1). When the adenovirus major late PC COLUMN (AdML) gene was used as a template, the PC-A, PC-C and PC-D fractions 1.0M were found to be necessary to reconKCI stitute specific initiation by pol II, 0.6M ] whereas the PC-B fraction was disPO-D pensable. This indicated that there PC-A PC-B I I are at least three factors required for [ TFIIB ~R2,, ] TFIID I pol II initiation, and these activities were named TFIIA, TFIIB and TFIID. With purified pol III, the PC-B and DE52 COLUMN the PC-C fractions were necessary and sufficient to reconstitute specific initiation at a tRNA and an adenovirus I O.1M ] 0.25MKCI VA gene, and these fractions were also required by a 5S rRNA gene. This TFIIB 3"~R~ ] showed that there are at least two general pol III initiation factors, which were named TFIIIB and TFIIIC. PTGR These were clearly minimum estiSimplified version of fractiohation scheme used in Refs 2 and 3 to separate class II mates, since any of the crude PC fracand class III general transcriptmn initiation factors from an S100 extract of cultured tions could have contained more than human KB cells. The KCI concentrations at which the fractions eluted are indicated. one factor; indeed, several additional Class II factors are italicized and class III factors are shadowed. PC and DE52 denote general initiation factors have since phosphocellulose and DEAE-cellulose column matrices, respectively.
Different sets o f initiation factors In 1980, Roeder and co-workers 23 fractionated a human cell extract and asked which of the fractions
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B o t h the adult nervous system and its development have many c o m m o n features in most animals. Some time after the initial cleavage divisions of early embryogenesis, neuron precursors arise and undergo limited proliferation. The resulting progeny differentiate into neurons and organize into a coherent nervous system. Once formed, the individual neurons are considered to be terminally differentiated, as they maintain the same phenotype for the lifetime of the organism. Similarly, the resulting nervous system, in terms of the organization of its neuron populations, is generally static. The nervous system of hydra, a freshwater coelenterate, provides a striking contrast in several respects. (1) Instead of neurons being generated only during embryogenesis, they are produced continuously in the adult. Since neurons are also continually being lost, the nervous system of the adult is not static, but is in a steady state. (2) Almost all neurons are constantly changing their axial or regional location in the animal. (3) Many neurons change phenotype as they are displaced from one region to another. (4) Despite this dynamic situation, the overall organization of the nervous system remains constant. In this review, I discuss these unusual features, some of the problems that arise in maintaining the nervous system, and how these problems have been solved.
23 Ozawa, M., Ringwald, M. and Kemler, R. (1990) Proc. Natl Acad. Sci. USA 87, 4246--4250 24 Ozawa, M., Baribault, H. and Kemler, R. (1989) EMBOJ.
8, 1711-1717 25 McCrea, P.D. and Gumbiner, B.M. (1991)./'i Biol. Chem. 266, 4514-4520 26 McNeill, H., Ozawa, M., Kemler, R. and Nelson, wo. (1990) Cell 62, 309-316 27 Fleming, T.P., Pickering, S.J., Qasim, F. and Maro, B. (1986) J. EmbryoL F,x'p.Morphol. 95, 169-191 28 Johnson, M.H. and Maro, B. (1985)J. Embryol. Exp. Morphol. 90, 311-334 29 Maro, B. and Pickering, S.J. (1984)J. Embo,ol. F~cp. Morphol. 84, 217-232 30 Houliston, E., Pickering, S0. and Maro, B. (1989) Dev. Biol. 134, 342-350 3 I Johnson, M.H. el al. (1988) Development 102, 143-158 32 Johnson, M.H. and Ziomek, C.A. (1981) Cell 24, 71-80 33 Pickering, S.J., Maro, B., Johnson, M.H. and Skepper, J. (1988) Development 103, 353-363 34 Goodall, H. and Maro, B. (1986)J. Cell Biol. 102, 568-575 35 Ziomek, C.A. and Johnson, M.H. (1981) Wilhelm Roux's Arch. Dev. Biol. 190, 287-296 36 Surani, M.A.H. and Barton, S.C. (1984) Dev. Biol. 102, 335-343 37 Johnson, M.H. and Ziomek, C.A. (1983) Dev. Biol. 95, 211-218
C. GUETH-HALLONET AND ~ MARO ARE IN THE LABORATOIRE DE PHYSIOLOG1E DU DI~VELOPPEMENT~ INSTITUT JACQUES
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Continuous conversion of neuron phen0type in
hydra HANS R. BODE All neurons in adult hydra are constantly changing their location. This poses interesting problems for the maintenance of the organization of the hydra nervous system, The solutions provide a different perspective on the development of nervous systems.
The structure of the nervous system is simple The body plan of hydra, whose ancestors appeared very early in metazoan evolution, is quite simple (Fig. 1). It is basically a tube with, at the apical end, a head consisting of the hypostome, the mouth region and a ring of tentacles. The basal disk, or foot, is at the other end. Throughout the animal, the body wall consists of two epithelial layers, the ectoderm and endoderm, separated by the mesoglea, a basement m e m b r a n e (Fig. 2). The nervous system is equally simple, consisting of a nerve net that extends throughout the animal ~ (part
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Overall structure of a hydra and the distribution of the FLI÷ subset of neurons. Arrows running along the body column indicate the direction of tissue displacement. The locations of the FLI+ neurons and their processes are indicated by the network of dots and connecting lines. Reproduced, with permission, from Ref. 1. of the net is shown schematically in Fig. 1). The cell bodies of neurons are found mostly near the basal sides of both tissue layers, while their processes weave among the epithelial cells (Fig. 2). The density of neurons varies, being sixfold higher in the head, and threefold higher in the foot than in the body column. The only neurons that are not part of the net form a nerve ring at the base of the hypostome in the ectoderm 2. The ring is connected to the net, as processes of neurons of the ring synapse with those of the net. The net consists of a mosaic of subsets of neurons, some defined by morphology, and others by staining with antisera against neuropeptides, or with monoclonal antibodies against u n k n o w n antigens 1. Many subsets, such as the four subsets of sensory neurons, have specific regional locations, while other subsets are found in several regions. Steady-state production and loss o f neurons The tissue dynamics .of adult hydra are unusual, and play an important role ,in many aspects of the development of the animal. The epithelial cells of the body column are constantly in the mitotic cycle3, and although the epithelial tissue is continuously growing,
the adult animal does not change in size because tissue is lost at the same rate as it is produced 4. About 80-90% of the loss is due to displacement of tissue into developing buds, which eventually detach 4. (Budding is hydra's form of asexual reproduction.) The remainder is sloughed at the ends of the tentacles, hypostome and basal disk. The production and loss of cells is similar to that seen in mammalian epithelia, such as the epidermis or the intestinal epithelium. What is unusual here is that the entire animal exhibits these tissue dynamics; there are no static tissues in hydra. A consequence of this steady state is that almost all epithelial cells are constantly changing their axial location 5. Those in the upper part of the b o d y column are displaced in an apical direction into the head, and are eventually sloughed (see Fig. 1). The remainder are displaced basally either into developing buds or into the foot, where they are also sloughed. Hence there must be a stationary zone where cells are not displaced in either direction. This zone does not correspond to any particular region or set of cells, and its location can be shifted by altering the feeding regime of the animal. The tissue dynamics of the epithelia raise a problem for the nervous system. With epithelial cells of the body column continuously dividing, the 'mesh size' of the associated nerve net should continuously increase, and neuron density should decrease. Further, when epithelial tissue is displaced into an extremity or a developing bud, the nerve net is displaced too, so neurons are lost continuously from the animal. Yet the number of neurons, and the regional densities of the nerve net, remain constant. How are these parameters maintained? Neurons differentiate continuously from the multipotent stem cells among the interstitial cells along the entire length of the animal 6. Thus, new neurons are constantly intercalated into the net at a rate appropriate to the rate of epithelial cell division, thereby mainraining the mesh size as well as neuron number. How these processes are coordinated is not known. The other problem is the maintenance of the higher densities of neurons in the head and foot compared to the body column. Higher rates of neuron differentiation take place in the extremities because greater numbers of stem cells enter the neuron pathways 6, and because neuron differentiation intermediates immigrate from the body column 7. Specification o f neuron p h e n o t y p e An even more intriguing problem concerns the mosaic of subsets of neurons of the nerve net. The regional location of individual subsets of neurons is maintained despite constant expansion of the tissue, and the constant displacement of all neurons of the net. For example, a subset defined by an antiserum against the neuropeptide FMRFamide, termed neurons with FMRFamide-like immunoreactivity, or FLI+ neurons, are found in the head, and in a narrow ring directly above the foot 8 (see Fig. 1). As a result of tissue displacement, the basal border of the subset of FLI÷ neurons in the head might be expected to move towards the apical ends of the tentacles and hypostome. Yet this does not
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h a p p e n . Instead, the location of the basal b o r d e r remains constant. To achieve this, n e w FLI+ neurons must be continuously ,~ a d d e d at the base of the tentacles and hypostome, either by new differentiation, or by conversion of FLI- into FLI+ neurons as sensory mesoglea they are displaced up the b o d y column into cells the base of the head. The location of the basal border remained u n c h a n g e d in animals in which all neuron endodermal precursors were r e m o v e d 9. Similarly, decapi.,sensory tation of animals without neuron precursors ~I¢ cell resulted in the regeneration of animals with an FLI+ subset in the head 9. Both results indicate that FLI+ cells can form without n e w differentiation, and thus that FLI- neurons convert to FLI+. The only other cells in the ectoderm of these animals were epithelial cells, which are k n o w n not to be converted ganglion cell j into neurons. Another subset of neurons in the head, defined by an antiserum against endoderm -~lzI-/~- ectoderm vasopressin, is also maintained b y p h e n o " type c o n v e r s i o n ° . Although these examples of p h e n o t y p e conversion may involve only a single peptide, another e x a m p l e clearly involves more extensive changes. During head regeneration, ganglion cells of the b o d y column b e c o m e part of the regenerating head, and some are converted into epidermal sensory cells, which are found only in the head. Two antibodies, one specific for each cell type, detected cells that stained with both antibodies and had intermediate FIGS] morphologies as part of the transition of Longitudinal section of a hydra showing the two tissue layers and the ganglion to sensory cell ~1. As the sensory location of the nerve net. The two enlargements of the regions indicated by cell has a c o m p l e x stereociliary apparatus boxes illustrate the location of the nerve cell bodies and their connecting processes within the tissue layers. All cell types except nerve cells and distinct from the cilia of ganglion cells 1, epithelial cells have been omitted. Reproduced, with permission, from Ref. 1. this e x a m p l e of p h e n o t y p e conversion required the synthesis of a large n u m b e r of subset from the base towards the apical tip of the tenmacromolecules as well as a complete alteration in tacles. After 10 days the subset has vanished con> shape. pletely. An analysis of the differentiation of this subset If the p h e n o t y p e of a neuron can change as the of neurons indicates they are formed at the base of the neuron is displaced from one region to another, it is tentacles. Hence, this subset is maintained by conplausible that more than one such conversion might tinuous differentiation ofJD1 + cells at the basal border occur as it moves along the b o d y axis. In the p e d of the subset. uncle, w h e r e a ring of FLI÷ neurons is located, the Thus, each subset is as dynamic as the whole implication is that some neurons displaced into this nerve net. Neurons are continuously lost from a subset region b e c o m e FLI+, and later switch to FLI w h e n either as they are displaced out of the territo W of that they are displaced out of the p e d u n c l e and into the subset or as they are sloughed. To maintain the subset, foot. By grafting tissue of the lower p e d u n c l e into the neurons are continuously a d d e d either by new differmiddle of the b o d y column it has been shown that entiation or by p h e n o t y p e conversion of displaced FLI÷ neurons remain FLI+ if the grafted tissue maintains neurons. For some subsets, both mechanisms p r o b a b l y the character of the lower peduncle9. However, w h e n operate, as p h e n o t y p e conversion would be insufficient the tissue takes on the character of the b o d y column in regions w h e r e the neuron density increases substanor the foot, all the neurons b e c o m e FLI- (Ref. 9). tially; however, this has not yet been demonstrated. These results are consistent with two sequential The critical factor in maintaining the subset is the conversions. addition of new neurons at the subset border facing Not all subsets are maintained by p h e n o t y p e conthe oncoming tissue. This implies that neurons a n d / o r version. In another species, Hydra oligactis, a subset neuron precursors respond to quite localized environof neurons found in the tentacles, and defined by the mental cues for specification of phenotype. If all submonoclonal antibody JD1, is maintained by differensets b e h a v e like the four examined so far. positional tiation ~e. Removal of the neuron precursors results in the displacement of the basal border of the JD1 + cues are an important factor in determining neuron TIG AUGUST1992 VOL. 8 NO. 8
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FIG[] Change in the regional distribution of FLI+ neurons as a result of daily treatment of animals with diacylglycerol (DAG) for six days. The solid, roughly triangular figure to the right of each hydra represents the head activation gradient in normal animals. The dotted extension in the second and third hydra represents the increase in head activation due to DAG treatment. The arrow indicates an assumed threshold concentration of head activation that elicits FLI expression. An antibody against the neuropeptide RFamide was used for these experiments. The regional distribution of the subset defined by this antibody is identical to that defined by the antibody against FMRFamide, and is probably the same subset. For purposes here, it is assumed to be the same subset. Reproduced, with permission, from Ref. 25. phenotype. Finally, the fact that neurons of three out of the four subsets examined can arise by phenotype conversion indicates that the phenotype of many neurons in hydra is only metastable, and not an irreversibly determined property of the cell. However, the phenotype cannot be solely a response to local positional cues. The FLI÷ and VLI+ (vasopressin-like immunoreactivity) subsets are separate subsets of neurons that are both found in the head. When a FLDVLI neuron is displaced into the base of the tentacle, for example, the position could influence this neuron to express either phenotype, or possibly yet a third one. H o w is the choice made? Plausibly, a FLI-VLI- neuron could be committed to one or other phenotype before reaching the head. This seems unlikely. Migratory neuron precursors or neurons moved to different regions of the body column express phenotypes that they would not normally express if left in their original locations 7,9. These results complement those already described, and suggest neurons are not determined to undergo specific differentiation before arriving in a particular location. It is more likely that a second mechanism determines the choice between FLI and VLI. A positiondependent influence could activate both possibilities, FLI or VLI, and perhaps lateral inhibition from existing neighboring FLI÷ or VLI÷ cells could dictate the choice of one or the other. Lateral inhibition can explain the spatial pattern of neuroblasts that appear in ventral ectoderm in the developing Drosophila embryo 13, as well as the spacing of the dopaminergic amacrine neurons of the developing amphibian retina TM.
Head activation gradient and positional information If the axial position or regional location plays a major role in determining the phenotype of the neuron, what is the basis of this position dependence? For the FLI÷ subset, evidence implicates the head activation gradient, a process governing the formation of the hydra head. Head activation is the ability of a piece of the body column to induce the formation of a head, w h e n trans-
planted into the b o d y wall of a host animal 15. This property, which is associated with epithelial cells 16, is graded down the column, with a higher fraction of transplants from the apical than the basal end of the column forming heads 15. Current evidence suggests that the head activation gradient involves the following elements. A signal produced and secreted by cells of the head 17 diffuses down the column, forming a concentration gradient. The signal is transduced by epithelial cells via the diacylglycerol/protein kinase C branch of the inositol phospholipid pathway, resulting in an increase of the head activation property of these cells TM. The level of head activation would correspond to the concentration of the signal, thereby generating the observed gradient. Head activation could plausibly be the synthesis or activation of a transcription factor for head-specific genes. For the FLI+ subset in the head there is evidence that a threshold concentration of head activation provides the local environmental cue establishing the basal border of the subset. Periodic treatment of animals with diacylglycerol for six days raises the head activation concentration proportionately along the body column 18. The treatment also results in the appearance of FLI+ neurons in the body column, thereby increasing the population size of this subset 19. The additional FLI÷ neurons appear in a specific pattern 19. After three days of treatment new FLI+ neurons are found in the upper quarter of the body column, thereby effectively displacing the basal border of this subset from the base of the head to the upper part of the body column. By 6 days, FLI+ neurons are found in the upper half of the b o d y column. One way to explain the progressive basal displacement of this border is shown in Fig. 3. If neurons respond to a threshold level of head activation, and that level normally exists in the head, then FLI÷ neurons would appear in the head. If the head activation levels rise with diacylglycerol treatment, the threshold concentration would shift down the animal, and neurons, or neuron precursors, further down
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would respond by forming FLI+ neurons. With time, the head activation levels would continue to rise, and the FLI+ border would move in a basal direction, as observed. It seems likely that the effects of the altered head activation gradient on neurons or neuron precursors in the b o d y column are mediated by epithelial cells whose head activation levels have been raised. The epithelial cells are exposed to the diacylglycerol, but it is unlikely that the neurons or neuron precursors are, as they lie in the interior of the tissue.
depending on feeding conditions 4, thereby resulting in quite different possible patterns of neuron conversion. Thus, although some transitions may indeed be unidirectional, the critical element in the type of phenotype conversion a neuron undergoes in hydra is the new location or local environment in which it resides. In hydra, the metastability of the differentiated state is not restricted to neurons. Other cell types undergo changes in their differentiated state due to displacement into new regions. Ectodermal epithelial cells displaced into the tentacles or the foot differentiate into battery cells and foot gland cells, respectively 23. Gland cells of the body column are probably converted to mucous cells upon displacement into the head, though this is not as well established 23. This behavior indicates that many cell types in hydra are not 'irreversibly committed' or 'terminally differentiated'. Instead, it suggests that the differentiated state of these cells is continuously regulated in an 'active' manner, as proposed recently by Blau and Baltimore 24. Thus, changing the local environment of any of these cell types by displacement would change the change the regulatory influences they receive, leading to an alteration in their differentiated state. As these authors suggest, the differentiated state of cells in general may be metastable and regulated in this manner. This metastability is more obvious in hydra than in other animals simply because of the unusual displacement behavior of all the cells in this organism.
Conclusions At first glance the nervous system of an adult hydra appears highly unusual, in that there is a steady state of production and loss of neurons, and every neuron is constantly changing its location. Yet the processes that generate and maintain this nervous system are not so different from those of more c o m m o n static nervous systems. The neuron progenitors in hydra are stem cells with extensive, probably unlimited, selfrenewal capacity, compared to progenitors with clearly circumscribed proliferation capacity in most animals. As in most nervous systems, location or position plays an important role in specifying neuron phenotype. Perhaps the most striking difference in hydra is the obvious plasticity of the differentiated state of neurons as they are displaced from region to region. This difference may be more apparent than real, as neurons of other nervous systems experimentally moved to other locations undergo changes in phenotype. Comparison of the plasticity of the differentiated state of neurons in hydra with that in other animals reveals c o m m o n features, and a notable difference. Neuron conversion occurs without the need for DNA replication or cell division, as also happens, for example, in the conversion among amphibian chromatophores 2°. And, instead of involving dedifferentiation to an earlier c o m m o n progenitor, conversion or transdifferentiation occurs directly from one type of neuron to another (e.g. ganglion cell -+ epidermal sensory cell), as also occurs, for example, in the transition of some mammalian endocrine cells into neurons2L Transdifferentiation, or phenotype conversion, is commonly considered to be unidirectional; a cell might shift from A--+B or A--+B--+C, but does not move in the reverse direction 20,21. It is not clear that this is true for neuron phenotype conversion in hydra. Neurons arising in the body column have no particular fixed fate, and do not follow a fixed sequence of differentiated states. Conversion can be reversible, since some ganglion cells displaced d o w n the column are converted from FLI- to FLI+, and later back to FLI- after further basal displacement 9. The same can be inferred from the distribution of neurons expressing the neuropeptide neurotensin 22. In another example, ganglion cells displaced d o w n the body column towards the foot will remain ganglion cells or may form sensory cells of the foot, but would never form epidermal sensory cells of the head. But when the animal is bisected, some of these ganglion cells will be incorporated into the regenerating head, and thereafter form epidermal sensory cells 9. Similarly, a particular ganglion cell of the body column will be displaced apically or basally
Acknowledgements My work is supported by the National Institute for Child Health and Human Development.
References 1 Bode, H.R, et al. (1988) Am. Zool. 28, 1053-1063 2 Grimmelikhuizjen, C.J.P. (1985) Cell Tissue Res. 241, 171-182 3 David, C.N. and Campbell, R.D. (1972) L Cell&i. 11. 557-568 4 Otto, J.J. and Campbell, R.D. (1977) J. Cell Sci. 28, 117-132 5 Campbell, R.D. (1967)J. MorphoL 121, 19-28 6 Bode, H.R. and David, C.N. (1978) Prog. Biophy~. Mol. Biol. 33, 189-206 7 Teragawa, C.K. and Bode, H.R. (1990) Dev. Biol. 138, 63-81 8 Grimmelikhuijzen, C0.P., Dockray, G.J. and Schot, L.P.C. (1982) Histochemistry 73, 499-508 9 Koizumi, O. and Bode, H.R. (1986) Dev. Biol. 116, 407-421 10 Koizumi, O. and Bode, H.R. (1991)./. Neurosci. 11, 2011-2020 11 Koizumi, O., Heimfeld, S. and Bode, H.R. (1988) Dev. Biol. 129, 358-371 12 Yaross, M.S., Westerfield, J., Javois, L.C. and Bode, tI.R. (1986) Dev. Biol. 114, 225-237 1.3 Doe, C.A. and Goodman, C.S. (1985) Dev. Biol. 111, 206-219 14 Reh, T.A. and Tully, T. (1986) Dev. Biol. 114, 463-469 15 MacWilliams, H.K. (1983) Dev. Biol. 96, 239-272 16 Nishimiya, C., Wanek, N. and Sugiyama, T. (1986) 1)ev. Biol. 115, 469-478 17 Wilby, O.K. and Webster, G. (1970)J. Emb~.,ol. Exp. Morphol. 24, 583-593 18 Muller, W.A. (1990) Differentiation 42, 131-143
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19 Muller, W.A. (1991) Dev. Biol. 147, 460-463 20 Okada, T.S. (1991) Transdifferentiation: Flexibility in Cell Differentiation, Oxford Scientific Publications 21 Anderson, D.J. (1989) Trends Genet. 5, 174-178 22 Grimmelikhuijzen, c.J.P., Carraway, R., Rokaeus, A. and Sundler, F. (1981) Histochemistry 72, 199-209 23 Bode, H. et al. (1986) Curr. Top. Dev. Biol. 20, 257-279 24 Blau, H.M. and Baltimore, D. (1991) J. CellBiol. 112, 781-783
T h e r e are three eukaryotic nuclear RNA polymerases, each with a distinct biological function: RNA polymerase I (pol I) synthesizes ribosomal RNA, RNA polymerase II (pol II) synthesizes messenger RNA and several small nuclear RNAs, and RNA polymerase III (pol Ill) synthesizes transfer RNA, 5S rRNA, and various other small cellular and viral RNAs (reviewed in Ref. 1). Although transcription is extremely specific within the cell, a purified RNA polymerase transcribes DNA essentially randomly. Specificity can be restored by the addition of a cell-free protein extract. This reflects the presence in such an extract of transcription initiation factors, which serve to recruit RNA polymerases to the correct start sites of the appropriate sets of genes. Until recently, it was believed that the genes transcribed by pols I, II and III (referred to as class I, II and III genes, respectively) utilized distinct and nonoverlapping sets of initiation factors. However, it now appears that one factor is involved in all cases.
25 Bode, H.R. in Determinants of Neural Identity (Shankland, S.M. and Macagno, E.R., eds), Academic Press (in press)
H.R. BODE IS IN THE DEVELOPMENTAL BIOLOGY CENTER AND I THE DEPARTMENT OF DEVELOPMENTAL AND CELL BIOLOGY, UNIVERSITY OF CALIFORNIA, IgRVINE, CA 9 2 7 1 7 , USA.
The TATA-binding protein: a central r01e in transcription by RNA p01ymerases I, II and Ill ROBERTJ. WHITEAND STEPHENP. JACKSON The TATA-box-binding protein, first notedfor its association with the general transcription factor T¥IID, has recently been shown to be required for t r a n s c r i p t i o n by all three classes of nuclear RNA polymerase found in eukaryotes. As such, it plays a unique and pivotal role in gene expression in higher organisms.
were required to direct specific initiation by purified pols II and III. In this way it was hoped to determine whether there are multiple initiation factors or just one, and whether the different polymerases use the same or different factors. The scheme began with chromatography on phosphocellulose (PC), to generate four HUMAN CELL EXTRACT fractions, A-D (Fig. 1). When the adenovirus major late PC COLUMN (AdML) gene was used as a template, the PC-A, PC-C and PC-D fractions 1.0M were found to be necessary to reconKCI stitute specific initiation by pol II, 0.6M ] whereas the PC-B fraction was disPO-D pensable. This indicated that there PC-A PC-B I I are at least three factors required for [ TFIIB ~R2,, ] TFIID I pol II initiation, and these activities were named TFIIA, TFIIB and TFIID. With purified pol III, the PC-B and DE52 COLUMN the PC-C fractions were necessary and sufficient to reconstitute specific initiation at a tRNA and an adenovirus I O.1M ] 0.25MKCI VA gene, and these fractions were also required by a 5S rRNA gene. This TFIIB 3"~R~ ] showed that there are at least two general pol III initiation factors, which were named TFIIIB and TFIIIC. PTGR These were clearly minimum estiSimplified version of fractiohation scheme used in Refs 2 and 3 to separate class II mates, since any of the crude PC fracand class III general transcriptmn initiation factors from an S100 extract of cultured tions could have contained more than human KB cells. The KCI concentrations at which the fractions eluted are indicated. one factor; indeed, several additional Class II factors are italicized and class III factors are shadowed. PC and DE52 denote general initiation factors have since phosphocellulose and DEAE-cellulose column matrices, respectively.
Different sets o f initiation factors In 1980, Roeder and co-workers 23 fractionated a human cell extract and asked which of the fractions
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