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Chapter 15 The differentiation and function of the touch receptor neurons of Caenorhabditis elegans
A.C.H. Yo. L.F. Eng, U.J. McMahan, H. Schulman, E.M. Shooter and A. Stadlin (Eds.) Progress in Brain Research, Vol. 105 0 1996 Elsevier Science BV. All rights reserved.
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CHAPTER 15
The differentiation and function of the touch receptor neurons of Caenorhabditis elegans Martin Chalfie Department of Biological Sciences, 1012 Sherman Fairchild Center, Columbia University, New York, NY 10027, USA
Introduction Research over the last twenty years on the nematode Caenorhabditis elegans has provided a wealth of information on the structure and development of the nervous system of this animal (reviewed by Chalfie and White, 1988; Bargmann, 1993). For example, complete descriptions of the cellular anatomy (including synaptic contacts) and of the lineage derivation of each of the 302 neurons of the adult C. eZeguns hermaphrodite are known (White et al., 1986; Sulston and Horvitz, 1977; Sulston et al., 1983). Moreover, numerous classical and molecular genetics techniques are available t o characterize genes that direct the development and function of cells within the nervous system. Research in my laboratory has used these techniques to study a set of six touch receptor neurons. In this chapter I will describe some of the lessons we have learned about how these cells acquire their specific characteristics (and other cells do not) and how the cells function as touch receptors.
Genes needed for touch cell differentiation These cells are the first in a three-neuron reflex circuit (touch receptor, interneuron, and motor)
that allows the animals to move away from a gentle touch stimulus (usually the stroking of a thin hair; Chalfie and Sulston, 1981; Chalfie et al., 1985). When these cells are killed by laser microsurgery, animals do not move when gently touched. The animals will move normally when stimulated more harshly, a stimulus that activates the interneurons. Our initial analysis of the development and function of the touch receptors began with the collection of touch-insensitive mutants, i.e., animals that respond to harsh, but not gentle, touch (Sulston et al., 1975; Chalfie and Sulston, 1981).At present we have over 450 such touch mutants representing defects in 17 genes (Chalfie and Au, 1989; unpublished data). The mutant phenotypes of these genes have allowed them to be characterized into four groups (Chalfie and Au, 1989;Way and Chalfie, 1989): generation, specification, maintenance, and function. The genes Zin-32 and unc-86 are needed for the generation of the touch receptors. When these genes are mutant the cell lineages giving rise to the touch receptors are altered so that the cells are never made (Chalfie et al., 1981; Chalfie and Au, 1989). The unc-86 gene encodes a POU-type homeodomain protein (Finney et al., 1988) that is not only expressed in the precursors of the touch recep-
180
tors, but also in the cells themselves (as well as in several other cells; Finney and Ruvkun, 1990). These properties suggested to us that UNC-86 (the protein encoded by the unc-86 gene) might act as a direct trans-activator of touch cell differentiation. A likely target for UNC-86 was the mec-3 gene, a gene that is required for touch cell specification. When mec-3 is mutant, cell lineages appear normal, but the cells that should become touch receptors fail t o do so. The mec-3 gene encodes a homeoprotein, but of the LIM type (Way and Chalfie, 1988; Way et al., 1991; Freyd et al., 1990;Xue et al., 1992).The expression of a mec-3ZacZfusion in wild-type animals and mec-3mutant animals suggested that mec3 is autoregulated, i.e. it is needed for its own expression (Way and Chalfie, 1989).These predictions were supported by DNA binding studies which showed that both UNC-86 and MEC-3 bind t o the promoter of the mec-3 gene (Xue et al., 1992, 1993). Two unexpected results arose from these studies: (1)UNC-86 appears to be needed for the continued expression of mec-3 and (2) UNC-86 and MEC-3 can form heterodimers that associate with the DNA in a more stable fashion than either protein alone. The increased stability of the binding, which is needed for the in vivo function of these proteins, probably accounts for the specificity of action of the proteins in determining touch receptor fate. In more recent work (A. Duggan and M. Chalfie, unpublished data) we have found that two targets of mec-3 action, mec-4 and mec-7 (see below) also have UNC-86:MEC3 heterodimer binding sites that are needed for proper expression. This result suggests that touch cell differentiation is probably not under the control of a linear progression of transcription factors (UNC-86 activating mec-3 and MEC-3 activating the downstream genes), but rather under an accumulative control (UNC-86 activates mec-3 and UNC-86 and MEC-3 together activate the downstream genes). Thus, UNC-86 and MEC-3 appear to act in a combinatorial fashion in touch cell develop-
ment. These genes (even with lin-32), however, do not explain why C. elegans has precisely six touch receptors, especially since unc-86 (Finney and Ruvkun, 1990) and mec-3 (Way and Chalfie, 1989) are expressed together in more cells than just the six touch receptor cells. Specifically, mec-3 is expressed in two other pairs of neurons (the FLP cells and the PVD cells); these cells also express unc-86. These obseniations indicate that other factors are required for touch receptor specification. By looking for the ectopic expression of touch cell-specific features or genes we have been able t o identify seven other genes that are needed to restrict the number of touch cells to six (Mitani et al., 1993). The PVD cells do not differentiate into touch receptor cells because they (or their precursors) fail t o express the lin-14 gene (Zin-14 itself is negatively regulated by the Zin-4 gene). The FLP cells express Zin-14 (G. Ruvkun, pers. comm.), but they do not become touch cells because of the action of the genes egl-44 and egl-46 (mutations in either of these genes results in these cells acquiring touch cell features in a lin-14-dependent fashion). Another pair of neurons in the tail can differentiate as touch receptors if the sem-4 gene is mutant. I t is likely that sem-4 negatively regulates mec-3, normally preventing its expression in these cells. Finally, programmed cell death, which requires the ced-3 and ced-4 genes (Ellis and Horvitz, 1986),causes the loss of four cells that could differentiate into touch receptors in the wild-type animal. Together positively- and negatively-acting genes as well a s genes needed for programmed cell death are needed t o produce the correct number of touch receptors within the animals.
Genes needed for touch cell function Mutations in the remaining genes (rnec-1,2, 4, 6 - 10,12,14,15,18) result in animals that have identifiable touch receptors that fail to respond to touch. These function genes are thus needed for the activity, but not the development of the
181
cells. Some of these genes (mec-7,12) affect the unique form of microtubule (15-protofilament) that is found in these cells. The mec-7 gene encodes a P-tubulin that is expressed at high levels only in the touch receptor cells (Savage et al., 1989; Hamelin et al., 1992; Mitani et al., 1993). The mec-12 gene has not been cloned. Two other genes (mec-I,5 ) affect an extracellular matrix (called the mantle) that is associated with the touch receptors and appears to secure it to the body wall (Chalfie and Sulston, 1981; Chalfie and Au, 1989). The remaining genes, when mutant, result in animals that have nonfunctioning, yet normal appearing, touch receptors. These latter genes are potentially quite exciting because they may encode products that are needed for mechanosensory transduction. Support for such a hypothesis began with the analysis of one of these genes, mec-4.Although the majority of mec-4 mutations (56 recessive mutations) produce this phenotype (touch insensitivity without apparent disruption of touch receptor morphology), three dominant mutants cause the vacuolated death of the touch receptor neurons (Chalfie and Sulston, 1981; Chalfie and Au, 1989; Driscoll and Chalfie, 1991). The mec-4 gene and a second gene, deg-1, that also mutates rarely to produce a similar appearing death but of different neurons (Chalfie and Wolinsky, 1990)encode putative membrane proteins (called degenerins) that were novel when they were first discovered. Recently, however, a mammalian homologue of these genes h a s been identified (Canessa et al., 1992; Lingueglia et al., 1993). This new protein produces an amiloride-dependent sodium conductance when expressed in Xenopus oocytes. It seems likely that the C. elegans proteins will have a similar function. If so, then the mec-4 gene may represent the first cloned component of a mechanosensory transduction apparatus. Interestingly, a second degenerin, from the mec-10 gene, is also expressed in the touch receptor cells (Huang and Chalfie, 1994). This
gene can be also be mutated to cause touch receptor death. Moreover, by testing for suppressor and enhancer mutations, we have been able to identify several other genes of the function class that are absolutely or partially required for the degenerin-caused deaths (Chalfie and Wolinsky, 1990; Huang and Chalfie, 1994).A n intriguing possibility is that some of these genes (e.g. rnec-6) are components of a channel complex with the mec-4 and mec-10 proteins while other modify the activity of the channel. The functional analysis of the products of these genes is one of the exciting challenges for the future.
Summary We have identified several genes required for four aspects of the differentiation and function of a set of six touch receptor neurons in the nematode Caenorhabditis elegans: (1)the generation of appropriate cells; (2) the specification of those cells to differentiate as touch receptors; (3) the maintenance of the differentiated state; and (4)the expression of products need for the cell function. Three major conclusions about the development of the touch cells arise from the analysis of these genes. First, specification of cell fate is a combinatorial process. At least seven genes, none of which are expressed solely in these cells, are needed to restrict the expression of touch-cell features in the appropriate cells. Second, the differentiated state must also be maintained. Three genes appear necessary for this maintenance function. Third, regulation of development is not strictly linear; at least one gene is needed at more than one stage of differentiation. In addition to being interested in the factor that determine cell fate, we are also interested in understanding the molecular basis of mechanosensory transduction. The function class genes are particularly important in this regard, especially those that when mutant result in the loss of the touch response without producing any obvious morphological defects in the touch cells.
182
References Bargmann, (2.1. (1993) Genetic and cellular analysis of behavior in C. elegans. Ann. Rev. Neurosci., 16: 47-71. Canessa, C.M., Horisberger, J.-D., and Rossier, B.C. (1993) Epithelial sodium channel related to proteins involved in neurodegeneration. Nature, 361: 467-470. Chalfie, M., and Au, M. (1989) Genetic control of differentiation of the C. elegans touch receptor neurons. Scienm, 243: 1027-1033. Chalfie, M., Horvitz, H.R. and Sulston, J.E. (1981) Mutations that lead to reiterations in the cell lineages of Caenorhabditis elegans. Cell, 24: 59-69. Chalfie, M., and Sulston, J. (1981) Developmental genetics of the mechanosensory neurons of Caenorhabditis eleguns. Develop. Biol., 82: 358-370. Chalfie, M., Sulston, J.E., White, J.G., Southgate, E., Thomson, J.N. and Brenner, S . (1985) The neural circuit for touch sensitivity in Caenorhabditis elegans. J. Neurosci., 5: 956-964. Chalfie, M. and White, J.G. (1988) The nervous system. In: W.B. Wood (Ed.), The Nematode Caenorhabditis eleguns. Cold Spring Harbor Laboratory, pp. 337-391. Chalfie, M, and Wolinsky, E. (1990) The identification and suppression of inherited neurodegeneration in Cneiiorhabditis elegans. Nature, 345: 410-416. Driscoll, M., and Chalfie, M. (1991) The mec-4 gene is a member of a family of Caenorhabditis elegans genes that can mutate to induce neuronal degeneration. Nature, 349: 588-593. Ellis, H.M., and Horvitz, H.R. (1986) Genetic control of programmed cell death in the nematode C. elegans. Cell, 44: 817-829. Finney, M. and Ruvkun, G. (1990)The unc-86 gene product couples cell lineage and cell identityin C. elegans. Cell, 63: 895-905. Finney, M., Ruvkun, G. and Horvitz, H.R. (1988). The C. elegans cell lineage and differentiation gene unc-86 encodes a protein with a homeodomain and extended similarity to transcription factors. Cell, 55: 757-769. Freyd, G., Kim, S.K. and Horvitz, H.R. (1990). Novel cysteine-rich motif and homeodomain in the product of the Caenorhabditis elegans cell lineage gene lin-11. Nature, 344: 876-879. Hamelin, M., Scott, I.M., Way, J.C. and Culotti, J.G. (1992) The mec-7 P-tubulin gene of C. elegans is expressed primarily in the touch receptor neurons. EMBO J., 11:
2885-2893. Lingueglia, E., Voilley, N., Waldmann, R., Lazdunski, M. and Barby, P. (1993). Expression cloning of an epithelial amiloride-sensitive Na' channel. A new channel type with homologies to Caenorhabditis elegans degenerins. FEBS Lett., 318: 95-99. Mitani, S., Du, H., Hall, D.H., Driscoll, M. and Chalfie, M. (1993) Combinatorial control of touch receptor neuron expression in Caenorhabditis elegans. Development, 119: 773-783, Savage, C., Hamelin, M., Culotti, J.G., Coulson, A., Albertson, D.G. and Chalfie, M. (1989) mec-7 is a P-tubulin gene required for the production of 15-protofilament microtubules in Caenorhabditis elegans. Genes Dev., 3: 870-881. Sulston, J.E., and Horvitz, H.R. (1977) Post-embryonic cell lineages if the nematode Caenorhabditis elegans. Develop. Biol., 56: 110-156. Sulston, J., Dew, M. and Brenner, S. (1975). Dopaminergic neurons in the nematode Caenorhabditis elegans. J. Comp. Neurol. 163: 215-226. Sulston, J.E., Schierenberg, E., White, J.G. and Thomson, J.N. (1983) The embryonic cell lineage of the nematode Caenorhabditis elegans. Develop. Biol., 100: 64-119. Way, J.C. and Chalfie, M. (1988) mec-3, a homeobox-containing gene that specifies differentiation of the touch receptor neurons in C. elegans. Cell, 54: 5-16. Way, J.C. and Chalfie, M. (1989) The mec-3 gene of Caenorhabditis elegans requires its own product for maintained expression and is expressed in three neuronal cell types. Genes Deu., 3: 1823-1833. Way, J.C., Wang, L.L., Run, J.Q. and Wang, A. (1991). The mec-3 gene contains cis-acting elements mediating positive and negative regulation in cells produced by asymmetric cell division in Caenorhabditis elegans. Genes Dev., 5: 2199-2211. White, J.G., Southgate, E., Thomson, J.N. and Brenner, S. (1986) The structure of t h e nervous system of Caenorhabditis elegans. Philos. Trans. R. SOC.Lond., B: B i d . Sci., 314: 1-340. Xue, D., Finney, M., Ruvkun, G. and Chalfie, M. (1992) Regulation of the mec-3 gene by the C. elegans homeoproteins UNC-86 and MEC-3. EMBO J., 11: 49694979. Xue, D., Tu, Y . and Chalfie, M. (1993) Cooperative interaction between the C. elegans homeoproteins UNC-86 and MEC-3. Science, 261: 1324-1328.
179
CHAPTER 15
The differentiation and function of the touch receptor neurons of Caenorhabditis elegans Martin Chalfie Department of Biological Sciences, 1012 Sherman Fairchild Center, Columbia University, New York, NY 10027, USA
Introduction Research over the last twenty years on the nematode Caenorhabditis elegans has provided a wealth of information on the structure and development of the nervous system of this animal (reviewed by Chalfie and White, 1988; Bargmann, 1993). For example, complete descriptions of the cellular anatomy (including synaptic contacts) and of the lineage derivation of each of the 302 neurons of the adult C. eZeguns hermaphrodite are known (White et al., 1986; Sulston and Horvitz, 1977; Sulston et al., 1983). Moreover, numerous classical and molecular genetics techniques are available t o characterize genes that direct the development and function of cells within the nervous system. Research in my laboratory has used these techniques to study a set of six touch receptor neurons. In this chapter I will describe some of the lessons we have learned about how these cells acquire their specific characteristics (and other cells do not) and how the cells function as touch receptors.
Genes needed for touch cell differentiation These cells are the first in a three-neuron reflex circuit (touch receptor, interneuron, and motor)
that allows the animals to move away from a gentle touch stimulus (usually the stroking of a thin hair; Chalfie and Sulston, 1981; Chalfie et al., 1985). When these cells are killed by laser microsurgery, animals do not move when gently touched. The animals will move normally when stimulated more harshly, a stimulus that activates the interneurons. Our initial analysis of the development and function of the touch receptors began with the collection of touch-insensitive mutants, i.e., animals that respond to harsh, but not gentle, touch (Sulston et al., 1975; Chalfie and Sulston, 1981).At present we have over 450 such touch mutants representing defects in 17 genes (Chalfie and Au, 1989; unpublished data). The mutant phenotypes of these genes have allowed them to be characterized into four groups (Chalfie and Au, 1989;Way and Chalfie, 1989): generation, specification, maintenance, and function. The genes Zin-32 and unc-86 are needed for the generation of the touch receptors. When these genes are mutant the cell lineages giving rise to the touch receptors are altered so that the cells are never made (Chalfie et al., 1981; Chalfie and Au, 1989). The unc-86 gene encodes a POU-type homeodomain protein (Finney et al., 1988) that is not only expressed in the precursors of the touch recep-
180
tors, but also in the cells themselves (as well as in several other cells; Finney and Ruvkun, 1990). These properties suggested to us that UNC-86 (the protein encoded by the unc-86 gene) might act as a direct trans-activator of touch cell differentiation. A likely target for UNC-86 was the mec-3 gene, a gene that is required for touch cell specification. When mec-3 is mutant, cell lineages appear normal, but the cells that should become touch receptors fail t o do so. The mec-3 gene encodes a homeoprotein, but of the LIM type (Way and Chalfie, 1988; Way et al., 1991; Freyd et al., 1990;Xue et al., 1992).The expression of a mec-3ZacZfusion in wild-type animals and mec-3mutant animals suggested that mec3 is autoregulated, i.e. it is needed for its own expression (Way and Chalfie, 1989).These predictions were supported by DNA binding studies which showed that both UNC-86 and MEC-3 bind t o the promoter of the mec-3 gene (Xue et al., 1992, 1993). Two unexpected results arose from these studies: (1)UNC-86 appears to be needed for the continued expression of mec-3 and (2) UNC-86 and MEC-3 can form heterodimers that associate with the DNA in a more stable fashion than either protein alone. The increased stability of the binding, which is needed for the in vivo function of these proteins, probably accounts for the specificity of action of the proteins in determining touch receptor fate. In more recent work (A. Duggan and M. Chalfie, unpublished data) we have found that two targets of mec-3 action, mec-4 and mec-7 (see below) also have UNC-86:MEC3 heterodimer binding sites that are needed for proper expression. This result suggests that touch cell differentiation is probably not under the control of a linear progression of transcription factors (UNC-86 activating mec-3 and MEC-3 activating the downstream genes), but rather under an accumulative control (UNC-86 activates mec-3 and UNC-86 and MEC-3 together activate the downstream genes). Thus, UNC-86 and MEC-3 appear to act in a combinatorial fashion in touch cell develop-
ment. These genes (even with lin-32), however, do not explain why C. elegans has precisely six touch receptors, especially since unc-86 (Finney and Ruvkun, 1990) and mec-3 (Way and Chalfie, 1989) are expressed together in more cells than just the six touch receptor cells. Specifically, mec-3 is expressed in two other pairs of neurons (the FLP cells and the PVD cells); these cells also express unc-86. These obseniations indicate that other factors are required for touch receptor specification. By looking for the ectopic expression of touch cell-specific features or genes we have been able t o identify seven other genes that are needed to restrict the number of touch cells to six (Mitani et al., 1993). The PVD cells do not differentiate into touch receptor cells because they (or their precursors) fail t o express the lin-14 gene (Zin-14 itself is negatively regulated by the Zin-4 gene). The FLP cells express Zin-14 (G. Ruvkun, pers. comm.), but they do not become touch cells because of the action of the genes egl-44 and egl-46 (mutations in either of these genes results in these cells acquiring touch cell features in a lin-14-dependent fashion). Another pair of neurons in the tail can differentiate as touch receptors if the sem-4 gene is mutant. I t is likely that sem-4 negatively regulates mec-3, normally preventing its expression in these cells. Finally, programmed cell death, which requires the ced-3 and ced-4 genes (Ellis and Horvitz, 1986),causes the loss of four cells that could differentiate into touch receptors in the wild-type animal. Together positively- and negatively-acting genes as well a s genes needed for programmed cell death are needed t o produce the correct number of touch receptors within the animals.
Genes needed for touch cell function Mutations in the remaining genes (rnec-1,2, 4, 6 - 10,12,14,15,18) result in animals that have identifiable touch receptors that fail to respond to touch. These function genes are thus needed for the activity, but not the development of the
181
cells. Some of these genes (mec-7,12) affect the unique form of microtubule (15-protofilament) that is found in these cells. The mec-7 gene encodes a P-tubulin that is expressed at high levels only in the touch receptor cells (Savage et al., 1989; Hamelin et al., 1992; Mitani et al., 1993). The mec-12 gene has not been cloned. Two other genes (mec-I,5 ) affect an extracellular matrix (called the mantle) that is associated with the touch receptors and appears to secure it to the body wall (Chalfie and Sulston, 1981; Chalfie and Au, 1989). The remaining genes, when mutant, result in animals that have nonfunctioning, yet normal appearing, touch receptors. These latter genes are potentially quite exciting because they may encode products that are needed for mechanosensory transduction. Support for such a hypothesis began with the analysis of one of these genes, mec-4.Although the majority of mec-4 mutations (56 recessive mutations) produce this phenotype (touch insensitivity without apparent disruption of touch receptor morphology), three dominant mutants cause the vacuolated death of the touch receptor neurons (Chalfie and Sulston, 1981; Chalfie and Au, 1989; Driscoll and Chalfie, 1991). The mec-4 gene and a second gene, deg-1, that also mutates rarely to produce a similar appearing death but of different neurons (Chalfie and Wolinsky, 1990)encode putative membrane proteins (called degenerins) that were novel when they were first discovered. Recently, however, a mammalian homologue of these genes h a s been identified (Canessa et al., 1992; Lingueglia et al., 1993). This new protein produces an amiloride-dependent sodium conductance when expressed in Xenopus oocytes. It seems likely that the C. elegans proteins will have a similar function. If so, then the mec-4 gene may represent the first cloned component of a mechanosensory transduction apparatus. Interestingly, a second degenerin, from the mec-10 gene, is also expressed in the touch receptor cells (Huang and Chalfie, 1994). This
gene can be also be mutated to cause touch receptor death. Moreover, by testing for suppressor and enhancer mutations, we have been able to identify several other genes of the function class that are absolutely or partially required for the degenerin-caused deaths (Chalfie and Wolinsky, 1990; Huang and Chalfie, 1994).A n intriguing possibility is that some of these genes (e.g. rnec-6) are components of a channel complex with the mec-4 and mec-10 proteins while other modify the activity of the channel. The functional analysis of the products of these genes is one of the exciting challenges for the future.
Summary We have identified several genes required for four aspects of the differentiation and function of a set of six touch receptor neurons in the nematode Caenorhabditis elegans: (1)the generation of appropriate cells; (2) the specification of those cells to differentiate as touch receptors; (3) the maintenance of the differentiated state; and (4)the expression of products need for the cell function. Three major conclusions about the development of the touch cells arise from the analysis of these genes. First, specification of cell fate is a combinatorial process. At least seven genes, none of which are expressed solely in these cells, are needed to restrict the expression of touch-cell features in the appropriate cells. Second, the differentiated state must also be maintained. Three genes appear necessary for this maintenance function. Third, regulation of development is not strictly linear; at least one gene is needed at more than one stage of differentiation. In addition to being interested in the factor that determine cell fate, we are also interested in understanding the molecular basis of mechanosensory transduction. The function class genes are particularly important in this regard, especially those that when mutant result in the loss of the touch response without producing any obvious morphological defects in the touch cells.
182
References Bargmann, (2.1. (1993) Genetic and cellular analysis of behavior in C. elegans. Ann. Rev. Neurosci., 16: 47-71. Canessa, C.M., Horisberger, J.-D., and Rossier, B.C. (1993) Epithelial sodium channel related to proteins involved in neurodegeneration. Nature, 361: 467-470. Chalfie, M., and Au, M. (1989) Genetic control of differentiation of the C. elegans touch receptor neurons. Scienm, 243: 1027-1033. Chalfie, M., Horvitz, H.R. and Sulston, J.E. (1981) Mutations that lead to reiterations in the cell lineages of Caenorhabditis elegans. Cell, 24: 59-69. Chalfie, M., and Sulston, J. (1981) Developmental genetics of the mechanosensory neurons of Caenorhabditis eleguns. Develop. Biol., 82: 358-370. Chalfie, M., Sulston, J.E., White, J.G., Southgate, E., Thomson, J.N. and Brenner, S . (1985) The neural circuit for touch sensitivity in Caenorhabditis elegans. J. Neurosci., 5: 956-964. Chalfie, M. and White, J.G. (1988) The nervous system. In: W.B. Wood (Ed.), The Nematode Caenorhabditis eleguns. Cold Spring Harbor Laboratory, pp. 337-391. Chalfie, M, and Wolinsky, E. (1990) The identification and suppression of inherited neurodegeneration in Cneiiorhabditis elegans. Nature, 345: 410-416. Driscoll, M., and Chalfie, M. (1991) The mec-4 gene is a member of a family of Caenorhabditis elegans genes that can mutate to induce neuronal degeneration. Nature, 349: 588-593. Ellis, H.M., and Horvitz, H.R. (1986) Genetic control of programmed cell death in the nematode C. elegans. Cell, 44: 817-829. Finney, M. and Ruvkun, G. (1990)The unc-86 gene product couples cell lineage and cell identityin C. elegans. Cell, 63: 895-905. Finney, M., Ruvkun, G. and Horvitz, H.R. (1988). The C. elegans cell lineage and differentiation gene unc-86 encodes a protein with a homeodomain and extended similarity to transcription factors. Cell, 55: 757-769. Freyd, G., Kim, S.K. and Horvitz, H.R. (1990). Novel cysteine-rich motif and homeodomain in the product of the Caenorhabditis elegans cell lineage gene lin-11. Nature, 344: 876-879. Hamelin, M., Scott, I.M., Way, J.C. and Culotti, J.G. (1992) The mec-7 P-tubulin gene of C. elegans is expressed primarily in the touch receptor neurons. EMBO J., 11:
2885-2893. Lingueglia, E., Voilley, N., Waldmann, R., Lazdunski, M. and Barby, P. (1993). Expression cloning of an epithelial amiloride-sensitive Na' channel. A new channel type with homologies to Caenorhabditis elegans degenerins. FEBS Lett., 318: 95-99. Mitani, S., Du, H., Hall, D.H., Driscoll, M. and Chalfie, M. (1993) Combinatorial control of touch receptor neuron expression in Caenorhabditis elegans. Development, 119: 773-783, Savage, C., Hamelin, M., Culotti, J.G., Coulson, A., Albertson, D.G. and Chalfie, M. (1989) mec-7 is a P-tubulin gene required for the production of 15-protofilament microtubules in Caenorhabditis elegans. Genes Dev., 3: 870-881. Sulston, J.E., and Horvitz, H.R. (1977) Post-embryonic cell lineages if the nematode Caenorhabditis elegans. Develop. Biol., 56: 110-156. Sulston, J., Dew, M. and Brenner, S. (1975). Dopaminergic neurons in the nematode Caenorhabditis elegans. J. Comp. Neurol. 163: 215-226. Sulston, J.E., Schierenberg, E., White, J.G. and Thomson, J.N. (1983) The embryonic cell lineage of the nematode Caenorhabditis elegans. Develop. Biol., 100: 64-119. Way, J.C. and Chalfie, M. (1988) mec-3, a homeobox-containing gene that specifies differentiation of the touch receptor neurons in C. elegans. Cell, 54: 5-16. Way, J.C. and Chalfie, M. (1989) The mec-3 gene of Caenorhabditis elegans requires its own product for maintained expression and is expressed in three neuronal cell types. Genes Deu., 3: 1823-1833. Way, J.C., Wang, L.L., Run, J.Q. and Wang, A. (1991). The mec-3 gene contains cis-acting elements mediating positive and negative regulation in cells produced by asymmetric cell division in Caenorhabditis elegans. Genes Dev., 5: 2199-2211. White, J.G., Southgate, E., Thomson, J.N. and Brenner, S. (1986) The structure of t h e nervous system of Caenorhabditis elegans. Philos. Trans. R. SOC.Lond., B: B i d . Sci., 314: 1-340. Xue, D., Finney, M., Ruvkun, G. and Chalfie, M. (1992) Regulation of the mec-3 gene by the C. elegans homeoproteins UNC-86 and MEC-3. EMBO J., 11: 49694979. Xue, D., Tu, Y . and Chalfie, M. (1993) Cooperative interaction between the C. elegans homeoproteins UNC-86 and MEC-3. Science, 261: 1324-1328.