The Worm's Sense of Smell Development of Functional Diversity

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Developmental Biology 265 (2004) 302–319 www.elsevier.com/locate/ydbio Review The worm’s sense of smell Development of functional diversity in the chemosensory system of Caenorhabditis elegans

Tali Melkmana and Piali Senguptaa,b,*

a Department of Biology, Brandeis University, Waltham, MA 02454, USA b Volen Center for Complex Systems, Brandeis University, Waltham, MA 02454, USA

Received for publication 23 January 2003, revised 23 July 2003, accepted 29 July 2003

Abstract

Animals sense their chemical environment using multiple chemosensory neuron types, each of which exhibits characteristic response properties. The chemosensory neurons of the nematode Caenorhabditis elegans provide an excellent system in which to explore the developmental mechanisms giving rise to this functional diversity. In this review, we discuss the principles underlying the patterning, generation, differentiation, and diversification of chemosensory neuron subtypes in C. elegans. Current knowledge of the molecular mechanisms underlying each of these individual steps is derived from work in different model organisms. It is essential to describe the complete developmental pathways in each organism to determine whether functional diversification in chemosensory systems is achieved via conserved or novel mechanisms. Such a complete description may be possible in C. elegans. D 2003 Elsevier Inc. All rights reserved.

Keywords: C. elegans; Chemosensory system; Functional diversity

You smell that? Do you smell that? Napalm, son. Nothing in information regarding the nature of their environment and to the world smells like that. I love the smell of napalm in the respond appropriately. Chemicals are first encountered by morning. (Kilgore, Apocalypse Now (1979)) tens to millions of chemosensory neurons (CNs) present in peripherally located sense organs. CNs include olfactory NOSE, n. The extreme outpost of the face. It has been observed that one’s nose is never so happy as when thrust neurons that sense volatile odorants and gustatory neurons into the affairs of others, from which some physiologists that sense water-soluble chemicals. Olfactory sensory neu- have drawn the inference that the nose is devoid of the sense rons are present in the olfactory epithelium lining the nasal of smell. (Ambrose Bierce, The Devil’s Dictionary (1911)) cavities of vertebrates, while insects sense odorants using CNs present in sensilla located on the antennae and maxillary palp. The nematode Caenorhabditis elegans responds Introduction to both volatile and water-soluble chemicals using sensory neurons in the amphid and phasmid sensory organs. Phys- As anyone who has driven by a landfill can attest, the iological and behavioral experiments dating back to the ability to smell many different molecules of diverse chem- 1960s indicated that individual olfactory neurons respond to ical structures does not always appear to be beneficial. distinct subsets of odorants at different concentrations However, it is this remarkable diversity of function in the (Gesteland et al., 1965; Getchell, 1974; Getchell and Shep- chemosensory system that enables animals to obtain detailed herd, 1978; O’Connell and Mozell, 1969), suggesting that the enormous discriminatory ability of the olfactory system is mediated primarily by diversity of function among * Corresponding author. Department of Biology, Brandeis University, olfactory neurons. In this review, we discuss how complex- 415 South Street, Waltham, MA 02454. Fax: +1-781-736-3107. ity of function is generated in the chemosensory nervous E-mail address: [email protected] (P. Sengupta). system of C. elegans. In particular, we focus on the

Fig. 1. Olfactory neuron diversity in C. elegans. (A) Location of the amphid and phasmid sensory neurons. A subset of amphid and phasmid sensory neurons fill with lipophilic dyes. White arrow indicates the cell bodies of dye-filling amphid sensory neurons in the head; yellow arrow indicates the cell bodies of the phasmid sensory neurons in the tail. Lateral view; dorsal is on the outside. (B) Amphid olfactory neurons exhibit distinctive sensory morphologies and connectivity. The AWA, AWB, and AWC olfactory neurons extend dendrites anteriorly to the tip of the nose where they end in specialized ciliary structures that are in contact with the external environment. Shown in detail are the unique ciliary endings of each neuron type (left) (adapted from Ward et al., 1975). The neurons representing the major synaptic outputs of each olfactory neuron type in the nerve ring are indicated (top). Each olfactory neuron mediates stereotypical attractive or avoidance responses to specific subsets of odorants. (C) Each olfactory neuron expresses unique subsets of signal transduction molecules. Multiple seven transmembrane domain chemosensory receptors are expressed in each neuron type (curved lines). The odorant diacetyl (hexagon) is known to interact with the ODR-10 olfactory receptor expressed in the AWA neurons. Signals are transmitted via different G proteins (circles). In the AWB and AWC olfactory neurons, receptor guanylyl cyclases (rectangle) are activated, leading to the gating of cGMP-gated channels (gray ovals). Different TRPV channels (colored ovals) have been implicated in both primary signal transduction in the AWA neurons and in adaptation to odorants in the AWC neurons. Although only the olfactory neurons are shown here, additional amphid chemosensory neuron types also exhibit diversity in morphology, function, and gene expression profiles. See text for references. developmental mechanisms by which CNs acquire and initially identified based on their bipolar morphology and modulate their unique sensory response profiles, enabling the direct or indirect exposure of their sensory endings to the C. elegans to navigate its aroma-rich environment. environment. Thirty-two neurons of 14 types—where a type is generally defined as a L/R bilateral pair—fit this defini- tion and are believed to mediate chemosensory functions in Defining diversity the C. elegans hermaphrodite (Ward et al., 1975; White et al., 1986). Of these, 11 pairs are present in the bilateral To discuss how CNs diversify during development, we amphid organs of the head, two pairs are present in the will begin by describing in brief what makes a CN in C. phasmid organs of the tail, and six neurons are present in the elegans different from its neighbors. CNs in C. elegans were inner labial (IL) organs of the head (Fig. 1A). The male 304 T. Melkman, P. Sengupta / Developmental Biology 265 (2004) 302–319 contains additional male-specific sensory neurons present in transduction mechanisms in their responses to chemicals. the sensory rays, spicules, hook and postcloacal sensilla in For instance, while cGMP signaling appears to be necessary the tail, and in the cephalic sensilla of the head (Sulston et for primary signal transduction by the AWC olfactory al., 1980; Ward et al., 1975). These sensory neurons are neurons, calcium may act as the second messenger in the required primarily for male mating behaviors (Liu and AWA olfactory neurons (Bargmann and Mori, 1997; Troe- Sternberg, 1995; Sulston et al., 1980). In this review, we mel, 1999b) (Fig. 1C). Thus, for a neuron to mediate its will restrict our discussion to the development of the amphid correct cell-type-specific functions, developmental mecha- CN types that are present in both hermaphrodites and males. nisms must act to ensure the coordinated expression of all Amphid CNs differ from each other in their sensory cell-type characteristics in each neuron type. functions, their morphologies, their synaptic connectivities, and in the expression of signal transduction genes (Fig. 1). For example, the AWA, AWB, and AWC neuron pairs in the Patterning chemosensory neurons amphid respond to distinct subsets of volatile attractants and repellents (Bargmann et al., 1993; Chou et al., 2001; The origin of chemosensory neurons Troemel et al., 1997; Wes and Bargmann, 2001); the ASE neuron pair responds to attractive salts (Bargmann and Developmentally speaking, where do C. elegans CNs Horvitz, 1991a; Pierce-Shimomura et al., 2001); the ASH come from? Lineage tracing experiments have shown that neuron pair is polymodal responding to volatile repellents, 96% of all neurons including all CNs in a just-hatched C. mechanical stimuli, and high osmolarity (Kaplan and Hor- elegans L1 larva are generated from the AB founder cell, vitz, 1993; Hart et al., 1995; Maricq et al., 1995; Troemel et which arises as the anterior daughter of the first asymmetric al., 1995); and the ADL neurons respond to both volatile and cell division of the zygote (Sulston et al., 1983). AB divides water-soluble chemicals (Bargmann and Horvitz, 1991a; to generate the anteriorly placed ABa and the posteriorly Troemel et al., 1997). Additional amphid neurons mediate placed ABp daughters, which divide again along the L–R minor responses to different sets of water-soluble and and the A–P axes to generate the eight great granddaughters volatile molecules (Bargmann and Horvitz, 1991a, 1991b; of the AB cells present in the 12-cell embryo. Five of these Schackwitz et al., 1996). Moreover, individual neuron types eight AB descendants give rise to all CNs of the amphid can be definitively identified based on the unique morphol- (Fig. 2). The left members of the AFD thermosensory, and ogy of their sensory endings and their axonal trajectories, the ASK, ADL, ADF, AWB, ASE, and ASJ CN types arise and each sensory neuron makes a unique set of synaptic from the ABalp blastomere, whereas their right counterparts connections to downstream interneurons (Perkins et al., arise from the nonhomologous ABpra cell. The left AWA, 1986; Ward et al., 1975; White et al., 1986) (Fig. 1B). ASG, and ASI neurons arise from ABpla and the right Not surprisingly, the specific functions of each CN are neurons from ABpra, and the left and right AWC and ASH directed by the expression of distinct sets of signaling and CNs arise from the ABplp and ABprp cells, respectively. structural genes in each neuron type (Fig. 1C). Similar to Thus, different types of CNs do not arise from a single vertebrates and Drosophila, each CN in C. elegans expresses blastomere type, nor do they arise from a dedicated sub- a unique set of seven transmembrane domain chemosensory lineage on either side of the animal. Instead, in each amphid receptor genes (Sengupta et al., 1996; Troemel, 1999a, organ, different neuronal members are generated from 1999b; Troemel et al., 1995, 1997). However, given the multiple lineages and from distinct precursors that do not small number of chemosensory neurons in C. elegans, and all share obvious symmetry, homology, or parallels in their the >600 chemosensory receptors encoded by the C. elegans cell division programs (Finney and Ruvkun, 1990; Sulston genome, unlike other organisms, each C. elegans CN is and Horvitz, 1977; White et al., 1976) (Fig. 2B). Moreover, predicted to express multiple receptor genes (Peckol et al., while all amphid neurons are generated between 280 and 2001; Robertson, 1998, 2000; Troemel, 1999a; Troemel et 400 min late in the first half of embryogenesis, each neuron al., 1995). Consistent with this, examination of the expres- type is generated at somewhat different developmental times sion patterns of receptors using promoter fusions to the gfp (Schnabel et al., 1997; Sulston et al., 1983). The exact reporter showed that at least 6, and possibly as many as 20 timing of cell divisions in each CN lineage varies signifi- receptors, were expressed in an individual CN type (Troe- cantly from embryo to embryo and even between bilaterally mel, 1999a; Troemel et al., 1995). Each CN in C. elegans homologous lineages (Schnabel et al., 1997). also expresses a unique subset of additional signal transduction genes including genes encoding G protein subunits, Generating chemosensory neurons transmembrane guanylyl cyclases, and channel subunits (Coburn and Bargmann, 1996; Colbert et al., 1997; Jansen What are the mechanisms that dictate when and where a et al., 1999; Komatsu et al., 1996; Roayaie et al., 1998; CN is generated? Although C. elegans development had Tobin et al., 2002; Troemel, 1999b; Yu et al., 1997).Asa previously been described as invariant and deterministic, consequence of this differential gene expression pattern, this view has changed radically (Schnabel, 1991; Wood, different groups of CNs in C. elegans utilize different signal 1991; Priess and Thomson, 1987). Experimental manipula- T. Melkman, P. Sengupta / Developmental Biology 265 (2004) 302–319 305 tions have shown conclusively that in the early embryo, a the ABalp blastomere give rise to the majority of left amphid series of inductive interactions mediated by the glp-1 Notch- sensory neurons, while the nonsymmetrical ABpra blasto- like receptor and the apx-1 Delta-like ligand are responsible mere generates 10 of 12 right amphid sensory neurons? for specifying the identities of the eight AB great grand- Although the generation of these cells may simply be due to daughters (Hutter and Schnabel, 1994, 1995a,b; Priess and an intrinsically programmed cell division pattern, it has been Thomson, 1987; Evans et al., 1994; Mango et al., 1994b; suggested that regional specification mechanisms may also Mello et al., 1994; Moskowitz et al., 1994). However, play a role in dictating the lineage patterns of individual development at stages later than the 12-cell stage is likely blastomeres (Schnabel, 1996; Schnabel et al., 1997). Each to be primarily cell autonomous based on several experi- blastomere may specify a nonoverlapping region of the mental observations. These include the lack of compensation embryo similar to segments or compartments in the body upon the ablation of individual cells, the variability of cell plans of Drosophila and vertebrate embryos (Garcia-Bellido positions from embryo to embryo, and the variability in the et al., 1973; Keynes and Lumsden, 1990; Lawrence and timing of cell divisions in different lineages (Hutter and Struhl, 1996; Lumsden, 1990). In this model, the number Schnabel, 1995b; Schnabel et al., 1997; Sulston et al., 1983). and types of progeny generated by a given blastomere is Asymmetric cell divisions primarily along the A–P axis determined by its regional identity, which then acts upon its generate the vast majority of cells in the nervous system lineage pattern to regulate the production of progeny appro- including the chemosensory nervous system (Sulston et al., priate for that region (Schnabel et al., 1997). Thus, depend- 1983). The POP-1 TCF/LEF transcription factor may play a ing on its position, a given blastomere produces cells of role in specifying the fates of the anterior daughters of all multiple types, as required to specify tissues and organs in these asymmetric cell divisions in the AB and other lineages that region. In other words and more relevant to the present (Lin et al., 1995, 1998). POP-1 is present at higher concen- discussion, the ABalp and the ABpra blastomeres may trations in the anterior daughters, which adopt the fates of generate the majority of amphid neurons since these blasto- their posterior siblings upon reduction of pop-1 activity (Lin meres specify the regions of the embryo fated to give rise to et al., 1995, 1998). The MOM-4 MAPKKK-related protein, the left and right amphid organs, respectively (Fig. 2A). the WRM-1 h-catenin, and the LIT-1 Nemo-like kinase act Taken together, we suggest that amphid neurons are in the posterior daughters to maintain POP-1 at low levels patterned primarily via lineage intrinsic mechanisms. Thus, (Ishitani et al., 1999; Meneghini et al., 1999; Rocheleau et once the blastomere cell fate and its regional identity has al., 1999; Shin et al., 1999).Inlit-1 and wrm-1 mutants, the been determined, intrinsic cell division patterns coupling posterior daughters adopt the fates of their anterior sisters A–P axis cues and POP-1 asymmetry with lineage-specific (Kaletta et al., 1997; Meneghini et al., 1999; Rocheleau et factors specify the expression of distinct programs of genes al., 1997). Asymmetric expression of POP-1 in the E and in each cell type. The factors present in a cell type act to MS blastomeres is initiated by MOM-2 Wnt and SRC-1 restrict its developmental potential in a predictable and signaling from the P2 blastomere (Bei et al., 2002; Lin et al., invariant manner, thereby ensuring that the correct number 1995, 1998; Rocheleau et al., 1997; Thorpe et al., 1997). and types of CNs are generated from invariant positions in However, mutations in mom-2 and ablation of P2 do not the lineage. Thus, lineal mechanisms as opposed to regula- appear to affect POP-1 levels in the AB lineages that give tory mechanisms are likely to be the primary determinants rise to the CNs (Hutter and Schnabel, 1995b; Gendreau et of the patterning and generation of CNs. al., 1994; Lin et al., 1998), suggesting that additional signaling molecules from other blastomeres may play a role in initiating POP-1 asymmetry in the AB lineage (Hutter Becoming a chemosensory neuron and Schnabel, 1995b). Asymmetry of pop-1 expression in the AB and other lineages may be subsequently maintained Elegant work in Drosophila has suggested a framework by intrinsic mechanisms, perhaps via propagation by other for how neuronal subtypes are specified. In this progressive transcription factors (Lin et al., 1998). These lineally regu- determination model, neuronal potential is successively lated factors may act with POP-1 to execute the fate of the restricted so as to ultimately result in the production of anterior cell, whereas in the absence of POP-1, the posterior defined cell types (Dambly-Chaudiere and Vervoort, 1998; daughter adopts a different fate (see below). POP-1 and LIT- Ghysen and Dambly-Chaudiere, 1989; Jan and Jan, 1994). 1 functions appear to be required repeatedly at each suc- For instance, a CN may acquire a generic neuronal and a cessive A–P cell division irrespective of lineage and ulti- generic sensory neuronal potential en route to acquiring its mate fates of the daughter cells (Kaletta et al., 1997; Lin et final subtype identity. Are CNs in C. elegans specified via al., 1998). Thus, globally acting genes such as pop-1 and lit- similar mechanisms? 1, together with lineage-specific genes, function to dictate the generation of specific cell types, including CNs, at Defining neuronal potential defined times and locations from individual blastomeres. This raises the question of how the lineage program of a Proneural genes such as members of the Achaete-scute, blastomere is determined. For instance, why and how does Atonal, and NeuroD subfamilies of bHLH domain con- 306 T. Melkman, P. Sengupta / Developmental Biology 265 (2004) 302–319

Fig. 2. Patterning chemosensory neurons via regional specification and the lineage. (A) Amphid chemosensory neurons arise from multiple blastomeres that specify the regions of the embryo fated to give rise to the left and right amphid organs. (Left) Shown are the positions of the blastomeres in a 12-cell embryo at approximately 47 min of development. The positions can be variable; shown locations are derived from the positions of the blastomeres in two different embryos (Schnabel et al., 1997). The larger space occupied by the ABpra blastomere reflects the variable position of this blastomere in these embryos. (Middle) The regions specified by the progeny of the AB descendants that give rise to the amphid chemosensory neurons are shown at 235 min of development. These regions are also derived from data on two embryos (Schnabel et al., 1997). The layers have been collapsed to provide a composite overlapping view. Note that regions can be specified by the progeny of multiple blastomeres. (Right) The locations of the amphid chemosensory neuron precursors at 260 min of development are shown. These precursors are color-coded to reflect their origin. Anterior is at left in all panels. L and R refer to the precursors of the CNs in the left and right amphids, respectively. Based on data in Schnabel et al. (1997). The right panel was adapted from Sulston et al. (1983). (B) Lineages giving rise to amphid chemosensory neurons of the left (L) and right (R) amphid organs. Cells are color-coded reflecting their blastomere origin as in A. Note that in each amphid organ, the lineages giving rise to individual chemosensory neuron types do not exhibit obvious symmetry or parallels in their cell division programs. However, L/R CN pairs exhibit bilaterally symmetrical lineage patterns either throughout their developmental histories or in later stages (shaded boxes). All divisions are along the A–P axis unless noted otherwise.

taining transcription factors have been shown to specify organs in different parts of the central and peripheral neuronal potential in both Drosophila and vertebrates nervous systems (Brunet and Ghysen, 1999; Goulding et (Brunet and Ghysen, 1999; Dambly-Chaudiere and Ver- al., 2000; Guillemot et al., 1993; Huang et al., 2000; voort, 1998; Jan and Jan, 1994; Kageyama and Nakanishi, Jarman et al., 1993, 1994; Lo et al., 1998).TheC. 1997; Lee, 1997; Modolell, 1997). However, in addition elegans genome is predicted to encode several bHLH to being neurogenic, these genes also confer neuronal type proteins (Ruvkun and Hobert, 1998). Several of these specificity such that each of these proneural genes plays a genes are expressed broadly in neuronal precursors and role in the generation of different neurons and sense their descendants, and have been implicated in the gener- T. Melkman, P. Sengupta / Developmental Biology 265 (2004) 302–319 307 ation and fate specification of multiple neuron types et al., 1986; Tabish et al., 1995). The DAF-19 RFX-type (Hallam et al., 2000; Krause et al., 1997; Portman and transcription factor appears to regulate the expression of Emmons, 2000; Zhao and Emmons, 1995). The daugh- general ciliary structural genes, such that the cilia of all terless homolog hlh-2 is initially broadly expressed, but sensory neurons are completely absent in daf-19 mutants becomes progressively restricted in its expression pattern (Perkins et al., 1986; Swoboda et al., 2000). However, to most neuronal precursors and neurons at the time of the expression of genes such as osm-3, and the expres- maximal neurogenesis (Krause et al., 1997). Expression sion of additional cell-type characteristics, including the appears to be maintained in a few cells, including the expression of cell-type specific signaling genes is unal- ADL and ASH amphid sensory neurons into early larval tered in daf-19 mutants (Swoboda et al., 2000). This stages. Similarly, the Achaete-scute homolog hlh-3 and the suggests that DAF-19 may act to confer a subset of NeuroD homolog cnd-1 are expressed in multiple neuro- generic sensory neuron features onto all CNs, and that blasts descended from the AB founder cells, including cell-type-specific specialized features may be acquired via those neuroblasts destined to give rise to amphid sensory independent mechanisms (see below). daf-19 may be neurons (Hallam et al., 2000; Krause et al., 1997; Portman activated by the same gene(s) in each CN, or may be and Emmons, 2000). The fates of the amphid neurons activated in different neurons by distinct sets of genes were reported to be unaltered in animals carrying a weak whose presence is dictated via lineal cues. It is interesting mutation in the lin-32 Atonal homolog (C. Kenyon and E. to note that the homologous Rfx gene in Drosophila is Hedgecock, personal communication), although the devel- also required for sensory cilia morphogenesis, although opment of these neurons has not been examined in unlike daf-19, Rfx may also affect additional aspects of animals carrying stronger lin-32 loss-of-function alleles neuronal differentiation (Dubruille et al., 2002). or in animals mutant for other proneural genes. Amphid- expressed genes were identified in a microarray-based Defining amphid organ identity screen for targets of LIN-32 and HLH-2 (D. Portman and S. Emmons, personal communication), suggesting that Organ identity genes have been shown to act in multiple these bHLH domain proteins may play a role in the lineages that give rise to the diverse cell types comprising an development of the amphid chemosensory neurons. The organ (Mango et al., 1994a; Page et al., 1997). For example, precise roles of these genes in the generation and/or fate the pharynx is composed of multiple cell types including specification of the amphid neuron lineages remain to be neuronal, epidermal, and muscle cells. Although pharyngeal clarified. Thus, it is currently unclear whether a single or cells are derived from different founder cells, in each multiple proneural gene(s) act in the diverse lineages that lineage, precursors are specified which gives rise to only give rise to amphid sensory neurons, and whether these pharyngeal cells (Sulston et al., 1983). The forkhead domain genes play roles both in early and later steps in the transcription factor PHA-4 is expressed in these precursors, developmental hierarchy. as well as in their pharyngeal daughters and acts as an organ identity gene (Horner et al., 1998; Kalb et al., 1998; Mango Defining sensory neuronal properties et al., 1994a). In the absence of PHA-4 function, the pharynx is not specified. Is there an analogous amphid Do CNs acquire a generic sensory or a CN potential, organ identity gene? Unlike the pharynx, no precursors in and is the acquisition of these characteristics separate the amphid neuron lineages give rise to only amphid cells from, sequential to, or concomitant with the acquisition (Labouesse and Mango, 1999; Sulston et al., 1983). Even of a specific chemosensory cell type identity? Sensory terminal divisions in these lineages give rise to interneurons, neurons including mechanosensory neurons in C. elegans motorneurons, and epidermal cells in addition to CNs are ciliated and the acquisition of ciliary structures may (Sulston et al., 1983). Thus, it appears unlikely that an be construed as a marker for a generic sensory neuron amphid organ gene acts similarly to PHA-4 early in these identity (Perkins et al., 1986; Ward et al., 1975; White et lineages to specify the generation of all amphid components. al., 1986). CNs generate cilia late in development, well It is possible, however, that an amphid organ identity gene after the neurons are born, implying that cilia represent a functions in each CN type by integrating multiple, diverse relatively late step in the differentiation process (Sulston lineage cues. et al., 1983). Although the ciliary structures of each Based on described models both in other organisms as sensory neuron are relatively unique, all express a com- well as in C. elegans, a cell likely becomes a CN via a series mon set of structural genes including osm-1 and osm-6, of lineage-driven intermediate states with increasingly re- which encode components of the intraflagellar transport stricted developmental potential. However, as is clear from complex (Cole et al., 1998; Collet et al., 1998; Orozco et the above discussion, neither the molecules nor the mech- al., 1999; Signor et al., 1999; Swoboda et al., 2000). anisms involved in this process have yet been characterized. However, subsets of CNs also express additional genes Therefore, it remains to be seen whether the determination such as the kinesin heavy chain gene osm-3, which plays of CNs utilizes well-conserved principles, or whether novel a role in defining cell-specific ciliary structures (Perkins mechanisms are employed. 308 T. 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Generating chemosensory neurons via asymmetric cell similar manner in the ADL and other lineages, although not divisions all CN lineages are affected in ham-1 mutants (Sarafi- Reinach, 2001). Since both UNC-130 and HAM-1 affect Intrinsic mechanisms asymmetric cell divisions in many lineages giving rise to multiple CN types, they may act only to segregate factors As discussed earlier, asymmetric expression of the POP- that regulate specific cell fates. Alternatively, they may 1 TCF/LEF transcription factor plays an important role in function along with lineally regulated subsets of proteins most, if not all, asymmetric cell divisions that generate to dictate distinct cell fates in the daughters generated from multiple cell types from the AB founder cell. POP-1 is likely the asymmetric cell division. to act with additional lineage-specific genes, which may themselves be asymmetrically localized and/or segregated to Extrinsic mechanisms regulate distinct daughter cell fates. Two genes affecting asymmetric cell divisions in the CN lineages have been Besides intrinsic factors, cell–cell interaction has also identified. UNC-130, a forkhead domain transcription fac- been implicated in regulating asymmetric cell division. In tor, controls the asymmetric division that gives rise to the the Drosophila PNS, cell–cell interaction between two sibling AWA and ASG CNs, such that the ASG neurons daughter cells mediated via Notch/Delta signaling interfaces adopt the AWA fate in unc-130 mutants (Sarafi-Reinach and with asymmetrically segregated factors to result in the Sengupta, 2000). Asymmetric division in additional CN generation of two distinct cell types (Guo et al., 1996; lineages are also affected in unc-130 mutants, albeit to a Hartenstein and Posakony, 1990; Spana and Doe, 1996; weaker degree than in the AWA/ASG lineage. As might be Zeng et al., 1998). The role of Notch/Delta signaling in the expected from an effector of the asymmetric division differentiation of CNs in C. elegans is not entirely clear, process, UNC-130 is expressed in the precursors to AWA/ although hypomorphic alleles of the lin-12 and glp-1 Notch ASG, but is either not expressed or only transiently receptor, and the lag-2 Notch ligand genes do not appear to expressed in the postmitotic AWA and ASG neurons. Since severely affect the differentiation of a subset of CNs (Sarafi- UNC-130 is localized to the nucleus and is itself not Reinach, 2001; Troemel et al., 1999). Mutations that affect asymmetrically distributed in the precursors, it may act by fate specification of a CN type also do not affect the fate of regulating the transcription of molecules required for the their sister cells concomitantly (Sarafi-Reinach et al., 2001; asymmetric segregation of other factors. Intriguingly, the Lanjuin et al., 2003), suggesting that cell–cell interaction Jumeaux forkhead domain transcription factor has been between daughter cells may not be required for the daugh- proposed to act in a similar manner to regulate asymmetric ters to adopt distinct fates. This is consistent with the cell division in the Drosophila embryonic CNS (Cheah et suggestion that intrinsic mechanisms may be the primary al., 2000). Jumeaux acts by mediating the correct asymmet- regulators of asymmetric cell divisions at later embryonic ric localization and segregation of the cell fate determinants stages. Numb and Partner of Numb in neural progenitors. A Numb homolog is encoded by the C. elegans genome (Ruvkun and Hobert, 1998) but has not been yet been implicated in Specifying chemosensory neuron identities asymmetric cell divisions. A possible target of a transcriptional regulator of asym- Genetic and behavioral screens for mutants with altered metric cell division is a tether molecule that is itself cell-specific marker expression and/or defective sensory asymmetrically localized, and acts to regulate the asymmet- behaviors have resulted in the identification of transcription ric localization and distribution of cell fate determinants. factors required for the specification of individual CN HAM-1, a novel protein, was proposed to be such a identities. These genes can be broadly categorized into molecule (Guenther and Garriga, 1996). Mutations in two classes. The first category includes genes, mutations ham-1 were found to affect the development of several in which affect only a subset of the differentiated functions neurons including the ADL amphid and PHB phasmid CNs. of sensory neurons. Genes in this class include the unc-3 O/ The role of ham-1 has been studied most extensively in the E transcription factor, which is expressed in and regulates PHB lineage. In this lineage, HAM-1 was shown to be only a subset of the differentiated characteristics of the ASI asymmetrically localized in the precursors, and is segregated CNs (Prasad et al., 1998). This relatively minor role of an O/ to only one of the daughter cells, the immediate PHB E transcription factor in the differentiation of a single CN precursors. Interestingly, in ham-1 mutants, the daughter type in C. elegans is somewhat surprising given the broad cell that does not inherit HAM-1 is affected by its loss, expression of O/E genes in the majority of immature and adopting the fate of its sister cell. This is consistent with the mature olfactory neurons of rodents, and the identification idea that HAM-1 acts as a tether for factor(s) specifying the of O/E binding sites upstream of olfactory signal transduc- fate of the daughter that inherits it. In ham-1 mutants, these tion genes including those encoding olfactory receptors factors may be distributed to both cells causing both to (Dugas and Ngai, 2001; Glusman et al., 2000; Kudrycki adopt the same fate. ham-1 is suggested to function in a et al., 1993; Mori et al., 2000; Sosinsky et al., 2000; Vassalli T. Melkman, P. Sengupta / Developmental Biology 265 (2004) 302–319 309 et al., 2002; Wang et al., 1997, 2002). Similarly, mutations transduction genes (Freund et al., 1997; Furukawa et al., in the LIM homeobox gene ceh-14 have been shown to 1997; Livesey et al., 2000). Alternatively, these ‘master’ affect the sensory morphology of the AFD thermosensory genes may act by promoting the expression of one or neurons of the amphid, without altering the expression of multiple additional downstream factors, which then function AFD-specific signaling genes (Cassata et al., 2000). Thus, either sequentially or in parallel to coordinate the expression genes such as unc-3 and ceh-14 are likely to represent of cell-specific features. It may reasonably be expected that members of suites of genes that act within a neuron type genes that act to directly regulate terminal features would be to specify multiple but not all aspects of cell identity. expressed throughout development. Expression of unc-42, Genes in the second class affect all aspects of terminally che-1, lin-11, ttx-1, and ceh-36 is maintained through adult differentiated cell-type identities, including cell-specific stages in the ASH, ASE, ASG, AFD, and AWC neurons, gene expression as well as cell-specific sensory morpholo- respectively (Baran et al., 1999; Sarafi-Reinach et al., 2001; gy. These genes can be thought of as ‘master’ regulatory Satterlee et al., 2001; Lanjuin et al., 2003). TTX-1 may act genes, which act at the apex of a hierarchical differentiation both directly and indirectly to regulate AFD-specific fea- cascade in each neuron type. Genes in this class include tures. TTX-1 binding sites have been identified upstream of members of several well-conserved families of transcription AFD-specific signal transduction genes (H. Kagoshima and factors including LIM and paired-type homeobox genes. Y. Kohara, personal communication). However, TTX-1 has Interestingly, as described below, investigation of the func- also been shown to regulate the expression of the LIM tions of these genes has revealed unexpected differences in homeobox gene ceh-14, which specifies some aspects of the mechanisms by which the sensory functions of individ- AFD sensory morphology (Satterlee et al., 2001).By ual CNs are specified. analogy, UNC-42, CHE-1, LIN-11, and CEH-36 may also act both directly and indirectly to specify terminal features Genes regulating sensory neuron identities of cell identity. In contrast, in the AWA and AWB neurons, expression of The paired-like homeobox gene unc-42 regulates all cell- lin-11 and ceh-37 is transient. Both genes are expressed specific characteristics of the ASH polymodal sensory early, but expression is abolished soon after hatching neurons, such that all ASH-mediated sensory functions are (Sarafi-Reinach et al., 2001; Lanjuin et al., 2003). This lost in unc-42 mutants (Baran et al., 1999). In addition, suggests that these genes may act by promoting the expres- mutations in unc-42 result in a loss of expression of ASH- sion of additional downstream gene(s), which may then expressed chemosensory receptor genes. Similarly, muta- regulate subtype identity. In addition, while both lin-11 and tions in the GLASS-like zinc finger transcription factor che- ceh-37 null mutants are partially penetrant for their pheno- 1 lead to the loss of all known cell-type characteristics and types, these mutants do not appear to exhibit partial expres- functions of the ASE chemosensory neurons (Dusenbery et sivity; that is, the affected amphid neurons are either wild al., 1975; Uchida et al., 2003). Mutations in each of the type or lose all aspects of cell identity, with no neurons three Otx-like homeobox genes encoded by the C. elegans exhibiting partial loss of cell-specific features. This obser- genome have now been shown to specify the identities and vation suggests that a specific threshold of LIN-11 and functions of distinct amphid neuron types. Mutations in ttx- CEH-37 or their partner protein(s) is sufficient to trigger a 1 result in a loss of differentiated characteristics of the AFD complete CN differentiation program. This may be mediated thermosensory neurons, including the expression of AFD- by either multiple aspects of the differentiation program specific signaling genes and AFD sensory morphology requiring the same threshold of LIN-11/CEH-37 or their (Hedgecock and Russell, 1975; Satterlee et al., 2001). partner gene product(s). However, a simpler and more Mutations in the ceh-37 and ceh-36 Otx-like genes result practical model would be that a given threshold is sufficient in a similar loss of cell-type identities of the AWB and AWC to activate a single target gene that then triggers the olfactory neurons, respectively (Lanjuin et al., 2003). The complete neuronal differentiation program in each neuron LIM homeobox gene lin-11 regulates the cell-type identities type. These target genes are likely to be the odr-7 nuclear of both the AWA olfactory and their sibling ASG chemo- hormone receptor gene in the AWA neurons, and the lim-4 sensory neurons (Sarafi-Reinach et al., 2001). lin-11 and LIM homeobox gene in the AWB neurons (Colosimo et al., ceh-37 mutants are partially penetrant for the AWA, ASG, in press; Sagasti et al., 1999; Sengupta et al., 1994). While and AWB defects, indicating that they act with as yet LIN-11 and CEH-37 are required to initiate the expression unidentified partner(s) to regulate cell identities. of odr-7 and lim-4, respectively, expression of both genes Identity genes such as unc-42, che-1, lin-11, and the three through adult stages is then maintained via autoregulation. Otx-like homeobox genes may specify a defined neuronal In odr-7 and lim-4 mutants, all known aspects of AWA and subtype by directly regulating the expression of cell-type- AWB cell-type identities are lost, including the expression specific terminal differentiated features. For example, the of cell-specific signal transduction genes and morphological Otx-related gene Crx is required for the differentiation of features. It is not yet clear whether ODR-7 and LIM-4 act photoreceptors in vertebrates, and has been shown to directly and/or indirectly to activate downstream target directly regulate the expression of photoreceptor signal genes. Thus, a temporally regulated cascade of transcription 310 T. Melkman, P. Sengupta / Developmental Biology 265 (2004) 302–319 factors acts to specify the identities of the AWA and AWB ciliary structures of the AWC neurons (Perkins et al., 1986; olfactory neurons. Satterlee et al., 2001). Moreover, the AWC marker misex- pressed most highly and consistently in odr-7, lin-11, and Consequences of losing cell-type identities ttx-1 mutants is the AWC-specific str-2 olfactory receptor gene. str-2 is expressed asymmetrically in one of two AWC Do neurons which have lost cell-type-specific features neurons in wild-type animals, and this expression is regu- as in the above mutants, retain generic neuronal or chemo- lated by axo-axonal contact and calcium signaling (see sensory neuronal features? Alternatively, upon loss of below) (Sagasti et al., 2001; Tanaka-Hino et al., 2002; identity, does a neuron type adopt the fate of another cell Troemel et al., 1999). Spatial expression of str-2 is also type? Unexpectedly, the AWB neurons in lim-4 mutants regulated by environmental signals and developmental adopt the characteristics of the lineally unrelated AWC stage-specific cues (Nolan et al., 2002; Peckol et al., olfactory neurons (Sagasti et al., 1999). The switch in cell 2001).Thus,str-2 may not represent a true AWC fate fate appears to be relatively complete, since the AWB marker. Nevertheless, it is important to determine whether neurons not only express all AWC markers examined in all CNs do indeed share a common default developmental the absence of LIM-4 function, but also adopt AWC-like identity (and what it is), which is expressed upon the loss of sensory morphology and possibly synaptic specificities. cell-type-specific characteristics, or whether the ultimate Conversely, misexpression of lim-4 in the AWC neurons identity of each neuron type is determined by the sequential is sufficient for conversion to an AWB fate. Thus, lim-4 layering of distinct neuronal identities dictated by individual appears to act as a true binary switch between the AWB sublineage programs. It is also possible that upon loss of and AWC cell fates. Single genes have been shown to act cell-specific identity, CNs may express features that repre- as binary switches between sibling cells generated as a sent a hybrid of other chemosensory cell properties. It is result of asymmetric cell division of their precursors important to note, however, that in all cases investigated to (Hawkins and Garriga, 1998; Jan and Jan, 2001; Lu et date, neurons that have lost cell-specific identity continue to al., 2000). In these cases, a molecule may be segregated or exhibit neuronal, and in particular, chemosensory neuronal expressed asymmetrically in one daughter cell; in the morphology. This suggests that genes such as lin-11, che-1, absence of this molecule, the cell may adopt the fate of odr-7, and the Otx-like genes play roles specifically in the its sibling. However, given that the AWB and AWC acquisition of cell-specific identities and not in the specifi- neurons are related only distantly by lineal ancestry cation of a CN fate. (Sulston et al., 1983), this raises the question of how (and why) an AWC-like fate is chosen as the default The importance of cellular context developmental state of the AWB neurons. Interestingly, no AWC-like characteristics are expressed in the AWB An interesting correlate to the above work is that the neurons in ceh-37 mutants or in ceh-37 lim-4 double same or related gene(s) can specify different identities in mutants, although the neurons retain generic sensory different cellular contexts. For instance, lin-11 specifies the neuronal features (Lanjuin et al., 2003). A simple model different identities of both the AWA and ASG neurons. In to explain this finding suggests that CEH-37 acts to this case, the distinct developmental pathways triggered by promote an AWC-like fate in the AWB neurons. However, lin-11 in the different cells are partly mediated by differen- in the presence of the CEH-37 partner protein(s) in the tial temporal regulation of its expression. Although lin-11 is AWB lineage, CEH-37 triggers the expression of lim-4, expressed in both the AWA and ASG neuron types, lin-11 which then both suppresses the AWC identity and pro- expression is transient in the AWA neurons, while expres- motes an AWB identity. Thus, an intermediate step in the sion is maintained throughout development in the sibling development of the AWB neurons is the adoption of ASG neurons (Sarafi-Reinach et al., 2001). Prolonged AWC-like characteristics that must then be acted on by expression in the AWA neurons results in a partial loss of LIM-4 to promote AWB-specific properties. AWA identity. The importance of the cellular context is This result also raises the intriguing possibility that an particularly evident in the case of the roles of the three Otx- AWC-like fate represents the elusive common denominator like genes. The Otx1 and Otx2 genes in mouse, and the otd for an amphid or a CN ‘identity’, such that all CNs adopt an gene in Drosophila, are functionally interchangeable, indi- intermediate AWC-like developmental state that is then cating a remarkable conservation of gene function across further modified by the expression of cell-specific genes. phyla (Acampora et al., 1998a, 1998b, 1999; Leuzinger et However, the issue turns out to be not quite so straightfor- al., 1998; Nagao et al., 1998). Similarly, we have recently ward. Unlike the AWB neurons in lim-4 mutants, the AWA shown that ttx-1 and ceh-37 (as well as rat Otx1) can neurons in lin-11 and odr-7 mutants misexpress only a functionally substitute for ceh-36 function in the AWC subset of AWC markers, and do not adopt an AWC-like neurons, while ceh-36, ttx-1, and Otx1 can substitute for morphology (Sagasti et al., 1999; Sarafi-Reinach et al., ceh-37 function in the AWB neurons (Lanjuin et al., 2003). 2001). Similarly, in ttx-1 mutants, the AFD neurons also The crucial observation here is that expression of an Otx misexpress only a few AWC markers and do not adopt the gene such as ceh-37 in the AWC lineage promotes only the T. Melkman, P. Sengupta / Developmental Biology 265 (2004) 302–319 311

Fig. 3. Transcription factor cascades specify chemosensory neuron identities. Mutations in the ‘master’ regulatory genes affect cell-type-specific differentiated characteristics of chemosensory neuron types (top shaded box). These ‘master’ genes regulate cell-specific features either directly or indirectly via the regulation of expression of sets of additional downstream genes, each of which is required for the specification of different aspects of cell identity (lower shaded box). See text for additional details and references. Arrows represent either direct or indirect regulation. Curved arrows represent autoregulation.

AWC identity, whereas expression of the same gene in the until recently, the L/R members of a CN pair were believed AWB lineage promotes only the AWB identity. These to be functionally identical, recent work suggests that this is experiments suggest that the distinct developmental pro- not always the case. As mentioned previously, the str-2 OR gram triggered by each of these genes in a particular cell is expressed stochastically in the left or the right AWC type is likely to result from the unique cellular context neuron (Troemel et al., 1999). In the ASE neurons, however, dictated by its lineage program, which constrains its devel- the gcy-5 guanylyl cyclase gene is always expressed in the opmental potential. right ASE (ASER) neuron, whereas the gcy-6 and gcy-7 In summary, genes necessary and sufficient to trigger the genes are expressed in ASEL (Yu et al., 1997). The str-2 complete cell-type-specific differentiation program have ‘ON’ AWC neuron mediates olfactory behaviors distinct been identified for several neuronal subtypes (summarized from the str-2 ‘OFF’ AWC neuron (Wes and Bargmann, in Fig. 3). These genes may act in regulatory hierarchies to 2001). Similarly, the ASEL and the ASER neurons are direct the successive layering of several intermediate states. required for responses to different water-soluble attractants The final expressed identities of each neuron type, and thus (Pierce-Shimomura et al., 2001). The mechanisms by which diversity of function, is ultimately determined by the com- L/R asymmetry is generated in C. elegans have been binatorial expression of transcription factors that act to discussed in a recent review (Hobert et al., 2002), and will specify a distinct cell identity from these intermediate cell be described only briefly here. fates. The challenge then becomes to define the mechanisms In the case of AWC, a str-2 ‘OFF’ fate appears to be the that direct the expression of these regulatory genes to initial fate of both AWC neurons. An unknown signal defined sublineages and to identify the molecules conferring between the two AWC neurons triggers calcium-mediated the lineage-driven cellular context. A lineage-based mech- signaling and str-2 expression in either AWCL or AWCR anism of cell determination anticipates that (i) cell fate will during late embryonic development, although the exact time be tightly coupled to the lineage division pattern; (ii) period has not been determined (Troemel et al., 1999). This expression of genes in a given cell and lineage will be is reminiscent of the phenomenon of lateral specification subject to extremely complex regulatory mechanisms; and where cell–cell signaling via the Notch ligand and the Delta that (iii) cell fate will be defined by the combinatorial action receptor allows one cell to stochastically adopt a different of multiple transcription factors. This is the case in the fate from a field of initially equivalent cells (Artavanis- development of multiple neuron types in C. elegans (Altun- Tsakonas et al., 1999; Greenwald, 1998). Whether genes Gultekin et al., 2001; Baumeister et al., 1996; Chalfie and other than str-2 are also expressed asymmetrically in the Au, 1989; Desai et al., 1988; Finney and Ruvkun, 1990; AWC neurons is unknown. Other signal transduction genes Finney et al., 1988). It is expected that the regulation of such as the guanylyl cyclase gene odr-1 and the G protein amphid neuron identity will be similarly complex. subunit odr-3 are expressed in both the AWC neurons and are taken to represent markers for a general AWC fate (L’Etoile and Bargmann, 2000; Roayaie et al., 1998). Increasing diversity via L/R asymmetry In the case of the ASE neurons, transcriptional complexes that include CEH-36, the Groucho-like transcription- C. elegans takes a unique approach towards maximizing al repressor UNC-37, the COG-1 homeodomain protein, and functional diversity in its chemosensory system. Although, the putative transcriptional cofactor LIN-49 act to diversify 312 T. Melkman, P. Sengupta / Developmental Biology 265 (2004) 302–319 the identities of the ASEL from the ASER neurons. The alone may be regulated by these mechanisms. Since the differential activities of these complexes in the left and right behavior elicited by a given odorant is dictated by the CN neurons is mediated in part by the differential expression type in which the corresponding receptor is expressed levels of COG-1 (Chang et al., in press). Similar to the (Troemel et al., 1997), and since each C. elegans CN AWC neurons, several other signaling genes are expressed expresses multiple receptor genes, modulation of spatial symmetrically in both ASE neurons, and they share overall and levels of expression of individual receptor genes may symmetry in morphology and connectivity (Coburn and represent a simple mechanism by which C. elegans can Bargmann, 1996; Komatsu et al., 1996; Li et al., 1999; rapidly alter specific sensory behaviors in response to Perkins et al., 1986; White et al., 1986). Thus, layered upon environmental or developmental cues (Albert and Riddle, the developmental mechanisms that specify an ASE or 1983; Colbert and Bargmann, 1997; L’Etoile and Barg- AWC fate are mechanisms that act to allow the further mann, 2000; Riddle and Albert, 1997). This represents a functional diversification of the left and right members. The novel method by which functional complexity among CNs signals that regulate asymmetric str-2 expression and the may be regulated. induction of asymmetry in the ASE lineage remain to be identified. Whether L/R asymmetry is a general feature of all CN pairs in C. elegans awaits the identification of Developing functional diversity in the chemosensory additional asymmetrically expressed sensory behaviors or systems of worms, flies, and mammals: a brief markers. comparison

Similarities Regulating chemosensory receptor gene expression The overall developmental mechanisms by which func- The C. elegans genome is predicted to encode over 600 tional diversity is generated in the chemosensory systems of functional chemosensory receptor genes that can be sub- C. elegans, Drosophila, and vertebrates are remarkably divided into at least six families (Robertson, 1998, 2000; similar. In all organisms, regional specification mechanisms Troemel et al., 1995). Phylogenetic analyses have shown dictate the types of chemosensory organs and neurons that these families have arisen as a result of extensive gene generated at particular locations albeit via distinct mecha- duplication and diversification (Robertson, 1998, 2000). nisms and operating at different developmental levels. In the Gene expression experiments using promoter fusions to olfactory epithelium of mammals, four distinct zones are the gfp reporter gene showed that each chemosensory patterned via intrinsic mechanisms (Cau et al., 1997; neuron expresses multiple receptor genes from multiple LaMantia et al., 2000; Norlin et al., 2001; Sullivan et al., families (Troemel, 1999a; Troemel et al., 1995). A priori, 1995; Whitesides et al., 1998). In each zone, olfactory developmental mechanisms that regulate the expression of sensory neurons are constrained to express a subset of cell-specific signaling and structural genes could also coor- olfactory receptors and other signaling genes, as well as dinately select the subset of chemosensory receptor genes to molecules that may regulate their axonal projection patterns be expressed in a given cell type. Consistent with this, (Alenius and Bohm, 1997; Norlin and Berghard, 2001; promoter sequences are shared between a chemosensory Ressler et al., 1993, 1994; Strotmann et al., 1992; Vassar receptor gene and signal transduction genes expressed in a et al., 1993, 1994; Yoshihara et al., 1997).InDrosophila, single neuron type (M. Colosimo and P. Sengupta, unpub- patterning mechanisms specify the generation of specific lished observations), and mutations that affect cell-specific types of sensilla and the types and numbers of component identities also affect the expression of chemosensory recep- olfactory neurons in restricted spatial domains of the third tor genes (Baran et al., 1999; Sagasti et al., 1999; Sarafi- antennal segment (de Bruyne et al., 2001; Gupta et al., Reinach et al., 2001; Sengupta et al., 1996). 1998; Jhaveri et al., 2000; Stocker, 1994). These patterning However, a posteriori, receptor genes appear to be mechanisms act at the level of sensory organ precursors in subject to additional modes of regulation. For instance, the Drosophila and blastomeres in C. elegans, and may also act expression of a subset of receptors in the ASI neurons is on chemosensory neuron progenitors in mammals (Fisher regulated by concentrations of a constitutively produced and Caudy, 1998; Gomez-Skarmeta et al., 1996; Gupta et pheromone, while the expression of the srd-1 and odr-10 al., 1998; Jan and Jan, 1994; Jhaveri et al., 2000; Schnabel, OR genes is regulated by neuronal activity (Nolan et al., 1996; Simpson, 1996; Skeath et al., 1992). Chemosensory 2002; Peckol et al., 2001; Tobin et al., 2002).TGF-h neuron precursors in all animals generate progeny via a signaling and a Ser/Thr kinase have also been implicated series of cell divisions in a relatively stereotyped manner in the regulation of subsets of chemosensory receptors (Calof and Chikaraishi, 1989; Ray et al., 1993; Sulston et (Lanjuin and Sengupta, 2002; Nolan et al., 2002). Invari- al., 1983). At each cell division, the combinatorial expres- ably, the overall known functions of the neurons are sion of transcription factors defines intermediate stages with unaffected and the expression of additional cell-specific changing developmental potentials. Proneural genes act to signaling genes is unaltered, indicating that receptor genes confer a neuronal competence and also neuronal identity, T. Melkman, P. Sengupta / Developmental Biology 265 (2004) 302–319 313 whereas neuronal selector genes act to define sensory organ neuronal precursors to determine their fates in both Dro- identity (Cau et al., 1997, 2002; Dambly-Chaudiere et al., sophila and vertebrates, but not yet in C. elegans (Cau et al., 1992; Goulding et al., 2000; Guillemot et al., 1993; Gupta et 1997, 2002; Goulding et al., 2000; Guillemot et al., 1993; al., 1998; Jan and Jan, 1994; Reddy et al., 1997). Finally, Gupta and Rodrigues, 1997; Jan and Jan, 1994; Jhaveri et al., different transcription factor subsets confer chemosensory 2000; Reddy et al., 1997). However, lateral specification neuronal subtype identity to generate complexity in the mechanisms involving Notch/Delta signaling have been chemosensory system. shown to play important roles in the neurogenesis of chemosensory organs of Drosophila and likely in the vertebrate Differences olfactory epithelium, as well as in the specification of the founder cells giving rise to the CNs of C. elegans (Bower- Despite these gross developmental similarities, there are man, 1995; Cau et al., 2000, 2002; Jan and Jan, 1994; Reddy clearly also both major and minor differences in the mech- et al., 1997). The process of asymmetric cell division has anisms by which chemosensory neurons are generated and been well described in Drosophila but not extensively in C. acquire their diverse functions. The most obvious difference elegans CN lineages. Cell–cell interactions between daugh- between the CNs of C. elegans and their Drosophila and ter cells mediated via Notch/Delta signaling and the asym- vertebrate counterparts lies in the fact that each olfactory metric segregation of molecules such as Numb and Prospero neuron in insects and vertebrates expresses a single or a few act to make a daughter cell different from its sibling in receptor gene(s), as opposed to the multiple receptor genes Drosophila (Jan and Jan, 1994, 1998; Lu et al., 2000). expressed per CN in C. elegans (Buck and Axel, 1991; Homologs of Numb and Prospero have been identified but Chess et al., 1994; Clyne et al., 1999b; Dobritsa et al., 2003; not implicated in asymmetric cell division of C. elegans CN Malnic et al., 1999; Troemel, 1999a; Troemel et al., 1995; lineages (Ruvkun and Hobert, 1998). Homologs of these Vosshall et al., 1999, 2000). Not only does the choice of molecules have also been shown to be required for asym- receptor gene expressed determine the chemical sensitivity metric cell divisions during neurogenesis in the vertebrate of the ORN, the OR also plays a role in refining the cortex (Petersen et al., 2002; Shen et al., 2002; Zhong et al., projection patterns of the OSN in vertebrates (Mombaerts 1996, 1997, 2000); whether they also act during neuro- et al., 1996; Ressler et al., 1994; Vassar et al., 1994; Wang et genesis in the olfactory epithelium is unknown. Olf-1/EBF al., 1998). Although regional patterning mechanisms may and the bHLH domain protein NeuroD have been proposed restrict the expression of subsets of ORs and other genes to to act as a general OSN differentiation factors in vertebrates, particular zones and areas of the chemosensory organs, an although the functions of O/E do not appear to be conserved additional layer of regulatory mechanisms must be postu- in either Drosophila or C. elegans (Cau et al., 2002; Dubois lated that allows each CN in these areas to express a single and Vincent, 2001; Wang et al., 1997). receptor gene from the expression-competent subset. Al- Do conserved sets of transcription factors act to confer though this choice within an area has been suggested to be distinct neuronal subtype identities? Molecules involved in stochastic, expression of a receptor gene within a zone is this process have not yet been well characterized in either punctate, indicating that some mechanism prevents the same Drosophila or vertebrates, again making this issue difficult gene from being expressed in neighboring neurons (Baier et to discuss. A LIM homeobox gene Lhx2 and a paired al., 1994; Ressler et al., 1993; Strotmann et al., 1996; homeobox gene Phd1 have been suggested to play roles Tirindelli et al., 1998; Vassar et al., 1993). In addition, in defining subtype identities in the vertebrate ORNs, olfactory and vomeronasal receptor genes are expressed suggesting that members of conserved families of transcrip- monoallelically in vertebrates (Chess et al., 1994; Rodriguez tion factors may define subtype identities in both vertebrates et al., 1999; Serizawa et al., 2000). Since neurogenesis in and C. elegans (Saito et al., 1996; Xu et al., 1993). Recently, the olfactory epithelium continues in the adult in vertebrates sequences required for both correct monoallelic and zone- (Graziadei and Monti Graziadei, 1978), these or similar specific expression of two olfactory receptor genes have pathways must operate at multiple developmental stages to been defined in the rodent (Vassalli et al., 2002). Analysis of maintain olfactory neuron diversity. the regulatory sequences has revealed the presence of both O/E binding sites, as well as homeodomain binding sites. It A comparison of the underlying molecular mechanisms is likely that zonal positional information is translated into the expression of distinct programs of transcription factor Are the molecular mechanisms of chemosensory neuron expression in the OSN lineages in each zone. These con- development in C. elegans, Drosophila, and vertebrates served factors may then regulate the expression of both OR conserved? Unlike C. elegans, transcription factors involved and other genes in each area, although the mechanisms by in initial neurogenesis and determination of chemosensory which a single allele of an OR is chosen to be expressed are neuron identity have been well described in both Drosophila likely to be novel. In Drosophila, the ACJ6 POU-domain and vertebrates. Members of the Achaete-scute, Atonal, and transcription factor has been shown to regulate the expres- Neurogenin subfamilies of bHLH domain transcription fac- sion of subsets of OR genes in the maxillary palp and the tors have been shown to act in chemosensory organ and third antennal segment (Clyne et al., 1999a, 1999b). Similar 314 T. Melkman, P. 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