DEVELOPMENTAL BIOLOGY 56, 110-156 (1977)

Post-embryonic Cell Lineages of the ,

J. E. SULSTON AND H. R. HORVITZ Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, England

Received August 23,1976; accepted November 4, 1976

The number of nongonadal nuclei in the free-living soil nematode Caenorhabditis elegans increases from about 550 in the newly hatched larva to about 810 in the mature and to about 970 in the mature male. The pattern of cell divisions which leads to this increase is essentially invariant among individuals; rigidly determined cell lineages generate a fixed number of progeny cells of strictly specified fates. These lineages range in length from one to eight sequential divisions and lead to significant developmental changes in the neuronal, muscular, hypodermal, and digestive systems. Frequently, several blast cells follow the same asymmetric program of divisions; lineally equivalent progeny of such cells generally differen- tiate into functionally equivalent cells. We have determined these cell lineages by direct observation of the divisions, migrations, and deaths of individual cells in living . Many of the cell lineages are involved in sexual maturation. At hatching, the hermaphrodite and male are almost identical morphologically; by the adult stage, gross anatomical differences are obvious. Some of these sexual differences arise from blast cells whose division patterns are initially identical in the male and in the hermaphrodite but later diverge. In the hermaphro- dite, these cells produce structures used in egg-laying and mating, whereas, in the male, they produce morphologically different structures which function before and during copulation. In addition, development of the male involves a number of lineages derived from cells which do not divide in the hermaphrodite. Similar postembryonic developmental events occur in other nematode species.

INTRODUCTION after hatching. As in embryogenesis, the The development of a multicellular or- pattern of these divisions is rigidly deter- ganism from a unicellular egg involves a mined; essentially invariant postembry- complex pattern of repeated cell divisions. onic cell lineages generate fixed numbers Classical observations of nematode embry- of neurons, glial cells, muscles, and hypo- ogenesis (reviewed by Chitwood and Chit- dermal cells of rigidly specified fates. wood, 1974) revealed that in these orga- These lineages reveal the ancestral rela- nisms early development follows a rigidly tionships among specific cells of known fixed program, i.e., an invariant pattern of structure and function; they thus comple- cell divisions produces specific progeny ment the classical embryology, which de- cells which, in turn, give rise to particular fined the ancestral relationships among parts of the organism. These and similar different organs. We have determined the studies led to the concept of “cell lineages,” postembryonic cell lineages by direct ob- in which the ancestry of different organs servation of living nematodes. can be traced back to specific progenitor C. elegans is an excellent organism for cells and, ultimately, to the egg (e.g., Wil- the study of cell lineages. It is small, easily son, 1925). cultured, and readily amenable to genetic In this paper, we extend these observa- manipulations (Brenner, 1973, 1974). Like tions from the embryonic to the postem- other nematodes (e.g., Chitwood and Chit- bryonic period. Many cell divisions occur wood, 1974), C. elegans is anatomically in the nematode Caenorhabditis elegans simple (Fig. 1). Its tubular body, consist- 110 Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved. ISSN 0012-1606 SULSTON AND HORVITZ Cell Lineages of a Nematode 111 112 DEVELOPMENTAL BIOLOGY VOLUME 56, 1977

ing of a hypodermal wall and an underly- ing larval development, we begin with a ing musculature, encloses its digestive and detailed account of the cellular anatomy of reproductive systems. Although it has the young nematode. This anatomy has most major differentiated tissue types been determined by a combination of No- (nerve, muscle, hypodermis, intestine, marski and electron microscopy. and gonad), C. elegans consists of rela- Readers who do not wish to become sub- tively few cells; as we show below, the merged in the detailed descriptions under adult contains fewer than 1000 nongonadal Materials and Methods and Results will nuclei. find a summary of our observations and The life cycle of C. elegans is rapid; in conclusions under Discussion. 3.5 days (at 20°C) it develops from a fertil- MATERIALS AND METHODS ized egg through four larval stages to a mature adult. Superficially, the newly (A) Nematode Strains hatched larva appears to be quite similar Caenorhabditis elegans var. Bristol to the adult. The most obvious develop- (strain N2) was obtained from Sydney mental change is in the size and complex- Brenner and grown on Escherichia coli ity of the gonad, which contains 4 nuclei in OP50 on petri dishes at 2O”C, as described the young larva and increases to about previously (Brenner, 1974). Hermaphro- 2500 in the mature adult (Hirsh et al., dite stocks were propagated by self-ferti- 1976). Gross morphological differences are lization; male stocks were propagated by also apparent in the nongonadal sexual crossing with . structures of the hermaphrodite and male; Aphelencoides blastophthorus, Longido- these structures are not present in young rus macrosoma, Panagrellus redivivus larvae (Figs. 1 and 2). (Panagrellus silusiae), and Turbatrix Recently, one of us (Sulston, 1976) de- aceti were obtained from David J. Hooper, scribed a technique with which it is possi- Rothamsted Experimental Station, Har- ble to determine cell lineages by observing penden, Hertfordshire, England. Ascaris living nematodes. When appropriately lumbricoides var. suis was obtained from mounted on a thin block of agar on a mi- the local slaughterhouse; eggs were croscope slide, a nematode proceeds squeezed out of adult uteri and develop- through its normal life cycle. Observation ment was initiated by incubation in 0.1 M of such under Nomarski differen- sulfuric acid at 25°C (Rogers, 1960); at ap- tial interference contrast optics in the propriate intervals, samples were washed light microscope allows one to directly fol- with tissue culture medium (Shields low the migrations, divisions, and deaths et al., 1975) and larvae were released from of individual cells. the eggs by gently rolling the tip of a Pas- Using this technique, we have extended teur pipet over them. the earlier study (Sulston, 1976) of devel- (B) Techniques opment in the ventral nervous system of C. elegans. In this paper, we report the (1) Study of Living Specimens complete postembryonic nongonadal cell (a) Mounting. An agar slab about 0.5 lineages of C. elegans. These lineages pro- mm thick was prepared by flattening a duce the accessory sexual structures of the drop of 5% agar on a microscope slide with hermaphrodite and male and lead to other a second, siliconized slide placed across it; significant developmental changes in the the siliconized slide was supported by neuronal, muscular, hypodermal, and “spacer” slides raised by one or two thick- digestive systems. nessesof adhesive tape. After the agar had To provide a framework for describing hardened, the siliconized slide was re- the anatomical changes which occur dur- moved and a small drop (about 2 ~1) of 10% .%.u%oN AND HORVITZ Cell Lineages of a Nematode 113 114 DEVELOPMENTAL BIOLOG Y VOLUME 56, 1977 polyvinylpyrrolidone (molecular weight Nuclei and large nucleoli were clearly re- 44,000; BDH Chemicals, Ltd., Poole, Eng- solved by Nomarski optics, which permits land) in S medium (Sulston and Brenner, visualization of changes in refractive in- 1974) was placed on the agar, care being dex and has a very shallow depth of field taken not to break its surface. A selected (e.g., Fig. 2). Cell boundaries, however, nematode was transferred to this drop, us- were not always visible. The nuclei of dif- ing either a sharpened wooden stick or a ferent types of cells were generally dis- human eyelash attached with Plasticine to tinguishable and are described under Re- a stick. An successfully transferred sults. would thrash wildly. The center of a 12 x This technique is nondestructive and al- 12-mm coverslip was very thinly coated lows the nematode to live under reasona- with E. coli OP50 scraped from a petri dish bly natural conditions. While mounted for with a wire loop. The coverslip was low- observation, the nematode moved freely ered gently onto the agar, care again being between the coverslip and the surface of taken not to break the agar surface. The the agar block. Attracted by the bacterial volume of liquid was such that the pres- lawn, it generally stayed near the middle sure of the coverslip prevented the worm of the coverslip. If the nematode strayed from thrashing without excessively in- out from under the coverslip, it could be hibiting its movement. All of these opera- transferred to a petri dish and remounted. tions were performed at a room tempera- C. elegans can only flex in a dorso-ven- ture of about 20°C. tral direction. Hence, because it was re- Excess agar protruding beyond the cov- stricted to the plane between the agar and erslip was removed with a razor blade. The the coverslip, the nematode was observed edges of the remaining agar block were lying on its side. A properly mounted ani- sealed with silicone grease or Vaseline to mal usually remained lying on the same prevent dehydration. Air bubbles which side. However, for a period just before formed when the coverslip was positioned molting, nematodes actively and repeat- were gradually absorbed by the agar. edly invert their positions with a rapid After a period of quiescence (less than 1 twisting movement. On occasion, such hr), the nematode generally moved into “flips” proved very inconvenient, as they the bacterial lawn and started to feed. made it difficult to continue observing Nematodes mounted for observation in cells which had moved from the side near- the light microscope could be safely re- est the coverslip to the side nearest the moved afterward for other types of study agar slab (because of interference intro- (e.g., electron microscopy). duced by the body of the nematode). (b) Observation. Nematodes were ob- (c) Cell lineages. Lineages were deter- served in either a Zeiss Universal or a mined by continually observing given nu- Zeiss Standard RA microscope equipped clei as they migrated, divided, and died with a Plan 100 objective and Nomarski during the course of postembryonic devel- differential interference contrast optics. opment. Because cell boundaries were of- To reduce heating, illumination was kept ten not visible, these lineages depict the as low as possible; a heat filter was rou- behavior of nuclei and not necessarily the tinely used and, occasionally, a broad- behavior of cells; it is possible that some band green interference filter was also em- nuclear divisions (or movements) are not ployed. Air temperature was maintained accompanied by concomitant cellular divi- between 19 and 22°C. sions (or movements). During periods of Because C. elegans is small and trans- nuclear activity, sketches of those nuclei of parent, internal structures in intact living interest and of appropriate adjacent land- specimens could be readily examined. marks (usually other nuclei) were made as SULSTON AND HORVITZ Cell Lineages of a Nematode 115

frequently as possible. During periods of ing them through to the adult stage, in nuclear quiescence, observations were re- which they have been identified by recon- corded at intervals of 0.5-2 hr. When line- struction from serial section electron mi- ages in a given nematode were to be fol- crographs available in this laboratory and lowed for more than 1 day, the animal was prepared as described by Ward et al. refrigerated overnight at 6-K during a (1975). suitable quiescent period. Nematodes were (3) Camera Lucida Drawings generally chilled during the early period of a given larval stage. (Individuals refriger- Drawings were made using a Zeiss cam- ated just prior to or during lethargus - a 2- era lucida. Specimens were mounted on hr period preceding each of the four larval slabs of 5% agar as described above, except molts during which there is little pharyn- (1) no bacteria were added, and (2) the geal pumping or body movement -often molten agar contained 1-phenoxy-2-pro- did not recover.) When returned to 2o”C, panol (Bird, 1971) as an anesthetic. The most individuals recommenced develop- concentrations of phenoxypropanol used ment after a 0.5 to 2-hr lag. Generally, were 0.2% v:v for Ll’s, 0.3% for L2’s and nematodes were refrigerated for no more L3’s, and 0.5% for L4’s and adults. (Ln than 15 hr. denotes animals of the nth larval stage.) When appropriately mounted, healthy The line drawings of nematodes pre- nematodes developed as rapidly as on petri sented below are based upon camera lu- dishes. Some individuals, particularly cida drawings. They show nuclei, nucleoli after refrigeration, developed more slowly. (when visible), and other appropriate mor- Occasionally, a younger worm was dam- phological landmarks. These drawings de- aged during the mounting process and pict typical individuals, but, as there are barely developed at all; such individuals minor variations from animal to animal, were discarded. To obtain a standard time they cannot always be used to identify nu- scale for the lineage charts presented be- clei in animals in which lineages have not low, observed time intervals were normal- been traced directly. Also, minor distor- ized according to the observed length of the tions in nuclear position sometimes are appropriate intermolt period. evident in anesthetized nematodes. On the lineage charts, the time of a molt is defined as the moment when the head of (4) Feulgen Staining the nematode breaks through the old cuti- A small drop of 10% ovalbumin was cle after lethargus. Lethargus is indicated placed on a glass slide coated with a thin by dotted regions along the time scales. layer of collagen prepared according to The time of a cell division reflects the time Bornstein (1958). A large number of nema- a metaphase plate is visible (see Results, todes were spread evenly over the surface Cell Division). The time of a cell death of the slide with a paper strip; alterna- indicates the time of maximal nuclear re- tively, single worms were transferred with fractility (see Results, Programmed Cell a wooden stick or platinum wire. When Death). necessary to prevent evaporation, the slide (d,l Photography. Photomicrographs was kept cool over ice. The slide was were taken with a Zeiss microflash illumi- placed in Carnoy’s fixative (Pearse, 1968) nator. All photomicrographs are of living for at least 1 hr; overnight fixation was specimens. preferable. After rehydration through 50 and 30% ethanol (10 min each), the slide (2) Cell Assignments was incubated in 1 N HCl at room temper- Nuclei observed with Nomarski optics ature for lo-20 min and then in 1 N HCl at were assigned to specific cell types by trac- 60°C for lo-12 min. It was transferred to 116 DEVELOPMENTAL BIOLOGY VOLUME 56. 1977

Schiffs reagent for 1.5-2 hr. Schiff’s re- produce M.d and M.v. Left-right divisions agent was prepared by bubbling sulfur are indicated by “1” and Y’; e.g., M.d di- dioxide gas through 1% basic fuchsin, es- vides to produce M.dl and M.dr. Anterior- sentially as described by Rafalko (1946). posterior divisions are indicated by an era” After staining, the slide was rinsed twice and a “p”; e.g., M.dl divides to produce in distilled water and dehydrated through M.dla and M.dlp. As indicated in these a graded series of alcohols (30, 50, 70, and examples, a period separates the name of 90%, 10 min each). After two 30-min baths the original blast cell from the labels in absolute ethanol and two 30-min baths which define specific progeny of subse- in xylene, the slide was mounted in Depex quent divisions. mounting medium (Gurrs, London, Eng- Within the lineage trees, “d,” “1,” and land). Stained specimens were examined “a” daughters are represented by left using bright field illumination through a branches, and “v,” “r,” and “p” daughters blue filter. are represented by right branches; these branches are labeled accordingly (except (5) Laser in lineages with most divisions anterior- A coumarin dye laser microbeam system posterior, in which the labels a and p are developed by J. G. White was used to kill omitted from the branches). An oblique specific cells in individual nematodes. division is indicated on the branches by This system produces a focused spot about labels with two (or three) characters; the 1.5 pm in diameter and can destroy a first character from these labels defines given cell with no apparent damage to its the branch assignments. For example, E.r neighbors. It is based upon a system devel- divides along an anterior/ventral-poste- oped by Berns (1972) and will be described rior/dorsal axis; hence, the left branch is in a future publication by Dr. White. labeled “av,” and the right branch is la- beled “pd” (Fig. 22). The daughters of such (C) Nomenclature a division are named by using only the In describing the anatomy and develop- first of the characters which indicate the ment of C. elegans, we have adopted the division axis, i.e., E.ra and E.rp. Thus, the following system to name specific cells (or number of characters which follow a blast nuclei) and their daughters. Upper-case cell name directly implies the number of letters indicate blast cells of unknown em- divisions which generated the progeny bryonic lineage; for example, M is a meso- cell; e.g., E.rap was produced by the third blast present in the newly hatched Ll. In division of E. some cases, a set of blast cells undergoes In the B lineage, the precise fates of similar patterns of cleavage and produces certain progeny cells are not predeter- morphologically similar groups of daugh- mined. For example, B.alaa and B.araa, ters; each member of such a set is denoted generated on the left and right sides re- with a common upper-case letter followed spectively, recentralize. Their relative an- by a specific numeric designation, e.g., six terior-posterior position after recentrali- blast cells on each side of the lateral hy- zation is indeterminate. The anterior cell podermis are Vl-V6. When a blast cell (“Ba”) follows one lineage program, divides, each daughter is named by adding whereas the posterior cell (“BP”) follows to the name of its mother cell a single another. In other words, either B.alaa or lower-case letter representing its position B.araa can become Ba. Thus a greek letter immediately after division relative to its following a blast cell name indicates a cell sister cell. A cell which divides along a derived from that blast cell via one of a dorso-ventral axis has its daughters indi- number of alternative lineage routes. cated by a “d” and a “v”; e.g., M divides to Entirely lower-case names are used as SUIBTON AND HORVITZ Cell Lineages of a Nematode 117 abbreviations for cell types in lineage oocytes. Two reflexed gonadal arms, each charts, camera lucida drawings, and pho- of which contains an ovary, oviduct, sper- tographs. For example, “bm” identifies a matheca, and uterus, terminate at the body muscle. In the lineage charts, this vulva, located midway along the ventral name follows the systematic name based side. The reproductive system of the adult on the lineage history of the cell, e.g., male consists of a single testis which emp- M.dlpp; bm. The abbreviations used are: ties into the vas deferens; the vas deferens bm, body muscle; cc, coelomocyte; dep, unites posteriorly with the rectum, form- anal depressor muscle; exe, excretory cell; ing a cloaca. Accessory sexual structures exe gl, excretory gland; g, neuron or glial are located in the male tail. cell; hsn, hermaphrodite-specific neuron; i, intestinal nucleus; im, intestinal muscle; (2) Newly Hatched Hermaphrodite plg, posterior lateral ganglion; rect gl, rec- (a) Ectoderm. The ectoderm, which tal gland; se, seam cell; set, tail seam cell; comprises the majority of cells in the Ll, sph, anal sphincter muscle; sy, syncytial can be broadly divided into the nervous nucleus; uml, type 1 uterine muscle; um2, system with its supporting cells and the type 2 uterine muscle; vcn, ventral cord hypodermis. Embedded in the hypodermis neuron; vh, ventral hypodermal nucleus; are various blast cells and a number of vml, type 1 vulva1 muscle; vm2, type 2 cells of specialized functions. vulva1 muscle. The hypodermis essentially consists of The nomenclature used in this paper is four longitudinal ridges (dorsal, ventral, somewhat different from that used previ- lateral right, and lateral left) joined cir- ously (Sulston, 1976) to describe the cell cumferentially by thin sheets of cytoplasm lineages of the ventral nervous system of which separate the muscle cells from the C. elegans. We believe that the revised cuticle. Hypodermal nuclei, generally nomenclature is easier to use in referring large and flat with large nucleoli (Fig. 3), to the relatively large number of cells de- are located in these four ridges. The dorsal scribed in this paper. ridge, which is filled with refractile gran- ules in the late embryo and the young Ll, RESULTS normally contains nuclei only in the head. IA) Anatomy The ventral ridge contains hypodermal nuclei in the head and tail but not along (1) Gross Morphology the body in the young Ll; as described Like other nematodes (e.g., Chitwood below (see Cell Lineages, Ectoderm), a and Chitwood, 19741, C. elegans has an number of hypodermal nuclei are produced elongated cylindrical body with tapered in the ventral ridge during later develop- ends (Figs. 1 and 2). An external cuticle ment. The lateral hypodermal ridges are covers the hypodermal body wall. Beneath almost bilaterally symmetrical and con- the hypodermis are four longitudinal rows tain nuclei throughout their lengths. of body muscles located subventrally and Twenty-four of these nuclei are arranged subdorsally. The mouth leads into the bi- in six similar ventro-lateral pairs located lobed muscular pharynx, which pumps on each side along the body; each pair food into the tubular intestine to the rec- includes one hypodermal precursor (V) tum and anus. The nervous system con- and one precursor (P) of the ventral nerv- sists of a circumpharyngeal nerve ring, ous system (Figs. 4 and 9). Anterior to the dorsal and ventral nerve cords, and a vari- first of these pairs on each side there are ety of sensory receptors and ganglia. two hypodermal precursors (Hl and H2). The reproductive system of the adult Each side of the tail has a single precursor hermaphrodite produces both sperm and (T). There are 11 other hypodermal nuclei 118 DEVELOPMENTAL BIOLOGY VOLUME 56, 1977

FIG. 3. Left lateral hypodermis, posterior region of young Ll hermaphrodite, lateral view; Nomarski optics. hsn, hermaphrodite-specific neuron; sy, syncytial hypodermal nuclei. Bar = 20 pm. on each side of the nematode. These nuclei the nucleus of the duct cell. Two gland are not bilaterally symmetrical in loca- cells (exe gl) which feed into the excretory tion; the set on the left appears to be dis- duct are located just posterior to the poste- placed posteriorly relative to that on the rior-most region of the ventral ganglion. right. Except for the precursor cells, al- Although normally quiescent, the duct most all of the lateral hypodermal nuclei pulsates in dauer larvae. [The lie in a single syncytium (White, 1974). is a developmental variant specialized for Some syncytial hypodermal nuclei do not dispersal and/or survival in harsh condi- occupy rigidly defined positions and seem tions (Cassada and Russell, 197511. The to be moved passively during develop- rate of excretory duct pulsation in dauer ment. larvae of other nematodes depends upon Three gland cells (rect gl) (Chitwood the osmolarity of the medium (e.g., Wein- and Chitwood, 1974) ring the junction be- stein, 1953, indicating that one function of tween the intestine and the rectum (Fig. the “excretory” system is osmoregulation. 4). Two other cells (B and F) are located In the Ll, the nuclei of the nervous sys- dorsal to the rectum; these cells divide tem are generally distinct morphologically only in the male. Another pair of cells is from those of the hypodermis. Neurons located laterally; the cell on the left (K) and glial cell nuclei are small and granu- divides in both hermaphrodites and males. lar with no visible nucleoli (Figs. 2, 7, and Two additional cells (C and E) are located 8); in older larvae and adults, these nuclei ventral to the rectum and posterior to the often acquire visible nucleoli. The adult pre-anal ganglion. C lies more ventrally anterior sensory nervous system has been and further right than E. Both C and E described in detail (Ward et al., 1975; divide only in the male. Ware et al., 1975); we have observed no The cells of the so-called excretory sys- postembryonic cell divisions in this sys- tem (Bird, 1971) lie in close association tem. Its cell bodies lie in groups anterior with the hypodermis. The nucleus of the and posterior to the ring of nerve fibers “II”-shaped excretory cell (exe) lies just left which runs circumferentially around the of the midline, ventral to the rear bulb of isthmus of the pharynx. The posterior the pharynx. Immediately anterior to it is groups, known as the lateral ganglia, also SULSTON AND HORVITZ Cell Lineages of a Nematode 119 120 DEVELOPMENTAL BIOLOGY VOLUME 56, 1977 include some interneuron and motoneuron neuroblasts: one on the left side (Ql) and cell bodies. In the young Ll, the amphid one on the right side (Q2). sheath cell bodies (Ward et al., 1975) lie (b) Mesoderm. At hatching, the Ll con- behind the pharynx and are embedded in tains 81 body muscle cells (bm) which con- the hypodermis; during larval develop- trol its locomotion. The nuclei of these ment, they gradually move anteriorly and cells are ovoid with a granular nucleo- medially (Figs. 4 and 9). The ventral gan- plasm surrounding a spherical nucleolus glion contains two blast cells (Gl and G2) (Fig. 6). During the L2 stage, the nucleo- and lies ventral and posterior to the nerve plasm becomes smooth and remains so ring, extending to the excretory glands. throughout the rest of development. The The sublateral ganglia lie dorsolateral to somatic muscle nuclei are distributed al- the excretory glands. The retrovesicular most symmetrically in four longitudinal ganglion, with 12 mature nuclei and 1 neu- rows located subventrally and subdorsally roblast (PO.a), lies ventral and posterior to (Figs. 4 and 23). Each dorsal quadrant con- the excretory cell and merges with the tains 21 somatic muscle cells, the ventral ventral nerve cord. The ventral nerve cord right quadrant contains 20, and the ven- is the major longitudinal fiber bundle in tral left quadrant contains 19. The ventral the nematode and runs along the right asymmetry appears to result from a gap on side of the ventral hypodermal ridge; the the left side somewhat posterior to the dorsal nerve cord contains fewer fibers and gonad primordium. runs along the left side of the dorsal hy- Four specialized muscle cells are present podermal ridge. In the Ll, 15 motoneuron in the body of the newly hatched larva. cell bodies are present in the ventral cord. Two are intestinal muscles (im). These At the posterior end of the ventral cord is muscles run subventrally along the poste- the pre-anal ganglion, which contains six rior-most region of the intestine and at- nuclei. Two lumbar ganglia are present in tach to it and to the thin layer of hypoder- the post-anal region; the ganglion on the mis just ventral to the lateral cords; their left contains eight nuclei, and that on the filaments are oriented longitudinally. The right contains nine nuclei. A small dorso- intestinal muscle nuclei are located rectal ganglion with two nuclei is also slightly dorso-laterally to the ventral body present in the tail. muscle rows and are morphologically simi- On each lateral side of the nematode, lar to but somewhat smaller than body four isolated cell bodies with neuronal-like muscle nuclei. Slightly posterior to these nuclei are embedded in the hypodermis cells is a single saddle-shaped muscle immediately beneath the cuticle (Figs. 4 (sph), which almost surrounds the poste- and 9). The small subventral neuron (hsn) rior end of the intestine and probably func- near the gonad primordium in the her- tions as a sphincter muscle to control defe- maphrodite is absent in the male (Fig. 5). cation. The nucleus of this sphincter mus- Two other lateral neuronal-like cells are cle is located somewhat left of the central located (a) just anterior to Vl and (b) sub- midline. An H-shaped “anal depressor” dorsally, near V3. A fourth nucleus with muscle cell (dep), with its nucleus located similar morphology is present between V3 centrally in the crossbar of the H, is lo- and V4, anterior to the hermaphrodite- cated posterior to the anus and opens the specific neuron; although this nucelus be- anus during defecation. comes more hypodermal-like during devel- A single mesodermal blast cell (M) is opment, the cell nonetheless appears to be present on the right side of the young Ll a neuron or supporting cell in the adult. somewhat posterior to the gonad primor- Also lying subdorsally in the lateral hy- dium (Fig. 6). This cell has a large, rela- podermis between V4 and V5 is a pair of tively flat nucleus and a large nucleolus. SUISTON AND HORVITZ Cell Lineages of a Nematode 121

cephalic I CO~p3JliO” cephalic HZ.an

FIG. 5. Anatomical differences between the young hermaphrodite and the young male. The top two figures show lateral views of the posterior halves of a young Ll male and hermaphrodite. The sex-specific characteristics shown are (a) position of a left coelomocyte (cc), (b) presence of hermaphrodite-specific neurons (hsn), and (c) size of the B and C nuclei. Four male-specific cephalic companion neurons, also present at hatching, are best seen during the L4 stage, as shown in the bottom figure (left lateral view). The position of H2.aa in the lateral ganglion is indicated.

Four coelomocytes (cc) (Chitwood and primordium (Fig. 5). We consider these Chitwood, 1974) are also present in the Ll. coelomocytes to be mesodermal in origin These glandular cells are located in the because, as described below (see Cell Line- pseudocoelom adjacent to the somatic mus- ages, Development), other coelomocytes culature. In the young Ll, their nuclei are are generated by blast cells which produce granulated and do not contain visible nu- muscles during larval development. cleoli (Fig. 6). During larval development, Located centrally, dorsal to the rear the cytoplasm of the coelomocytes acquires bulb of the pharynx, is a single cell with a both granules (of high refractive index) muscle-like nucleus and a nucleolus and vacuoles (of low refractive index), giv- slightly smaller than that of a body mus- ing these cells a very characteristic ap- cle. This cell is often squashed in appear- pearance (Fig. 6). In the Ll hermaphro- ance. Electron micrographs reveal that dite, all four coelomocytes are located sub- this head mesodermal cell forms gap junc- ventrally between the pharynx and the tions (White et al., 1976) with adjacent gonad primordium; the two on the right body muscles. are anterior to the two on the left (Figs. 4 (c) Intestine. The intestine of the Ll is a and 23). In the male, one of the left coelom- hollow tube which normally contains 20 ocytes is located posterior to the gonad large, round nuclei with large nucleoli 122 DEVELOPMENTALBIOLOGY VOLUME 56, 1977

FIG. 6. Mesoderm, left lateral views; Nomarski optics. Bar = 20 Wm. (a) Body muscle (bm) and mesoblast (M) nuclei in young Ll hermaphrodite; go, gonad. (b) Ventral left coelomocyte (cc) in young Ll hermaphro- dite. i, intestinal nucleus; nut, nucleus; vat, vacuole. (c) Dorsal right coelomocyte (cc, M.drpa) in L4 hermaphrodite. Note opacity of the well-fed intestine.

(Fig. 2); its cytoplasm is usually filled with posterior part, slightly right-is caused by refractile granules. Along most of the this rotational twist. length of the intestine, nuclei are located Pale at hatching, the cytoplasm of the dorsal and ventral to the lumen; in the intestine becomes filled with refractile anterior-most region, however, a ring of droplets as the nematode feeds. Lethargus four nuclei surrounds the lumen. This an- (or starvation), causes the intestine to terior-most region has a thinner internal again become relatively pale. Subsequent boundary than the rest of the intestine; feeding restores the refractile droplets, electron micrographs reveal that the mi- which are therefore probably involved in crovilli in this region are approximately energy storage. Because of the opacity of half the length of those found elsewhere. the well-fed intestine, hypodermal nuclei The number of intestinal nuclei is not rig- on the lower side of the nematode are diffr- idly determined, as individuals with 19, cult to observe between molts. 21, and 22 nuclei have been observed. The (d) Gonad. The gonad primordium con- intestine has a left-handed twist of 180” sists of four cells in the young Ll (Fig. 4). about the longitudinal axis of the nema- The two larger cells are adjacent; the two tode in the region of the primordial gonad. smaller cells are slightly anterior and pos- As such a twist can introduce a left- terior to them, respectively. All four nuclei handed superhelical twist, we suspect that have relatively large nucleoli (Fig. 2). The the bilateral asymmetry of the position of gonad primordium lies obliquely, with its the intestine -its anterior part is dis- anterior cells located slightly right and its placed slightly left of the midline, and its posterior ones located slightly left. This SULSTON AND HORVITZ Cell Lineages of a Nematode 123 bilaterally asymmetric positioning is prob- of a blast cell generally has a prominent ably related to the twist in the intestine nucleolus and a smooth nucleoplasm. (see above, Intestine). The gonad primor- About 10 min before division, the nucleo- dium of the male is morphologically iden- plasm becomes coarsely granular, often tical to that of the hermaphrodite. producing a “rosette” around the nucleo- (e) Pharynx. The morphology of the lus. In rapid succession during the next pharynx of the adult hermaphrodite has few minutes, the nucleolus disappears, the recently been described in detail (Albert- nuclear membrane breaks down, and the son and Thomson, 1976). No postembry- metaphase plate forms. During early ana- onic cell divisions or deaths have been seen phase, the plate splits and the two halves in the pharynx. begin to move toward the poles; individual chromosomes are usually not visible. For a (3) Newly Hatched Male short time, nuclear components cannot be At hatching, the Ll male is very similar seen; the daughter nuclei then gradually in structure to the Ll hermaphrodite. We appear. Finally, in some cells, daughter have found three criteria by which the sex nucleoli are formed. of a young nematode can be determined (2) Programmed Cell Death. (Fig. 5): (1) In the male, a left coelomocyte lies posterior to the gonad primordium. (2) In a number of the ectodermal cell line- The male is missing a neuronal-like nu- ages, certain cells undergo a series of mor- cleus embedded in each side of the lateral phological changes which we interpret as hypodermis near the gonad primordium. programmed cell death (Saunders, 1966) (3) Ectodermal nuclei B and C near the (Fig. 8). Blobs appear at the perimeter of anus enlarge soon after hatching in the the nucleus. The nucleoplasm becomes male; these nuclei divide only in the male. less granular and somewhat more refrac- In addition, the male head contains four tile. There is then a marked increase in neuronal-like nuclei which are absent in the refractility of the nucleus, giving it a the hermaphrodite. These nuclei are lo- characteristic flattened appearance. The cated close to the cell bodies of the cephalic internal refractility diminishes slightly, neurons and presumably are the cell bod- leaving a refractile shell at the surface of ies of the four male-specific sensory proc- the nucleus. The nucleus shrinks and it esses in the cephalic sensilla discovered by gradually disappears. Generally, other nu- Ward et al. (1975). Although present in the clei completely obliterate the site, al- newly hatched larva, these “cephalic com- though in some cases a permanent gap panion” cell nuclei are more easily distin- remains. Either way, no subsequent trace guished in the L4, in which they have of the dead cell can be detected either by relatively large distinct nucleoli (Fig. 5), Feulgen staining or by electron micros- possibly reflecting increased activity at COPY. this stage of development, Most cell deaths are of posterior daugh- ters from anterio-posterior divisions. (B) Cell Lineages As described below (see Cell Lineages), the pattern of cell deaths in the’male dif- (1) Cell Division fers from that in the hermaphrodite. Although the size and shape of different blast cells vary, the overall process of cell (3) Development division as observed with Nomarski optics To the best of our knowledge, the results during the postembryonic development of described in this section include all non- C. elegans is quite uniform (Fig. 7). For gonadal cell divisions and deaths that oc- several hours before division, the nucleus cur during the postembryonic develop- FIG. 7. Cell division. Sequential photographs of an Ll hermaphrodite, lateral view; Nomarski optics. vcn, ventral cord neurons. 0 min, interphase; 16 and 21 min, PlO prophase; 24 min, PlO metaphase; 26 min, PlO anaphase; 27 min, PI0 telophase; 29 min, P9 prophase; 33 and 34 min, P9 metaphase. Bar = 20 pm. 124 SULSTON AND HORVITZ Cell Lineages of a Nematode

FIG. 8. Cell death. Sequential photographs of an Ll hermaphrodite, lateral view; Nomarski optics. The arrow points to the dying cell, Pll.aap. Bar = 20 Frn. ment of C. elegans. However, very late main separate from the syncytium; to- developmental events in the male tail gether with one head hypodermal cell (on could have been missed. Because the de- each side) present at hatching, the poste- tailed anatomy of the adult male tail is not rior daughters form a distinct row of cells known, many of its nuclei seen with the embedded in each lateral hypodermal light microscope could not be assigned to ridge. Such cells have been named “seam” specific cell types. cells. Seam cells display a characteristic Cell lineages not specifically described periodic activity. Toward the end of each for the male are identical to those which intermolt period, the cytoplasm of the occur in the hermaphrodite. seam cells fills with refractile blobs (Fig. (a) Ectoderm. V cells, hermaphrodite 11). This activity continues until midway (Figs. 9 and 10): All 12 of the ventrolateral through lethargus. That this activity is hypodermal precursor cells (V) divide seen prior to the L4 molt (when the seam about midway through the Ll stage. The cells do not divide; see below) as well as anterior daughters enter the hypodermal prior to the other three molts (when the syncytium. The posterior daughters re- seam cells do divide) suggests that it is she,.th

FIG. 9. H, V, and T.a; hermaphrodite, lateral views; development of the left lateral hypodermis. Seam cells are indicated by dotted lines. hsn, hermaphrodite-specific neuron; plg, posterior lateral ganglion.

128 DEVELOPMENTAL BIOLOGY VOLUME 56, 1977

wood and Chitwood, 1974) and contain the cell bodies associated with these sensilla. The proposed homology (Chitwood and Chitwood, 1974) between the postdeirids and the deirids in the head is supported by the observation that each of these sensilla has a single neuron containing the neuro- transmitter dopamine (Sulston et al., 1975). We have now identified the dopaminergic cell in each posterior lateral ganglion as being V5.paaa. This assignment was sug- gested by correlating the formaldehyde- induced fluorescence of the dopaminergic cell with the position of the VEi.paaa cell body as visualized by Feulgen staining, using methods described previously (Sul- FIG. 11. Seam cell. Cytoplasmic activity in Hl.aa of late L4 hermaphrodite, lateral view; No- ston et al., 1975); it was confirmed by kill- marski optics. The seam is outlined with a dotted ing selected cells of known lineage with a line. Bar = 20 pm. laser microbeam and showing that the death of V5.paaa or of any of its ancestors (but not of any other cell) leads to loss of related to molting rather than to cell divi- the formaldehyde- induced fluorescence. sion. These experiments failed to reveal any The Vn.p seam cells undergo two rounds regulative capacity of the cells involved in of division at about the time of the Ll molt; the development of the posterior lateral each produces a longitudinal row of four ganglia; no extra divisions occurred to re- small nuclei. With the exception of place those cells destroyed with the laser. Kpap, the posterior daughters of the sec- H cells, hermaphrodite (Figs. 9 and 10): ond round of divisions are themselves The lineages of the hypodermal precursors seam cells that divide at the L2 and L3 of the head (Hl and H2) appear to be re- molts. The posterior daughter of each of lated to those of the V cells. Both generate these divisions is itself a seam cell. All of seam cells as well as hypodermal nuclei in the other hypodermal progeny nuclei be- the syncytium, and both utilize similar come part of the syncytium. In the L4 and stem cell patterns of cell division. How- adult, the seam cells form a continuous ever, there is an inversion of the pattern of lateral band. the divisions of Hl compared to that of the During the L2 stage, the lineage of V5 V cells; the anterior daughter H1.a is the becomes distinct from that of the other V blast cell of the Hl lineage, whereas the cells. On each side of the nematode, posterior daughter Vn.p is the blast cell of V5.paa and V5.pap undergo a series of the V lineage. It should be noted that H1.a divisions to produce four daughter nuclei. and H1.p slowly reverse their anterior- Together with two other nuclei on the left posterior positions; however, this posi- side (Ql.paa and Ql.pap; see below, Q tional reversal is probably not the cause of Cells), these nuclei compose the posterior the inversion of the pattern of divisions, lateral ganglia, which consist of six cells because H1.a has the morphological ap- on the left side and four cells on the right pearance of a blast cell before any move- side. Ultrastructural studies of the pos- ment of its nucleus occurs. Hl.aa has the terior lateral ganglia show that they are appearance of a seam cell; unlike the positioned beneath the “postdeirids” (Chit- “equivalent” Vn.pa cells, however, Hl.aa SULSTON AND HORVITZ Cell Lineages of a Nematode 129 does not divide. The lineage of H2, which its fate is weakly sex specific. is related to those of Hl and the V cells, P cells, hermaphrodite (Figs. 12, 15, 16, produces seam cells, syncytial hypodermal 17, and 18): The P precursor cells are re- nuclei, and one neuron-like cell (HB.aa), sponsible for the postembryonic develop- which is positioned in the mass of cell bod- ment of the ventral nerve cord and its ies posterior to the nerve ring (Figs. 5 and associated ganglia. One of us has described 9). That Hl and H2 together produce only the development of these systems in detail half the number of syncytial hypodermal elsewhere (Sulston, 1976). For complete- nuclei expected from two V cells may re- ness, we summarize these observations be- late to the fact that postembryonic growth low. of the head is much less than that of the About the middle of the Ll stage, the body (Hodgkin, 1974). ventro-lateral P cells appear to be “loos- T cell, hermaphrodite (Figs. 9, 10, and ened” from their hypodermal neighbors. 12): The tail precursor-cell T generates Cytoplasmic extensions of the P cells grow both hypodermal and nerve cells. Its ante- into the ventral cord. The nuclei of Pl and rior daughter T.a produces a lineage ini- P2 migrate into these extensions; the re- tially like that of a V cell; however, this sidual cytoplasm soon follows (Fig. 15). similarity in the pattern and timing of cell After these movements of Pl and P2, simi- divisions is not reflected in the morphology lar migrations of successively more poste- of the progeny. In the neuronal lineage of rior pairs occur roughly in order along the T.p (Figs. 10 and 12), there are two ante- cord; Pll and P12 migrate into the pre- rior-posterior positional inversions. First, anal ganglion. about 1 hr after it is formed, T.pp begins to There is some variability in the ante- move slowly past T.pa. This migration ap- rior-posterior order of a given left-right pears to be an active process; neighboring pair of P cells after they have entered the cells seem to hinder rather than assist the ventral cord, in at least some cases, the movement. Later, T.pppap moves anterior subsequent development of a cell depends to its sister; its nucleus becomes consider- not upon its side of origin, but rather upon ably deformed as it squeezes past other its anterior-posterior position. For this cells to reach its final position. reason, we number the P cells after their The fates of the progeny of the T cells in migration into the cord (Fig. 16). We have the adult have not yet all been established; determined the extent of this variability at least some of these progeny appear to be for the following pairs. Pl generally ar- associated with the phasmids, post-anal rives from the right side (in 14 out of 16 sensilla located on each side of the nema- individuals examined), Pll arrives from tode (Chitwood and Chitwood, 1974). the left (7 out of 7), and the entries of P3 Q cells (Figs. 13 and 14): The left and and P5 are approximately random. right lateral neuroblasts, Ql and Q2, dis- Soon after its migration, each P cell di- play bilaterally symmetrical patterns of vides. All 12 P cells follow identical initial cell division. However, their progeny are programs of division, each producing five not symmetrical in their movements: neurons and one hypodermal cell (Fig. 17). Q2.a, Q2.p, and Q2.ap all migrate ante- In addition, a single neuroblast in the re- riorly, while Ql.ap migrates posteriorly. trovesicular ganglion follows a program The fate of Q2.pp is variable. In herma- identical to that of the anterior daughters phrodites, it becomes less and less distinct of the P cells (Pna); by analogy, we have and usually disappears, although in one named this neuroblast P0.a. individual it was observed to survive into As cell divisions continue, the neuro- the adult. In two males, Q2.pp underwent blasts and their progeny move freely past typical programmed cell death. Perhaps the juvenile cells and the new hypodermal DEVELOPMENTAL BIOLOGY VOLUME 56, 1977

T.apaa K P

\

50 nr

FIG. 12. T, K, Pll, and P12; hermaphrodite, left lateral views; development of the hermaphrodite tail Only left and central nuclei are drawn. 4 hr

/ I Q2.W 92 paa

FIG. 13. Q, left lateral views; development of the lateral neuroblasts. Sometimes, QZ.paa and Q2.pap move directly to their final positions, g without circling around one another. +

- - 132 DEVELOPMENTAL BIOLOGY VOLUME 56, 1977 kg”91 92 GI G2 K In

Ll 10

.;. ?'::r 20 1 L2

FIG. 14. Q, G, K, and I lineages. Ql and Q2 are lateral neuroblasts; Gl and G2 are in the ventral ganglion; K is in the tail; In are intestinal nuclei. Divisions are anterior-posterior unless otherwise indicated. g, neuron or glial cell; i, intestine.

FIG. 15. Ventral cord and muscle development. Ll hermaphrodite, left lateral view, central plane; Nomarski optics. The nucleus of PlO is just entering the cord. The intestine is pinched between M.d and M.v. Bar = 20 km. cells (Pn.p); the extent of this movement is an extra division of the most posterior hy- variable, leading to different patterns of podermal cell (P12.p), followed by the intercalation of new neurons with the ju- death of its posterior daughter (P12.p~). venile and hypodermal cells in different Late during the L3 stage, the six ventral individuals. Gradually, the ventral cord hypodermal nuclei (P3.p, P4.p, P5.p, P6.p, becomes evenly filled with nuclei. Toward P7.p, P&p) in close proximity to the devel- the end of the period of division, the pat- oping gonad (Fig. 18) divide. The daugh- tern of nerve cells is modified by a fixed ters of P5.p, P6.p, and P7.p move closer program of cell deaths in the anterior and together and continue to divide to produce posterior regions of the cord. There is also the 22 cells which form the vulva. The SULSTON AND HORVITZ Cell Lineages of a Nematode 133 _-___,-“___r- I..-.~ ..-- _( ..- II I -

P” aap.g P”.aaap,g -G r%.aaas.e SUMTON AND HORVITZ Cell Lineages of a Nematode

FIG. 18. P3.p-P8.p; hermaphrodite, left lateral views midway along ventral side; development of the vulva. daughters of P3.p, P4.p, and P8.p become length of the cord. The number of cells of ventral hypodermal nuclei. In the adult, each class is constant in different individ- the ventral hypodermal nuclei formed dur- uals, but slight variations in the relative ing the development of the ventral cord lie positions of different classes occur. These in the hypodermal syncytium (White et anatomical observations can be explained al., 1976). by the developmental processes described The fine-structure neuroanatomy of the above according to the following model. ventral cord of the adult C. elegans herma- Each morphological class consists of those phrodite has been elucidated by White et cells with an equivalent lineage history al. (1976). The neurons in the ventral cord (Table 1); the positional variations result can be assigned to morphologically distinct from the differential migrations which oc- classes which recur periodically along the cur during development. The correlation of 136 DEVELOPMENTAL BIOLOGY VOLUME 56, 1977

TABLE 1 stage to produce daughters with neuronal- CORRELATION OF MORPHOLOGICAL CLASS WITH like nuclei. Also in the ventral ganglion, LINEAGE HISTORY OF MOTOR NEURONS IN THE G2 divides during the L2 stage. Like Gl, VENTRAL NERVOUS SYSTEM OF THE ADULT G2.a then divides transversely and pro- HERMAPHRODITE a duces two daughters with neuronal-like (1) (2) (3) nuclei. Morpho- Lineage Confirmed by EM as- logical history K cell (Figs. 12 and 14): The solitary K class (model) signment precursor cell in the tail divides once about VA Pnaaaa P2, P3, P4, P5, P6 VB Pnaaap PO, Pl, P2, P3, P4, P5, P6 3.5 hr before the Ll molt. K.p joins the two vc Pnaap P3, P4, P5, P6 other neurons in the dorso-rectal ganglion. DAS Pn.apa Pl, P2, P3, P4, P5, P6 K.a is a hypodermal cell which completes VD Pn.app PO, Pl, P2, P3, P4, P5, P6 a bilaterally symmetrical pair with a cell DA, DB, DD Juvenile Eight anterior-most juve- located right of the midline and present in nile cells of ventral cord the young Ll. (three anterior-most es- tablished in individuals Other cell deaths: On two occasions, of known lineage). other cell deaths were observed early dur- a Column 1 indicates morphological classes as ing the Ll stage. One was immediately defined by White et al. (1976). Column 2 shows the anterior to the excretory cell and the other predicted lineage history of cells of each morphologi- was in the dorsal region of the lateral gan- cal class according to the model. Column 3 indicates glion. These two cell deaths may normally neurons of each morphological class which, by their be embryonic events which, in some indi- positions and neighbors, are consistent with having the lineage listed; to date, morphological assign- viduals, are delayed until after hatching. ments have been completed only for the anterior Male: Below, we first describe those ec- ventral cord (containing the progeny of PO.a-P6). todermal lineages which occur in the her- Assignments for neurons in boldface in Column 3 maphrodite and are modified in the male have been established in individuals of known li- (V, T, P); we then describe male-specific neage. Three neurons located in the retrovesicular ganglion are not allocated as expected from this lineages derived from cells present in the model: PO.aaaa and Pl.aaaa are nonmotor neurons, newly hatched Ll which do not divide in and PO.apa is of morphological class VA. Three the hermaphrodite (B, F, C, E). progeny (PO.aap, Pl.aap, PS.aap) undergo pro- V and T cells, male (Figs. 10 and 19): grammed cell death and thus are not found in the The V and T lineages of the male are iden- adult. All electron microscopy was done by White et tical to those of the hermaphrodite until L2 al. (1976). lethargus; thus, the male carries all tail cell type with lineage history has been and posterior lateral cell bodies found in directly confirmed by using serial-section the hermaphrodite. Further divisions in electron micrographs to reconstruct the re- the V5, V6, and T lineages then generate trovesicular ganglion and part of the ven- nine similar sets of neuronal-like nuclei on tral cord of individuals of known lineage each side of the nematode as well as a (Table 1). However, three daughters lo- number of larger hypodermal-like nuclei. cated in the retrovesicular ganglion are Numerically, these nine sets correspond to not allocated as expected from their li- the nine “rays” found on each side of the neage history. As discussed below (see male tail (Figs. 1 and 20). These rays lie in Discussion, Mechanisms of Determina- a noncellular cuticular webbing, which tion), they may reflect end effects which forms a leaf-shaped fan. The entire struc- seem to affect a number of aspects of the ture, consisting of fan and rays, probably postembryonic development of C. elegans. functions as a mechanosensory organ dur- G cells (Figs. 4 and 14): Gl, located near ing copulation. the anterior end of the ventral ganglion, Preliminary experiments with the laser divides transversely late during the Ll microbeam suggest how the V and T cell SULSTON AND HORVITZ Cell Lineages of a Nematode 137 V6.PPPP T..PPS T

FIG. 19. V5, V6, and T, male, left lateral views of the left side; development of the rays of the male tail. There is extensive rearrangement of the cells later, during L4 lethargus. 138 DEVELOPMENTAL BIOLOGY VOLUME 56, 1977

3

FIG. 20. Male tail, ventral view. One spicule is extended and one is retracted. As indicated, the sensory tips of the rays lie either on the ventral (inside) surface (indicated by a solid circle) or on the dorsal (outside) surface (dotted circle).

lineages lead to the development of the dopaminergic cell. These results suggest rays. Destruction of the precursor (e.g., that, in each set, aaa is the neuron and VG.ppppp) of one of the nine sets of prog- app forms the structural element. Destruc- eny cells with the laser results in the loss tion of any one of the hypodermal nuclei of a particular ray. In this way, the ances- “p” (e.g., VG.pppppp) associated with these try of the nine rays has been assigned sets produced no lesion detectable in the (Fig. 10). Destruction of the precursor of light microscope. As in the development of ray 5, 7, or 9 also resulted in the loss of one the posterior lateral ganglia (see above, V of the three dopaminergic cells on that side cells, hermaphrodite), laser experiments of the male tail (Sulston et al., 1975). That failed to reveal any potential for regula- only 6 of 18 morphologically similar rays tion. have dopaminergic neurons suggests why Although 18 similar lineages generate mutant males lacking dopamine are still 18 morphologically similar rays, lineally capable of mating (Sulston et al., 1975): at equivalent daughters do not all differen- least some of the 12 other rays may act as tiate identically: Only six of the aaa prog- alternative sensory elements. eny are dopaminergic. It will be interest- The precise lineages of the six dopami- ing to determine if the other aaa progeny nergic cells were then determined by des- are indeed neurons and, if so, to identify troying some of the progeny of the precur- what transmitter(s) they employ. sors of rays 5, 7, and 9. Usually, destruc- P cells, male (Figs. 17 and 21): In the tion of “aaa” (e.g., VG.pppppaaa) elimi- development of the male ventral nerve nated the dopaminergic cell, but did not cord, there are fewer programmed cell eliminate the ray; destruction of “apa” deaths during the Ll stage and extra cell (e.g., VG.pppppapa) eliminated neither the divisions during the L3 stage; both aspects dopaminergic cell nor the ray; and destruc- of this “reprogramming’ involve only the tion of “app” (e.g., VG.pppppapp) elimi- Pnaap daughters. In these ways, the male nated the ray, but did not eliminate the produces 14 extra cells in the ventral cord. SULSTON AND HORVITZ Cell Lineages of a Nematode 139

In the male, ventral hypodermal nuclei (whether it was derived from B.al or B.ar), P3.p-P&p (which generate the vulva in but rather by its newly assumed position the hermaphrodite) do not divide; instead, (anterior or posterior). In other words, po- Pl0.p and Pl1.p divide to produce 16 male- sitional influences play a determining role specific nuclei in the region of the pre-anal in the developmental fates of these cells. ganglion. We do not know the fate of these E cell, male (Figs. 21 and 22): E.lp and nuclei in the adult male; preliminary ob- E.rp are associated with the vas deferens servations suggest that some may be neu- where it joins the cloaca. E.rap and E.raa rons. In 4 out of 17 individuals observed, are in the pre-anal ganglion. Usually, E.la P9.p divided once, yielding two ventral hy- remains posterior to the preanal ganglion, podermal nuclei. but, in two individuals, it behaved like B and F cells, male (Figs. 21 and 22): E.ra and migrated anteriorly and divided. Some of the descendants of the B and F C cell, male (Figs. 21 and 22): The C cell cells form the cloaca; others produce the generates compact, neuronal-like nuclei. copulatory spicules, elongated cuticular C.a remains in the pre-anal ganglion, structures used to open the vulva of the while C.p divides transversely and ulti- hermaphrodite during mating (Figs. 1 and mately produces a pair of five-celled gan- 20). The specific fates of the daughters of glia which flank the cloaca. these lineages have not been established. (b) Mesoderm. M cell, hermaphrodite Most of these progeny cells are probably (Figs. 6, 23, 24, and 25): All of the postem- structural, as they have clear nucleo- bryonic mesodermal cell lineages derive plasms with prominent nucleoli; a few, from a single mesoblast, M. M divides designated “g” in Fig. 22, are more com- dorso-ventrally about midway through the pact with granular nucleoplasms and seem Ll stage (Fig. 15). Three subsequent divi- to be more like neurons or glial cells. How- sions generate 16 daughter cells, 4 in each ever, unlike the neurons of the ventral of the muscle quadrants. Two of the ven- cord and hermaphrodite tail, these cells tral cells (M.vlpa and M.vrpa) divide lon- often contain visible nucleoli soon after gitudinally once more, so that by the Ll they are formed (possibly reflecting rela- molt 18 progeny cells are present. During tively rapid growth). Because of these am- the L2 stage, two of the dorsal cells biguities, any distinctions made between (M.dlpa and M.drpa) differentiate into coe- neurons and structural cells for these line- lomocytes. It is interesting that all four of ages are rather subjective. these “specialized” cells are lineally equiv- B divides obliquely late during the Ll alent daughters from the four muscle stage. During the L2 stage, its anterior- quadrants. Two of the daughters of the dorsal daughter (B.a) divides transversely; “extra” ventral divisions (M.vlpaa and B.al and B.ar divide twice each, producing M.vrpaa) migrate anteriorly. The other 14 a row of four nuclei on the right and left progeny cells (6 dorsal, 8 ventral) become sides of the tail. Both the anterior-most normal body muscles and are used for loco- (B.alaa and B.araa) and posterior-most motion. They intercalate with juvenile (B.alpp and B.arpp) of these nuclei recen- body muscles posterior to the gonad pri- tralize. One member of each of these pairs mordium. The precise pattern of this inter- becomes located anterior to the other, but calation varies among different individ- there is no fixed pattern as to the order uals. A typical arrangement is shown in these cells assume; all four possible final Fig. 24. The mature adult thus contains 95 configurations have been observed. The body muscle cells; the ventral right quad- subsequent lineage of each of these four rant and each dorsal quadrant contains 24 cells is determined not by its ancestry of these cells, and the ventral left quad- 140 DEVELOPMENTAL BIOLOGY VOLUME 56, 1977

FIG. 21. B, C, E, F, PlO.p, and Pl1.p; male, lateral views; ectodermal development in the male tail. There is extensive rearrangement of the cells later, during L4 lethargus. Only left and central cells are shown. 141

cf hrs F o-

Ll lo-

20- L2

30-L3

40-L4 A-2 FIG. 22. B, C, E, and F lineages, male; ectodermal development in the male tail. g, neuron or glial cell. rant contains 23. gin a series of three longitudinal divisions. M.vlpaa and M.vrpaa continue their Each cell produces 2 sets of 4 daughters by movement until late during the L2 stage, the L3 molt, generating a total of 16 new stopping when they reach a position mid- cells located in 4 sectors around the dorso- way along the developing gonad. Late dur- ventral axis of the developing vulva. In ing the L3 stage, these sex myoblasts be- each set, the cell nearest the vulva (e.g., 142 DEVELOPMENTAL BIOLOGY VOLUME 56, 1977

FIG. 23. M; hermaphrodite, lateral views; mesodermal development during the Ll stage. bm, body muscle; cc, coelomocyte; dep, anal depressor muscle; im, intestinal muscle; sph, anal sphincter muscle.

M.vlpaaapp) becomes a type 1 vulva1 mus- of their filaments in a circumferential ori- cle (vml); its sister (M.vlpaaapa) becomes entation. urn1 attaches to the ventral side a type 2 vulva1 muscle (vm2). vml at- of the uterus close to the vulva and to the taches to the vulva more ventrally than lateral ridge of the body wall. urn2 at- does vm2 and attaches to the body wall taches solely to the uterus in a region fur- more dorsally than vm2; vml is located ther from the vulva; in contrast to uml, more superficially than vm2. The cell far- urn2 extends to the dorsal side of the thest from the vulva in each set (e.g., uterus. M.vlpaaaaa) becomes a type 2 uterine During the L4 stage, the nuclei of the muscle (um2); its sister (m.vlpaaaap) be- sex muscles assume their final positions comes a type 1 uterine muscle (uml). Both around the developing gonad. During the urn1 and urn2 form thin sheets with most L4 molt, all of the 16 sex muscle cells SVLSTON AND HORVITZ Cell Lineages of a Nematode 143

FIG. 24. M; hermaphrodite, left lateral views of the left side; mesodermal development. cc, coelomocyte. twitch in characteristic directions. In the dermal cell lineage in the male begins ex- adult, these muscles are used in laying actly as in the hermaphrodite. However, ekxs. cells homologous with those which in the M cell, male (Figs. 25 and 26): The meso- hermaphrodite differentiate into coelomo- DEVELOPMENTAL BIOLOGY VOLUME 56, 1977

hrs

cf hrs M Or I

FIG. 25. M lineage, hermaphrodite and male; mesodermal development. Divisions are anterior-posterior unless otherwise indicated. bm, body muscle; cc, coelomocyte; uml, type 1 uterine muscle; urn& type 2 uterine muscle; vml, type 1 vulva1 muscle; vm2, type 2 vulva1 muscle. cytes (M.dlpa and M.drpa) are blast cells stage, these 6 cells begin a series of divi- in the male. The six male sex mesoblasts sions, producing a total of 42 daughters. grow in size and, during the L3 stage, Most of the progeny of the ventral cells migrate posteriorly. Late during the L3 move to positions along the ventral side of SULSTON AND HORVITZ Cell Lineages of a Nematode 145

FIG. 26. M; male tail, left lateral views of the left side; mesodermal development. cc, coelomocyte. The muscles continue to migrate in the L4. the nematode. Some of the daughters of rection. These migrations, which have not the posterior dorsal cells migrate poste- yet been followed in detail, often occur riorly into the post-anal region of the tail; concomitantly with the divisions which some of the others move ventrally. Most of generate these cells; there is substantial the daughters of the anterior dorsal cells variation among individuals regarding the migrate to the ventral side; most then con- precise order of events as well as the ori- tinue their movements, some in an ante- entation of the division planes of the mi- rior direction and some in a posterior di- grating cells. For example, sometimes a 146 DEVELOPMENTAL BIOLOGY VOLUME 56, 1977

given cell will migrate from the dorsal to have studied the postembryonic develop- the ventral side and then divide, whereas ment of a variety of nematode species in sometimes this same cell will divide on the two ways: (1) observation of Feulgen- dorsal side and then both of its daughters stained specimens to determine whether will migrate to the ventral side. or not a postembryonic increase occurs in We have not yet identified these 42 the number of cell bodies in the ventral daughter cells in the adult male, but we cord, and (2) examination of living speci- believe they must include a variety of mens using Nomarski optics to determine male-specific muscles located in the tail whether or not cell divisions could be ob- and, usually, one coelomocyte (located served directly. Based upon these criteria, near the dorsal midline somewhat anterior postembryonic nongonadal cell divisions to the anus). Approximately 16 of the mus- occur in all nematodes we have studied. cles are located anterior to the anus and Panagrellus redivivus (the “sour-paste run diagonally in a ventral-posterior to nematode”) and Turbatrix aceti (the “vine- lateral-anterior direction. Other male-spe- gar eelworm”) are free-living nematodes in cific muscles control the movements of the a different family from that of C. elegans spicules. (de Coninck, 1965). Aphelencoides blasto- cc) Intestine. I cells (Figs. 14 and 16): phthorus is plant parasitic and in a differ- Fifteen minutes after the beginning of Ll ent order (de Coninck, 1965). All three of lethargus, the intestinal divisions take these species showed development of their place. Normally, the six anterior-most nu- ventral nerve cords and cell divisions in clei do not divide; any of the four posterior- other tissues that appeared to be very sim- most nuclei may also fail to divide. Thus, ilar to these events in C. elegans. A. blas- division of lo-14 of the 20 juvenile intes- tophthorus may contain slightly more ven- tinal nuclei produces the 30-34 nuclei nor- tral cord neurons than C. elegans. mally found in the adult. Ascaris lumbricoides var. suis, an in- Other patterns of division occasionally testinal parasite of pigs, is in an order occur. Once, for example, an intestinal nu- different from both C. elegans and A. blas- cleus located immediately behind the an- tophthorus (de Coninck, 1965). It also terior six appeared to die during division; showed development in its ventral cord after anaphase, no daughter nuclei were similar to that seen in C. elegans. For formed. Concomitantly, the adjacent ante- microscopy, embryos and Ll larvae were rior nucleus divided, the only time this artificially released from eggs at different nucleus has been observed to do so. developmental stages as described under (d) Gonad. The cell lineages of the Materials and Methods. (Normally, A. gonad have not been determined. As sug- lumbricoides larvae do not hatch until the gested by Hirsh et al. 11976), the morphol- L2 stage.). The first cleavage of the egg ogy of the gonad offers a convenient means occurred on Day 4; by Day 14, the embryos of staging the postembryonic development had reached the “comma” or “tadpole” of C. elegans. For comparison with the stage (Chitwood and Chitwood, 1974). At timing of other postembryonic events, var- Day 17, the eggs contained moving larvae ious stages of gonad development in both with nuclei sparsely distributed in the the hermaphrodite and the male can be ventral cord. By Day 21, the number of seen in Figs. 9, 24, and 26. nuclei in the ventral cord had increased severalfold to a number comparable to (4) Other Nematodes that in C. elegans L2 larvae. Some speci- Postembryonic developmental events mens with intermediate cord counts con- similar to those we have observed in C. tained cells in the ventral cord in the proc- elegans also occur in other nematodes. We ess of division. Because of difficulties in SULSTON AND HORVITZ Cell Lineages of a Nematode 147

visibility, cell divisions in A. lumbricoides both the anterior sensory nervous system were not observed using Nomarski optics. and the pharynx appear to be complete at Longidorus macrosoma, a raspberry hatching. ecto-parasite, is in a different subclass In the development of the hypodermis, from the other nematode species examined eight blast cells on each lateral side (Hl, (de Coninck, 1965). Its ventral cord was H2, Vl-V6) follow similar programs of cell quite distinctive, containing about 140 division to generate a total of 92 syncytial neuron-like nuclei in the Ll. As in the hypodermal nuclei and 28 seam cells. other nematodes, substantial development (Seam cells form a distinct cellular band occurs after the Ll stage, increasing the embedded in the lateral hypodermal number OS ventral cord neurons to about ridge.) These hypodermal cell divisions oc- 1000 by the beginning of the L3 stage. Cell cur around the time of the first three lar- division was not observed using Nomarski val molts. Two lateral tail blast cells (T) optics. produce two seam and six syncytial nuclei. We have not traced lineages in any of Twelve other syncytial hypodermal nuclei these other nematodes. are produced in the ventral cord by precur- Postembryonic increases in the number sors (Pl-P4, P&P121 primarily responsible of cells in the ventral cord, hypodermis, for neuronal development. The other three and intestine of the free-living nematode ventral cord precursors (P5-P7) generate anomala have been described the 22-cell vulva, the hypodermal struc- by Wessing (1953). The increase in the ture through which eggs are laid. In the number of ventral cord nuclei in P. rediui- young larva, the ventral cord precursors uus has been previously reported by Hech- form an integral part of the hypodermal ler (1970). body wall. DISCUSSION Most of the neuronal development oc- curs in the ventral nerve cord and its asso- (A) Summary ciated ganglia. Identical patterns of divi- The number of nongonadal nuclei in the sion during the first larval stage of 12 pre- hermaphrodite of C. elegans increases cursor cells (Pl-P12) and 1 neuroblast from about 550 at hatching to about 810 in (P0.a) followed by a fixed program of cell the mature adult (Table 2). Essentially deaths add 56 neurons to the ventral motor invariant cell lineages generate a fixed nervous system. Two of the lateral hypo- number of progeny cells of rigidly deter- dermal blast cells (V5) produce small gan- mined fates. These postembryonic cell lin- glia as well as the hypodermal nuclei men- eages range in length from one to eight tioned above. Two other lateral hypoder- sequential divisions. ma1 blast cells (T) and the lateral neuro- A number of these lineages produce the blasts (Ql, Q2) also generate neurons and/ accessory sexual structures of the her- or glial cells. maphrodite: the vulva (through which Mesodermal development derives exclu- eggs are laid), vulva1 and uterine muscles, sively from a single cell (M) present in the and neurons which innervate these mus- newly hatched larva. A series of divisions cles. New neurons are also added to the during the first larval stage produces 14 motor nervous system. Almost one-third of body muscles, 2 coelomocytes, and 2 myo- all postembryonic progeny nuclei become blasts. These myoblasts migrate ante- part of the hypodermal syncytium of the riorly until they reach the position where nematode body wall. The number of intes- the vulva will form. They divide shortly tinal nuclei almost doubles. There is a before the third molt to produce 16 sex small increase in the number of body mus- muscles, which function in egg-laying. cles. Few cell divisions occur in the head; Most of the intestinal nuclei (In) divide 148 DEVELOPMENTAL BIOLOGY VOLUME 56, 1977

TABLE 2 NUCLEAR COUNTS, HERMAPHRODITE

Present in young Ll Derived from postembryonic lineages

Nondivid- Blast Suhv~l” Cell Present in ing nuclei cells deaths adult Lateral hypodermis Seam 2 - 30 - 32 Syncytial 20 - 98 - 118 Neuronal or glial 8 - 28 - 36 Total 30 20 (H,V,T,Q) 156 8 186

Ventral cord and associated ganglia Neuronal or glial 33 - 56 9 89 Hypodermal 0 - 12 1 12 Vulva 0 - 22 0 22 Total 33 13 (P) 90 10 123

Mesoderm Body muscles 81 - 14 0 95 Sex muscles 0 - 16 0 16 Coelomocytes 4 - 2 0 6 Digestive tract muscles 4 - 0 0 4 Head mesodermal cell 1 - 0 0 1 Total 90 1 00 32 0 122

Intestine 6” 14” (I) 28 0 34

Head Neuronal, glial, small structural 201” - 4 0 205* Hypodermal (dorsal, ventral) 15 - 1 0 16 Pharynx 80 - 0 0 80 Pharyngeal-intestinal valve 6 - 0 0 6 Excretory system 4 - 0 0 4 Total 306 2 (G) 5 0 311

Tail Neuronal or glial 19 - 1 0 20 Hypodermal 7 - 1 0 8 Rectal glands 3 - 0 0 3 Total 29 1 W 2 0 31

Other tail ectoderm (B,C,E,F) 4 0 0 0 4

Total, excluding gonad 498 51 313 18 811 o The Ll intestine contains 20 nuclei. Of these, 6 never divide; in a given individual, lo-14 of the others divide. b Estimate. once, before the first molt. about 970 (Table 3). Most of the additional, A number of other cell divisions also male-specific cells are located in the spe- occur in the hermaphrodite. cialized structures of the tail used by the The postembryonic development of the male during copulation. Many cell line- male differs from that of the hermaphro- ages are similar in the male and herma- dite. In the male, the number of nongona- phrodite; the two sexes display virtually da1 nuclei increases from about 550 to identical changes in the lateral hypoder- SULBTON AND HORVITZ Cell Lineages of a Nematode 149

TABLE 3 NUCLEAR COUNTS, MALE

Present in young Ll Derived from postembryonic lineages

Nondivid- Blast SWl~~~~” Cell Present in ine nuclei cells deaths adult Lateral hypodermis Seam 2 - 36 - 38 Syncytial 20 - 104 - 124 Neuronal or glial 6 - 28 - 34 Rays 0 - 54 - 54 Total 28 20 MV,T,Q) 222 26 250

Ventral cord and associated ganglia Neuronal or glial 33 - 70 4 103 Hypodermal 0 - 10 1 10 Unknown 0 - 16 0 16 Total 33 13 (P) 96 5 129

Mesoderm Body muscles 81 - 14 0 95 Sex muscles 0 - Coelomocytes 4 - 42 0 46 Unknown 0 - > 1 > Digestive tract muscles 4 - 0 0 4 Head mesodermal cell 1 - 0 0 1 Total 90 1 (M) 56 0 146

Intestine 6” 14” (I) 28 0 34

Head Neuronal, glial, small structural 2056 - 4 0 209* Hypodermal (dorsal, ventral) 15 - 1 0 16 Pharynx 80 - 0 0 80 Pharyngeal-intestinal valve 6 - 0 0 6 Excretory system 4 - 0 0 4 Total 310 2 (G) 5 0 315

Tail Neuronal or glial 19 - 1 0 20 Hypodermal 7 - 1 0 8 Rectal glands 3 - 0 0 3 Total 29 1 (K) 2 0 31

Other tail ectoderm 0 4 (B,C,E,F) 66 5 66

Total, excluding gonad 496 55 475 36 971 (1 The Ll intestine contains 20 nuclei. Of these, 6 never divide; in a given individual, lo-14 of the others divide. b Estimate. mis, body musculature, and intestine. podermal lineages (V5, V6, T) produce the Some male-specific cells are produced by 18 rays of the male tail. The mesodermal lineages which are initially identical to lineage (M) generates the male-specific those seen in the hermaphrodite. For ex- sex muscles instead of the vulva1 and uter- ample, extra divisions in the lateral hy- ine muscles of the hermaphrodite. The de- 150 DEVELOPMENTAL BIOLOGY VOLUME 56, 1977

velopment of the male ventral nerve cord of the ventral cord. In the male, related involves extra cell divisions as well as ectodermal, mesodermal, and neuronal fewer programmed cell deaths. Two poste- lineages generate some of the structures of rior ventral cord hypodermal cells (PlO.p, the tail which function during copulation. Pl1.p) divide to form part of the male tail, The other male-specific structures are pro- whereas the three cells homologous with duced by lineages derived from cells which those which produce the vulva in the her- in the hermaphrodite do not divide. maphrodite (P5.p, P6.p, P7.p) do not divide Some of the postembryonic cell divisions in the male. The other male-specific struc- may relate to growth. For example, four tures (the cloaca, spicules, and associated rounds of hypodermal cell divisions during ganglia) are derived from cells (B, C, E, F) the first three larval stages substantially that are present in all Ll larvae but divide increase the number of hypodermal nuclei. only in males. The increases in the numbers of intestinal The postembryonic development of C. nuclei and body muscle cells may similarly elegans involves the sequential addition of reflect growth, although it is unclear why new structures onto a preexisting larval there are cell divisions in these tissues edifice; no destruction of larval-specific only during the first larval stage. cells occurs. However, there are structural Explanations for other postembryonic modifications of larval cells: The “dorsal cell divisions are not obvious. For exam- D” motoneurons of the ventral cord reverse ple, the Ll moves quite adequately using their polarity during postembryonic devel- its juvenile locomotory ventral nervous opment; in the Ll, they receive innerva- system; yet, the number of neurons in this tion from the dorsal cord and synapse onto system is almost tripled during early lar- ventral body muscles, whereas in the val development. Perhaps for mechanical adult they receive innervation from the reasons the larger adult requires a more ventral cord and synapse onto dorsal body intricate nervous system to control its loco- muscles (J. G. White, personal communi- motion. Alternatively, the adult may be cation). capable of behaviors more complex than those of the Ll. (B) Functions of Postembryonic Cell Divisions A number of the postembryonic cell line- (C) Invariance ages are related to sexual maturation. At The postembryonic somatic cell lineages hatching, the hermaphrodite and the male of C. elegans are generally invariant, with are almost identical with respect to cell a fixed pattern of cell divisions and a fixed number, position, and (presumably) func- developmental program defined for every tion. Yet, by the adult stage, gross ana- daughter cell. However, essentially five tomical differences are obvious in nongo- types of variations in the details of this nadal tissues. Most of the cell divisions developmental program have been ob- which give rise to these sex-specific struc- served, as follows. (1) Variation in the pat- tures occur shortly before the third molt. tern of cell divisions: The greatest incon- In the hermaphrodite, ectodermal lineages stancy appears in the single round of divi- produce the 22 cells which form the vulva, sions of the intestinal nuclei; the four pos- the structure through which eggs are laid. terior-most nuclei sometimes divide and Mesodermal lineages generate the 16 mus- sometimes do not. Slight variations occur cle cells which control the vulva and in the patterns of divisions of the hypoder- uterus during egg-laying. Neurons which ma1 cells of the ventral cord. Among innervate at least some of these muscles males, P9.p occasionally divides; among (White et al., 1976) are also formed post- hermaphrodites, P3.p sometimes fails to embryonically from the neuronal lineages divide. Similarly, E.la occasionally di- SULSTON AND HORVITZ Cell Lineages of a Nematode 151 vides in males. (2) Variation in the pattern anterio-posterior asymmetry of develop- of cell deaths: &2.pp, which normally ap- mental determinants located either inside pears to die, has once been observed to or outside the parent cell. That transverse survive into the adult. (3) Variation in divisions are symmetrical (see below, which of two alternative lineage programs Symmetry) suggests that the distribution a given cell will follow: These cases appear of such determinants may be bilaterally to involve positional effects and are dis- symmetric. The few dorsal-ventral divi- cussed below (see Mechanisms of Determi- sions appear to be asymmetrical. How- nation). (4) Variation in the precise order ever, the examples in the male-specific B, of specific events: For example, in meso- C, and F lineages are difficult to interpret, dermal development, sometimes M.vla di- because divisions in these lineages gen- vides before M.vra, whereas sometimes erally fail to conform to the orthogonal M.vra divides before M.vla. Similar tim- coordinate system seen elsewhere. The ing variations occur between lineages as only other example (the first division of well as within a given lineage. Migrations the mesoblast, M) produces progeny which and divisions also are not rigidly ordered, follow identical lineages for three further because sometimes a cell will migrate and divisions; the subsequent different fates of then divide, whereas sometimes it will di- a few of the lineally equivalent dorsal and vide and its daughters will migrate (see ventral cells (e.g., M.dlpa and M.vlpa) Results, Mesoderm). (5) Variation in the could arise from local environmental in- precise positions of cell nuclei: For exam- fluences. ple, the development of both the ventral Comparison of the various cell lineages nerve cord and the somatic musculature reveals that there is no standard type of leads to variable intercalation of new prog- cell lineage utilized for generating groups eny cells with the juvenile cells present at of new cells. Although most lineages in- hatching. For this reason, our diagrams volve both symmetrical and asymmetrical cannot be used to identify all cells in an cell divisions, two exemplify extreme types animal which has already developed. of lineage logic. In the mesodermal lineage (Fig. 251, a series of symmetrical divisions (0) Patterns of Cell Divisions adds a relatively large number of new cells Two distinguishable types of cell divi- of a particular type at one time. In the sions occur during the postembryonic de- lateral hypodermal lineages (Fig. lo), a velopment of C. elegans. Some divisions series of asymmetrical divisions adds se- are symmetrical, producing daughters quentially individual progeny which dif- which are equivalent in morphology and, ferentiate into types distinct from the par- often, in subsequent development. Other ent cell; in such stem cell lineages, one divisions are asymmetrical, producing two daughter always maintains the morphol- distinctly different types of daughter cells; ogy of the original mother cell. in at least some instances, asymmetric di- A variation of this “stem cell logic” is visions generate a posterior daughter that thought to produce the ventral nervous is morphologically like its mother cell and system of melanogaster (Poul- an anterior daughter of a new cell type. son, 1950; Seecoff et al., 1973) and may also Both types of divisions can be seen in the be used by C. elegans. In Drosophila, each development of the lateral hypodermis of a set of neuroblast stem cells generates (Fig. 10); for example, Vnp divides sym- another stem cell and a “predifferentiated” metrically, whereas Vn.pa and Vn.pp di- cell that divides once before differentiat- vide asymmetrically. Asymmetric divi- ing. The lineage of the C. elegans ventral sions almost always occur along an an- nervous system (particularly in the male; terio-posterior axis and could reflect an Fig. 17) is similar. Perhaps this type of 152 DEVELOPMENTAL BIOLOGY VOLUME 56, 1977

lineage is common in neuronal develop- ous system produce 5 daughters each, 10 ment. lateral hypodermal stem cells produce 9 Another type of lineage that has been daughters each, 4 sex myoblasts produce 4 observed in may be utilized by C. daughters each, and 18 ectodermal blast elegans. In a number of insects, similar cells in the male tail produce 5 daughters cell lineages generate scales, hairs, bris- each. In all of these cases, the lineally tles, and sensilla (Lawrence, 1966); many equivalent progeny of different blast cells of these insect lineages involve pro- differentiate into functionally equivalent grammed cell death. In C. elegans, we find cells. In effect, each precursor cell gener- possible analogs in the development of the ates one of a number of repeating “units” rays (probably mechanosensilla), the pos- present in, in these examples, the ventral terior lateral ganglia (associated with the cord, lateral hypodermis, sex muscula- postdeirid sensilla), and the lumbar gan- ture, and male tail. The logic utilized in glia (partially associated with the phasmid these lineages may be contrasted with an a sensilla) (Fig. 27). Although there are dif- priori plausible alternative in which func- ficulties in interpretation (see the legend tionally equivalent cells are all produced to Fig. 271, we feel that the similarity of from a given blast cell and then reposi- these lineages may reflect a fundamental tioned with respect to the daughters from subprogram of development. other lineages to form an appropriate final Frequently, several blast cells follow the configuration. same asymmetric program of divisions. The relationship between lineage his- Thus, 13 neuroblasts of the ventral nerv- tory and developmental fate suggests that

nematode

FIG. 27. Comparison of lineages found in insects and C. eleguns. Insects: Generalized from Lawrence (1966). The lineage tree does not reflect the division axes. Ray: The neuron assignment is based on the identification of dopamine in this cell. The assignment of the structural element is based upon experiments with the laser (see Results, Cell Lineages). Postdeirid: Assignments were made by comparing nuclear morphology and arrangement as seen with Nomarski optics with that observed in serial-section electron micrographs. Because individuals of known lineage have not been examined by electron microscopy, the assignments of the sheath and socket cells (defined by Ward et al., 1975) conceivably could be switched. Lumbar ganglion: assignments were made by tracing nuclei observed with Nomarski optics through to the adult stage and identifying these nuclei in serial-section electron micrographs. The wing-shaped accessory cell and the socket cell are associated with the phasmid. However, the three neurons apparently are not closely associated with this sensillum. Furthermore, the phasmid has another associated accessory cell which is present in the newly hatched larva. In this case, we would propose that the lineage defines the general functions of cells which subsequently assort into different sensory elements. The fates of some sister cells in the lumbar ganglion lineage are reversed compared to the fates of lineally equivalent cells in the ray and postdeirid lineages, possibly reflecting local environmental influences (see Mechanisms of Determina- tion). SIJLSTON AND HORVITZ Cell Lineages of a Nematode 153 specific types of cells may arise only from B6) -two cells of different lineage histories an appropriate series of divisions of a par- appear to be equally competent to follow ticular type of blast cell. Hence, when only either of two alternative developmental some progeny cells of a given lineage are programs; their subsequent lineages are needed, it may be necessary to generate correlated with their relative positions and and later destroy the other daughters. not with their previous histories. Simi- Such a mechanism provides one explana- larly, ventral cord hypodermal blast cells tion for programmed cell death, common P5.p and P6.p follow distinct programs in in both the nematode (see Results) and the development of the vulva, despite the other developmental systems (Saunders, fact that either of two cells present in the 1966). young Ll can become either P5 or P6; in other words, the developmental program (E) Mechanisms of Determination that each of these cells follows is correlated The strong correlation between lineage with the position it assumes in the ventral and function could arise in essentially two cord. Two observations suggest that the distinct (but not necessarily exclusive) gonad may be responsible for this posi- ways. On the one hand, the ultimate dif- tional influence on P5 and P6: (a) The line- ferentiation of a cell could be determined ages of the ventral hypodermal cells Pn.p extrinsically according to its position; al- are arranged essentially symmetrically ternatively, its fate could be determined around the midpoint of the developing intrinsically according to its lineage his- gonad (Fig. 181, as if this point were acting tory. Our observations are consistent with as the source of a positional determinant, the hypothesis that much of the develop- and (b) the Pnp cells which divide are ment of C. elegans is based upon a lineal those immediately adjacent to the gonad. determination of cell function. The high Other developmental events - e.g., the degree of invariance of these lineages sug- Pn.aap cell deaths in the ventral cord and gests such a mechanism. Furthermore, ex- the differentiation of the sex muscles- periments with the laser microbeam sys- also occur symmetrically around the de- tem (see Results) suggest that the fates of veloping gonad of the hermaphrodite and specific cells in the posterior lateral gan- similarly may reflect its influence. glia, the ventral cord, and the rays of the Other aspects of the postembryonic de- male tail are autonomously determined; velopment of C. elegans possibly may be these fates remain invariant despite the affected by positional influences: (1) Those fact that the undamaged cells sometimes lineages which utilize sets of “equivalent” differentiate in positions which normally blast cells show apparent end effects; such would be occupied by cells with different effects include logical inversions (in which fates. Preliminary experiments (J. White, the fates of anterior and posterior daugh- J. Sulston, N. Thomson, E. Southgate, ters are reversed) and/or positional inver- and R. Horvitz, unpublished results) uti- sions (in which daughters migrate past lizing mutants with abnormal cell lineages one another), as well as modifications of have also confirmed these observations; the pattern of cell divisions itself. For ex- blocking one branch of the cell lineages of ample, such alterations are apparent in the posterior lateral ganglia or the ventral the H and T lineages (the head and tail cord does not alter the developmental fates equivalents of the V lineage; Fig. 10) and of cells generated by another branch. in the divisions, deaths, and ultimate cell There are, however, some develop- fates at the ends of the ventral cord (Fig. mental events in which position appears to 17; Table 1). These various modifications of have an influence. In the male tail-in cell lineages in the head and tail regions each of two cases (Ba: and B/3; By and may reflect local environmental influ- 154 DEVELOPMENTAL BIOLOGY VOLUME 56, 1977 ences. (2) Six apparently equivalent devel- podermal nucleus on the right side near opmental “regions” can be defined longitu- the head and another on the left side in the dinally along the lateral hypodermis of the tail. (5) There is an extra ventral body nematode. In five of these regions, identi- muscle nucleus on the right side posterior cal lineages are produced by the hypoder- to the gonad primordium. (6) The lumbar ma1 blast cells. However, hypodermal ganglion on the right contains one more blast cell V5 follows a modified develop- nucleus than that on the left. (7). The anal mental program to generate part of the sphincter muscle nucleus is located on the posterior lateral ganglion. This region of left. (8) The excretory cell is displaced left the nematode is also unique in that it con- of the midline. tains the lateral neuroblasts (Q) as well as Bilateral symmetry is reflected in the the single mesoblast (M). Perhaps posi- transverse divisions which occur during tional factors-possibly defined by one or postembryonic development. Virtually all more of these cells - determine the unique transverse divisions produce morphologi- developmental aspects of this area of the cally identical progeny which follow iden- nematode. tical programs of development. The only Although the examples described above indications of exceptions to this rule- (1) indicate that cell lineages may be affected B.alapaav dies, B.arapaav does not; (2) by positional influences, we do not know if By.ald dies, By.ard does not; (3) E.ra di- the nematode is capable of regulation per vides, E.la sometimes does not-may well se. In experiments involving the posterior arise from positional influences at the end lateral ganglia, the rays of the male tail, of identical lineage programs. and the ventral cord, those structures nor- The left and right lateral neuroblasts, mally derived from a cell which had been Ql and Q2, are bilaterally symmetrical in eliminated either by the laser or by muta- their initial positions and in their division tion, were unequivocally absent in the programs. However, their progeny mi- adult; no regulatory processes induced ex- grate differently. Ql produces three prog- tra divisions to compensate for the laser- eny in the posterior half of the left side and or mutation-induced deficiencies. The Q2 produces three (or four) progeny in the unique observation of an abnormal death anterior half of the right side. in the intestine followed by a compensa- The obliquely oriented gonad primor- tory abnormal division (see Results, Intes- dium of the young Ll shows twofold rota- tine) is a possible example of regulation. tional symmetry around a dorso-ventral axis. The subsequent development of the (F) Symmetry hermaphrodite gonad, vulva, and sex mus- Although the young Ll is essentially cles is also symmetrical about this dorso- bilaterally symmetrical, there are a vari- ventral axis through the midpoint of the ety of minor differences between its left gonad. and right sides which can be distinguished in the light microscope: (1) The gonad pri- (G) Migrations mordium lies obliquely, with its anterior Cellular migrations occur frequently portion displaced to the right and its poste- during the postembryonic development of rior portion displaced to the left. In the C. elegans. Because Nomarski optics does region of the gonad primordium, the intes- not allow clear visualization of cellular tine is reciprocally displaced (anterior, boundaries, we cannot always be certain left; posterior, right). (2) The mesoblast that whole cells- as opposed to nuclei - (Ml is on the right. (3) The two coelomo- are migrating. However, in most migra- cytes on the right are anterior to those on tions, nucleus and cytoplasm do appear to the left. (4) There is an extra lateral hy- move together. The migrations of the pre- SULSTON AND HORVITZ. Cell Lineages of a Nematode 155

cursor (P) cells into the ventral cord are is rigidly determined; specific cells follow distinctive; a cytoplasmic extension moves precise developmental programs at well- into the cord and is followed by the nucleus defined times. This system can now be and, finally, by the remaining cytoplasm. used to obtain a detailed analysis at the Some migrations -such as those in the fine-structure level of such developmental posterior half of the ventral cord - appear events as cell migration and synapse for- to be passive events that depend solely on mation as well as other types of cellular the activities of neighboring cells; often differentiation. For example, the morpho- such movements are variable in extent logical changes associated with pro- from individual to individual. Other mi- grammed cell death could be studied for a grations are rigidly determined, with cells specific cell; the sequence of events could traversing relatively long distances from be traced by examining individuals at dif- reproducible starting and stopping sites. ferent stages from the time that cell is The most striking of the migrations in- born to the time it disappears. (2) The volve progeny of the lateral neuroblasts laser system developed by J. G. White will (Ql and Q2) and the mesoblast (M): (1) allow exploration of possible intercellular Three of the progeny of Q2 (Q2.a, Q2.p, communications during the processes of Q2.ap) travel rapidly along the left lateral cell divisions, migration, death, and differ- hypodermis; they move about 50 pm entiation. In addition, this laser system (about 16 nuclear diameters) in 2 hr. Ql.ap can be used to determine additional cell shows a similar, but somewhat shorter, assignments [as described above (see Re- migration. (2) The two sex myoblasts of sults) for the posterior lateral ganglia and the hermaphrodite (M.vlpaa, M.vrpaa) the rays in the male tail] and cell func- migrate from their origin among the poste- tions. (3) Mutants which affect the cell rior ventral body muscles to the anterior- lineages of postembryonic development posterior center of the developing gonad; may similarly be used to examine regula- they move about 65 pm (about 16 nuclear tive effects and to determine cell assign- diameters) in 6 hr. (3) Most of the daugh- ments and functions. Furthermore, such ters of the dorsal anterior sex mesoblasts mutants might provide clues concerning of the male (M.dlpaa, M.drpaa) migrate the logical bases of the genetic program for from the region of the dorsal body mus- the postembryonic development of C. ele- cles around to that of the ventral body gans. muscles; they move about 20 pm (about 5 nuclear diameters) in 1 hr. The move- We are indebted to Donna Albertson, Lois Edgar, ments of these male-specific mesoblasts Nichol Thomson, and John White for providing elec- tron micrographs with which we could compare our occur independently of the concomitant observations from the light microscope. We thank divisions of these cells. As described above David Hooper for providing us with various nema- (see Results, Mesoderm), sometimes a tode strains, Marilyn Dew for assistance, John given cell will migrate and then divide; White for use of the laser microbeam system, and sometimes it will divide and then both Sydney Brenner for stimulating our interest in C. elegans. We also thank our many colleagues for daughters will migrate; and sometimes it much detailed background information and stimu- will start to migrate, divide, and then the lating discussions. H.R.H. was supported by a re- two daughters will complete the migra- search fellowship from the Muscular Dystrophy As- tion. sociations of America.

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