left-right asymmetry in c. elegansintestine …notch signaling and organ left-right asymmetry 3431...

INTRODUCTION

Although most animals have bilaterally symmetrical surfacefeatures, their internal organs often show pronounced left-rightasymmetries in morphology and placement. For example, thetube of embryonic cells that forms the vertebrate heart loopsto the right, and organs like the stomach and liver arepositioned on the left and right, respectively. Although little isknown at the molecular level about how left-right asymmetryoriginates during animal embryogenesis, or how this leadsto subsequent asymmetries in organ morphology, recentstudies have identified a few genes that show early left-rightasymmetries in expression. In chick, frog, mouse and zebrafishembryos, an asymmetry in TGF-β signaling results in theasymmetric expression of transcription factors, such as Pitx2,within organ primordia (reviewed in Ramsdell and Yost, 1998;Capdevila et al., 2000). Through mechanisms that are notyet understood, Pitx2 and related factors may regulatemorphogenetic events that result in organ asymmetry.

The nematode C. elegansdisplays numerous left-rightasymmetries in tissues and organs, the most prominent beingthe asymmetrically positioned, interwoven helices of theintestine and gonad in adults (reviewed in Wood, 1998). Theseand other asymmetries result in an overall body pattern that has

been designated arbitrarily as dextral handedness. Wild-type C.elegansare invariably dextral when cultured using standardlaboratory conditions; however, culturing C. elegansat coldtemperatures can result in some sinistral animals, with reversedleft-right body axes (Wood et al., 1996). Sinistral animals alsocan be generated experimentally through micromanipulation ofspecific early embryonic blastomeres (Wood, 1991). The firstapparent left-right asymmetry is observed during the 4-cellstage of embryogenesis when two blastomeres divide obliquelywith respect to the left-right axis. If the left-right asymmetryof this division axis is reversed by micromanipulation, theresulting animal is sinistral. Changing the positions of the earlyblastomeres can alter the pattern of certain cell-cell interactionsthat determine some cell fates. The blastomeres that divideobliquely, and the descendants of these blastomeres, all expressGLP-1, a receptor related to Notch (Evans et al., 1994). Thedivision asymmetries cause only a subset of these blastomeresto contact a signaling cell; the resulting GLP-1 mediatedinteractions result in left blastomeres adopting differentlineages from right blastomeres (Gendreau et al., 1994; Hutterand Schnabel, 1994, 1995). It is not understood whether, orhow, these early interactions lead to specific organasymmetries.

The development of the C. elegansintestine provides a very

3429Development 127, 3429-3440 (2000)Printed in Great Britain © The Company of Biologists Limited 2000DEV3218

The C. elegansintestine is a simple tube consisting of amonolayer of epithelial cells. During embryogenesis, cellsin the anterior of the intestinal primordium undergoreproducible movements that lead to an invariant,asymmetrical ‘twist’ in the intestine. We have analyzed thedevelopment of twist to determine how left-right andanterior-posterior asymmetries are generated within theintestinal primordium. The twist requires the LIN-12/Notch-like signaling pathway of C. elegans. All cellswithin the intestinal primordium initially express LIN-12,a receptor related to Notch; however, only cells in the lefthalf of the primordium contact external, nonintestinal cellsthat express LAG-2, a ligand related to Delta. LIN-12 andLAG-2 mediated interactions result in the left primordialcells expressing lower levels of LIN-12 than the right

primordial cells. We propose that this asymmetricalpattern of LIN-12 expression is the basis for asymmetry inlater cell-cell interactions within the primordium that leaddirectly to intestinal twist. Like the interactions thatinitially establish LIN-12 asymmetry, the later interactionsare mediated by LIN-12. The later interactions, however,involve a different ligand related to Delta, called APX-1. Weshow that the anterior-posterior asymmetry in intestinaltwist involves the kinase LIT-1, which is part of a signalingpathway in early embryogenesis that generates anterior-posterior differences between sister cells.

Key words: Left-right asymmetry, Anterior-posterior asymmetry,Intestinal organogenesis, Notch signaling, APX-1, GLP-1, LAG-1,LAG-2, LIN-12, LIT-1, POP-1, SPN-1

SUMMARY

Left-right asymmetry in C. elegans intestine organogenesis involves a

LIN-12/Notch signaling pathway

Greg J. Hermann 1, Ben Leung 1,2 and James R. Priess 1,2,3,4,*1Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA2Molecular and Cellular Biology Program and 3Department of Zoology, University of Washington, Seattle, Washington 98195, USA4Howard Hughes Medical Institute, Seattle, Washington 98109, USA*Author for correspondence (e-mail: [email protected])

Accepted 31 May; published on WWW 20 July 2000

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simple model of organ morphogenesis, suggesting that amolecular explanation for asymmetry in the intestine should bepossible. The fully formed intestine is an epithelial tubecontaining only 20 clonally derived cells (Sulston et al., 1983).During embryogenesis, cells within the intestinal primordiumbecome polarized with respect to a central, anterior-posterioraxis through the primordium (Leung et al., 1999). In cross-section, the early primordium displays radial symmetry;however, cells in the left half of the primordium intercalatewith other left cells, and cells in the right half intercalate withother right cells. These cell movements result in a bilaterallysymmetrical tube composed of a single row of left cells thatabut a single row of right cells; the intestinal lumen forms atthe interface between the left and right rows.

Asymmetry in the intestine first becomes evident shortlyafter cell intercalation. Three of the anterior cells in the rightrow of the primordium move counterclockwise to the left,crossing the plane of bilateral symmetry, as the contralateralthree left cells move simultaneously toward the right (see Fig.1; Sulston et al., 1983; Leung et al., 1999). This movementinitiates a left-handed ‘twist’ in the anterior of the intestine; bythe time the larva hatches, the twist is about 180° (Sulston andHorvitz, 1977). The superhelical twist resulting from these cellmovements has been proposed to displace the anterior end ofthe intestine to the left of the larval body, and the posterior endto the right (Sulston and Horvitz, 1977). This asymmetry mayinfluence the subsequent morphogenesis of the gonad as itwraps around the intestine during larval development.

The development of the intestinal twist must involve both aleft-right, rotational asymmetry, as well as an anterior-posteriorasymmetry that limits the twist to the anterior end. In thisreport, we describe a genetic and cellular analysis of howasymmetry is generated in the intestine during embryogenesis.We show that the Notch-like signaling pathway of C. elegansis required for the intestinal twist. This pathway involves thereceptor LIN-12, the ligands LAG-2 and APX-1, and thenuclear protein LAG-1. We show that lin-12(+) function isrequired for intestinal twist, and that the LIN-12 protein isexpressed asymmetrically in the right half of the intestinalprimordium. The asymmetrical expression of LIN-12 requireslag-1(+) and lag-2(+) functions, and involves interactionsbetween cells in the intestinal primordium and cells external tothe primordium. Finally, we show that a separate polaritypathway, involving the genes pop-1 and lit-1, is required tospecify the anterior-posterior boundary between cells thatundergo twist and cells that do not.

MATERIALS AND METHODS

Strains and allelesN2 was used as the wild-type strain. Mutant alleles used are listedby chromosome: linkage group I (LGI): spn-1(it143), unc-40(e271),unc-40(e1430); LGIII: ceh-13(sw1), glp-1(e2141ts), glp-1(e2142ts),glp-1(q224ts), lin-12(n302sd), lin-12(n676n930ts), lin-12(n941),lin-12(n950sd), lin-39(n1760), lit-1(t1512ts), mab-5(e1239); LGIV:lag-1(q385), unc-5(e53); LGV: apx-1(zu347ts), lag-2(q387), lag-2(q411), lag-2(q420ts); LGX: elt-2(ca15), unc-6(e78), unc-6(ev400). Strains containing lag-2::GFP, JK2003 (qEx233) andJK2822 (qIs19) (Blelloch et al., 1999), were provided by JudithKimble (University of Wisconsin, Madison, USA). The JR662(wIs47) hs-end-1strain was provided by Joel Rothman (UC, Santa

Barbara, USA) (Zhu et al., 1998). C. elegansstrains were handledas described (Brenner, 1974).

MicroscopyWe scored intestinal twist in 1.5-fold embryos by Nomarskimicroscopy. Only embryos that had a lateral presentation such that therectum was present in the same focal plane as the intestine wereanalyzed.

Al Candia and Stuart Kim (Stanford University) provided theaffinity-purified rat anti-LIN-12 antiserum used in this study. Embryoswere immunostained with the LIN-12 antiserum using theimmunofluorescence protocol described in Leung et al. (1999).Immunostaining with mAb 1CB4 and mAb RL2 was performed asdescribed (Lin et al., 1998; Leung et al., 1999).

Laser ablations were performed as described (Leung et al., 1999).For immunofluorescence, embryos that developed to the E8 or E16

stage were transferred to 0.1% polylysine-coated slides andimmunostained for LIN-12. JR662 hs-end-1embryos were heat-shocked as described (Zhu et al., 1998) and fixed and immunostainedfor LIN-12 after 2.5 hours. To quantify the number of LIN-12expressing cells following heat shock, immunostained embryos wereoptically sectioned using a Delta Vision microscope. The resultingimages were analyzed to determine the total number of nuclei andtotal number of LIN-12 positive cells in each embryo.

RESULTS

Left-right asymmetry in the intestineThe embryonic cell that produces the C. elegansintestine iscalled the E blastomere, and the various developmental stagesof the intestinal primordium are named with reference to thenumber of E descendants present; E, E2, E4, E8, E16 and E20.The development of the intestine has been described in detailpreviously (Sulston et al., 1983; Leung et al., 1999). Once cellintercalation shapes the primordium into a bilaterally polarizedtube, it is convenient to refer to specific left-right pairs ofintestinal cells as intestinal rings (int ring I through int ring IX;Fig. 1A). Asymmetrical movements of cells in the int II, IIIand IV rings occur during stages E16 to E20. When viewed fromthe posterior end of the intestine, cells in the right half of theprimordium are seen to move counterclockwise toward the left,as cells on the left move toward the right (Fig. 1A). Thisinvariant pattern of cell movement can be summarized as arotation of the int II, III and IV rings, and for convenience werefer to this rotation as intestinal twist. Since several of themutants analyzed in the present study do not complete normaldevelopment and hatch, we selected the embryonic stage calledthe 1.5-fold (Fig. 1D) to score twist in all experiments; at thisstage, twist is about halfway complete (90°). Twist can bescored in fixed embryos after immunostaining with an antibodythat recognizes the intestinal cells (Fig. 1B), and in livingembryos by following the positions of intestinal nuclei by lightmicroscopy (Fig. 1D).

A Notch-like pathway is required for intestinal twistAn isolated E blastomere grown in culture produces a relativelysymmetrical cyst of intestinal cells, suggesting that cell-cellinteractions are likely to play a role in intestinal twist (Leunget al., 1999). Since a Notch-like signaling pathway mediatesseveral cell-cell interactions in the early embryo (reviewed inSchnabel and Priess, 1997), we began by asking whether eitherof the two Notch-related receptors in C. eleganswere required

G. J. Hermann, B. Leung and J. R. Priess

3431Notch signaling and organ left-right asymmetry

for twist. These receptors are called GLP-1 and LIN-12(Kimble and Simpson, 1997; Greenwald, 1998). Mutations thatdisrupt glp-1(+) functions after the 4-cell stage ofembryogenesis appeared to have no effect on intestinal twist(Table 1). In contrast, mutations in thelin-12 gene resulted inprominent twist defects. All of the embryos fromhermaphrodites homozygous for the null allelelin-12(n941)had defective twist, as did over 80% of the embryos fromhermaphrodites homozygous for the conditional allele lin-12(n676n930ts)(Table 1, Fig. 1C,E). In addition, we found thatwild-type embryos had a fully penetrant defect in twist whenlin-12(+) function was inhibited with double-stranded RNA(lin-12(RNAi); Table 1). We found that 19% (n=57) of theprogeny of heterozygouslin-12(n941)/+ hermaphrodites had atwist defect, indicating that zygotic expression of lin-12(+),rather than maternal expression, is required for twist. In thecourse of these experiments we noticed that some of thehomozygouslin-12(n941) and lin-12(RNAi) larvae and adultshad aberrant positioning of the gonad relative to the intestine;although this defect was not analyzed in detail, it is consistentwith the view that intestinal twist contributes to subsequentgonad morphogenesis (see Introduction).

We found that twist requires the lag-1 and lag-2genes (Table1), which encode proteins related, respectively, to theDrosophilaNotch pathway proteins Suppressor of Hairless, atranscription factor (Christensen et al., 1996), and Delta, aNotch ligand (Henderson et al., 1994; Tax et al., 1994). Since92% of embryos homozygous for lag-1(q385)had a twistdefect, we initially were surprised to find that only 43% ofembryos homozygous for lag-2(q411), a null allele, had a twistdefect. We discovered, however, that 100% of embryoshomozygous for lag-2(q387) had a twist defect; the lag-2(q387) mutation is a deletion that removes lag-2 as well asthe neighboring gene apx-1, which encodes a second Delta-

related ligand (Henderson et al., 1994; Mello et al., 1994).APX-1 is capable of substituting for LAG-2 in certain cell-cellinteractions (Fitzgerald and Greenwald, 1995; Gao andKimble, 1995), suggesting that LAG-2 and APX-1 might haveeither partially redundant, or distinct, functions required forintestinal twist. Consistent with these hypotheses, we foundthat 85% of the self-progeny of apx-1(zu347ts)homozygousmothers cultured at the semipermissive temperature (23°C) hada defect in intestinal twist (Table 1). We found that 24% (n=50)of the self-progeny of apx-1(zu347ts)/+ heterozygous motherscultured at the nonpermissive temperature (26°C) lacked twist,indicating that zygotic expression of apx-1(+) is required fortwist. Therefore, a Notch-like pathway consisting of thereceptor LIN-12, the effector protein LAG-1, and the ligandsLAG-2 and APX-1, is required for intestinal twist.

LIN-12 is expressed asymmetrically in the intestinalprimordiumWe examined the expression pattern of LIN-12 before andduring intestinal twist using an affinity-purified antiserumagainst the LIN-12 protein (A. Candia and S. Kim,unpublished). The patterns we describe are not observed inembryos from lin-12(n941) hermaphrodites or from lin-12(RNAi) hermaphrodites, and are thus specific for LIN-12(data not shown; K. Mickey and J. Priess, unpublished). LIN-12 expression is first detected at very low, but abovebackground, levels in each of the cells in the E4 intestinalprimordium (arrows and arrowheads; Fig. 2A). By the E8 stage,LIN-12 appears most abundant in cells in the right half of theprimordium (arrowheads; Figs 2B, 5E), and is present at loweror undetectable levels in cells in the left half (arrows; Figs 2B,5E). The left-right asymmetry in LIN-12 expression persists tothe E16 stage, when dynamic changes in LIN-12 expressionoccur along the anterior-posterior axis of the primordium (Figs

Fig. 1. Intestinal twist in wild-type and lin-12 embryos. (A) Diagram of the dorsal surface of a wild-type intestine showing the development oftwist. Before twist, left (light green) and right (dark green) cells are organized with bilateral symmetry. Each int ring contains two cells, exceptint ring I, which contains four cells (not shown). The int rings rotate in the sequence III, II, then IV. The rotation initially is 90°, but in thenewly hatched larvae the anterior intestine is twisted about 180°. (B,C) Dorsal views of embryos at the 1.5-fold stage immunostained with mAb1CB4 to show intestinal cells (green) and DAPI to show nuclei (red); orientation as in A. (D,E) Lateral views of living embryos at the 1.5-foldstage. Black arrowheads indicate the anterior end of the intestinal primordium, and white arrowheads in B and D indicate the posteriorboundary of twist. Embryos are 50 µm in length; in D and E anterior is to the left and dorsal is up. Bar, 5 µm.

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2C, 4A; see Fig. 2 legend). At the E16 stage, LIN-12 isprominent in punctate structures near the midline of theprimordium (Figs 2C, 4A); these structures resemble in shape,position and timing of appearance the ‘apical vesicles’described in an ultrastructural study of the primordium (Leunget al., 1999).

Regulation of LIN-12 expression in the intestinalprimordiumA previous analysis of LIN-12 expression in nonintestinal,embryonic cells showed a requirement for GLP-1 mediated,cell-cell interactions (Moskowitz and Rothman, 1996). Briefly,LIN-12 expression was shown to occur only in cells that hadprior expression of GLP-1, and that were in contact withligand-expressing cells. In contrast, we found that LIN-12 wasexpressed in the E8 primordium in mutants lacking glp-1(+)function (Table 2; Fig. 5F). We asked whether cell-cellinteractions were required for LIN-12 expression by using alaser microbeam to kill all of the blastomeres except for E. TheE descendants were allowed to develop to the equivalent of theE8 or early E16 cell stage, then fixed and stained for LIN-12.In 8/8 cases, LIN-12 appeared to be expressed in all of the Edescendants (Fig. 3A). Thus neither glp-1(+) function nor cell-cell interactions after the birth of E appear to be required forcells in the intestinal primordium to express LIN-12.

Several aspects of intestine-specific differentiation arecontrolled by a small group of ‘endoderm-determining’transcription factors that are expressed very early in the Elineage. For example, the transcription factor END-1 isexpressed in the E blastomere itself (Zhu et al., 1997), and ELT-2 is expressed in the daughters of E (Fukushige et al., 1998).Ectopic, heat-shock-induced expression of an end-1 transgeneis sufficient to cause a majority of embryonic cells to undergoat least partial intestine-specific differentiation (Zhu et al.,1998). To determine whether END-1 expression could causeectopic expression of LIN-12, we fixed and immunostainedembryos after heat-shock induction of the end-1transgene. Thepattern of cells expressing LIN-12 in these transgenic embryos(Fig. 3B,B′) appeared very different from the pattern in normalembryos at approximately equivalent stages (Fig. 2C). Afterheat shock, 41% of the cells in the transgenic embryosexpressed LIN-12 (range, 18-65%; n=8 embryos), compared toonly 18% of the cells in heat-shocked, wild-type controls(range, 14-21%, n=4 embryos). We looked for, but did notobserve, a requirement for ELT-2 in LIN-12 expression; elt-2(ca15) homozygous embryos expressed LIN-12 in theintestinal primordium, and appeared to have normal intestinaltwist (data not shown). We conclude that LIN-12 expression inthe E lineage is controlled by some of the endoderm-determining transcription factors, rather than by the GLP-1activation pathway described previously.

LAG-2-expressing cells induce LIN-12 asymmetry inthe intestinal primordium Although the E blastomere appears to have an ‘autonomous’ability to express LIN-12, we did not observe any evidenceof LIN-12 asymmetry between the E descendants whenneighboring blastomeres were killed (Fig. 3A). Thus cell-cellinteractions appear to be required for LIN-12 asymmetry. Sincelin-12(+) activity can alter the levels of LIN-12 expression incertain postembryonic cells (Wilkinson et al., 1994), we asked


Table 1. Intestinal twist

Embryo type Temperature (°C) n

Wild type 15 0 2120 0 2323 0 2926 0 33

glp-1 (e2142ts)1 26 0 26glp-1 (e2141ts)2 26 0 17lin-12(n941)3 20 100 30lin-12(RNAi)4 20 100 21lin-12(n676n930ts)5 15 92 25

20 85 2726 87 15

lin-12(n302sd)6 20 0 23lin-12(n950sd)6 20 0 19lag-1(q385)7 20 92 24lag-2(q411)7 20 43 21lag-2(q420ts)8 15 54 24

23 55 20lag-2(q387)7 20 100 24apx-1(zu347ts)9 15 0 27

20 23 2623 85 27

MSap ablation 20 36 11unc-5 (e53)11 20 0 23unc-6 (e78)11 20 5 20unc-6 (ev400)11 20 10 20unc-40 (e271)11 20 8 25unc-40 (e1430)11 20 11 27ceh-13 (sw1)12 20 6 18lin-39(n1760) 20 0 24mab-5(e1239) 20 0 21

% embryos withtwist defect

10

12

12

The rotation of int rings II, III, and IV was examined in 1.5-fold embryos by Nomarski microscopy. Embryos were scored as having a twist defect when 2-3 rings did not rotate. 1glp-1(e2142ts) is defective for glp-1(+) functions at, and after, the 12-cell stage of embryogenesis (Priess et al., 1987; Kodoyianni et al., 1992). Embryos were raised at 26°C throughout embryogenesis. 2glp-1(e2141ts) is defective for glp-1(+) functions at, and after, the 4-cell stage of embryogenesis (Hutter and Schnabel, 1994; Mello et al. 1994; Moskowitz et al., 1994). Embryos were shifted to 26°C between the 8 and 24-cell stage (E to E2 stages of the intestinal primordium, respectively). 3lin-12(n941) embryos examined were the rare self-progeny of semisterile hermaphrodites homozygous for the null allele of lin-12(n941) (Greenwald et al., 1983). 4lin-12(RNAi) embryos were from parents injected with double stranded RNA generated using the eighth exon of lin-12 as a template. 5The lin-12(n676n930ts) allele is temperature-sensitive for only a subset of lin-12(+) functions (Sundaram and Greenwald, 1993). 6n302sd and n950sd are semi-dominant hypermorphic alleles of lin-12 (Greenwald et al., 1983). 7Homozygous lag-1 and lag-2 embryos were identified at the 1.5-fold stage by the absence of an excretory cell and rectum; the Lag phenotype (Lambie and Kimble, 1991). 8At 15°C, 21 embryos were Lag; of these, 9 did not have a twist defect. Of the 3 embryos at 15°C that were not Lag, one had a twist defect. At 23°C, all embryos scored were Lag. 9Intestinal twist was only scored in apx-1(zu347ts) embryos where APX-1/GLP-1 signaling at the 4-cell stage was not disrupted (Mickey et al., 1996). 10MSap was ablated using a laser microbeam. 11All are severe loss-of-function alleles (Hedgecock et al., 1990). 12sw1, n1760, and e1239 are, or appear to be, null alleles (Kenyon, 1986; Clark et al., 1993; Brunschwig et al., 1999); the posterior boundary of twist appeared normal in all mutants examined.


whether the LIN-12 signaling pathway was required forLIN-12 asymmetry. Although neither of two semidominant,activating mutations oflin-12 affected the expression ofLIN-12 (lin-12(sd); Table 2) or intestinal twist (Table 1),mutations in either lag-1or lag-2 affected LIN-12 asymmetry.21% of the F2 self-progeny oflag-1(q385)/+ heterozygoushermaphrodites had approximately equal levels of LIN-12expression in the left and right halves of the E8 and E16

primordia (Fig. 4B and Table 2). Similar results were observedin 27% and 22% of the F2 self-progeny of hermaphroditesheterozygous for lag-2(q387)/+ and for lag-2(q411)/+,respectively (Fig. 4C and Table 2). lag-2(q420ts)embryosshowed symmetrical LIN-12 expression in the intestinalprimordium at all temperatures (Table 2), consistent with ourfinding that this allele of lag-2 results in intestinal twist defectsat all temperatures (Table 1).

The lag-1 and lag-2 genes could function in cell-cellinteractions within the intestinal primordium that lead to LIN-12 asymmetry, or in interactions between nonintestinal cellsand cells in the primordium. To distinguish between thesepossibilities, we examined the expression patterns of LAG-1

and LAG-2 using an antiserum and a Green Fluorescent Protein(GFP) reporter, respectively (J. Kimble, unpublished; Blellochet al., 1999). LAG-1 appeared to be expressed in the nuclei ofall embryonic cells during the stages that the intestine forms,and was thus not informative (data not shown). We could notdetect lag-2::GFP expression in any of the cells in theintestinal primordium; however, strong expression wasobserved in a row of 2-4 nonintestinal cells outside the left ofthe E4 (arrows, Fig. 5B) and E8 primordia (data not shown). Aprevious study reported that several embryonic cells expressLAG-2, including the daughters and granddaughters of anembryonic blastomere named MSap (Moskowitz andRothman, 1996). Through lineage analysis of the lag-2::GFPstrain, we identified the cells adjacent to the intestinalprimordium as the MSap descendants. To determine if theMSap descendants were required for LIN-12 asymmetry andintestinal twist, we used a laser microbeam to kill MSap. Ineach of eight operated embryos, LIN-12 was expressedsymmetrically on both sides of the intestinal primordium (Fig.4D and Table 2). We observed twist defects in 4/11 of theoperated embryos, which is comparable to the frequency oftwist defects observed in lag-2 mutants (Table 1).

glp-1 mutant embryos, like wild-type embryos, lack or havevery low levels of LIN-12 in the left half of the E8 primordium(see above). On the right half of the primordium, however,there are two additional cells that fail to express LIN-12 in 98%of glp-1(e2141ts)and glp-1(q224ts)embryos (asterisks, Fig.5F; Table 3). We therefore examined LAG-2 expression in theglp-1 mutants. In addition to the normal pattern of LAG-2expression by MSap descendants outside the left of the

Fig. 2. LIN-12 expression. Embryos at various stages of intestinaldevelopment (E4, E8, E16) stained for LIN-12 (top row) and withDAPI (bottom row). Intestinal cells in the left and right halves of theprimordium are indicated with white arrows and arrowheads,respectively; the anterior end of the primordium is indicated withblack arrowheads for the E8 and E16 stages. During the E16 stage, thepattern of LIN-12 expression in the right half of the primordiumchanges markedly (also see Fig. 4A). Using the numbering systemdescribed in Leung et al. (1999), LIN-12 is detected in all of the rightprimordial cells except numbers 5 and 8 in the early E16 stage, thenonly in numbers 3 and 4 (arrowheads in C; note that the LIN-12expressing cell at the posterior (bottom) of the embryo is not anintestinal cell), then only in number 4. At the time that the int ringsbegin to rotate, LIN-12 is not detected in the primordium.

Fig. 3.Control of LIN-12 expression. (A) LIN-12 staining of an E8

primordium derived from an ‘isolated’ E blastomere. All 8 of the E descendants showed LIN-12 expression; 7 are visible. Cellslacking LIN-12 expression are the laser-killed blastomeres, visible inthe DAPI-stained image in (C). (B,B′) Two different focal planes of asingle, heat-shocked hs-end-1embryo stained for LIN-12. Theembryo was allowed to develop to an age corresponding toapproximately the E16 stage. The corresponding DAPI images areshown in (D,D′).

3434 G. J. Hermann, B. Leung and J. R. Priess

Fig. 5. LAG-2 and LIN-12 asymmetry. (A-D) Nomarski micrographs of a wild-type embryo (A) and a glp-1mutant embryo (C) containing alag-2::GFP transgene at the E4 stage of intestinal development (the intestinal primordium is outlined). LAG-2 expression (GFP fluorescence) inthe embryos in A and C is shown in B and D, respectively. The MSap daughters (two arrows in D) and granddaughters (four arrows in B) eachexpress LAG-2 during the E4 stage. Note the ectopic, LAG-2 expressing cell (arrowhead) in D. (E-J) E8 stage embryos prepared as in Fig. 2;genotypes listed above each panel. Labeling is as in Fig. 2 except that the two anterior, right intestinal cells are indicated with asterisks in eachpanel for comparison.

Fig. 4. Control of LIN-12 asymmetry. All images show E16 stage embryos prepared and labeled as in Fig. 2; genetic or cellular defects arelisted above each panel.


primordium (arrows, Fig. 5D), we observed an additionalexpressing cell outside the right of the E4 primordium in 14/14glp-1(e2141ts)and 8/8 glp-1(q224ts)mutants (arrowhead; Fig.5D). We identified this ectopic cell as ABpraap, and found thatit was in contact with the E4 primordial cell that is the parentof the two, right E8 primordial cells that lack LIN-12expression. lag-2(+) activity was required for the lack of LIN-12 in the two right primordial cells, since these cells expressed

LIN-12 in glp-1(e2141ts); lag-2(q411)and glp-1(q224ts); lag-2(q411)double mutants (Fig. 5G; Table 3). Thus, in both wild-type and in mutant embryos, contact with a LAG-2 expressingcell causes primordial cells to develop reduced levels ofimmunodetectable LIN-12 (see Discussion).

Initiation of left-right asymmetryLAG-2 is expressed by MSap descendants outside the left ofthe intestinal primordium; however, the contralateral MSppdescendants, outside the right of the primordium, do notexpress LAG-2 (Fig. 6A). We found that the descendants ofMSap, but not MSpp, expressed lag-2::GFP in lag-1(q385)mutants (5/5 embryos) and in glp-1(e2141ts) mutants (4/4embryos), suggesting that the Notch-like signaling pathway isnot required for the difference between MSap and MSpp. Toinvestigate whether any cell-cell interactions were required forthis difference, we used a laser microbeam to kill all of theearly embryonic blastomeres except for MS, the precursor toboth MSap and MSpp. In 5/5 experiments, lag-2::GFPwasexpressed only in the MSap daughters (arrows, Fig. 6C) andgranddaughters (data not shown), and not in the MSppdescendants.

Since MSap and MSpp are descendants of sister cells thatare born in an anterior-posterior division, we considered thepossibility that the LIT-1/POP-1 polarity pathway was involvedin regulating LAG-2 expression. In early embryogenesis, allsister cells that are born in anterior-posteror divisions appearto differ in their levels of POP-1, a transcription factor that isrelated to vertebrate TCF/LEF1 and Drosophila Pangolin(Lin et al., 1995, 1998; Brunner et al., 1997). LIT-1 is aserine/threonine protein kinase that is required for POP-1asymmetry (Meneghini et al., 1999; Rocheleau et al., 1999).Mutations in lit-1 cause anterior-posterior sisters to both adoptthe anterior fate, and mutations in pop-1cause the reciprocaltransformation (Lin et al., 1995, 1998; Kaletta et al., 1997). Weconstructed a strain containing the temperature-sensitive allelelit-1(t1512ts)and the lag-2::GFP marker, and shifted embryos

Fig. 6. Control of lag-2expression.(A) Diagram showing the earlydivisions of the MS and E blastomeres.Dashes link sister cells. (B) Nomarskimicrograph showing five of the firsteight descendants of an ‘isolated’ MSblastomere; the daughters of MSap arelabeled (arrows). (C)lag-2::GFPexpression in the embryo shown in B.(D) Nomarski micrograph of atemperature-shiftedlit-1 mutantembryo showing a subset of the first 16descendants of the MS blastomere inaddition to other cells; thegranddaughters of MSap (arrows) andMSpp (arrowheads) are labeled.(E) lag-2::GFP expression in theembryo shown in D.

Table 2. Asymmetric expression of LIN-12 in the intestine

Parentalgenotype

% embryos expressing LIN-12

n

Wild type 15 0 100 0 65 20 0 100 0 173 26 0 100 0 76

glp-1 (e2142ts)1 26 0 100 0 80glp-1 (e2141ts)1 26 0 100 0 74glp-1 (q224ts)1 26 0 100 0 51lin-12( n302sd) 20 0 100 0 28lin-12(n950sd) 20 0 100 0 21lag-1(q385)/+2 20 0 79 21 107lag-2(q411)/+2 20 0 73 27 171lag-2(q420ts) 15 0 2 98 119

23 0 0 100 107lag-2(q387)/+2 20 0 78 22 294apx-1 (zu347ts) 15 0 100 0 60

23 0 100 0 92MSap ablation 20 0 0 100 8spn-1(it143) 20 17 83 0 244

Temperature

(°C)Both

halvesLefthalf

Righthalf

3

LIN-12 expression was scored in E8 and E16 intestinal primordia. For allele descriptions, see Table 1. 1Embryos were raised at 26°C throughout embryogenesis. 2Embryos scored were F2 progeny from single heterozygous parents. Since lag-1 and lag-2 cause L1-lethality, 17% of the F2 progeny are predicted to be homozygous for lag-1 or lag-2. 3Since spn-1(it143) is semi-embryonic lethal (Bergmann et al., 1998), onlyembryos with wild-type morphology were scored.

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to the nonpermissive temperature (25oC) after the birth of theMS blastomere. The level of lag-2::GFP expression varied ineach of four embryos examined, presumably from theincomplete penetrance of the lit-1(t1512ts) mutation (seeKaletta et al., 1997); however, all showed expression in thegranddaughters of both MSap (arrows, Fig. 6E) and MSpp(arrowheads, Fig. 6E). We thus infer that LAG-2 expression inwild-type embryos is determined by an anterior-posterior cell-fate decision that requires lit-1(+) activity.

Although MSap and MSpp are descendants of sister cellsthat are born in an anterior-posterior division, this division isskewed slightly along the left-right axis of the embryo (Fig.6A). This asymmetry results in MSap descendants contactingthe left side of the intestinal primordium while MSppdescendants contact the right side. Mutations in several geneshave been shown to disrupt the normally invariant division axesof the early embryonic cells (see Discussion). While most ofthese mutations result in grossly disorganized embryos, amutation in the spn-1gene can result in a few embryos thathave a simple inversion of the first left-right asymmetries inblastomere divisions; 19% of viable spn-1(it143) embryosgrow into adults that have a reversal of left-right asymmetry(Bergmann et al., 1998). We examined LIN-12 expression atthe E8 and E16 stages of the intestinal primordium in embryosfrom spn-1(it143)homozygous hermaphrodites. Although themajority of embryos had the normal pattern of LIN-12asymmetry in the intestinal primordium, 17% (n=244) had aleft-right reversal of LIN-12 asymmetry (Fig. 4E; Table 2).

APX-1 functions after LIN-12 becomes asymmetricOur genetic analysis of twist indicates that the ligand APX-1,in addition to LAG-2, is essential for intestinal twist (Table 1).In contrast to the defects in LIN-12 asymmetry observed in lag-2 mutants, LIN-12 appeared to be expressed with normal left-right asymmetry in the intestinal primordia of apx-1(zu347ts)mutant embryos cultured under conditions that cause thesesame embryos to show a highly penetrant twist defect (Tables1, 2). This result suggested that apx-1(+) functions in twistafter LIN-12 asymmetry is established at the E8 stage.Consistent with this hypothesis, we found that nearly all of theapx-1(zu347ts) mutant embryos that were shifted at the earlyE16 stage to the nonpermissive temperature had twist defects(Table 4).

We have not yet been able to determine whether APX-1 isexpressed asymmetrically in the intestinal primordium. Thecurrently available APX-1 antiserum (Mickey et al., 1996)stains at or near the apical surfaces of cells in the E16

primordium (our unpublished results). The immunostaining,however, does not appear to be specific because it persists inapx-1mutant embryos, as well as in wild-type embryos afterdsRNA inhibition of apx-1 (our unpublished results).

Cell movements in intestinal twistBecause twist involves the circumferential migrations of cellsin the anterior of the intestine, and the intestine is surroundedby a basement membrane when twist occurs (our unpublishedobservations), we addressed the possibility that the UNC-6guidance system was required for twist. UNC-6, a laminin-related basement membrane component, acts as a guidancesignal for circumferential migration by a wide variety of celltypes in C. elegansincluding axons, mesodermal cells and


Fig. 7. LIT-1/POP-1 and twist boundary. (A,D) Nomarskimicrographs of living embryos oriented as in Fig. 1D. Black andwhite arrowheads indicate the anterior end of the intestinalprimordium and the posterior boundary of twist, respectively. Thecells in int VI are the posterior sisters of cells in int IV (Sulston et al.,1983; Leung et al., 1999). (B, E) Comparably staged wild-type (B) and lit-1mutant (E) embryos immunostained for POP-1;anterior-posterior sister cells in the E8 primordium are indicated bydouble-headed arrows. (C, F). DAPI images of embryos shown in B and E, respectively.


gonadal cells (Hedgecock et al., 1990; Ishii et al., 1992). UNC-5 and UNC-40 are cell-surface receptors that guide cellmigrations away from or towards UNC-6 cues, respectively(Hedgecock et al., 1990; Leung-Hagesteijn et al., 1992; Chanet al., 1996). Almost all of the embryos produced by unc-5,unc-6 or unc-40 mutants appeared to have normal twist,indicating that these genes do not play a major role in the cellmovements associated with twist (Table 1). Some unc-6 andunc-40 mutant embryos, however, had subtle and variabledefects in the movements and positions of anterior intestinalcells (Table 1 and data not shown).

Control of the posterior boundary of intestinal twistThe twist in the wild-type embryonic intestine extends to, butdoes not include, int ring V (Fig. 1A). Cells in int ring V areunique in that two nonintestinal cells, the germ cell precursors,invariably insert processes into their ventral surfaces (Sulstonet al., 1983; our unpublished observations). Attachment of thegerm cell precursors, however, does not appear to prevent twistin int ring V; we found that twist did not extend into int ring Vin all experiments where either the parent (n=6) or grandparent(n=16) of the germ cell precursors was killed with a lasermicrobeam. Although in most experiments the laser-killed cellremained in contact with some region of the intestine, in twocases the grandparent was extruded from the embryo duringgastrulation and so did not contact the intestine. We alsoexamined the possibility that the C. eleganshomeotic complexgenes, ceh-13, lin-39 and mab-5, could have a role inspecifying anterior-posterior differences between intestinalcells that determined the posterior boundary of twist. Theposterior boundary appeared normal in strains with mutationsin each of these genes (Table 1).

Since POP-1, the downstream effector of the LIT-1/POP-1polarity, is expressed with anterior-posterior asymmetry in atleast some cells in the intestinal primordium (Lin et al., 1998),we asked whether lit-1(+) function was required to stop twistat int ring V, and whether lit-1(+) was required for POP-1asymmetry. We found that twist extended beyond int ring V, in100% (n=21) of lit-1(t1512ts) embryos shifted to thenonpermissive temperature at the E8 stage (Fig. 7D), and in 5%(n=22) of the embryos shifted at the E16 stage. Twist occurredin cells up to, and including, int ring VI. These temperature-shift experiments did not appear to affect the pattern of LIN-12 expression in the E8 or E16 primordia (data not shown), but

had a marked effect on the expression of POP-1. In 98% ofwild-type control embryos (n=62; Fig. 7B), each anterior-posterior pair of sister cells in the E8 primordium showedhigher levels of POP-1 immunostaining in the anterior sisterthan in the posterior. In contrast, all pairs of sisters hadapproximately equivalent levels of POP-1 in lit-1(t1512ts)embryos (n=32; Fig. 7E) that were shifted to the nonpermissivetemperature at about the E4 stage. Thus lit-1appears tofunction to regulate POP-1 asymmetry in the intestinalprimordium, as it does in the early embryo, and lit-1(+)function is required to define the posterior boundary ofintestinal twist.

DISCUSSION

Development of the C. elegansintestine provides a simplemodel system for a genetic and molecular analysis oforganogenesis (Leung et al., 1999). In this report, we haveaddressed how asymmetry is generated in the intestinalprimordium by analyzing the asymmetrical ‘twist’ thatdevelops at the anterior of the intestinal tube. Our results showthat the LIN-12 signaling pathway of C. elegans, which is usedrepeatedly during development in cell-fate decisions, also hasa role in controlling morphogenetic differences betweenotherwise identical intestinal cells. Anterior-posteriorasymmetry in the intestine appears to be controlled by a cell-polarity pathway involving the lit-1 gene.

Initial events in left-right asymmetryWe have shown that lin-12(+)activity is essential for cells inthe intestinal primordium to undergo the asymmetric, patternedmovements we call intestinal twist. All of the cells in the E4

primordium express LIN-12, apparently in response toendoderm-determining transcription factors like END-1. Anonintestinal cell, MSap, plays an important role in intestinaltwist, since about half of the embryos completely lack twistwhen MSap is killed. MSap descendants express LAG-2 andcontact the left side of the primordium. Since loss of lag-2(+)activity causes a twist defect comparable to that caused bykilling MSap, we propose that interactions between the LAG-2-expressing cells outside the primordium and LIN-12-expressing cells in the primordium initiate the events that leadto twist.

Table 3. LIN-12 expression in glp-1 mutants

Embryo type n

Wild type 6 34glp-1 (e2141ts) 98 53glp-1 (e2141ts); lag-2(q411) 0 29glp-1 (q224ts) 98 44glp-1 (q224ts); lag-2(q411) 0 12

% embryos lacking LIN-12expression in anterior,right E descendants*

Embryos were raised at 26°C throughout embryogenesis and scored for LIN-12 expression in the E8 primordium. The homozygous mutant progeny from glp-1; lag-2/+ parents were identified by the expression of LIN-12 on both halves of the intestinal primordium. *These cells (see asterisks in Fig. 5F) are daughters of the E descendant called Ear.

E2 100 12E4 96 25E8 90 40Early E16 72 25Late E16 4 27

Table 4. Temperature-shift analysis ofapx-1 (zu347ts)

Stage ofprimordiumat shift

% embryos with twist defect n

Embryos were shifted from 15°C to 26°C at the indicated stage and scored for the rotation of int rings II, III, and IV at the 1.5-fold stage. Embryos were scored as having a twist defect when 2-3 rings did not rotate. Early E16 is prior to, and late E16 is after, migration of intestinal nuclei (Leung et al., 1999).

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The lit-1 gene, and presumably other genes in the LIT-1/POP-1 polarity pathway (see Rocheleau et al., 1997; Thorpeet al., 1997), appears to create an anterior-posterior differencebetween the sister cells MSa and MSp that leads to an MSadaughter (MSap) expressing LAG-2, while the correspondingMSp daughter (MSpp) does not. We propose that this anterior-posterior difference is transduced into a left-right difference bythe slight skewing of the division axis of the MS blastomere.Cells surrounding MS appear to influence the pattern of MSdivision, since this pattern is not normal when the surroundingcells are killed (unpublished observations) or when theirpositions are inverted (see Introduction; Wood, 1991). Thesurrounding cells could provide specific signals that alter theMS division orientation, such as the effect of the C. elegansWnt-like pathway on the division of the early blastomere ABar(Rocheleau et al., 1997; Thorpe et al., 1997), or could simplyprovide steric constraints on division.

The development of left-right asymmetry in intestinalorganogenesis has some similarity to the mechanism thatestablishes left-right asymmetry in the lineages of certain earlyblastomeres in C. elegans (see Introduction). Both eventsinvolve a Notch-related signaling pathway, although LIN-12,rather than GLP-1, is used as the receptor for intestinalasymmetry. Early lineage asymmetry results from skeweddivision axes that position receptor-expressing cellsasymmetrically with respect to a signaling cell. In contrast,intestinal asymmetry appears to result from skewed divisionaxes that position a signaling cell asymmetrically with respectto receptor-expressing, intestinal cells. In this case, divisionasymmetry changes what is fundamentally a LIT-1 and POP-1mediated, anterior-posterior difference between MS daughtersinto a left-right difference.

Left-right asymmetry in the primordiumAlthough LIN-12 expression appears at low, uniform levelsthroughout the E4 primordium, by the E8 stage LIN-12 appearsat much higher levels in the right half than in the left. Theasymmetry appears to result both from an increase in the levelof LIN-12 in the right half of the primordium, and a decreasein the low levels in the left half. We have shown that this LIN-12 asymmetry requires MSap, and the wild-type functions ofthe genes lag-2 and lag-1; defects in any of these componentslead to uniform, relatively high levels of LIN-12 throughoutthe E8 primordium. We propose, therefore, that a LAG-2mediated activation of LIN-12 in the left half of the E4

primordium leads to low levels, or lack of, LIN-12 in the lefthalf of the E8 primordium, and this hypothesis is supported byour analysis of the effect of ectopic LAG-2 on LIN-12expression in glp-1mutants.

After exposure to ligand, receptors in the Notch familyundergo proteolytic cleavage events that are essential for signaltransduction (reviewed in Annaert and De Strooper, 1999;Artavanis-Tsakonas et al., 1999; Chan and Jan, 1999).Nevertheless, it is unusual for ligand exposure to cause avisible decrease in level of receptor. For example, GLP-1 levelson the surfaces of early embryonic blastomeres are notnoticeably different between blastomeres that either do, or donot, interact with ligand-expressing cells (Evans et al., 1994).Moreover, LIN-12 expression appears to increase, rather thandecrease, after exposure to ligand in the interaction betweenthe AC and VU cells during postembryonic development of C.

elegans (see below; Wilkinson et al., 1994). Similarly,Drosophila embryonic cells that are exposed to ligand canincrease their relative levels of Notch (Huppert et al., 1997).The proteolytic cleavage of LIN-12 that normally results fromligand exposure is, by itself, unlikely to account for the lowlevels of LIN-12 in the left half of the E8 primordium, sincethe transcription factor LAG-1, the downstream effector of theactivated receptor, is required for these low levels.

We propose that LIN-12 asymmetry in the E8 primordiumserves to pattern a second, subsequent, LIN-12 mediatedinteraction that leads to twist asymmetry. Our temperature-shiftexperiments demonstrate that the ligand APX-1 is essential forintestinal twist during the early E16 stage of the intestinalprimordium, after LIN-12 asymmetry has been establishedthrough the earlier, LAG-2 mediated, interaction. Since LIN-12, but not GLP-1, is required for twist, we infer that APX-1must be interacting with LIN-12 at the E16 stage. In addition,the observation that lag-1 mutations cause a complete loss oftwist, while lag-2mutations cause a variable and partial loss,supports the hypothesis that there are two distinct LIN-12mediated interactions.

We consider it likely that apx-1(+)function is requiredwithin the cells of the intestinal primordium for twist. Theanterior primordial cells, which are the cells that undergo twist,express LIN-12 but do not contact any nonintestinal cellsthat express LIN-12, suggesting that APX-1 interacts withLIN-12 on the intestinal cells. Furthermore, the surfaces of theintestinal cells in the E16 primordium are separated from thesurface membranes of all surrounding, nonintestinal cells bytwo extracellular basement membranes (our unpublishedresults). Thus we consider it likely that APX-1 and LIN-12interactions occur between cells within the E16 primordium,rather than between primordium cells and surrounding cells.

In principal, the asymmetric, high levels of LIN-12 in theright half of the E8 and E16 primordia could provide a basis forreproducible, asymmetric cell-cell interactions, irrespective ofwhether APX-1 is expressed in all, or only a subset of, theprimordial cells. If APX-1 is expressed asymmetrically, oneinteresting possibility is that lin-12(+) activity determines theexpression pattern of APX-1. A well-characterized example ofcell interactions affecting LAG-2 and LIN-12 expression in C.elegansis the AC/VU cell-fate decision during postembryonicdevelopment (Wilkinson et al., 1994). An interaction betweentwo initially equivalent cells, each expressing both LIN-12 andLAG-2, leads to one cell adopting the AC fate and the otheradopting the VU fate. A feedback mechanism appears toincrease the expression of LAG-2 in whichever cell initiallyhas slightly less lin-12(+)activity. Since cells in the left halfof the E8 and E16 primordia have low levels of LIN-12, asimilar feedback mechanism could increase the levels ofAPX-1 in those cells. This general model would not explainhow LIN-12 asymmetry is generated between the E4 and E8

stages, since the LAG-2 mediated interaction leads to adecrease, rather than an increase, in the levels of LIN-12.

We have shown that lag-2 mutants have a twist defect thatis only partially penetrant. Interestingly, rotation of the variousint rings appears to be variable in the abnormal intestines oflag-2 mutants; often one or two of the three int rings appear torotate (our unpublished observations). Since lag-2 mutantshave equivalent levels of LIN-12 in the right and left halvesof the E8 primordium, one possibility is that the feedback



mechanism described above could lead to variable asymmetryin APX-1 and LIN-12 mediated interactions betweenprimordial cells that initially have equivalent levels of bothAPX-1 and LIN-12. In future studies it will be important todevelop methods for detecting APX-1 expression, and LIN-12activation, in the primordium to analyze this second LIN-12mediated interaction directly.

Anterior-posterior asymmetry in the primordiumThe Homeotic Complex (HOM-C) genes, and the anterior-posterior differences in the expression patterns of these genes,appear to be highly conserved in most animals, including C.elegans. Anterior-posterior differences observed in tissues ororgans often depend on HOM-C function, and the C. elegansHOM-C genes are required during postembryonicdevelopment for numerous, position-specific differencesbetween epithelial cells (reviewed in Krumlauf, 1994;Capecchi, 1997; Kenyon et al., 1997; Beck et al., 2000; Grapin-Botton and Melton, 2000). However, the only anterior-posterior asymmetries that have been characterized thus far inthe intestine (our present study; Schroeder and McGhee, 1998)are not determined by HOM-C genes, but rather by a distinctpolarity pathway involving LIT-1 and POP-1. Our resultspaired with those of Lin et al. (1998) demonstrate that sistercells at each anterior-posterior division of the intestinal lineageshow POP-1 asymmetry, and that lit-1(+) activity is requiredfor this asymmetry. In the analysis by Schroeder and McGhee(1998) of a transgene that is expressed in the anterior of theintestine, expression was found to be regulated by pop-1(+)activity. In the present report, we have shown that lit-1(+)activity is required to limit twist to the anterior of the intestine,and that lit-1(+) function is required at or after the E8 stage.Thus the LIT-1/POP-1 pathway appears to function late inembryogenesis, and appears to be a major source of anterior-posterior asymmetry in the intestine.

Asymmetric left-right morphogenesisOnce the anterior-posterior boundary of twist is specified, whatis the basis for the circumferentially oriented movements ofintestinal cells that result in twist? Since the intestinalprimordium is covered with an extracellular matrix at the timeof rotation, it is possible that cells recognize, and respond to,guidance cues in the matrix. The circumferential migration ofseveral other cell types in C. eleganshas been shown to becontrolled by the well-characterized UNC-6 guidance system(Hedgecock and Norris, 1997). We found, however, thatmutants defective in this pathway had only minor, if any,defects in twist. A second possibility is that intestinal cellsmove in response to changes in the adhesive properties of otherintestinal cells. Prior to the development of twist, there is aperiod of cell intercalation in the primordium. The behavior ofcells during intercalation suggests that there may be left-rightdifferences in cell adhesivity (Leung et al., 1999): cells in theright half of the primordium intercalate with other right cells,but never with left cells, and vice versa. The development oftwist requires that some of the left cells exchange ipsilateralcontacts with other left cells for new contacts with right cells,and vice versa. For example, the right int IV cell establishesnew contacts with the left int V cell, while reducing ipsilateralcontacts with the right int V cell (Fig. 1A). In theory, twistcould result by creating heterotypic interactions between left

and right cells that were stronger than homotypic interactions.Studies of Drosophilasupport the general hypothesis that theNotch pathway might play a role in controlling adhesivedifferences between otherwise equivalent cells. Notch appearsto have a role in the epithelial cell movements that result indorsal closure of the embryo (Zecchini et al., 1999), and in theaxonal outgrowth of some neurons (Giniger et al., 1993). It willbe interesting to determine if other developmental events thatrequire Notch function involve changes in adhesivity, forexample in the control of ommatidial rotation in the Drosophilaeye.

We thank Stuart Kim, Al Candia, and Judith Kimble for allowingus to use their antisera against LIN-12 and LAG-1 prior to publication.We thank members of the Priess laboratory for helpful discussion andKatie Mickey for sharing unpublished observations. For strains andantibodies we thank Dominique Bergmann, Tetsunari Fukushige, DaliGao, Judith Kimble, Jim McGhee, Joel Rothman, Jim Thomas, BillWood and Jiangwen Zhu. Some nematode strains used in this workwere provided by the CaenorhabditisGenetics Center, which isfunded by the NIH. G. Hermann is supported by a postdoctoralfellowship from the Damon Runyon-Walter Winchell Foundation(DRG1561); B. Leung is supported by a training grant from theNational Cancer Institute (CA09657-09); J. Priess is supported by theHoward Hughes Medical Institute.

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