Left–right asymmetry in Drosophila melanogaster gut development

Develop. Growth Differ. (2001) 43, 239–246

Introduction

In recent years, there has been spectacular progressin the study of the mechanism of left–right (LR) asym-metric development in vertebrate (deuterostome)embryos. Since the discovery of LR asymmetric geneexpression at the node of the chick embryo (Levin et al. 1995,1997), various genes involved in establish-ing LR asymmetry have been identified and an outlineof the gene regulatory pathway that leads to LR asym-metry seems to have been revealed (Esteban et al.1999; Yokouchi et al. 1999; for reviews, see Yost 1999;Burdine & Schier 2000). The earliest event of LRdetermination has been suggested to be mediated bymictotubule-based cytoskeletal components, such askinesin superfamily proteins (Nonaka et al. 1998;Takeda et al. 1999) and the left/right dynein heavy chain(Supp et al. 1997), and the subsequent genetic eventsare primarily mediated by a cascade of LR asymmetricexpression of extracellular signaling molecules(reviewed by Esteban et al. 1999; Yokouchi et al. 1999).The LR asymmetric gene expression finally leads to themorphologic asymmetry of internal organs.

In protostomes, however, only a small number ofstudies have been carried out on the mechanism of LRasymmetric development. Nematodes have been themost extensively exploited protostome animals for LRstudies (for a review, see Wood 1998), in which thebiased position of spindles has been demonstrated tocause the LR asymmetric arrangement of the earlyblastomeres (Wood 1991). Recently, Hermann et al.(2000) have demonstrated that the LR asymmetricarrangement of blastomeres of Caenorhabditis elegansresults in asymmetric cell-to-cell interaction, leading toasymmetric gene expression.

In Drosophila melanogaster embryos, only a fewstudies have been performed with regard to LR asym-metry, with only very subtle or no LR asymmetry at allof outer body structures having been suggested(Tuinstra et al. 1990; Klingenberg et al. 1998). There hasbeen neither a systematic description of the LR asym-metry of internal organs nor a report on LR asymmetricgene expression. During the study of Drosophila gutdevelopment, we (and perhaps other fly researchers)have found that the gut is invariantly LR asymmetric(see the atlas by Hartenstein 1993). The LR asymmetryof the Drosophila gut is generated by twisting of theinitially LR symmetric gut tube in particular orientations,which results in an invariant LR asymmetric pattern ofconvolution. The invariant convolution pattern of the gut tube is also quite common to many vertebrates,suggesting the existence of some common basic

*Author to whom all correspondence should be addressed.Email: [email protected] 1 December 2000; revised 16 December 2000;

accepted 25 December 2000.

Left–right asymmetry in Drosophila melanogastergut development

Tomomi Hayashi and Ryutaro Murakami*Department of Physics, Biology and Informatics, Yamaguchi University, Yamaguchi 753-8512, Japan.

While left–right (LR) asymmetric morphogenesis is common to various animal species, there have been nosystematic studies of the LR asymmetry of body structures of Drosophila melanogaster. In the present paperthe LR asymmetric development of the Drosophila gut is described, in which three major parts, the foregut,midgut and hindgut, show almost invariant LR asymmetry. The asymmetry is generated by a twist of each partin particular orientations, resulting in a left-handed (sinistral) convolution as a whole. The frequency of spon-taneous reversal of LR orientations is very low (< 0.6%) and reversal of each part of the gut occurs indepen-dently. The bicoid mutation causes duplication of the posterior half of the gut, essentially keeping the left-handedtwist, suggesting that the LR asymmetry may depend on some intrinsic nature of the cells or tissues rather thana graded distribution of morphogens in the egg. The handedness of particular gut parts was randomized orbecame symmetric in mutants of brachyenteron, huckebein and patched, suggesting that different gene path-ways can interfere in determining LR asymmetry of the gut. It is noteworthy that all of these genes are expressedLR symmetrically.

Key words: bicoid, byn, Drosophila, gut, hkb, left–right asymmetry.

240 T. Hayashi and R. Murakami

mechanism. The present report provides the first systematic description of LR asymmetric developmentof the Drosophila gut. In addition, we report mutationsthat affect the handedness of the gut.

Materials and Methods

Fly strains

Enhancer-trap strains (PY1, PY258, PY282) that havelacZ expression in specific regions of the gut (Murakamiet al. 1994) were used for the observation of normal LRasymmetric morphogenesis. Embryos were stained forthe histochemical or immunohistochemical detection of�-galactosidase and were examined under a dissec-tion microscope or with a Nomarski differential inter-ference microscope. Immunostaining with anti-Crumbs(Crb) antibody, which stains luminal surface of thehindgut epithelium (Tepass et al. 1990), and with anti-Engrailed (En) antibody, which stains dorsal domain ofthe hindgut, was also carried out. y w and Canton-Sstrains, as well as the enhancer-trap strains, were usedfor in situ hybridization for the observation of normalgut development. Mutants with morphologic defects orhomeotic transformation of particular regions of the gutwere also used to investigate influences on LR asym-metric morphogenesis. The following mutants wereexamined: abd-AM1, Antp25, bcd12, hkbA, bynapro (a null

allele), Df(2R)enE, dppH46, dpphr92, hh21, ptcIN108, Ubx1

and wgPY40. All of these alleles are null or strong hypo-morphic. Embryos were staged according to Campos-Ortega and Hartenstein (1985).

Results

Disruption of LR symmetry in the developing gut

The Drosophila embryonic gut is composed of threedifferent parts, the foregut, midgut and hindgut. Theseparts initially arise from invaginations of the anterior andposterior terminals of the blastoderm, largely with mirrorimage symmetry, and form a continuous tube by fusion(for reviews, see Skaer 1993; Lengyel & Liu 1998;Murakami et al. 1999). Until early stage 13, all parts areLR symmetric, with some dorsoventral bends. The firstLR asymmetric morphogenesis begins in the hindgutafter late stage 13. Until early stage 13, the hindgut hasLR symmetric morphology, with a dorsoventral bend(Fig. 1A). The orientation of the hindgut can be shownby staining with anti-En antibody, which marks dorsaldomain of the hindgut (Fig. 1A, lateral view; Takashimaand Murakami 2001). At late stage 13, the hindguttwists 90° in a left-handed orientation (Fig. 1B), resultingin the original dorsal and ventral domains coming toface the left and right sides of the body, respectively.During stage 15, the foregut, which also has bent

Fig. 1. Left-handed twist of the (A,B) hindgut, (C,D) foregut and (E,F) midgut of Drosophila melanogaster embryos. (A) Hindgut of astage 13 embryo stained for the En protein that marks the dorsal domain of the hindgut (arrow in the lateral view), which is situatedon the midline, bending dorsoventrally. The arrowhead indicates the ventral domain of the hindgut. (B) The hindgut twists in a left-handed (sinistral) orientation at stage 14. (C) Foregut of an enhancer-trap marker strain (PY1) at early stage 15 shows a dorsoventralbending. (D) The foregut twists in a left-handed (sinistral) orientation during stage 15. (E) The midgut of an enhancer-trap marker strain(PY258) at stage 16 is stained in brown. At early stage 16, the midgut is composed of four chambers that are tandemly aligned onthe midline, with dorsoventral bends. (F) The second chamber of the midgut shifts to the right at late stage 16.

Left–right asymmetry in the Drosophila gut 241

dorsoventrally in previous stages (Fig. 1C, lateral view),twists in a left-handed orientation (Fig. 1D), resulting ina sinistral helix (counterclockwise as viewed from themouth). The LR asymmetry of midgut morphogenesisbegins after late stage 16. The midgut primordium,which is formed by fusion of the anterior and posteriorendoderms that invaginate separately from both polesof the blastoderm, appears as four tandemly alignedchambers at stage 16, with constrictions at the junc-tions of the chambers resulting in dorsoventral bends(Fig. 1E). The LR asymmetry of the midgut first appearsas a skew of the relative position of the four chambers,with the second chamber shifting to the right (Fig. 1F).Except for the most posterior portion, the convolution

of the midgut tube becomes largely left-handed (sinis-tral) at stage 17. The posterior-most part of the fourthchamber bends both upward and forward, connectingto the hindgut (see Fig. 2).

This process of LR asymmetric gut formation is dia-grammatically illustrated in Fig. 2. Note that the orien-tation of the convolution of each part of the gut tube islargely left-handed (sinistral).

Spontaneous reversal of the LR axis of the gut

The LR asymmetric morphogenesis of the gut parts wasalmost invariant and a spontaneous reversal of thehandedness was very rare. The frequency of LR rever-sal of the foregut was 0.2% (four of 1735); that of themidgut, 0.4% (five of 1318); and that of the hindgut,0.6% (17 of 2658; Table 1). Except for the LR reversal,morphologic abnormalities were not found in theLR-reversed embryos (Fig. 3A–C). It could not be deter-mined whether the embryos undergoing LR reversal of

Fig. 2. Illustration of the process of left–right asymmetries in thedeveloping gut. Each panel is a dorsal or slightly oblique viewof the embryo. The gut is delineated with bold lines. The ante-rior side of embryo is to the left. Left panels illustrate the devel-opment of the foregut and hindgut. Right panels illustrate thedevelopment of the midgut. All parts of the gut form a left-handedconvolution, except for the posterior-most portion of the midgut,which bends both upward and forward and connects to thehindgut. St., stage.

Table 1. Frequency of the reversal of the twist orientation of gut parts in wild-type, bcd, hkb, byn and ptc embryos

Wild-type bcd* hkb† bynapro ptcForegut Midgut Hindgut Duplicated Hindgut Midgut Foregut Midgut

hindgut

No. embryos with normal orientation 1731 1313 2641 373 220 61 71 38(%) (99.8%) (99.6%) (99.4%) (68.9%) (58.0%) (45.5%) (66.4%) (59.4%)No. embryos with reversed orientation 4 5 17 109 159 73 36 26(%) (0.2%) (0.4%) (0.6%) (20.1%) (42.0%) (54.5%) (33.6%) (40.6%)

*There were 59 duplicated hindguts (11.0%) with an intermediate orientation. †Hindguts located in the right side of the yolk are arbi-trarily defined as ‘normal orientation’ in this table.

Fig. 3. Spontaneous left–right reversals of the (A) foregut, (B)midgut and (C) hindgut, as viewed from the dorsal side.Compare the orientation of each part to that in Fig. 1D, Fig. 1Band Fig. 1F, respectively.


the gut were viable or lethal. Because the develop-mental stages in which the LR handedness of each gutpart becomes recognizable are different among the gutparts, it was technically difficult to examine the correl-ation between LR reversals among the gut parts in allobserved reversal cases. All five embryos showing anLR reversal of the midgut exhibited normal LR handed-ness in the foregut and hindgut, and four embryosshowing an LR reversal of the hindgut had a normalforegut. These results, together with differences infrequencies of spontaneous reversals among gut parts, indicate that the reversals of each gut part occurindependently.

Conserved handedness in the duplicated hindgutof the bicoid mutant

The bicoid (bcd) mutation has been reported to showa transformation of the anterior terminus (acron) into theposterior one (telson), maintaining largely normalpolarity of the cuticle in the trunk. We examined theorientation of the twist of the duplicated hindgut in bcdembryos. The majority of duplicated hindguts, whichare formed in the anterior, showed a left-handed twist,resulting in rotational symmetry with original hindgut inthe posterior, although the incidence of reversal wasquite high (20.1%) when compared with that of spon-taneous reversal (0.6%). The numbers of bcd embryosshowing rotational symmetry (Fig. 4A), mirror-imagesymmetry (Fig. 4B) and intermediate orientations of theduplicated hindgut (Fig. 4C) were 373, 106 and 59,respectively (Table 1).

Left–right asymmetry in mutants of genesexpressed in the gut

In order to identify genes involved in determining theLR handedness of the Drosophila gut, we examined theeffects of mutations in various genes that have beenreported to be expressed in the gut (see Materials andMethods). In wg and dpp mutants, the overall mor-phology of the gut was strongly affected; hence, it wasdifficult to judge specific effects on the LR asymmetryin these mutants. Some genes of the homeotic genecomplex (HOM-C) are expressed in the visceral meso-derm (circular muscle precursor) of the midgut in a non-overlapping adjacent manner, and are essential forregional differentiation (for a review, see Bienz 1994).In HOM-C mutants, particular midgut constrictions failto form: Antp is necessary for the anterior constriction,Ubx and abd-A for the middle constriction and abd-Afor the posterior constriction (Tremml & Bienz 1989;Reuter & Scott 1990). We examined the handednessof the midgut in HOM-C mutant embryos. Although the

overall morphology of the midgut was abnormal,because of the lack of particular constriction(s), the LRasymmetry was not affected in any of the mutantsexamined (Antp, Ubx, and abd-A). The hh mutant, inwhich the posterior-most portion of the hindgut degen-erates (Takashima & Murakami 2001), showed normalhandedness of the gut parts. The en mutant(Df(2R)enE), in which the ventral domain of hindguttransforms into the dorsal domain (Takashima andMurakami 2001), also showed normal handedness ofthe hindgut. Among the mutants examined, hkb, byn,and ptc mutants showed reversal of particular parts ofthe gut, as described below.

No twist of the hindgut in the hkb mutant

hkb is expressed (LR symmetrically) in the prospectiveendoderm and its mutation causes transformation ofthe midgut into part of the hindgut (Brönner et al. 1994;

Fig. 4. Orientation of the duplicated hindgut in bcd embryos.The hindgut is stained in brown. Duplicated hindguts, which canbe judged by the cuticular pattern of outer body segments, areto the left. A majority of the duplicated hindguts show rotationalsymmetry with original hindgut when viewed dorsally, keeping anormal left-handed twist (A). A smaller number of embryos showa (B) mirror-image symmetry or (C) intermediate orientation ofthe duplicated hindgut.


Reuter & Leptin 1994). The transformed portion of thehindgut becomes incorporated into the innate hindgutprimordium and together they form an elongatedhindgut (T. Hamaguchi and R. Murakami, unpubl. data,1999). In homozygous hkb embryos, the position of thehindgut with respect to the yolk appeared randomized(Table 1; Fig. 5A,B; L:R � 159:220). However, immuno-staining for the En/Inv proteins, which are expressedin dorsal subdomains of the hindgut, revealed that thehindgut of hkb embryos does not twist in either a leftor a right orientation (Fig. 5C). The morphology of thehindgut of heterozygous embryos (hkb/+) was normal.

Randomized handedness of the midgut in the bynmutant

byn (synonymous with apro) is an ortholog of the ver-tebrate Brachyury gene (Kispert et al. 1994; Murakamiet al. 1995; Singer et al. 1996). byn is expressed LRsymmetrically in the primordium of the hindgut and alsoin the longitudinal visceral muscle precursors of themidgut, and is essential for the development of thesetissues (Kusch & Reuter 1999). In homozygous bynmutants, the hindgut and the longitudinal visceralmuscles of the midgut fail to form, but the circularmuscles of the midgut do form. Development of themidgut of byn embryos appeared largely normal in thelate stages, and the LR asymmetric convolution wasrecognizable. However, the handedness of the convo-lution of the midgut of the byn embryos (which is left-handed in wild-type embryos) was found to be almost

randomized (Fig. 5D). The numbers of left-handed(normal) and right-handed (reversal) midguts were 61and 73, respectively (Table 1). The midguts of hetero-zygous embryos (byn/+) showed a normal convolutionpattern.

Left–right reversal of the foregut and midgut in theptc mutant

ptc is expressed in subdomains of the foregut andhindgut LR symmetrically (S. Takashima et al., unpubl.data, 2000). The handedness of the foregut andmidgut of homozygous ptc embryos was found to bereversed at high frequency (~34% and 41%, respec-tively; (Fig. 5E,F; Table 1). Although ptc is alsoexpressed in the hindgut, the orientation of the hindguttwist was normal. Heterozygous embryos showednormal hindgut morphology.

Discussion

The twist of the gut tube results in LR asymmetry

In the present study, the process of LR asymmetricmorphogenesis of the Drosophila gut was examined indetail. The most important findings are that each partof the gut; that is, the foregut, midgut, and hindgut,shows invariant LR asymmetry after forming tubularstructures, and that the asymmetry emerges as a twistof the dorsoventrally bent tube to particular orientations,rather than resulting from a different growth pattern orcell differentiation between the left and right sides of

Fig. 5. Disorder of the left–right (LR) asymmetry of the (A–C) hindgut of hkb embryos, (D) midgut of a bynapro embryo and (E,F) foregutand midgut of ptc embryos. When viewed dorsally, the left–right positions of the hindguts of hkb embryos appear to be completelyrandomized (A,B), but anti-En immunostaining, which stains the dorsal domain of the hindgut, reveals that the hindgut does not rotatein either the left or the right orientation. (C) The outline of the hindgut domains is marked with white lines. Arrow, dorsal domain; arrow-head, ventral domain. Approximately 55% of byn embryos show LR reversal of the midgut (D, compare with Fig. 1B). In ptc embryos,the foregut (E) and midgut (F) show LR reversal at high frequencies, ~34% and 41%, respectively.


the gut tube. The LR asymmetry of internal organs hasbeen studied in C. elegans, in which the LR axis isdetermined at very early cleavages essentially as aresult of a biased skew of the position of cytoskeletalcomponents, including spindles (Wood 1998).Recently, it has been reported that a LR asymmetriccell-to-cell interaction in the C. elegans embryo causesLR asymmetric gene expression that leads to anoriented twist of the gut (Hermann et al. 2000). InDrosophila, LR asymmetry of the gut appears in the latestages of organogenesis, only after the gut primordiaform a continuous tube. Because there is no experi-mental or genetic evidence, it cannot thus far be deter-mined whether the LR asymmetry of the Drosophila gutis determined at an early phase of nuclear division orat later stages of organogenesis. In Xenopus laevis, thetreatment of late neurula embryos with calciumionophores or Activin protein causes an LR reversal ofthe rotation of the gut and heart (Toyoizumi et al. 1997;Toyoizumi et al. 2000), indicating that a critical periodfor LR determination also exists at a late stage oforganogenesis.

Conserved LR asymmetry in the duplicatedhindgut

The orientation of the convolution of the Drosophila gutas a whole is left-handed (sinistral). In most of the dupli-cated hindguts of bcd embryos, the left-handed orien-tation of the twist was found to be maintained. Thisresult suggests that the LR asymmetry of the Drosophilagut originates from some intrinsic nature of the cells ortissues, as has previously been proposed as a gener-alized hypothesis (Brown & Wolpert 1990), rather thanan LR asymmetric distribution of some morphogen inthe egg. It is noteworthy that the frequency of LR rever-sal of the duplicated hindgut was very high (~20%).Some cytoplasmic condition that is established by theposterior group genes may also be required for normalLR handedness.

Several genes may independently affect LRasymmetry of the gut

By examining various mutants, we found that the threemutations, hkb, byn, and ptc, affect LR asymmetry ofparticular parts of the gut. The spatiotemporal expres-sion patterns of these genes in the gut are quite differ-ent from each other and, at present, it is difficult tohypothesize a common mechanism or pathway of geneaction for these genes. In the present study, we did notenter into the mechanism of LR reversals in thesemutants. The following are speculations on the LR dis-orders of these mutants.

In hkb embryos, the prospective posterior endodermtransforms into the ectodermal hindgut, being incor-porated into the innate hindgut tissues. The anterior endof the hindgut, which connects to the midgut in wild-type embryos, loses its mechanical foothold becauseof the lack of fusion with anteriorly invaginated gut parts(Murakami et al. 1995). This morphologic defect mayresult in the loss of twist. Another possibility is that incor-poration of the transformed midgut cells into the innatehindgut may disorganize the normal tissue arrange-ment that may be required for generating a biasedrotation.

In the case of byn mutants, the handedness of themidgut was randomized. byn has been identified as agene essential for determination of the hindgut (Kispertet al. 1994; Murakami et al. 1995; Singer et al. 1996).Recently, Kusch and Reuter (1999) have reported thatbyn is also essential for the development of the longi-tudinal visceral muscles of the midgut, while circularmuscles, another component of the visceral muscles,form normally in byn mutants. Contraction of the circu-lar visceral muscles has been suggested to be impor-tant in generating midgut constrictions (Reuter & Scott1990). It is tempting to speculate that the circular mus-cles are responsible for defining the sites of bending inthe midgut tube and that the longitudinal muscles restrictthe orientation of convolution by their contractility. Thepossibility, however, that degeneration of the hindgutcauses a mechanistic disorder that affects the normalmorphogenesis of the midgut cannot be excluded.

The ptc mutants showed very high frequencies of LRreversals of the foregut and midgut. Except for the LR reversals, the gross morphology of the gut tubeappears largely normal in these mutants. The LRreversal was not observed in the hindgut, although ptcis expressed in a portion of the hindgut as well. Atpresent, it is difficult to figure out a mechanism of LRdisorder in ptc mutants.

The phenotypes of the three mutations describedcould potentially be important clues for the study of LRasymmetry. At this moment, it can be said that variousdifferent mutations seem independently to affect the LRasymmetry of the Drosophila gut. Analysis of the struc-tural aspects of the cells and tissues, such as possiblebiased arrays of cytoskeletal components or biasedarrangements of cells, seems to be critical to under-standing the mechanism of the unidirectional twist ofthe gut tube.

Left–right asymmetry without asymmetric geneexpression?

It is noteworthy that, despite their influence on LRasymmetry, the hkb, byn and ptc genes are all


expressed LR symmetrically in the gut. After theinvention of the enhancer-trap method, which enablesus to visualize the spatial expression pattern ofenhancer activities (O’Kane & Gehring 1987), a tre-mendous number of expression patterns of unidenti-fied genes have been analyzed in Drosophila embryosall over the world. However, there has been no previ-ous report regarding LR asymmetric gene expression.It seems likely that the LR asymmetry of the Drosophilagut may be regulated by genes that are expressed LR symmetrically, as in the case of the products of the inv, iv and kinesin superfamily genes in mice embryos (Supp et al. 1997; Mochizuki et al. 1998;Morgan et al. 1998; Nonaka et al. 1998; Takeda et al.1999).

Because the gut has a very simple tissue architec-ture, being composed of an epithelial tube and the sur-rounding visceral muscles, this organ provides an idealsystem for the structural analysis of LR asymmetricmorphogenesis. The Drosophila gut will provide asimple and powerful system for genetic analysis of LRasymmetry.

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Left–right asymmetry in Drosophila melanogaster gut development