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Keywords:

  • bicoid;
  • byn;
  • Drosophila;
  • gut;
  • hkb;
  • left–right asymmetry

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

While left–right (LR) asymmetric morphogenesis is common to various animal species, there have been no systematic studies of the LR asymmetry of body structures of Drosophila melanogaster. In the present paper the 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 part in particular orientations, resulting in a left-handed (sinistral) convolution as a whole. The frequency of spontaneous reversal of LR orientations is very low (< 0.6%) and reversal of each part of the gut occurs independently. The bicoid mutation causes duplication of the posterior half of the gut, essentially keeping the left-handed twist, suggesting that the LR asymmetry may depend on some intrinsic nature of the cells or tissues rather than a graded distribution of morphogens in the egg. The handedness of particular gut parts was randomized or became symmetric in mutants of brachyenteron, huckebein and patched, suggesting that different gene pathways can interfere in determining LR asymmetry of the gut. It is noteworthy that all of these genes are expressed LR symmetrically.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

In recent years, there has been spectacular progress in the study of the mechanism of left–right (LR) asymmetric development in vertebrate (deuterostome) embryos. Since the discovery of LR asymmetric gene expression at the node of the chick embryo (Levin et al. 1995,1997), various genes involved in establishing LR asymmetry have been identified and an outline of the gene regulatory pathway that leads to LR asymmetry 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 LR determination has been suggested to be mediated by mictotubule-based cytoskeletal components, such as kinesin 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 events are primarily mediated by a cascade of LR asymmetric expression of extracellular signaling molecules (reviewed by Esteban et al. 1999; Yokouchi et al. 1999). The LR asymmetric gene expression finally leads to the morphologic asymmetry of internal organs.

In protostomes, however, only a small number of studies have been carried out on the mechanism of LR asymmetric development. Nematodes have been the most extensively exploited protostome animals for LR studies (for a review, see Wood 1998), in which the biased position of spindles has been demonstrated to cause the LR asymmetric arrangement of the early blastomeres (Wood 1991). Recently, Hermann et al. (2000) have demonstrated that the LR asymmetric arrangement of blastomeres of Caenorhabditis elegans results in asymmetric cell-to-cell interaction, leading to asymmetric gene expression.

In Drosophila melanogaster embryos, only a few studies have been performed with regard to LR asymmetry, with only very subtle or no LR asymmetry at all of outer body structures having been suggested (Tuinstra et al. 1990; Klingenberg et al. 1998). There has been neither a systematic description of the LR asymmetry of internal organs nor a report on LR asymmetric gene expression. During the study of Drosophila gut development, we (and perhaps other fly researchers) have found that the gut is invariantly LR asymmetric (see the atlas by Hartenstein 1993). The LR asymmetry of the Drosophila gut is generated by twisting of the initially LR symmetric gut tube in particular orientations, which results in an invariant LR asymmetric pattern of convolution. The invariant convolution pattern of the gut tube is also quite common to many vertebrates, suggesting the existence of some common basic mechanism. The present report provides the first systematic description of LR asymmetric development of the Drosophila gut. In addition, we report mutations that affect the handedness of the gut.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

Fly strains

Enhancer-trap strains (PY1, PY258, PY282) that have lacZ expression in specific regions of the gut (Murakami et al. 1994) were used for the observation of normal LR asymmetric morphogenesis. Embryos were stained for the histochemical or immunohistochemical detection of β-galactosidase and were examined under a dissection microscope or with a Nomarski differential interference microscope. Immunostaining with anti-Crumbs (Crb) antibody, which stains luminal surface of the hindgut epithelium (Tepass et al. 1990), and with anti-Engrailed (En) antibody, which stains dorsal domain of the hindgut, was also carried out. y w and Canton-S strains, as well as the enhancer-trap strains, were used for in situ hybridization for the observation of normal gut development. Mutants with morphologic defects or homeotic transformation of particular regions of the gut were also used to investigate influences on LR asymmetric morphogenesis. The following mutants were examined: 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 hypomorphic. Embryos were staged according to Campos-Ortega and Hartenstein (1985).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

Disruption of LR symmetry in the developing gut

The Drosophila embryonic gut is composed of three different parts, the foregut, midgut and hindgut. These parts initially arise from invaginations of the anterior and posterior terminals of the blastoderm, largely with mirror image 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 are LR symmetric, with some dorsoventral bends. The first LR asymmetric morphogenesis begins in the hindgut after late stage 13. Until early stage 13, the hindgut has LR symmetric morphology, with a dorsoventral bend (Fig. 1A). The orientation of the hindgut can be shown by staining with anti-En antibody, which marks dorsal domain of the hindgut (Fig. 1A, lateral view; Takashima and Murakami 2001). At late stage 13, the hindgut twists 90° in a left-handed orientation (Fig. 1B), resulting in the original dorsal and ventral domains coming to face the left and right sides of the body, respectively. During stage 15, the foregut, which also has bent dorsoventrally in previous stages (Fig. 1C, lateral view), twists in a left-handed orientation (Fig. 1D), resulting in a sinistral helix (counterclockwise as viewed from the mouth). The LR asymmetry of midgut morphogenesis begins after late stage 16. The midgut primordium, which is formed by fusion of the anterior and posterior endoderms that invaginate separately from both poles of the blastoderm, appears as four tandemly aligned chambers at stage 16, with constrictions at the junctions of the chambers resulting in dorsoventral bends (Fig. 1E). The LR asymmetry of the midgut first appears as 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 (sinistral) at stage 17. The posterior-most part of the fourth chamber bends both upward and forward, connecting to the hindgut (see Fig. 2).

image

Figure 1. Left-handed twist of the (A,B) hindgut, (C,D) foregut and (E,F) midgut of Drosophila melanogaster embryos. (A) Hindgut of a stage 13 embryo stained for the En protein that marks the dorsal domain of the hindgut (arrow in the lateral view), which is situated on 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 dorsoventral bending. (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 on the midline, with dorsoventral bends. (F) The second chamber of the midgut shifts to the right at late stage 16.

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image

Figure 2. Illustration of the process of left–right asymmetries in the developing gut. Each panel is a dorsal or slightly oblique view of the embryo. The gut is delineated with bold lines. The anterior side of embryo is to the left. Left panels illustrate the development of the foregut and hindgut. Right panels illustrate the development of the midgut. All parts of the gut form a left-handed convolution, except for the posterior-most portion of the midgut, which bends both upward and forward and connects to the hindgut. St., stage.

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This process of LR asymmetric gut formation is diagrammatically illustrated in Fig. 2. Note that the orientation of the convolution of each part of the gut tube is largely left-handed (sinistral).

Spontaneous reversal of the LR axis of the gut

The LR asymmetric morphogenesis of the gut parts was almost invariant and a spontaneous reversal of the handedness was very rare. The frequency of LR reversal of the foregut was 0.2% (four of 1735); that of the midgut, 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 the LR-reversed embryos (Fig. 3A–C). It could not be determined whether the embryos undergoing LR reversal of the gut were viable or lethal. Because the developmental stages in which the LR handedness of each gut part becomes recognizable are different among the gut parts, it was technically difficult to examine the correlation between LR reversals among the gut parts in all observed reversal cases. All five embryos showing an LR reversal of the midgut exhibited normal LR handedness in the foregut and hindgut, and four embryos showing an LR reversal of the hindgut had a normal foregut. These results, together with differences in frequencies of spontaneous reversals among gut parts, indicate that the reversals of each gut part occur independently.

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*hkbbynaproptc
 ForegutMidgutHindgutDuplicatedHindgutMidgutForegutMidgut
    hindgut    
  1. *There were 59 duplicated hindguts (11.0%) with an intermediate orientation. Hindguts located in the right side of the yolk are arbitrarily defined as ‘normal orientation’ in this table.

No. embryos with normal orientation173113132641373220617138
(%)(99.8%)(99.6%)(99.4%)(68.9%)(58.0%)(45.5%)(66.4%)(59.4%)
No. embryos with reversed orientation4517109159733626
(%)(0.2%)(0.4%)(0.6%)(20.1%)(42.0%)(54.5%)(33.6%)(40.6%)
image

Figure 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. 1B and Fig. 1F, respectively.

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Conserved handedness in the duplicated hindgut of the bicoid mutant

The bicoid (bcd) mutation has been reported to show a transformation of the anterior terminus (acron) into the posterior one (telson), maintaining largely normal polarity of the cuticle in the trunk. We examined the orientation of the twist of the duplicated hindgut in bcd embryos. The majority of duplicated hindguts, which are formed in the anterior, showed a left-handed twist, resulting in rotational symmetry with original hindgut in the posterior, although the incidence of reversal was quite high (20.1%) when compared with that of spontaneous reversal (0.6%). The numbers of bcd embryos showing rotational symmetry (Fig. 4A), mirror-image symmetry (Fig. 4B) and intermediate orientations of the duplicated hindgut (Fig. 4C) were 373, 106 and 59, respectively (Table 1).

image

Figure 4. Orientation of the duplicated hindgut in bcd embryos. The hindgut is stained in brown. Duplicated hindguts, which can be judged by the cuticular pattern of outer body segments, are to the left. A majority of the duplicated hindguts show rotational symmetry with original hindgut when viewed dorsally, keeping a normal left-handed twist (A). A smaller number of embryos show a (B) mirror-image symmetry or (C) intermediate orientation of the duplicated hindgut.

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Left–right asymmetry in mutants of genes expressed in the gut

In order to identify genes involved in determining the LR handedness of the Drosophila gut, we examined the effects of mutations in various genes that have been reported to be expressed in the gut (see Materials and Methods). In wg and dpp mutants, the overall morphology of the gut was strongly affected; hence, it was difficult to judge specific effects on the LR asymmetry in these mutants. Some genes of the homeotic gene complex (HOM-C) are expressed in the visceral mesoderm (circular muscle precursor) of the midgut in a non-overlapping adjacent manner, and are essential for regional differentiation (for a review, see Bienz 1994). In HOM-C mutants, particular midgut constrictions fail to form: Antp is necessary for the anterior constriction, Ubx and abd-A for the middle constriction and abd-A for the posterior constriction (Tremml & Bienz 1989; Reuter & Scott 1990). We examined the handedness of 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 LR asymmetry was not affected in any of the mutants examined (Antp, Ubx, and abd-A). The hh mutant, in which the posterior-most portion of the hindgut degenerates (Takashima & Murakami 2001), showed normal handedness of the gut parts. The en mutant (Df(2R)enE), in which the ventral domain of hindgut transforms into the dorsal domain (Takashima and Murakami 2001), also showed normal handedness of the hindgut. Among the mutants examined, hkb, byn, and ptc mutants showed reversal of particular parts of the gut, as described below.

No twist of the hindgut in the hkb mutant

hkb is expressed (LR symmetrically) in the prospective endoderm and its mutation causes transformation of the midgut into part of the hindgut (Brönner et al. 1994; Reuter & Leptin 1994). The transformed portion of the hindgut becomes incorporated into the innate hindgut primordium and together they form an elongated hindgut (T. Hamaguchi and R. Murakami, unpubl. data, 1999). In homozygous hkb embryos, the position of the hindgut with respect to the yolk appeared randomized (Table 1; Fig. 5A,B; L:R = 159:220). However, immunostaining for the En/Inv proteins, which are expressed in dorsal subdomains of the hindgut, revealed that the hindgut of hkb embryos does not twist in either a left or a right orientation (Fig. 5C). The morphology of the hindgut of heterozygous embryos (hkb/+) was normal.

image

Figure 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) foregut and midgut of ptc embryos. When viewed dorsally, the left–right positions of the hindguts of hkb embryos appear to be completely randomized (A,B), but anti-En immunostaining, which stains the dorsal domain of the hindgut, reveals that the hindgut does not rotate in either the left or the right orientation. (C) The outline of the hindgut domains is marked with white lines. Arrow, dorsal domain; arrowhead, 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.

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Randomized handedness of the midgut in the byn mutant

byn (synonymous with apro) is an ortholog of the vertebrate Brachyury gene (Kispert et al. 1994; Murakami et al. 1995; Singer et al. 1996). byn is expressed LR symmetrically in the primordium of the hindgut and also in the longitudinal visceral muscle precursors of the midgut, and is essential for the development of these tissues (Kusch & Reuter 1999). In homozygous byn mutants, the hindgut and the longitudinal visceral muscles of the midgut fail to form, but the circular muscles of the midgut do form. Development of the midgut of byn embryos appeared largely normal in the late stages, and the LR asymmetric convolution was recognizable. However, the handedness of the convolution 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 61 and 73, respectively (Table 1). The midguts of heterozygous embryos (byn/+) showed a normal convolution pattern.

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

ptc is expressed in subdomains of the foregut and hindgut LR symmetrically (S. Takashima et al., unpubl. data, 2000). The handedness of the foregut and midgut of homozygous ptc embryos was found to be reversed at high frequency (~34% and 41%, respectively; (Fig. 5E,F; Table 1). Although ptc is also expressed in the hindgut, the orientation of the hindgut twist was normal. Heterozygous embryos showed normal hindgut morphology.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

The twist of the gut tube results in LR asymmetry

In the present study, the process of LR asymmetric morphogenesis of the Drosophila gut was examined in detail. The most important findings are that each part of the gut; that is, the foregut, midgut, and hindgut, shows invariant LR asymmetry after forming tubular structures, and that the asymmetry emerges as a twist of the dorsoventrally bent tube to particular orientations, rather than resulting from a different growth pattern or cell differentiation between the left and right sides of the gut tube. The LR asymmetry of internal organs has been studied in C. elegans, in which the LR axis is determined at very early cleavages essentially as a result of a biased skew of the position of cytoskeletal components, including spindles (Wood 1998). Recently, it has been reported that a LR asymmetric cell-to-cell interaction in the C. elegans embryo causes LR asymmetric gene expression that leads to an oriented twist of the gut (Hermann et al. 2000). In Drosophila, LR asymmetry of the gut appears in the late stages of organogenesis, only after the gut primordia form a continuous tube. Because there is no experimental or genetic evidence, it cannot thus far be determined whether the LR asymmetry of the Drosophila gut is determined at an early phase of nuclear division or at later stages of organogenesis. In Xenopus laevis, the treatment of late neurula embryos with calcium ionophores or Activin protein causes an LR reversal of the rotation of the gut and heart (Toyoizumi et al. 1997; Toyoizumi et al. 2000), indicating that a critical period for LR determination also exists at a late stage of organogenesis.

Conserved LR asymmetry in the duplicated hindgut

The orientation of the convolution of the Drosophila gut as a whole is left-handed (sinistral). In most of the duplicated hindguts of bcd embryos, the left-handed orientation of the twist was found to be maintained. This result suggests that the LR asymmetry of the Drosophila gut originates from some intrinsic nature of the cells or tissues, as has previously been proposed as a generalized hypothesis (Brown & Wolpert 1990), rather than an LR asymmetric distribution of some morphogen in the egg. It is noteworthy that the frequency of LR reversal of the duplicated hindgut was very high (~20%). Some cytoplasmic condition that is established by the posterior group genes may also be required for normal LR handedness.

Several genes may independently affect LR asymmetry of the gut

By examining various mutants, we found that the three mutations, hkb, byn, and ptc, affect LR asymmetry of particular parts of the gut. The spatiotemporal expression patterns of these genes in the gut are quite different from each other and, at present, it is difficult to hypothesize a common mechanism or pathway of gene action for these genes. In the present study, we did not enter into the mechanism of LR reversals in these mutants. The following are speculations on the LR disorders of these mutants.

In hkb embryos, the prospective posterior endoderm transforms into the ectodermal hindgut, being incorporated into the innate hindgut tissues. The anterior end of the hindgut, which connects to the midgut in wild-type embryos, loses its mechanical foothold because of the lack of fusion with anteriorly invaginated gut parts (Murakami et al. 1995). This morphologic defect may result in the loss of twist. Another possibility is that incorporation of the transformed midgut cells into the innate hindgut may disorganize the normal tissue arrangement that may be required for generating a biased rotation.

In the case of byn mutants, the handedness of the midgut was randomized. byn has been identified as a gene essential for determination of the hindgut (Kispert et al. 1994; Murakami et al. 1995; Singer et al. 1996). Recently, Kusch and Reuter (1999) have reported that byn is also essential for the development of the longitudinal visceral muscles of the midgut, while circular muscles, another component of the visceral muscles, form normally in byn mutants. Contraction of the circular visceral muscles has been suggested to be important in generating midgut constrictions (Reuter & Scott 1990). It is tempting to speculate that the circular muscles are responsible for defining the sites of bending in the midgut tube and that the longitudinal muscles restrict the orientation of convolution by their contractility. The possibility, however, that degeneration of the hindgut causes a mechanistic disorder that affects the normal morphogenesis of the midgut cannot be excluded.

The ptc mutants showed very high frequencies of LR reversals of the foregut and midgut. Except for the LR reversals, the gross morphology of the gut tube appears largely normal in these mutants. The LR reversal was not observed in the hindgut, although ptc is expressed in a portion of the hindgut as well. At present, it is difficult to figure out a mechanism of LR disorder in ptc mutants.

The phenotypes of the three mutations described could potentially be important clues for the study of LR asymmetry. At this moment, it can be said that various different mutations seem independently to affect the LR asymmetry of the Drosophila gut. Analysis of the structural aspects of the cells and tissues, such as possible biased arrays of cytoskeletal components or biased arrangements of cells, seems to be critical to understanding the mechanism of the unidirectional twist of the gut tube.

Left–right asymmetry without asymmetric gene expression?

It is noteworthy that, despite their influence on LR asymmetry, the hkb, byn and ptc genes are all expressed LR symmetrically in the gut. After the invention of the enhancer-trap method, which enables us to visualize the spatial expression pattern of enhancer activities (O’Kane & Gehring 1987), a tremendous number of expression patterns of unidentified genes have been analyzed in Drosophila embryos all over the world. However, there has been no previous report regarding LR asymmetric gene expression. It seems likely that the LR asymmetry of the Drosophila gut 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 architecture, being composed of an epithelial tube and the surrounding visceral muscles, this organ provides an ideal system for the structural analysis of LR asymmetric morphogenesis. The Drosophila gut will provide a simple and powerful system for genetic analysis of LR asymmetry.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References