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Keywords:

  • left–right asymmetry;
  • Myosin I;
  • Myosin31DF;
  • Myosin61F;
  • Drosophila melanogaster

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

In Drosophila, Myosin31DF (Myo31DF), encoding a Myosin ID protein, has crucial roles in left–right (LR) asymmetric development. Loss of Myo31DF function leads to laterality inversion for many organs, including the embryonic gut. Here, we found that Myo31DF was required before LR asymmetric morphogenesis in the hindgut, suggesting it functions in LR patterning instead of directly in hindgut morphological changes. Myosin61F (Myo61F) encodes another Myosin I, and Myo31DF or Myo61F overexpression reverses the laterality of different organs. Myo31DF and Myo61F have domains conserved in Myosin proteins, particularly in the proteins' head regions. We studied the roles of these domains in LR patterning using overexpression analysis. The Actin-binding and ATP-binding domains were essential for both proteins, but the IQ domains, binding sites for Myosin light chains, were required only by Myo31DF. Our results also suggest that the organ specificities of the Myo31DF and Myo61F activities depended on their head regions. Developmental Dynamics 237:3528–3537, 2008. © 2008 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Although bilateral animals appear to have LR symmetry from the outside, their internal organs often show directional and stereotypical LR asymmetry. The mechanisms by which the LR axis is established in vertebrates have been extensively studied. For example, in mice, the initial LR symmetry is broken by a directional extracellular flow (nodal flow) in the embryonic node (Nonaka et al.,1998; Okada et al.,1999). The nodal flow is generated by the clockwise rotation of monocilia, called the nodal cilia (Nonaka et al.,1998). Nodal cilia are found in various vertebrates, suggesting that their function in LR patterning is conserved among vertebrates (Essner et al.,2002). On the other hand, in invertebrates, the mechanisms of LR asymmetric development remain largely unknown, although some excellent studies have been conducted in nematodes and fresh water snails (Wood,1991,1998; Bergmann et al.,2003; Ueshima and Asami,2003; Shibazaki et al.,2004; Sagasti,2007).

Recently, developmental and genetic analyses of the directional LR asymmetry in Drosophila melanogaster were initiated (Spéder et al.,2007; Taniguchi et al.,2007a). This species has several organs that develop with stereotypical LR asymmetry, including the gut, genitalia, testes, spermiduct, and adult brain (Strasburger,1932; Gleichauf,1936; Miller,1950; Hayashi and Murakami,2001; Ligoxygakis et al.,2001; Ádám et al.,2003; Pascual et al.,2004; Hayashi et al.,2005; Hozumi et al.,2006; Spéder et al.,2006). LR patterning probably takes place during embryogenesis, because the forced reversal of the anterior–posterior axis of the egg relative to that of the oocyte does not affect the laterality of the embryo (Hayashi et al.,2005). In addition, several mutations that affect the laterality of some of these organs have been identified (Ádám et al.,2003; Hozumi et al.,2006; Spéder et al.,2006; Maeda et al.,2007; Taniguchi et al.,2007b). For example, the handedness of the Drosophila embryonic gut is affected in single-minded (sim) mutant embryos, which lack the ventral midline structure, suggesting that the midline structures are involved in its LR asymmetric development (Maeda et al.,2007). In addition, mutant embryos of puckered (puc), which encodes a Drosophila Jun N-terminal kinase (D-JNK) phosphatase, show random laterality of the anterior midgut, indicating that D-JNK signaling is involved in the LR asymmetric looping of this organ (Taniguchi et al.,2007b).

Among the Drosophila mutations that affect directional LR asymmetry, Myosin31DF (Myo31DF), which encodes a type ID Myosin (Morgan et al.,1995; Gillespie et al.,2001), is the only one that causes the reversal of both embryonic and adult laterality (Hozumi et al.,2006; Spéder et al.,2006). For example, in adult Myo31DF mutant flies, the laterality of the testis, hindgut, spermiduct, and genitalia is reversed (Hozumi et al.,2006; Spéder et al.,2006). Thus, Myo31DF is required for the normal laterality of Drosophila organs at all life-cycle stages, even though most adult organs are reconstructed during the pupal stage. Myo31DF homozygotes are viable and fertile, suggesting that Myo31DF functions specifically in LR asymmetric development (Hozumi et al.,2006; Spéder et al.,2006). Myo31DF is required in the epithelial cells of the embryonic hindgut, where its product colocalizes with Actin filaments, for normal LR asymmetry (Hozumi et al.,2006). Furthermore, the overexpression in the hindgut epithelium of a dominant-negative form of Rho1 or Rac1, which are known to control the organization of Actin filaments, causes LR defects in the hindgut (Hozumi et al.,2006). Based on these observations, we previously proposed that the Actin-based functions of type I Myosins play critical roles in generating LR asymmetry.

Besides Myo31DF, two other Myosin I family genes have been identified in the Drosophila melanogaster genome, Myosin61F (Myo61F) and Myosin95E (Morgan et al.,1994,1995; Tzolovsky et al.,2002). Of the two, Myo61F, which is also referred to as Myosin IC, shares the most homology with Myo31DF (Morgan et al.,1994). Although the involvement of Myo61F in LR asymmetric development has not been studied using a loss-of-function Myo61F mutant, its overexpression results in the reversal of the LR asymmetry of the hindgut (Hozumi et al.,2006). In contrast, the overexpression of Myo31DF in embryos reverses the LR asymmetry only of the foregut (Hozumi et al.,2006). Thus, Myo31DF and Myo61F have distinct organ-specific activities in LR asymmetric development, even though all of the identified domains are well conserved between these two Myosin I proteins.

In this study, first, we further analyzed the developmental roles of Myo31DF in LR asymmetric development of the embryonic gut. The developmental period during which Myo31DF is required for normal laterality was determined. Second, using an overexpression approach, we sought to determine the roles of the conserved domains in Myo31DF and Myo61F in the organ-specific LR asymmetry reversal activities of these proteins. Various mutant forms of Myo31DF and Myo61F that lacked the conserved domains were overexpressed. We showed that these domains had essential and distinct roles in the activities of the two proteins. In addition, we examined the activities of chimeric forms of the proteins in which their head and IQ/tail regions were swapped. Our results suggest that the respective head regions of these two Myosin I proteins are responsible for the organ-specificity of their LR-reversing activity.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Myo31DF Function Is Essential for Normal Laterality Before LR Asymmetric Morphogenesis

Two possible models for how Myo31DF might function can explain the inversion of laterality in this mutant. First, Myo31DF might act to establish LR polarity in the embryo, organs, or cells. We previously showed that the zygotic function of Myo31DF is required and sufficient for normal LR asymmetry of the embryonic gut (Hozumi et al.,2006). Therefore, we speculate that Myo31DF is required for normal laterality during embryogenesis, but before the LR asymmetric morphogenesis. Second, Myo31DF may play a crucial role in the LR asymmetrical morphogenesis of the embryonic midgut and hindgut directly, instead of in the LR pattern formation. In this case, Myo31DF would probably be required during LR asymmetric morphogenesis. To distinguish between these two possibilities, we determined the phenocritical period at which Myo31DF is required for the normal handedness of the embryonic gut.

The TARGET system, which involves a temperature-sensitive GAL80 (GAL80ts) and GAL4-based overexpression system, allows a responder gene to be misexpressed in a spatially and temporally controlled manner (McGuire et al.,2003). We previously showed that the expression of Myo31DF-GFP, which encodes a GFP-tagged version of otherwise wild-type Myo31DF, from UAS-Myo31DF-GFP in the hindgut and posterior midgut driven by byn-GAL4, is sufficient to rescue the LR defects associated with the Myo31DF mutant (Hozumi et al.,2006). Thus, in this study, we overexpressed Myo31DF-GFP in a spatially and temporally controlled manner using byn-GAL4 and the TARGET system.

Embryos were collected for either 30 (yellow dots) or 60 (blue dots) min, and cultured further at 18°C, followed by a temperature shift to 32°C for 60 min at various time points after egg laying (AEL; Fig. 1A) to drive the expression of Myo31DF-GFP. Based on the fluorescence from the Myo31DF-GFP protein, 60 min was sufficient to induce its synthesis at 32°C (data not shown). Even in control embryos that were raised at 18°C throughout embryogenesis, we observed a slight rescue of LR inversion (45%) in animals carrying Myo31DF-GFP (Fig. 1A, green square), because UAS-Myo31DF-GFP expression is leaky in the absence of GAL4 driver (6/15, 40%). We found that a 60-min temperature shift at 10–12.75 hr AEL effectively rescued the LR defects of the Myo31DF mutant (Fig. 1A). However, it was difficult to determine precisely when Myo31DF became essential for normal laterality, because the efficiency of rescuing the LR defects gradually increased up to 12 hr AEL (Fig. 1A). Nevertheless, the LR defects of embryos that underwent the temperature shift at 12 hr AEL were almost completely suppressed (Fig. 1A). In contrast, the ability of Myo31DF-GFP expression to rescue the LR defects decreased rapidly after 13 hr AEL, indicating that Myo31DF is dispensable for the normal LR asymmetric development after 13 hr AEL (Fig. 1A). Considering that a 60-min temperature shift was sufficient for producing Myo31DF-GFP, it is likely that Myo31DF is required up to 13 hr AEL, at the latest.

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Figure 1. Determination of the phenocritical period for Myo31DF function in normal left–right (LR) asymmetric development of the hindgut. A,B: Using the TARGET system, GAL80ts is expressed ubiquitously, and the specific production of GAL4 in the hindgut and posterior midgut primordia is driven by byn-GAL4. In this experiment, the expression of UAS-Myo31DF-GFP was suppressed at 18°C and induced at 32°C. A: The embryos were collected for either 30 (yellow dots) or 60 min (blue dots) and cultured at 18°C, followed by a temperature shift to 32°C for 60 min at various times after egg laying (AEL), indicated on the horizontal axis. Embryos that were cultured at 18°C throughout embryogenesis served as a negative control (green square). The mean deviation is indicated by vertical lines. B: The developmental stage at which the expression of Myo31DF showed the efficient rescue of LR defects was determined. The developmental stages of embryos subjected to a temperature shift to 32°C for 60 min at 12.75 hr AEL are shown in the bar graph. Results obtained from two independently performed experiments are shown as two bars. CF: Lateral views of embryos of the corresponding stages: early stage 12 (C), early germband retraction (D), late germband retraction (E), and stage 13 (F) are shown. Anterior is to the left.

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To reveal the developmental stage at which Myo31DF is required for normal LR asymmetric development, we studied the embryos at 12.75 hr AEL in detail. This was the latest period at which initiating a 60-min temperature shift resulted in the efficient rescue of the laterality defects by Myo31DF-GFP expression (Fig. 1A). After this temperature shift at 12.75 hr AEL, the embryos were stained with an anti-Crumbs antibody to visualize the outline of the embryos and determine their developmental stages (Fig. 1B–F). We found that most of these embryos were at the late stage of germband retraction (Fig. 1B,E). These results suggest that Myo31DF-GFP was required for normal LR asymmetry before the completion of germband retraction, which precedes LR asymmetric morphogenesis of the hindgut, at late stage 13. We, therefore, speculate that Myo31DF does not contribute directly to the LR asymmetric morphogenesis.

Visceral Mesoderm of the Hindgut Is Not Required for Its Normal Laterality During LR Asymmetric Morphogenesis

The hindgut is composed of ectodermal epithelium and visceral muscles (Lengyel and Iwaki,2002). We previously showed that the expression of Myo31DF in the epithelium, but not in the visceral mesoderm of the hindgut or any other embryonic organ, is required for normal LR asymmetry of the midgut and hindgut (Hozumi et al.,2006). Therefore, we speculated that the hindgut epithelium alone is sufficient for normal LR asymmetric development. To test this hypothesis, we examined LR defects in embryos homozygous for the heartless (htl) mutation, which have a defect in the development of the hindgut visceral mesoderm (San Martin and Bate,2001). In wild-type embryos, the primordium of the hindgut visceral muscles was detected by anti-Connectin (Con) antibody staining (Fig. 2A–D,A′–D′,I). In contrast, in htls1-28 homozygous embryos, the hindgut epithelial tube was not covered by visceral mesoderm, although Con-positive cells were detected before the early germband retraction stage, at lower levels than in wild-type (Fig. 2E–H,E′–H′, arrowhead). The lack of visceral muscles was confirmed by anti-Con antibody staining at late stage 13, when LR asymmetric development of the hindgut was completed (Fig. 2J). However, we found that these htls1-28 homozygous embryos showed normal handedness in the hindgut (Fig. 2J). These results suggest that the visceral mesoderm of the hindgut is not required for the normal laterality of this organ during its LR asymmetric morphogenesis.

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Figure 2. Hindgut epithelium is sufficient for normal left–right (LR) asymmetric development. Top: Visceral mesoderm of wild-type and htls1-28 mutant embryos. AH: Dorsal views of the wild-type (A–D) and htls1-28 mutant embryos (E–H) at various stages were stained with an anti-Con antibody. A′–H′: Schematic diagrams of embryo lateral views showing the Con-positive cells in A–H (blue). A,A′,E,E′: Stage 12. B,B′,F,F′: Early germband retraction. C,C′,G,G′: Late germband retraction. D,D′,H,H′: Stage 13. Bottom: The frequency of wild-type and htls1-28 mutant embryos showing normal LR asymmetry at late stage 13. The numbers of examined embryos are shown in parentheses. I,J: The visceral mesoderm surrounding the hindgut was detected by anti-Con staining at the late stage 13 in wild-type (I) and htls1-28 mutant (J) embryos.

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Conserved Domains of Myo31DF and Myo61F Are Essential for Their LR Inversion-Inducing Activities

Myosin I family proteins are composed of a highly conserved head region and a relatively divergent tail region (Fig. 3A) (Morgan et al.,1994). The head region contains several small conserved domains: an ATP-binding domain (which includes a P-loop, switch1-loop, and switch2-loop), and an Actin-binding domain (Walker et al.,1982; Pollard et al.,1991; Shimada et al.,1997; Sasaki et al.,1998). The neck region contains IQ domains, which provide binding sites for Calmodulin and other Myosin light chains, although the binding of Myo31DF and Myo61F to these proteins has not been reported in Drosophila (Morgan et al.,1995). Myo31DF has two IQ domains, and Myo61F has three (Fig. 3A). To determine whether these domains are required for the functions of Myo31DF and Myo61F in LR asymmetric development, we synthesized wild-type and various mutant forms of these two proteins in vivo (Fig. 3A). The production of these proteins in the transgenic Drosophila lines was confirmed by Western blot analysis (Fig. 3B). Almost equivalent amounts of the proteins were produced in these lines, except for Myo31DFtail-HA and Myo61Ftail-HA, which were synthesized in lower amounts (Fig. 3B, asterisk).

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Figure 3. Functional analysis of the domains in Myo31DF and Myo61F. A: Schematic drawings of Myo31DF-HA, Myo61F-HA and various mutant forms of these two proteins. Myosin I family proteins are mainly composed of three major parts: the head, neck, and tail regions. The head region includes an ATP-binding domain (green) and an Actin-binding domain (red). The neck region contains two or three IQ domains (yellow), which are potential binding sites for Calmodulin or Myosin light chains. The tail region contains the Tail Homology (TH1) domain, which is less well conserved among myosin family proteins. The HA-tag was introduced at the C-terminus of the wild-type and mutant variants of Myo31DF and Myo61F. B: Western blot analysis for the wild-type and mutant derivatives of Myo31DF and Myo61F expressed in the transgenic Drosophila lines. The hs-GAL4 line was used for the misexpression of these proteins in whole larvae. Note that almost equivalent amounts of the proteins were produced, except for Myo31DFtail-HA and Myo61Ftail-HA, which were produced at lower levels than the others (asterisks).

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The Drosophila embryonic gut is composed of three major parts: the foregut, midgut, and hindgut. The da-GAL4-driven overexpression of Myo31DF-HA, but not of Myo61F-HA, induced LR inversion in the foregut (Figs. 4C, 5B′). In contrast, the byn-GAL4-driven overexpression of Myo61F-HA, but not of Myo31DF-HA, induced LR inversion in the midgut and hindgut (Figs. 4D, 5D,D′). Each domain in these two Myosin proteins was then analyzed for its ability to induce LR inversion under the overexpression conditions stated above. Myo31DFmATP bs-HA and Myo61FmATP bs-HA had amino acid substitutions in the P-loop, switch1-loop, and switch2-loop, all of which are known to be crucial for ATP binding (Fig. 3A, asterisks; Morgan et al.,1994; Shimada et al.,1997; Sasaki et al.,1998). We found that these two mutants failed to induce any of the LR defects under the conditions in which their wild-type counterparts induced LR inversion (Fig. 4A,B). In addition, the overexpression of Myo31DFΔP-loop-HA and Myo61FΔP-loop-HA, which had deletions only in the P-loop, did not induce the LR defects either (Fig. 3A, 4A, B). These results suggest that the ATP-binding ability of Myo31DF and Myo61F is essential for their activities in LR asymmetrical development.

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Figure 4. The frequency of left–right (LR) defects of the foregut and hindgut in embryos overexpressing wild-type and mutant forms of Myo31DF and Myo61F. A–D: The frequency of the LR defect in either the foregut or hindgut is shown in bar graphs. A: Overexpression of Myo31DF wild-type and mutant variants driven by da-GAL4, which caused the ubiquitous production of these proteins in embryos. B: Overexpression of Myo61F wild-type and mutant variants driven by byn-GAL4, which caused the production of these proteins in the hindgut. C,D: Overexpression of the wild-type Myo31DF and Myo61F and the chimeric proteins with the IQ/tail regions swapped between Myo31DF and Myo61F. Expression was driven by da-GAL4 (C) and byn-GAL4 (D).

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Figure 5. Left–right (LR) inversion of the embryonic gut induced by the overexpression of wild-type or chimeric Myo31DF and Myo61F proteins. A–E,A′–E′: The foregut and midgut (A′–E′) and hindgut (A–E), at stage 16 are shown. A,A′: Wild-type embryo. B,B′: Embryo overexpressing UAS-Myo31DF-HA driven by da-GAL4. C,C′: Embryo overexpressing UAS-Myo31DF-61FIQ/tail-HA driven by da-GAL4. D,D′: Embryo overexpressing UAS-Myo61F-HA driven by byn-GAL4. E,E′: Embryo overexpressing UAS-Myo61F-31DFIQ/tail-HA driven by byn-GAL4. In B′ and C′, the foregut showed reversed laterality (pink arrowhead, compare with that of the wild-type embryo in A′). In D, D′, E, and E′, the midgut and hindgut showed reversed laterality (midgut, blue brackets; hindgut, yellow brackets; compare with those of the wild-type embryo in A and A′). The chambers composing the midgut are labeled 1–4. A–E, dorsal view; A′–E′, ventral view. fg, foregut; mg, midgut; hg, hindgut; L, left; R, right.

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Next, we examined the activity of Myo31DFΔActin bs-HA and Myo61FΔActin bs-HA, both of which lack the Actin-binding domain (Fig. 3A). In agreement with a previous report demonstrating an interaction between these proteins and Actin in Drosophila (Morgan et al.,1995), Myo31DFΔActin bs-HA and Myo61FΔActin bs-HA were unable to induce the LR inversion (Fig. 4A,B). We also found that the overexpression of Myo31DFΔIQ-HA, which lacks the IQ domains, failed to induce the LR defects (Figs. 3A, 4A). In contrast, Myo61FΔIQ-HA retained the ability to induce the LR reversion, which was comparable to the activity of wild-type Myo61F-HA (Figs. 3A, 4B). These observations suggest that Myo31DF and Myo61F may have distinct requirements for Calmodulin or other Myosin light chains that are capable of binding the IQ domains.

In addition to the requirements of these domains for the activity of Myo31DF and Myo61F, we found that both the head and tail regions were essential for the activity of these two proteins. Myo31DFΔtail-HA and Myo61FΔtail-HA, which lack the entire tail region (Fig. 3A), failed to induce any LR defects (Fig. 4A,B). Furthermore, the overexpression of Myo31DFtail-HA and Myo61Ftail-HA, which lack the entire neck and head region, also did not induce LR defects (Figs. 3A, 4A,B).

Head Regions of Myo31DF and Myo61F Are Responsible for Their Organ-Specific LR Asymmetry-Reversing Activity

Given that Myo31DF and Myo61F have organ-specific LR asymmetry-reversing activities, it was important to determine whether the head or the tail region contains the information responsible for these specificities. To address this issue, chimeric proteins were overexpressed in which the head and IQ/tail regions were exchanged between Myo31DF and Myo61F. In Myo31DF-61FIQ/tail-HA and Myo61F-31DFIQ/tail-HA, the polypeptides from the IQ domain to the C-terminus were exchanged between Myo31DF and Myo61F (Fig. 3A). The overexpression of Myo61F-31DFIQ/tail-HA did not induce LR inversion in the foregut (Fig. 4C). However, the overexpression of Myo61F-31DFIQ/tail-HA reversed the laterality of the midgut and hindgut, as efficiently as the wild-type Myo61DF-HA (Fig. 4D, compare Fig. 5E,E′ with 5A,A′ and 5D,D′). We also found that the overexpression of Myo31DF-61FIQ/tail-HA induced the LR reversion in the foregut but not in the midgut and hindgut, although its activity in the foregut was significantly weaker than that of Myo31DF-HA (Fig. 4C, compare Fig. 5C′ with 5A′ and 5B′). These results together showed that the organ-specific properties associated with the LR polarity are contained in the head regions of Myo31DF and Myo61F. The IQ/tail region of Myo31DF may have specific functions that affect the activity of the Myo31DF head region, because the activity for inducing LR inversion was reduced when Myo31DF-61FIQ/tail-HA was overexpressed (Fig. 4C).

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Myo31DF Acts Before the LR Asymmetric Development of the Hindgut

In our previous report, we showed that Myo31DF is required in the epithelium of the hindgut but not the visceral mesoderm, which overlies the hindgut epithelial tube, for LR asymmetric development of the hindgut (Hozumi et al.,2006). However, the developmental stage at which the function of Myo31DF is required for normal LR asymmetric development of the embryonic hindgut had not been studied. We found that Myo31DF is required before the LR asymmetric morphogenesis of the hindgut. It is, therefore, unlikely that Myo31DF is directly involved in the Actin-dependent cell rearrangement that occurs during the LR asymmetric morphogenesis of the hindgut epithelium. Thus, the function of Myo31DF is different from that of the Drosophila zipper gene, which encodes Drosophila nonmuscle Myosin II and directly contributes to the cell rearrangement occurring during morphological changes of the embryonic epithelium (Bertet et al.,2004). We speculate that Myo31DF plays an important role in LR patterning in the epithelium of the hindgut. However, although it was previously shown that Myo31DF physically and genetically interacts with β-catenin (Spéder et al.,2006), the molecular nature of the LR patterning events is largely unknown.

Consistent with the notion that Myo31DF is essential for LR patterning of the hindgut epithelium, we found that the epithelial tube of the hindgut developed a normal LR asymmetric structure in the absence of the visceral mesoderm that overlies the hindgut epithelium. On the other hand, we previously demonstrated that the visceral mesoderm is required for LR asymmetric development of the anterior midgut (Taniguchi et al.,2007b). In this organ, LR asymmetric cell rearrangement of the visceral mesoderm cells is observed before LR asymmetric morphogenesis (Taniguchi et al.,2007b). Therefore, the contribution of the visceral mesoderm to the LR asymmetric morphogenesis is different in different parts of the embryonic gut in Drosophila.

Conventional Actin-Dependent Motor Activities Are Required for Myo31DF and Myo61F to Induce LR Inversion

Myo31DF and Myo61F share conserved domains with other Myosin family proteins (Morgan et al.,1994). Although our analysis relied on overexpression experiments, we also found that the IQ domains of Myo31DF were indispensable for its induction of LR inversion. Spéder et al. also showed that the IQ domains of Myo31DF are essential for its function in the LR asymmetric development of the male genitalia (Spéder et al.,2006). In contrast, the IQ domains of Myo61F were dispensable for its activity. The IQ domains of Myosin family proteins are known to be binding sites for Calmodulin or Myosin light chains (Cheney and Mooseker,1992; Bähler and Rhoads,2002). Therefore, a simple explanation for these distinct requirements for IQ domains is that the two Myosin proteins have different dependencies on such binding partners. However, embryos homozygous for a mutant Drosophila Calmodulin gene did not show any LR defects (R. Maeda, K. Taniguchi, and K. Matsuno, unpublished data).

In contrast to the different IQ domain requirements for the activities of Myo31DF and Myo61F, we found that the Actin-binding and ATP-binding domains were essential for the activities of both proteins. This finding suggests that the conventional Actin-based motor functions of these two Myosin I proteins are necessary for their LR asymmetry-reversing activities. In addition, mutant forms of the Myo31DF and Myo61F proteins lacking either the entire head or tail region lost their activity. Therefore, in addition to the motor activity, some functions associated with the tail region are essential for the LR inversion induced by wild-type Myo31DF and Myo61F. It was previously shown that the tail region of Myosin IC, which is homologous to Myo61F, interacts with the exocyst complex, which plays central roles in exocytosis (Sokac et al.,2006; Chen et al.,2007). The tail regions of Myosin I family members are also known to interact physically with membranes or anionic phospholipids through their basic tail domain (Adams and Pollard,1989; Hayden et al.,1990; Doberstein and Pollard,1992; Tang et al.,2002). Therefore, it is likely that the tail regions of Myo31DF and Myo61F include binding sites for some cellular components. Current studies aimed at identifying proteins that interact with these tail domains are in progress.

Organ-Specific Properties Associated With LR Polarity Are Contained in the Head Regions of Myo31DF and Myo61F

The chimeric protein composed of the Myo61F head and Myo31DF IQ/tail had an activity equivalent to that of the wild-type Myo61F. Thus, the IQ/tail region of Myo61F was interchangeable with that of Myo31DF for the function of the Myo61F head region. In addition, the head region of Myo31DF fused with the Myo61F IQ/tail retained the activity of wild-type Myo31DF, although this activity was significantly reduced. Therefore, the IQ/tail regions are not responsible for the organ-specific abilities of Myo31DF and Myo61F to reverse the laterality of the gut. Our results also suggest that the organ-specific properties associated with LR polarity are contained in the head regions. This result was unexpected, because the head regions of Myo31DF and Myo61F are highly conserved throughout their entire structure, whereas their IQ/tail regions are much more divergent. However, we found that several short stretches of amino acid sequence within the head region are specific for each protein. Therefore, it is possible that these short specific regions influence the activities or subcellular localizations of Myo31DF and Myo61F or may provide a binding site for organ-specific regulatory proteins.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Fly Stocks

Canton-S was used as the wild-type Drosophila strain. htlS1-28 is a hypomorphic allele of htl (Shishido et al.,1993), and Myo31DFL152 is a null allele of Myo31DF (Hozumi et al.,2006). The following GAL4 drivers were used: byn-GAL4 drives GAL4 expression in the hindgut and posterior midgut primordia at stage 8 and in the longitudinal visceral mesoderm at stage 11 (Iwaki and Lengyel,2002). da-GAL4 drives uniform GAL4 expression (Wodarz et al.,1995). The UAS lines UAS-Myo31DF-GFP (Spéder et al.,2006) and UAS-Myo61F (Hozumi et al.,2006) were described previously. TubP-GAL80ts was used for the TARGET method (McGuire et al.,2003). The genotypes of each embryo were determined using the appropriate blue-balancers, such as CyO, P{en1}wgen11 and TM3, ftz-lacZ. All crosses, except those used for the TARGET analysis, were performed at 25°C on standard Drosophila medium.

Histological Analyses of Embryos

Antibody staining of Drosophila embryos was performed as previously described (Sullivan et al.,2000). Embryos were photographed using a Zeiss Axioskop2 (Zeiss) plus or Pascal (Zeiss). The images were processed using the Zeiss LSM Image Browser Version 3, 2, 0, 104 (Zeiss) and Adobe Photoshop 7.0 (Adobe Systems). The primary antibodies were mouse anti–β-galactosidase (Promega, 1:1,000 dilution), rabbit anti-GFP (MBL, 1:1,000 dilution), rat anti-HA (3F10; Roche Diagnostics, 1:1,000 dilution), mouse anti-Connectin [C1.427, Developmental Studies Hybridoma Bank (DSHB), 1:5 dilution; Meadows et al.,1994], mouse anti-Crumbs (Cq4, DSHB, 1:30 dilution; Tepass et al.,1990), and mouse anti-FasIII (7G10, DSHB, 1:100 dilution; Patel et al.,1987). Secondary antibodies were Cy3-conjugated anti-mouse IgG (Jackson ImmunoResearch, 1:500) and biotinylated anti-mouse IgG (Vector Labs, 1:200 dilution) antibodies. The biotin-staining reaction was performed using the Vectastain ABC kit (Vector Labs).

Expression Constructs Producing Various Mutant Forms of Myo31DF and Myo61F

The amino acids of Myo31DF and Myo61F were numbered according to Morgan et al. (1994). The P-loop, Actin-binding domain, IQ domain, and head region were described previously (Morgan et al.,1994). The consensus sequence and mutations of the switch1 and switch2 loop were cited from Shimada et al. and Sasaki et al. (Shimada et al.,1997; Sasaki et al.,1998).

UAS-Myo31DF-HA produces a full-length [1-1011 amino acids (aa)] Myo31DF, UAS-Myo31DFmATP bs-HA produces a full-length Myo31DF with amino acid substitutions from the 106th K, 155th N, and 382th F to A. UAS-Myo31DFΔP-loop-HA produces a mutant form of Myo31DF lacking aa 103-107. UAS-Myo31DFΔActin bs-HA produces a mutant form of Myo31DF lacking aa 579-589, UAS-Myo31DFΔIQ-HA produces a mutant form of Myo31DF lacking aa 692-737. UAS-Myo31DFΔtail-HA produces a mutant form of Myo31DF lacking aa 1-737. UAS-Myo31DFtail-HA produces the tail region of Myo31DF (aa 738-1011). UAS-Myo31DF-61FIQ/tail-HA produces a chimeric protein composed of the Myo31DF head (aa 1-691) and the Myo61F IQ/tail (aa 696-1026). UAS-Myo61F-HA produces a full-length (aa 1-1026) Myo61F. UAS-Myo61FmATP bs-HA produces a full-length Myo61F with amino acid substitutions from the 111th K, 157th N, and 386th F to A. UAS-Myo61FΔP-loop-HA produces a mutant form of Myo61F lacking aa 105-112. UAS-Myo61FΔActin bs-HA produces a mutant form of Myo61F lacking the Actin-binding domain (aa 581-591). UAS-Myo61FΔIQ-HA produces a mutant form of Myo61F lacking the IQ domains (aa 696-765). UAS-Myo61DFΔtail-HA produces a mutant form of Myo61F lacking the tail region (aa 1-765). UAS-Myo61Ftail-HA produces the tail region of Myo61F (aa 766-1026). UAS-Myo61F-31DFIQ/tail-HA produces a chimeric protein composed of the Myo61F head (aa 1-695) and the Myo31DF tail (aa 692-1011). All proteins had an HA-tag (YPVDVPDYA) at the C-terminus.

Transgenic Lines

The germline transformations and subsequent crosses have been described (Sawamoto et al.,1994).

Stage-Specific Expression of Myo31DF in Myo31DF Mutant Embryos

Myo31DFL152; TubP-GAL80ts virgin females were mated with Myo31DFL152; byn-GAL4, UAS-Myo31DF-GFP males in a 50-ml tube, and the eggs were collected for 30 min or 60 min at 18°C. The midpoint of the egg collection was defined as “0” AEL. The eggs were incubated at 18°C, followed by a temperature shift to 32°C for 60 min. The midpoints of the temperature shift periods were plotted as blue (eggs were collected for 60 min) and yellow (eggs were collected for 30 min) dots in Figure 1A. The embryos were cultured at 18°C for up to 20 hr AEL and then were fixed with 4% paraformaldehyde in phosphate buffered saline and n-heptane. The LR asymmetric structure of the hindgut was examined at stages 14 to 16. Two or three independent experiments were performed and the mean values are indicated in Figure 1A. For Figure 1B, eggs were collected for 30 min at 18°C and incubated further at 18°C. Incubation at 18°C was followed by an incubation at 32°C for 60 min at 12.75 hr AEL. The embryos were fixed and stained with an anti-Crumbs antibody.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

We thank members of the Matsuno laboratory for critical comments and discussions. We also thank the Developmental Studies Hybridoma Bank (University of Iowa) for antibodies, and the Bloomington Drosophila Stock Center (Indiana) and the Drosophila Genetic Resource Center, Kyoto Institute of Technology (Kyoto) for fly stocks.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES