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Keywords:

  • transcription factor;
  • vestigial;
  • imaginal disc;
  • central nervous system;
  • peripheral nervous system;
  • muscle

Abstract

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

Background: The scalloped (sd) and vestigial (vg) genes function together in Drosophila wing development. Little is known about sd protein (SD) expression during development, or whether sd and vg interact in other developing tissues. To begin to address these questions, we generated an anti-SD antibody. Results: During embryogenesis, SD is expressed in both central and peripheral nervous systems, and the musculature. SD is also expressed in developing flight appendages. Despite SD expression herein, the peripheral nervous system, musculature, and dorsal limb primordia appeared generally normal in the absence of sd function. SD is also expressed in subsets of ventral nerve cord cells, including neuroblast 1–2 descendants and ventral unpaired median motor neurons (mVUMs). While sd function is not required to specify these neurons, it is necessary for the correct innervation of somatic muscles by the mVUMs. We also show that SD and vg protein (VG) are co-expressed in overlapping and distinctive subsets of cells in embryonic and larval tissues. Conclusions: We describe the full breadth of SD expression during Drosophila embryogenesis, and identify a requirement for sd function in a subset of motor neurons. This work provides the necessary foundation for functional studies regarding the roles of sd during Drosophila development. Developmental Dynamics 242:874–885, 2013. © 2013 Wiley Periodicals, Inc.


INTRODUCTION

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

The classic Drosophila scalloped (sd) mutant wing phenotype was first described in 1929 (Gruneberg, 1929), and the gene was subsequently cloned over 60 years later (Campbell et al., 1991). It was established that sd encodes a protein with a TEA family DNA-binding domain (Campbell et al., 1992; Jacquemin and Davidson, 1997; Anbanandam et al., 2006) and binds DNA in a sequence-specific manner (Halder et al., 1998b; Guss et al., 2001). SD acts as a transcription factor with two coactivators, vestigial (VG) and yorkie (YKI), functioning in wing development and the control of cell proliferation. With VG (Williams et al., 1991), SD functions as the selector (Mann and Carroll, 2002) for wing identity, forming a complex that is necessary and sufficient to direct wing development (Kim et al., 1996; Halder et al., 1998b; Simmonds et al., 1998; Halder and Carroll, 2001). SD binds to and directly regulates the wing-specific cis-regulatory elements for genes controlling many aspects of wing morphogenesis (Halder et al., 1998b; Guss et al., 2001; Lunde et al., 2003). With YKI, SD functions as an activator of growth and proliferation, in a complex regulated by the Hippo signaling pathway (reviewed in Bandura and Edgar, 2008).

Despite these extensive investigations of sd function, it has not before been possible to examine the expression of the SD protein. To address this challenge, we made an anti-SD antibody and used it to characterize the full breadth of SD expression during Drosophila development. In addition to the expected distribution of SD in the wing and haltere imaginal discs, we show that SD is strongly expressed in subsets of neuronal and muscle cells, where it colocalizes with VG. Based on this characterization, we present data that SD functions in neuronal pathfinding to muscle innervation of the ventral unpaired median motor neurons. This work lays the foundation for the systematic study of SD function in the development of the neuromusculature.

RESULTS AND DISCUSSION

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

Synthesis and Specificity of the SD Antibody

To define the cellular expression of SD during Drosophila development, we generated an anti-SD antibody. To test the specificity of this probe, its immunoreactivity was assessed in embryos showing loss of expression, or overexpression, of sd (Fig. 1). The SD antibody failed to label any tissues in Df(1)Exel6251/Y embryos, which carry an X chromosome deficiency that uncovers the sd locus (Parks et al., 2004; Fig. 1B). Expression was also assessed in embryos carrying the recessive lethal sd3L allele (data not shown). The molecular lesion associated with this allele is a nonsense mutation in the first leucine codon within the Vestigial Binding Domain (Campbell et al., 1991; Srivastava et al., 2004), Very low, diffuse signal was observed in the central nervous systems of sd3L embryos suggesting it is a strong hypomorphic mutation. Conversely, the antibody labeled additional cells in embryos in which patched-GAL4 drove UAS-sd in many cells in every segment (Fig. 1D). Taken together, these data show that our antibody recognizes specifically the SD protein.

image

Figure 1. Specificity of the anti-SD antibody (scalloped protein, SD). A,B: Detection of the guinea pig anti-SD antibody in stage 14 embryos of genotypes Df(1)Exel6251 or FM7c act-lacZ/FM7c act-lacZ (A) and Df(1)Exel6251/Y (B). While the anti-SD antibody is detectable in embryos carrying the balancer chromosome (A; βGAL expression not shown), no detection is observed in embryos missing the sd locus (B). C,D: Detection of the guinea pig anti-SD antibody in stage 12 embryos in which sd is expressed at normal levels (C) or overexpressed (D). The pattern of anti-SD antibody detection in embryos carrying patched-GAL4 is comparable to that observed in wild-type embryos (C; compare with Fig. 2I), while ectopic expression is detected in embryos that additionally carry UAS-sd (D; note that this image was collected using lower laser settings than panel C). Our rat anti-SD antibody showed the same results in sd loss of expression and overexpression experiments (data not shown). Anterior is to the left in all panels.

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SD Expression During Embryonic Development

We observe close correlation between the patterns of expression the sd RNA and SD protein, which further supports the specificity of our antibody (Fig. 2). The sd RNA is detectable in the central nervous system (CNS; Fig. 2A,D,E; see also Campbell et al., 1992) and the peripheral nervous system (PNS; Fig. 2B,C). SD protein is expressed in the ventral nerve cord (VNC) detectable beginning at stage 10 (Fig. 2F). The expression in the CNS continues through stages 11 (Fig. 2G), 12 (Fig. 2H), 14 (Fig. 2I), and 16 (Fig. 2J). Expression in the PNS is also observed at stage 14 (Fig. 2I), while expression in the muscles is discernible at stage 16 (Fig 2J). We also observe strong SD expression in adepithelial cells (Bate et al., 1991) located between the dorsal and ventral limb primordia (arrows, Fig. 2H; see also Fig. 3P–R).

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Figure 2. Scalloped expression during embryogenesis. A–E: Whole-mount in situ hybridization showing sd RNA distribution (purple) in embryos at stages 11 (A,B), 13 (C), and 15 (D,E). sd is expressed in the central nervous system at stages 11 (A) and 15 (D,E) and is also detectable in the peripheral nervous system of embryos at stages 11 (B) and 13 (C). F–J: SD protein expression (brown). SD is detectable in the ventral nerve cord at stages 10 (F), 11 (G), 12 (H) and becomes localized to subsets of cells by stage 16 (J). SD is also expressed in the peripheral nervous system at stage 14 (I), the developing somatic mesoderm and cells between the dorsal and ventral limb primordia (G–I; arrows, H). Anterior is to the left in all panels, and stages of development are in the lower left.

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image

Figure 3. SD (scalloped protein) expression in the embryonic central nervous system (CNS), peripheral nervous system (PNS), limb primordia, and somatic muscles. A–F: Expression of SD in the ventral nerve cord (VNC) dissected from stage 12 (A,B), stage 15 (C,D), and stage 16 (E,F) embryos. For each stage, dorsal (A,C,E) and ventral (B,D,F) focal planes are shown. SD is initially broadly expressed in the VNC, and expression resolves to subsets of cells as development proceeds. G–I: SD (green) and ELAV (Embryonic Lethal Abnormal Vision, magenta) in three abdominal segments of a stage 12 embryo, showing that most SD-expressing cells are sensory neurons. These images are maximum projections of multiple confocal slices. J–L: Four abdominal segments of a late stage 16 embryo showing SD (green) expression in muscles ventral lateral muscles 1–4 (VL1–4) and lateral longitudinal muscle 1 (LL1), which are marked by Myosin Heavy Chain (MHC, magenta). M–O: SD (green) is coexpressed with Esgargot (ESG, magenta) in the dorsal limb primordia of the second and third thoracic segments of a stage 17 embryo. These images are maximum projections of multiple confocal slices. P–R: Three thoracic segments and first abdominal segment of a stage 12 embryo. SD (green) is coexpressed with βGAL driven by twist-GAL4 in the three thoracic segments. Specimens are oriented with anterior up (A–F) or to the left (G–R).

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We examined more closely the expression of SD during development (Fig. 3). In the VNC, SD appears to be initially expressed in many cells (Fig. 3A,B), although the expression resolves to higher levels in a subset of cells as embryogenesis proceeds (Fig. 3C–F). To more thoroughly characterize the SD expression in other tissues during embryogenesis, we performed colocalization studies with tissue or cell type specific molecular markers. In the PNS, SD is expressed in most sensory neurons, based on close coexpression with Embryonic Lethal Abnormal Vision (ELAV; Robinow and White, 1991; Fig. 3G–I). This coexpression continues through embryogenesis, although the level of SD expression in cells in the PNS is reduced. SD is also expressed in the somatic bodywall muscles during embryogenesis. At stage 12, SD is detectable in the ventral lateral muscles 1–4 (VLs 1–4, muscle nomenclature per Bate, 1993). This expression continues through stage 16, with higher expression in the muscle nuclei (Fig. 3J–L). By this stage, SD is also detectable in ventral acute muscles 1 and 2 (VA1 and 2) and lateral longitudinal muscle 1 (LL1). In late stage 16 embryos, additional dorsal muscles also express SD. Therefore, expression of SD occurs in the most ventral muscles first, then expands to more dorsal muscles as development proceeds. Deng et al. (2009) observed broader sd expression, earlier in development, by using sd RNA and enhancer trap detection methods, with reduced expression by late stage 16, whereas we observed the most extensive expression at this late embryonic stage.

SD is also expressed at low levels in the embryonic wing and haltere primordia, based on colocalization with Esgargot (ESG; Fig. 3M–O). The cells expressing the higher level of SD in the lateral regions of the three thoracic segments are progenitors of the adult flight muscles (Fig. 3P–R). We make this conclusion based on the coexpression of SD and βGAL driven by twist-GAL4, a marker for these adepithelial cells (Bate et al., 1991). Our observation correlates well with those made by Bernard et al. (2003), who showed that VG and sd-lacZ are coexpressed in these cells later in development.

Scalloped Expression in the Larval Imaginal Discs

We also examined SD expression in tissues of the wandering third instar larva (Fig. 4). In the wing imaginal disc, SD is expressed in the wing pouch and margin (Fig. 4A), with expression extending into the hinge and notum (Cohen, 1993). SD is expressed in the corresponding structures in the haltere disc (Fig. 4B). The strong expression of SD in the wing disc correlates well with its documented role in wing development. In comparison, SD is expressed at much lower levels in the leg (Fig. 4C) and eye antennal (Fig. 4D) imaginal discs. We also examined the expression of sd RNA in the imaginal discs (Fig. 5). Concordant with the high levels of the protein in the wing and haltere, strong RNA expression is observed in these imaginal discs (Fig. 5A,B). We also observe expression, at lower levels, in the leg and eye antennal discs (Fig. 5C,D). In the leg and antenna, sd appears to be expressed in rings that may correspond to different locations along the proximal–distal axes of these tissues.

image

Figure 4. A–D: SD (scalloped protein) expression in larval imaginal discs. SD (green) is expressed in the wing (A) and haltere (B) imaginal discs, and at low levels in the leg (C) and eye antennal (D) discs.

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Figure 5. sd (scalloped gene) expression in larval imaginal discs. A,B: sd (blue) is expressed in the wing (A) and haltere (B) imaginal discs. C,D: It is also expressed at lower levels in the leg (C) and eye antennal (D) discs.

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Our observations generally agree with the expression pattern of sd that has been reported using the sd enhancer trap line ETX4 (Campbell et al., 1992), although we do see some differences. For example, we report here what appear to be higher levels of expression of sd and SD in cells in the embryo using direct detection of these gene products vs. those levels reflected by ETX4 (Campbell et al., 1992 and our own observations). In addition, Campbell et al. (1992) show that ETX4 drives high expression posterior to the morphogenetic furrow in eye imaginal disc, whereas we observe both RNA and protein at higher levels to the anterior of this landmark. Such discrepancies may be due to subtle differences in regulation, and/or perdurance, of the reporter protein in the enhancer trap line.

Coexpression of Scalloped and Vestigial in Developing Tissues

SD and VG have a well-established role working together in wing development (Halder et al., 1998b; Simmonds et al., 1998). Based on a characterized VG antibody, the identity of many VG-expressing cells have been determined in developing tissues (Williams et al., 1991; Landgraf et al., 2003; Guss et al., 2008). Therefore, using this tool, we next examined the coexpression of SD and VG in both embryos and larvae. This approach allowed us to determine the identity of SD-expressing cells, and provides the necessary foundation to identify tissues in which SD and VG may function together, as well as tissues in which they may function independently. A summary of these data is shown in Figure 6.

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Figure 6. SD (scalloped protein) and VG (vestigial protein) coexpression in the ventral nerve cord and imaginal discs. A–F: Dorsal (A–C) and ventral (D–F) focal planes of a stage 16 embryonic ventral nerve cord (VNC) showing expression of SD (green) and VG (magenta). These images are maximum projections of multiple confocal slices. SD is expressed at high levels in cells that also express VG, including descendants of neuroblast 1–2 (NB1–2; arrowheads, A–C) and the ventral unpaired median motor neurons (mVUMS; arrows, D–F). G–I: SD (green) and VG (magenta) expression in the somatic muscles of abdominal segments of a stage 15 embryo. At this stage, both proteins are expressed in ventral lateral muscles 1–4 (VLs 1–4; arrowheads, G–I), but VG is expressed in dorsal muscles as well (arrows; G–I). Specimens are oriented with anterior up (A–F) or to the left (G–I). J–L: Wing, haltere, and leg imaginal discs from a wandering third instar larva. SD (green) and VG (magenta) are coexpressed at high levels in the wing and haltere imaginal discs.

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SD is coexpressed with VG in subsets of cells in both the embryo and in the wing and haltere imaginal discs (Fig. 6). As discussed above, SD expression appears to be initially broad in the ventral nerve cord, and to resolve to high expression in a clear subset of cells. Interestingly, these same identified neurons also express VG (Fig. 6A–F). These cells include neuroblast 1–2 (NB1–2) descendants (Fig. 6A–C; referred to as mVg in Landgraf et al., 2003; Guss et al., 2008) and the ventral unpaired median motor neurons (mVUMs; Fig. 6D–F). With respect to the somatic muscles, both SD and VG are expressed at elevated levels in the ventral muscles VLs 1–4 and LL1 (Fig. 6G–I) At this stage, VG is additionally expressed in dorsal acute muscles (DA)1–3 (Landgraf et al., 1999). In the larval imaginal discs, consistent with their shared role as selectors for wing development, both SD and VG are expressed in the wing and haltere imaginal discs (Fig. 6J–L).

Functional Requirements for Scalloped in the Neuromusculature

To begin to investigate the function of Scalloped in embryonic tissues, we surveyed general morphological markers of the nervous systems and muscle in sd3L (strong hypomorph) and Df(1)Exel6251 (null) embryos (Fig. 7). It appears that sd function is not required for cell fate specification in the CNS, including in the SD-expressing NB1–2 descendants and mVUMs. Based on labeling for DVSX1 (Erclik et al., 2008), which is expressed in the NB1–2 descendants and mVUMs, we found no consistent difference between control and Df(1)Exel6251 null embryos (Fig. 7A–H). Although SD is broadly expressed in the PNS, we did not observe a detectable change in the number/position of neuronal cell bodies or the gross morphology of the sensory neuron axons. This conclusion is based on similar expression of microtubule-associated Futsch/22C10 (Hummel et al., 2000) in control and sd mutant embryos (Fig. 7I,J). Similarly, we observed no detectable change in embryonic muscle patterning or differentiation in the absence of SD function. Based on labeling for Myosin Heavy Chain (MHC), we observed a subtle change in the distribution of muscle nuclei in sd mutant vs. control embryos (Fig. 7K,L). We focused on VLs 1–4 and LL1 because we observed these muscles to consistently express SD from stage 12 through later embryogenesis. For this reason, we did not examine the ventral oblique muscles and cardiac cells, reported by Deng et al. (2009) to show defects in sd mutants.

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Figure 7. sd (scalloped gene) function is not required for global patterning of the peripheral nervous system (PNS), central nervous system (CNS), muscles, and limb primordia. A–H: sd is not required for cells fate specification of the ventral unpaired median motor neurons (mVUMs) and neuroblast 1–2 (NB1–2) descendants in the ventral nerve cord. SD (green, A–D) is coexpressed with DVSX1 (magenta, A,B,E–H) in cells in the ventral nerve cord, including the mVUMs and NB1–2 descendants. The distribution of these cells is normal in Df(1)Exel6251 embryos, which are null for sd. I,J: Antibody 22C10 was used to assess the distribution of cell bodies and axons in control (I) and sd3L embryos (J); these images are maximum projections of multiple confocal slices. We observed no reproducible difference between the wild-type and mutant, showing that sd is not required for general PNS patterning. K,L: Muscle structure was assessed using myosin heavy chain (MHC) in control (K) and Df(1)Exel6251 (L) embryos. Muscle structure is generally normally in sd mutant embryos, although altered distribution of nuclei was observed. M,N: Distribution of Esgargot (ESG) -expressing cells in the dorsal and ventral limb primordia of control (M) and Df(1)Exel6251 (N) embryos. The normal distribution of these cells in sd mutant embryos confirms the observation that sd function is not required for the establishment of the limb primordia or the dorsal migration of the flight appendage cells (marked w, h) from the leg primordia (marked T1, T2, T3). A–N: Specimens are oriented with anterior up (A–H) or to the left (I–N).

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Finally, we observe that sd function is not required for the establishment of the flight appendage primordia. This conclusion is based on the same distribution of ESG-expressing cells in the dorsal and ventral flight appendage primordia in the thoracic segments of sd mutant and control embryos (Fig. 7M,N), and is consistent with the observation that the wing disc forms in sd mutants (Williams et al., 1993). We conclude that SD is either not required for the specification of these tissues, or that its effects are subtler or address different aspects of differentiation than are accessible with the markers we used.

The fact that sd is not required for the cell fate specification in the neuromusculature led us to ask whether it may function in a later differentiation stage. Because the mVUM gene expression profile (Wheeler et al., 2006) and target muscles (Landgraf et al., 1997) have been characterized, we tested for sd phenotypes in this select cell population. Collectively, the three mVUMs innervate dorsal oblique muscles 3–5 (DO 3–5) by means of the intersegmental nerve (ISN), lateral transverse muscles 1 and 2 (LT1 and 2) by means of segmental nerve a (SNa), and ventral oblique muscles 1–6 (VO 1–6) by means of SNb and SNd (Landgraf et al., 1997). More recent accounts (e.g., Ruiz-Canada and Budnik, 2006) refer to only the ventral muscles VO 1–5 as being innervated by mVUMs, so we have focused on these muscles, and the structures of SNb and SNd. The monoclonal antibody ID4 for Fasciclin II (FasII; Van Vactor et al., 1993) was used to label all the motorneuron axons in control and sd mutant embryos. We observed disruptions of the segmental nerve branches in the absence of sd (Fig. 8). Because SNd could not consistently be identified and it was difficult to identify the stereotyped branches of SNb (Halpern et al., 1991), we focused on SNb length, from its projection from ISN to its distal tip. The length of SNb is shorter in sd mutant embryos than in controls (mean = 43.52; SEM = 4.38 vs. mean = 68.73; SEM = 5.38; P = 0.0034). This result indicates that sd function is required for the normal innervation of the ventral muscles.

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Figure 8. sd is required for the innervation of ventral muscles. A,B: Embryos of genotypes w1118 (A) and sd3L (B) showing expression of FASII in three posterior abdominal segments of late stage 16 embryos. Intersegmental nerve (ISN) and segmental nerves (SN) a–d are labeled in a representative control segment (A) and SNb is labeled in a segment in the sd mutant (B). These images, which are maximum projections of multiple confocal sections, are oriented anterior to the left. C: SNb length is shorter in the sd mutant than the control. Error bars represent the SEM, and P < 0.01.

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SUMMARY

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

Here, we use a new antibody probe to characterize the expression of the TEA domain transcription factor SD during Drosophila development. Although this protein is known for its role in wing development and growth control, SD is expressed in many tissue types during embryogenesis, including the central and peripheral nervous systems, and the muscles. We report a new role for sd, in the innervation of the ventral muscles. We also examined the coexpression of SD with VG, its cofactor in wing development. These proteins are distributed in similar but not identical expression patterns, presenting the possibilities that they may fine tune each other's expression, function together outside the wing, and/or function independently.

EXPERIMENTAL PROCEDURES

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

Drosophila Stocks

The genotype w1118 was used as wild-type. Df(1)Exel6251 is a deficiency that uncovers the sd locus (FlyBase), and sd3L is a recessive lethal allele (Campbell et al., 1991; Srivastava et al., 2004). Both mutations were maintained over FM7c act-lacZ (Bloomington Drosophila Stock Center) and male embryos carrying the mutation were identified by the absence of beta-galatosidase (βGAL) expression. Additional stocks used were patched-GAL4, twist-GAL4, UAS-lacZ.nls (all from Bloomington Drosophila Stock Center) and UAS-sd (Sean Carroll).

SD Antibody Generation

Both known SD isoforms span from N-terminal MVDSK through C-terminal RLIKE (FlyBase R5.8, 2008). This 375 amino acid SD sequence was subjected to the Jameson-Wolf antigenic index (Lasergene, Madison, WI). Polymerase chain reaction (PCR) primers were designed to generate a form of SD containing 369 amino acids, and had the following sequences, including restriction enzyme recognition sequences (bold) and end sequences to facilitate enzyme activity (underline):

SD_7_28: 5′-CTACATATGGATAGCAAAAACCTGGATGTCG-3′

SD_1113_1092: 5′-CTAAAGCTTACGGTATATGTGATGGGTGGTG-3′

Samples containing as template the sd open reading frame and the thermostable polymerase PfuTurbo were subjected to 30 cycles of amplification using an annealing temperature of 65°C. The PCR products of triplicate reactions were processed using the QIAQuick PCR purification protocol. The products were then digested with NdeI and HindIII for cloning. Following digestion, the products were again processed using the QIAQuick PCR purification protocol. The insert was cloned in-frame by means of NdeI and HindIII sites in the pET29a expression vector. The construct was transformed into competent BL21 cells, with expression induced by addition of IPTG to 1 mM to bacterial culture with OD600 approximately 0.5, followed by an additional 2.5 hr growth. The 46 kDa protein was recovered as described by Williams et al. (1995). Inclusion bodies recovered by treatment with lysozyme followed by recovery with a French press were solubilized in 10% sodium dodecyl sulfate, then dialyzed with phosphate buffered saline (PBS). The protein at ∼2 mg/ml concentration was injected by Pocono Rabbit Farm and Laboratory, Inc. (Canadensis, PA) into three rats and three guinea pigs. The final exsanguinations were affinity purified against the SD protein.

Whole-Mount In Situ Hybridization

Detection of RNA in Drosophila embryos was performed as described by Small (2000), and in third instar larval imaginal discs as described by Sturtevant and Bier (1997). A DNA construct containing the sd open reading frame cloned into pBluescript (Anj Hudson and Sean Carroll) was used as template to synthesize T3-primed sense or T7-primed antisense RNA probes using digoxigenin-labeled RNA synthesis materials from Roche. No signal was observed in embryos or imaginal tissues processed with the sense RNA probes (data not shown).

Immunocytochemistry

Embryos from overnight collections were washed with 0.4% NaCl/0.1% Triton X-100, dechorionated by incubating in bleach for 2.5 min, and rinsed with dH2O. Embryos were then fixed by agitating for 20 min in equal volumes heptane and fix buffer comprised of 0.1 M PIPES, 2 mM EGTA, 1 mM MgSO4, and 4% formaldehyde. Following fixation, embryos were devitellinized and washed with methanol. For immunolabeling, embryos were rehydrated into PBS with 0.1% Triton X-100 and 1% bovine serum albumin, which was used for all antibody incubations and washes. Wandering third instar larval imaginal discs were dissected, fixed, blocked, and processed with antibodies as described by Halder et al (1998a).

Antibodies

SD was detected using either guinea pig anti-SD (1:500 or 1:1,000 in embryos, 1:1,000 in larvae) or rat anti-SD (1:5 in embryos). Guinea pig anti-DVSX1 (1:750; Erclik et al., 2008) was provided by Howard Lipshitz. Rabbit anti-VG (1:25 in embryos, 1:100 in larvae; Williams et al., 1991) was the gift of Sean Carroll and in embryos was detected with biotinyl tyramide amplification as described in Guss et al. (2008). Rat anti-ESG (1:500; Fuse et al., 1994) was provided by Shigeo Hayashi. Rabbit anti MHC (1:500) was provided by Dan Kiehart by means of Emma Rushton. Rat anti-ELAV (7E8A10, 1:200), mouse anti FUTSCH (22C10; 1:100), mouse anti-FASII (ID4, 1:20) were developed by Gerald Rubin, Seymour Benzer, and Corey Goodman, respectively, and obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology (Iowa City, IA 52242). βGAL was detected using antibodies raised in either rabbit (Invitrogen) or mouse (Promega) and diluted 1:1,000. Primary antibodies were detected by either Alexa Fluor-conjugated secondary antibodies (1:300, Molecular Probes) or biotin-conjugated secondary antibodies (1:400, Jackson) using the ABC kit (Vector Laboratories) and diaminobenzidine (DAB) Substrate Kit (Pierce). All antibodies were routinely preadsorbed against fixed embryos before use. Samples were incubated overnight in primary antibodies at 4°C, and 2 hr at room temperature in secondary antibodies. For imaging, embryos were suspended and mounted in VECTASHIELD (Vector Laboratories) or 70% glycerol.

Image Collection and Presentation

Fluorescent specimens were imaged using Zeiss LSM 510 META and Olympus Fluoview 500 inverted laser scanning confocal microscopes. Data were collected using sequential scans to avoid artifacts from overlapping emission spectra, with image slices of 0.5 μM. Except as noted, each figure panel shows a single confocal section. Nonfluorescent specimens were collected using Zeiss Axioplan and Nikon E800 compound microscopes equipped for differential interference contrast illumination. Images were merged, cropped, and adjusted for brightness, contrast, and levels using Adobe Photoshop versions 10.0.1 and 13.0.1. Except as noted, images of specimens being directly compared (e.g., wild-type vs. mutant, wing vs. leg imaginal discs) were collected using the same microscope settings, and subjected to the same image adjustments.

Characterization of SNb Length

To assess the length of segmental nerve projections, maximum projections of Z-series were collected of FASII expression in posterior abdominal segments of late stage 16 embryos. The length of SNb was measured from the branch from the ISN to the most distal tip of SNb using MetaMorph Software. In sd3L embryos, eight segments in three animals were measured, while in controls, seven segments in three animals were measured. The data was analyzed using the Student's t-test with a two-tailed distribution assuming unequal variance. The standard error of the mean (SEM) is reported.

ACKNOWLEDGMENTS

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

We thank Anj Hudson for the DNA construct containing the sd open reading frame, John Bell, Sean Carroll, Shigeo Hayashi, Howard Lipshitz, and Emma Rushton for antibodies and/or fly stocks, Yi Zhu and Beth Wilson for advice and assistance during the SD antibody synthesis, Cheryl Gatto for advice on statistical analysis of SNb length, Erik Boczko for translating the first sd reference from the original German, and past and present members of the Skeath and Broadie labs for hospitality during research visits and sabbatical stays. As undergraduates at Dickinson College, Casey Delconte, Chelsea Gurvis, Evan Hennessy, Aaron Kopp, Kelly LaRue, Brandon Lee, Liz Norris, and Rachel Yonker assisted in preliminary characterization of the sd RNA or protein expression in wild-type or sd mutant embryos, and Elise Fiala and Matthew Weddig processed the samples shown in Figure 3, panels P–R. Several monoclonal antibodies used in the course of this work were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242. We also thank the Bloomington Drosophila Stock Center for providing fly stocks. J.B.S. and K.A.G. were supported by NSF and K.B. was funded by the NIH.

REFERENCES

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