Beat II and Side IV keep migrating longitudinal visceral muscle precursor cells on their substratum in Drosophila
Na Huang, Jaqueline C. Kinold, Niklas W. G. Weiß, Nargis Piroddi, Iris Fey, Hermann Aberle
TL;DR
This study shows how specific proteins help muscle cells migrate properly in fruit fly embryos, which is important for forming the gut's muscle structure.
Contribution
The novel contribution is identifying Beat II and Side IV as a ligand–receptor pair guiding CVM cell migration in Drosophila midgut development.
Findings
Beat II and Side IV mutants show migration defects in CVM cells.
Beat II proteins interact with Side IV in trans, guiding cell migration.
Ectopic Side IV expression in trachea aligns migrating CVM cells.
Abstract
The proper formation of tissues and organs relies on precisely regulated differentiation and migration processes. In Drosophila embryos, the development of the midgut musculature requires coordinated movement of caudal visceral mesoderm (CVM) cells across the trunk visceral mesoderm (TVM). Here, we report that Beaten path (Beat) II proteins (Beat IIa and Beat IIb) are specifically expressed in CVM cells, whereas Sidestep IV (Side IV) is expressed in their substrate, the TVM. Both beat II and side IV mutants show similar migration defects, characterized by irregular migration patterns and abnormal cell distributions. At the end of embryogenesis, the midgut contains areas devoid of longitudinal muscles, resulting in a reduced number of longitudinal muscle fibres at larval stages. In cell–cell aggregation assays, Beat II proteins specifically interact with Side IV in trans. Moreover,…
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Fig. 8- —Heinrich Heine University Düsseldorfhttp://dx.doi.org/10.13039/501100003484
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Taxonomy
TopicsDevelopmental Biology and Gene Regulation · Hippo pathway signaling and YAP/TAZ · Wnt/β-catenin signaling in development and cancer
INTRODUCTION
Cell migration is the directional movement of cells over various substrates and along diverse tissues. It ensures that cells meet at specific locations and are appropriately arranged to support cell differentiation and organogenesis. For migration, cells require adhesion to and traction on a substrate (Paluch et al., 2016). As cellular environments can be diverse, cells in general accept basal laminae, extracellular matrices (ECM) or the surfaces of other cells as substrates (Rørth, 2009). The Drosophila embryonic midgut musculature serves as a model system to investigate the molecular mechanisms of cellular migration and how cells are guided toward their targets (Sun et al., 2020).
The fully differentiated midgut musculature of Drosophila consists of longitudinal visceral muscles (LVM), which arise from the caudal visceral mesoderm (CVM), and circular visceral muscles (CirVM), which develop from the trunk visceral mesoderm (TVM) (Azpiazu and Frasch, 1993; Lee et al., 2006). The CVM and TVM originate from migratory mesodermal precursor cells that differentiate over time via a highly controlled gene network (Azpiazu and Frasch, 1993; Georgias et al., 1997; Sun et al., 2020).
At embryonic stage 10, when the CVM cells are specified by the basic helix-loop-helix (bHLH) transcription factor HLH54F at the most posterior tip of mesoderm, TVM cells are specified by Biniou (Bin) and the homeodomain transcription factor Bagpipe (Bap) in segmental cell clusters along the body axis (Azpiazu and Frasch, 1993; Sun et al., 2020; Zaffran et al., 2001). Once TVM cells cluster in a continuous band, CVM cells establish close contact with them and use them as a substrate for migration (Macabenta and Stathopoulos, 2019; Sun et al., 2020; Zaffran et al., 2001). During stage 12, CirVM precursors (CirVMps) develop from the TVM (Martin et al., 2001), and the CVM cells migrate precisely along the dorsal and ventral margins of them (Macabenta and Stathopoulos, 2019). At stage 13, when the first CVM cells reach the region of foregut–midgut transition, their anterior migration is not only completed but they now start to fuse with TVM-derived fusion-competent myoblasts to form LVM precursors (LVMps) (Martin et al., 2001).
At stage 14, in concert with the midgut endoderm expansion, CirVMps start to elongate dorsoventrally, and the LVMps spread concomitantly to cover them. During this dorsal-to-ventral migration, LVMps extend dynamic filopodia, contacting the underlying CirVMps (Frasch et al., 2023). Upon midgut fusion, at stage 15, LVMps are evenly distributed around the entire chamber. They then extend into anterior-posterior directions by fusing with remaining myoblasts of the TVMs to form long and thin fibres. At late stage 16, these fibres further mature, and each of them stretches along the length of the midgut (Frasch et al., 2023). They interweave evenly with the internal CirVM fibres, which are now ring-shaped and surround the midgut. After hatching, this grid-shaped musculature is responsible for transporting food through the gut tube by peristaltic contractions (Bodenstein, 1965).
The TVM is crucial for the development of the longitudinal muscles, as it provides not only mechanical support but also survival and guidance cues for CVM cells (Sun et al., 2020). For example, the FGF ligands Pyramus (Pyr) and Thisbe (Ths) are expressed in the TVM and activate their receptor Heartless (Htl) in the CVM to support CVM survival and migration (Kadam et al., 2012; Reim et al., 2012). In htl mutants, as well as in a deficiency line lacking both ths and pyr, CVM cells display a disordered migration pattern followed by extensive cell death at stage 13. In either ths or pyr single mutants, they show a similar but less severe phenotype, suggesting that both ligands act redundantly in this process (Kadam et al., 2012; Reim et al., 2012). Meanwhile, when apoptosis in CVM cells is suppressed by expressing p35, a caspase inhibitor, surviving CVM cells regain a high migration ability, but the migration path is still irregular and fewer cells reach the anterior foregut–midgut border compared to the number in wild-type embryos (Kadam et al., 2012; Reim et al., 2012). Moreover, TVM cells express the α-integrin subunit PS2, which is required to assemble a proper ECM for CVM migration (Urbano et al., 2011). In the absence of PS2 integrin, CVM migration is delayed, and the accumulation of an ECM component, Nidogen is impaired (Urbano et al., 2011).
Two independent searches for genes that might have key-regulatory functions in migrating CVM cells, identified beaten path II genes (beat IIa and beat IIb), because they are highly and specifically expressed in these cells (Bae et al., 2017; Ismat et al., 2010). Beat II proteins (hereafter Beat IIs) belong to the family of Beaten path-like proteins, which themselves belong to the immunoglobulin superfamily. This family contains 14 members, which are divided into seven clusters according to their phylogenetic relationships (Li et al., 2017; Pipes et al., 2001). Most Beat proteins are prominently expressed in subsets of neurons in the central nervous system (Pipes et al., 2001). They are thought to function with members of the Sidestep (Side) family as ligand–receptor pairs to facilitate neurogenesis. For example, Beat Ia-expressing motor axons recognize and follow Side-expressing cellular substrates to reach their target areas (Fambrough and Goodman, 1996; Siebert et al., 2009; Sink et al., 2001). Expression of Side II and Beat VI in pre- and post-synaptic partners, respectively, promotes synaptic specificity and inhibits miswiring in the visual system (Carrier et al., 2024; Yoo et al., 2023). Similarly, Beat IIb interacts with Side IV to form adhesive complexes that trigger synapse formation in the lamina of the optic neuropile (Osaka et al., 2024). Thus, as Beat IIs are also highly expressed in CVM cells, they might play a role in the migration of non-neuronal cells, too.
To this end, we first confirm that Beat IIa and Beat IIb are expressed in CVM cells as well as in LVMps. We find that the potential interaction partner, Side IV, is expressed in the TVM. In addition, we observe in beat II double mutants and side IV single mutants that LVMp cells display abnormal orientations and morphologies during their dorsoventral migration and fail to fully ensheath the midgut chamber. Cells that lost contact with the TVM show smaller cell shapes and undergo apoptosis. As a result, third-instar larvae show fewer LVM fibres. Interestingly, Side IV interacts in trans with Beat II in vitro and ectopic expression of Side IV in trachea of side IV mutant embryos attracts CVM and LVMp cells to migrate along tracheal branches. These findings suggest that Side IV functions together with Beat IIs as a ligand–receptor pair to guide CVM and LVMp cells along the TVM. Thus, Beat and Side proteins might also function in guiding the migration of non-neural cells.
RESULTS
Beat IIb is specifically expressed in migrating longitudinal visceral muscle precursor cells
To search for restricted tissue expression of specific members of the Beat family, we screened publicly available exon trap lines that fuse GFP to endogenous proteins. Interestingly, line Beat IIb::GFP, also known as Beat IIb^MI03102-GFSTF.0^ (Nagarkar-Jaiswal et al., 2015), showed specific expression in a group of cells located at the posterior tip of the germ band at stage 10 (Fig. 1A,A′). Subsequently, these Beat IIb-positive cells rearranged into two migratory cell streams towards anterior regions (Fig. 1B,B′), a migrating pattern highly reminiscent of migrating CVM cells, which are the precursors of LVMs (Georgias et al., 1997). We then examined these cells in the background of a CVM marker, which expresses cytoplasmic RFP under control of the HLH54Fb promoter (*HLH54Fb-*cytoRFP) (Hollfelder et al., 2014). Stainings with anti-GFP to visualize Beat IIb::GFP and anti-RFP (Shaner et al., 2004) to reveal CVM cells showed that both signals largely colocalized (Fig. S1G–L′). The colocalization indicates that Beat IIb is expressed in CVM cells, and this expression spans the entire period of migration and differentiation of these cells. As Beat Ia, a conserved paralog of Beat IIb, functions in migrating motor axons (Fambrough and Goodman, 1996; Siebert et al., 2009), it was intriguing to investigate whether Beat IIb might have a role in migrating, non-neuronal CVM cells.
Beat IIb::GFP-expressing cells migrate specifically along the trunk visceral mesoderm. (A–F′) Confocal images (projections) of Beat IIb::GFP embryos at the indicated embryonic stages (St) immuno-stained using anti-GFP antibodies (green) to detect GFP-tagged Beat IIb and anti-Fas III antibodies (magenta) to reveal cells of the trunk visceral mesoderm (TVM). (A,A′) At stage 10, Beat IIb-expressing cells are born at the posterior tip of the visceral mesoderm. (B,B′) At stage 12, Beat IIb-expressing cells migrate anteriorly along the TVM band. (C,C′) Once they reach the foregut-midgut transition at stage 13, they start to fuse with fusion-competent myoblasts to form longitudinal visceral muscle precursors (LVMps). (D,D′) During stage 14, LVMps migrate dorsoventrally along with the expansion of the midgut. (E,E′) At stage 15, LVMps are evenly distributed around the entire surface of the midgut. (F,F′) Beginning with stage 16, LVMps connect with each other to form long and thin longitudinal visceral muscles (LVMs). (G–L′) HLH54Fd-GAL4 driving expression of Histone–RFP in CVM and LVMp cells (visualized by anti-RFP antibodies; magenta). To demonstrate nuclear fusion, these cells were also labelled with HLH54Fb-LacZ (visualized by anti-βGal antibodies, green). The first binucleated cells are detected at stage 13 (H′) and most LVMps contain five or six nuclei at the end of stage 15 (J′). Images G-K′ have been reused as representative images for the same genetic background in Fig. S3B–F′. Images in A–L′ are representative of three experimental repeats. (M,N) Scheme of cell migration and cell fusion during the formation of LVMs. (M) CVM and LVMp cells (magenta) migrate along and spread over the TVM (green). (N) Fusion of CVM founder cells with TVM-derived myoblast to form LVMs in three distinguishable steps. CVM, caudal visceral mesoderm; LVMp, longitudinal visceral muscle precursor; LVM, longitudinal visceral muscle; TVM, trunk visceral mesoderm. Schemes according to Rudolf et al., 2014; Sun et al., 2020. Scale bars: 50 µm, 25 µm, 10 µm (as labelled). White squares are enlarged areas in corresponding images. Arrowheads highlight nuclei. Anterior is to the left and dorsal is up in this and other figures.
To obtain more details of the CVM migration pattern, we marked the migration substrate, the TVM with anti-Fasciclin III (Fas III) in Beat IIb::GFP embryos (Fig. 1A–F′). Starting with late stage 11, when TVM cells formed a continuous longitudinal band, CVM cells migrated as a loose cellular stream along the dorsal and ventral margins of this band until they reached the foregut–midgut transition, where their anterior migration came to a halt (Fig. 1C,C′). Beginning with stage 14, when expression of Fas III is downregulated, Beat IIb-expressing cells started to migrate dorsal-ventrally along with the expansion of the developing midgut (Fig. 1D,D′). Following dorsal closure, at stage 15, Beat IIb-expressing cells were distributed evenly over the midgut chamber (Fig. 1E,E′). They then connected with their anterior and posterior neighbours, forming parallel rows that extended along the midgut tube (Fig. 1F,F′).
Given that downregulation of Fas III prevented further characterization of Beat IIb-positive cells, we employed the CVM-specific driver line HLH54Fd-GAL4 (Ismat et al., 2010) and UAS-Histone-RFP, where RFP is fused to histone 2A (Emery et al., 2005), to follow the number of nuclei in their differentiation process. Differentiated LVMs, like somatic muscle fibres, form syncytia generated by myoblast fusion, involving CVM-derived founder cells within the CVM population and fusion-competent myoblasts from the TVM (see scheme in Fig. 1M,N) (Rudolf et al., 2014). As shown in Fig. 1G–L′, initial fusion events have already occurred by stage 13 when the first group of binucleated cells appear, which are called LVMps (Fig. 1H′, arrowheads indicate nuclei). At early stage 14, most LVMps had two to three nuclei, whereas at late stage 14, LVMps containing three to four nuclei became predominant (Fig. 1I′). At the end of dorsal-ventral migration, five to six nuclei were found in LVMps (Fig. 1J′). From late stage 15 onwards, the multinucleated LVMps elongated and connected anteriorly and posteriorly with neighbouring cells, forming long and twisted fibres with evenly distributed nuclei (Fig. 1K–L′).
Beat IIa is expressed in CVM and LVMp cells
To test whether beat IIa, a close family member of beat IIb, exhibits a similar expression pattern to beat IIb, we applied fluorescence in situ hybridization (FISH) in an *HLH54Fb-*GFP background. We found that GFP-expressing cells in these embryos (Fig. 2A–J′, depicted in green) overlapped with cells labelled with both beat IIa and beat IIb antisense probes (depicted in magenta in Fig. 2A–J′). Although beat IIa mRNA was initially only weakly detected in a subset of CVM cells (Fig. 2A,A′), the signal increased in migrating CVM cells and was strongest at stage 13 (Fig. 2B,B′). From late stage 13 to 14, when LVMps develop and start to migrate dorsoventrally, beat IIa mRNA expression started to decline (Fig. 2C,C′). Afterwards, the signal decreased even further and was no longer detectable at stage 16 (Fig. 2D–E′). In addition, we also compared embryos from a GFP-tagged Beat IIa line (Carrier et al., 2024) crossed to HLH54Fb-cytoRFP (Fig. S1A–F′). There, Beat IIa was clearly detected in CVM and LVMp cells and overlapped with the cytoRFP marker (Fig. S1A–F′). The expression pattern of beat IIa was thus quite similar to that of beat IIb. However, we observed the following differences. First, beat IIb was upregulated earlier in CVM cells, already increasing by late stage 10 (Fig. 1A) and being readily detectable at stage 11 (Fig. 2F,F′), thereby confirming prior observation using probes for in situ hybridizations (Sun et al., 2024). Second, unlike beat IIa, which could not be detected at stage 16, beat IIb mRNA remained expressed in LVM cells until the end of embryogenesis (Fig. 2G–J′). Based on these observations, both Beat IIs are expressed in CVM and LVMp cells and might play a role during their development.
beat IIa and beat IIb are expressed in CVM and LVMp cells, and side IV is expressed in TVM cells. (A–J′) Confocal images (projections) of embryos expressing GFP in CVM and LVMp cells (HLH54Fb–GFP) stained with beat IIa or beat IIb antisense probes (magenta), followed by immunofluorescence using anti-GFP antibodies (green). White squares indicate regions enlarged in A′–J′. Arrows in D indicate expression in heart cells. (A–E′) beat IIa mRNA is expressed in CVM cells starting with stage 11. Expression levels increase strongly during anterior migration, but commence to decline after late stage 13. (F–J′) beat IIb mRNA is already detectable in CVM cells at stage 10. Expression levels then increase and are highest during migration at stages 13 and 14. Levels decrease then again, but transcripts are still detectable in LVMs at stage 16. (K–N′) Fluorescent in situ hybridization (FISH) using side IV antisense probes (cyan) in embryos expressing mCD8GFP under control of bap-GAL4 (Zaffran et al., 2001) in TVM cells (red, stained with anti-GFP antibodies). At stage 11, side IV mRNA is detectable in clusters of TVM cells and continues to be expressed during the migratory phase of CVM and LVMp cells. Images are representative of two experimental repeats. Scale bars: 50 µm (A–N); 25 µm (A′–N′).
Side IV is expressed in cells of the TVM
As Beat proteins are believed to function via Side proteins, we searched for potential binding partners from the Side family. In two biochemical assays, Side and Side IV had been shown to interact with Beat IIs (Li et al., 2017; Özkan et al., 2013). Given that Side functions in motor axon guidance and has no reported expression in the midgut (Siebert et al., 2009; Sink et al., 2001), we selected Side IV as a candidate. Using fluorescently labelled side IV antisense probes, we hybridized embryos expressing a transmembrane form of GFP under control of bagpipe (bap)-GAL4 (Zaffran et al., 2001), a specific marker of TVM cells (Fig. 2K–N′). Our findings suggested that side IV mRNA (Fig. 2K–N′, depicted in cyan) is transcribed in TVM cells (Fig. 2K–N′, depicted in red). It was first detected in segmental TVM clusters at stage 11 and lasted until stage 15 (see also single channel images in Fig. S5A–D′ for clarity). Afterwards, expression decreased in TVM cells.
To further verify expression in these cells, we employed the independent TVM marker Fas III in a Side IV–GFP knock-in line (also called Side IV-FsF-GFP) (Osaka et al., 2024). Co-stainings of Fas III and GFP confirmed that Side IV is detectable in the TVM starting with stage 12 (Fig. S1M–R). It is worth mentioning that from stage 15 on, Side IV–GFP was also observed in LVMps and LVM fibres (Fig. S1P–R, see also single-channel images in Fig. S5E–J for clarity). This expression pattern resembles Fas III expression, which is also first detected in the TVM and later in LVMps and LVMs (Klapper et al., 2002).
CVM and LVMp cells exhibit similar migration defects in the beat II and side IV mutant embryos
As Beat IIs are expressed in migrating CVM and LVMp cells and Side IV in their substrate, they might function together to assist the migration and differentiation of these cells. According to protein structure predictions, both beat II and side IV genes encode proteins of the immunoglobulin superfamily (Fig. 3A,B,D). Although both Beat IIa and Side IV are most likely transmembrane proteins, Beat IIb is predicted to be anchored at the plasma membrane via a GPI anchor. To investigate their functional role, we examined CRISPR/Cas9-induced single and double mutants carrying preliminary stop codons over their respective deficiencies (Fig. 3C,E). We first analysed beat II single mutants (beat IIa^+2^ and beat IIb^Δ14^) (Osaka et al., 2024) and found that the migration pattern of CVM and LVMp cells appeared rather unaltered (Fig. S2A–L). We only observed minor defects during the transition phase from stage 14 to 15, when the arrangement of LVMps seemed to be disturbed. Rather than being evenly aligned to ensheath the entire midgut, LVMps in each single mutant failed to fully cover its central region and left a gap between their dorsal and ventral belts (Fig. S2G,K, asterisks indicate gaps). Interestingly, this phenotype was much more evident in beat IIb^Δ14^ mutants. At stage 16, no significant abnormalities were found in the beat IIa^+2^ embryos, whereas in beat IIb^Δ14^, gaps devoid of LVM fibres were still observed (Fig. S2L).
CVM and LVMp cells show migration defects in beat II and side IV mutants. (A–E) Schemes of the domain structures of Beat IIa, Beat IIb and Side IV. Identified mutations and positions of non-complementing deficiencies are indicated. The numbers below the schemes indicates the range of amino acids (aa) of each domain. (A) According to our domain predictions (see Materials and Methods), Beat IIa contains two immunoglobulin domains (IG) and two transmembrane domains (TM). The beat IIa+2 mutation results in an insertion of two base pairs, leading to a frame shift (see Materials and Methods). (B) Domain predictions of Beat IIb forecasts two IG-domain and a site (aa 376) for the posttranslational addition of a GPI anchor. beat IIbΔ14 exhibits a deletion of 14 base pairs. beat IIbΔ5+3 consists of deletion of 5 bp and insertion of 3 incidental base pairs during the repair process, resulting in a frame shift that causes a premature stop codon (see Materials and Methods). (C) Df(3R)ED5780 deletes both beat IIa and beat IIb genes, as well as at least 20 additional genes. (D) Side IV is predicated as a 1001 aa transmembrane protein having five IG domains and one FN3 domain. CRISPR-induced mutations delete 64 bp or 28 bp leading to frame shifts and premature stop codons in the first IG domain after 2 or 14 different amino acids, respectively. (E) Df(3R)Exel7320 deletes the side IV gene as well as at least 15 additional annotated genes. (F–I) HLH54Fb-cytoRFP control embryos showing regular CVM and LVMp cell migration patterns. (J–M) In transheterozygous beat II double mutant embryos, CVM cells initiate anterior migration and reach the foregut–midgut transition, but they are more densely connected and do not migrate in a loose cell stream (compare arrows in J with F). They also have abnormal cell morphologies and orientations (arrowheads in K). LVMps fail to cover the entire midgut (green asterisks in L indicate empty areas). By the end of embryogenesis, large midgut areas are devoid of longitudinal muscles (asterisks in M). (N–Q) Transheterozygous side IV mutant embryos showing similar migration defects as in the beat II double mutant. Images are representative of three experimental repeats. Scale bar: 50 µm.
To analyse potential compensatory effects, we created beat II double mutants using CRISPR/Cas9 in the beat IIa^+2^ background (see Materials and Methods). When both beat genes were interrupted, CVM and LVMp cell migration was more drastically affected compared to that in wild-type embryos (Fig. 3F–M; Fig. S2M–P). In transheterozygous beat IIa^+2^beat IIb^Δ5+3^ mutants, although CVMs did initiate migration and reached their anterior destination, they were more densely clustered compared to controls, where cells arranged in a loose stream (compare arrows in Fig. 3F and J). These abnormalities progressively worsened over time. LVMps exhibited irregular migration patterns and showed abnormal cell morphologies and orientations (Fig. 3K, arrowheads). At stage 15, the midgut chamber was not evenly covered (asterisks in Fig. 3L), and large spaces were still devoid of LVM fibres by the end of embryogenesis (asterisks in Fig. 3M). Interestingly, side IV mutant embryos harbouring preliminary stop codons (see Materials and Methods) phenocopied beat II double mutant phenotypes (compare Fig. 3N–Q with J–M; Fig. S2Q–T). Hence, Beat IIs and Side IV appear to be required for proper spacing and orientation of migratory CVM and LVMp cells.
Broader and fewer LVM fibres in the beat II and side IV mutant larvae
The viability of beat II double mutant and side IV mutant larvae made it necessary to examine whether the aberrant migration patterns persist into postembryonic stages and reveal a permanent, non-compensatory phenotype in third-instar larvae (L3), that is, in number and/or morphology of LVM fibres. We, therefore, isolated midguts from w^1118^ and mutant L3 larvae and stained them with fluorescently labelled phalloidin. As shown in Fig. 4A–C, phalloidin stained both circular and longitudinal visceral muscle fibres. In the w^1118^ larvae, there were ∼18 longitudinal fibres in total covering the midgut (Fig. 4D, only one side is shown in Fig. 4A–C), and the average width of each fibre was ∼3.2±0.1 µm (mean±s.d.) (Fig. 4E). In beat II double mutant larvae, the number of fibres dropped to 12, whereas the average width increased to 4.3±0.3 µm (Fig. 4D,E). Compared to wild-type larvae, the number of LVMs was similarly decreased in side IV mutant larvae but showed no difference to beat II double mutants. However, the average width of LVM fibres increased to 4.9±0.3 µm. These broader fibres with a simultaneous decrease in their numbers might be due to improper migration and differentiation of CVMs and LVMps during embryonic development.
*Fewer and broader LVM fibres in beat II and side IV mutant larvae. (A–C) Confocal images of isolated midguts of third-instar larvae of the indicated genotypes stained with phalloidin conjugated with Texas Red-X. Arrows indicate LVM fibres. Only one side of the midgut is shown. Scale bars: 50 µm. (D) Quantitative analysis of LVM fibre number (counted on both sides of the midgut). w1118 control larvae have on average 18 LVM fibres (ranging from 12–23). beat II and side IV larvae show on average 10–12 LVM fibres (ranging from 4 to 16). (E) Quantitative analysis of LVM fibre width. In the control group, average width of the fibres is 3.2±0.13 µm. It is significantly increased in beat II mutants (4.2±0.28 µm, mean±s.d.) and side IV mutants (4.9±0.25 µm). In box plots, the box represents the 25–75th percentiles, and the median is indicated. The whiskers show the complete data range. ns, not significant; *P<0.05; **P<0.001 (two-tailed unpaired Welch's t-test; test for normal distribution was Kolmogorov–Smirnov with Lilliefors correction). n, number of biological replicates.
Restored Beat IIa in the CVM and LVMp cells rescues the mutant phenotype
To verify the function of beat II in the CVM and LVMp cell migration, we generated a UAS-Beat IIa-Cherry construct and used it for rescue experiments in beat II mutant embryos carrying a homozygous deficiency [Df(3R)ED5780]. This deficiency deletes both beat II genes and severely disrupted the migration patterns of CVMs and LVMps, as shown by the HLH54Fb-LacZ marker and anti-βGal staining (Fig. 5A–D).
*Restored expression of Beat IIa in CVM and LVMp cells partially rescues mutant phenotypes. (A–H″) CVM and LVMp cells are marked with HLH54Fb-LacZ and visualized using anti-βGal (magenta). (A–D) Homozygous beat II deficiency embryos serve as controls. (E–H″) Beat IIa–Cherry was overexpressed in CVM and LVMp cells in the beat II deficiency background using HLH54Fd-Gal4 and stained with anti-RFP antibodies (green). Merged images (E″,F″,G″,H″) show that the beat II mutant phenotype is largely rescued, when compared to the deficiency (A–D). First, at stage 13, migrating CVM cells arrange in an orderly cell stream (compare E with A). Second, LVMp cells show a more regular dorsoventral migration pattern than in deficiency embryos (compare F,G with B,C). Third, at stages 15–16, midgut areas are more extensively covered by LVMp cells than in controls (compare H with D). (I–K) Phalloidin-stained and dissected midguts of third-instar larvae, highlighting visceral muscles. Single LVM fibres are marked (arrows). Only one side of the midgut is shown. (I) beat IIa+2 beat IIbΔ5+3/Df(3R)ED5780 midguts display a reduced number of LVM cells when compared to those in wild-type embryos (K). (J) Overexpression of Beat IIa–Cherry in this background using HLH54Fd-GAL4 partially rescues LVM fibre numbers. Scale bars: 50 µm. (L,M) Quantitative analysis of LVM cell number and their width on both sides of the midgut. In wild-type animals, the number of LVM fibres is on average 18 (median, ranging from 14–21), whereas it is reduced to 10 (ranging from 7–13) in mutant animals (beat IIa+2 beat IIbΔ5+3 / Df(3R)ED5780). Expression of Beat IIa-Cherry in this mutant background [HLH54Fd>Beat IIa-Cherry in beat IIa+2 beat IIbΔ5+3 / Df(3R)ED5780 background] partially rescues LVM numbers to a median of 12.5 (ranging from 11–14) of fibres. The mean width (±s.d.) of LVM fibres in wild-type midguts is 4.8±0.9 µm compared to 6.5±1.1 µm in mutants, which is significantly restored in rescued larvae (5.2±0.7 µm). In box plots, the box represents the 25–75th percentiles, and the median is indicated. The whiskers show the complete data range. ns, not significant; **P<0.01; **P<0.001 (two-tailed unpaired Welch's t-test; test for normal distribution was Kolmogorov–Smirnov with Lilliefors correction). n value: number of biological replicates.
We then expressed Beat IIa–Cherry under control of HLH54Fd-GAL4 in this background (Fig. 5E–H″). At stage 13, when anterior migration is completed, cells were clearly more organized and arranged in a stream-like pattern in rescued embryos compared to the disordered pattern seen in controls (compare Fig. 5A with E–E″). During stages 14–15, although there were still some uncovered gaps on the midgut, LVMps were more regularly arranged and showed fewer irregular morphologies (compare Fig. 5B,C with F–G″). Similarly, more LVMps were differentiated and covered most of the surface of the midgut at later stages (compare Fig. 5D with H–H″). In addition, the rescued phenotype appeared similar to the beat IIb single mutant phenotype (compare Fig. 5E-H with Fig. S2I–L). To quantify these rescue abilities, we turned to transheterozygous third-instar larvae, as terminally differentiated LVMs are more readily accessible compared to irregularly migrating CVM and LVMp cells (Fig. 5I–M). Whereas ectopic Beat IIa was not able to fully rescue beat II mutant phenotypes (compare Fig. 5K with I,J), quantification showed that LVM number (Fig. 5L) and their thickness (Fig. 5M) had substantially improved compared to control larvae, suggesting that Beat IIa is partially sufficient to restore the mutant phenotypes.
LVMps lose contact with the TVM and undergo apoptosis
As large areas of the midgut lacked LVMps in mutant embryos and contained fewer differentiated LVM fibres in L3 larvae, this raised the question of what causes these phenotypes. To address this, we first monitored the longitudinal myoblast fusion process in side IV mutant embryos. Myoblast fusion appeared to be accelerated in side IV mutants, as more cells were detected that harboured more nuclei than in wild-type embryos at similar developmental stages (Fig. S3, compare A–D′ with G–J′). However, at stage 15, most LVMps contained five or six nuclei, which was identical to wild type, indicating that myoblast fusion might occur slightly earlier, but the fusion process per se is unaffected in the long term (Fig. S3, compare E–F′ with K–L′). We also used a marker for fusion-competent myoblasts, Sticks-and-stones (Sns) fused with GFP (Sns::GFP) in the endogenous locus (Lang et al., 2022), to track fusion events. Sns::GFP signals were spatially colocalized in single focal planes with LVMps even at stage 15, suggesting that they can fuse with fusion-competent myoblasts in the absence of Side IV (Fig. S3M–N″).
Because Side IV was expressed in the TVM, we surveyed the morphology of this cell population in side IV mutant embryos. Interestingly, although no abnormalities were observed in the TVM itself, the contact zone towards migrating LVMps was altered. During dorsal-ventral migration, wild-type LVMps kept tight contact with TVM cells to promote spreading (Fig. 6A,B). However, in side IV mutants, instead of being well-organized along the substratum, subsets of LVMps appeared to have lost their contact to the TVM. These off-track cells were frequently found in groups (Fig. 6E, circles) and displayed a rounded and condensed shape (Fig. 6D,E, arrows), indicating that they might undergo apoptosis. A similar phenomenon was also observed in wild-type embryos but limited to a small portion of cells (Fig. 6A,B, arrows) (Macabenta et al., 2022). These observations lead to the idea that cell loss might be responsible for the decreased number of LVM fibres.
*LVMps lose contact to the substrate and undergo apoptosis in the absence of Side IV. (A–F′) HLH54Fb-cytoRFP in control or side IV mutant embryos immuno-stained with anti-RFP to mark CVM and LVMp cells (magenta). (A,B,D,E) TVM cells are marked with anti-Fas III (cyan). (A,B) LVMp cells are in close contact with the TVM. (D,E) In side IV mutants, dorsal-ventral migration of LVMps is severely affected. Cells lose their contact with the TVM. Those off-track cells are often smaller and roundish in shape and irregularly distributed (arrows). White circle indicates grouped cells. (C,F) TUNEL stainings of apoptotic cells are shown in green. White squares indicate areas enlarged in C′ and F′. (C,C′) TUNEL signals are detected in the epidermis, head and ventral region of control embryos but only rarely in LVMps. Arrow highlights a TUNEL-positive cell. (F,F′) In side IV mutants, TUNEL signals are present in LVMps, which appear roundish and small (arrows in F′). (G–L) CVM and LVMp cells are marked with HLH54Fb-LacZ and stained with anti-βGal (magenta). (G,H) When p35 is expressed in CVM and LVMp cells in a wild-type background, migration patterns appear normal. (I,J) Migration patterns are disturbed in side IV mutants. (K,L) When p35 is overexpressed in a side IV mutant background, however, fewer cells with smaller and irregular shapes are observed (compare K,L with I,J; arrows indicate cells with abnormal morphology). (M–O) Phalloidin-stained, dissected midguts of third-instar larvae. Arrows mark single LVMs on one side of the midgut. (M) HLH54Fd>p35 served as wild-type control. (N) side IV mutant (side IVΔ64/side IVΔ28) midguts show reduced LVM fibre numbers. (O) This phenotype is partially rescued when p35 is overexpressed (HLH54Fd>p35) in a side IV mutant background. Scale bars: 25 µm (A–L); 50 µm (M–O). (P–R) Quantitative analysis of control (HLH54Fd>p35), mutant (side IVΔ64/side IVΔ28) and rescued (HLH54Fd>p35 in side IVΔ64/side IVΔ28) midguts. (P) Evaluation of abnormal LVMp cell shapes in side IV mutants. Six embryos at stages 13 and 14 from each genotype were analysed. Expression of p35 significantly reduced the number of irregular migrating cells. (Q,R) Evaluation of LVM fibre number and width (both sides of the midgut were counted). In control midguts, the median number of LVM fibres is 19.5 (ranging from 16–25). In mutant midguts, the median is reduced to 8 (ranging from 5–12), whereas rescue leads to 11 LVM fibres in median (ranging from 8–13). The mean fibre width (±s.d.) in wild type is 5.8±0.5 µm, which increases to 7.4±1.2 µm in mutants. Rescue leads to a mean of 7.1±1.6 µm. In box plots, the box represents the 25–75th percentiles, and the median is indicated. The whiskers show the complete data range. ns, not significant; *P<0.05; **P<0.01; **P<0.001 (two-tailed unpaired Welch's t-test; test for normal distribution, Kolmogorov–Smirnov with Lilliefors correction). n value, number of biological replicates.
To confirm apoptotic processes in LVMps during dorsal-ventral migration, we employed TUNEL assays that detect apoptotic DNA fragmentation. In control embryos, TUNEL signals were detected in the epidermis, head, and ventral regions, but only a few specific signals were detected in LVMps at stage 14 (Fig. 6C,C′). In comparison, TUNEL signals were prominent in abnormally shaped mutant LVMp cells in side IV mutants, suggesting they are undergoing apoptosis (Fig. 6F,F′).
If the death of these off-track cells is induced by apoptosis, then the expression of an apoptosis inhibitor, for example the baculovirus p35 protein (Hay et al., 1994), should prevent the death of these cells. Firstly, to exclude potential effects of p35 overexpression on CVM and LVMp migration, p35 was expressed using HLH54Fd-GAL4 in HLH54Fb-LacZ control embryos, but no significant abnormalities were observed in subsequent anti-βGal stainings (Fig. 6G,H). Then, p35 was overexpressed using the same driver in a side IV mutant background (Fig. 6K,L), and the results were compared with the side IV mutant migration pattern without p35 overexpression (Fig. 6I,J). We found that the number of off-track cells with round and shrunken shapes were significantly decreased upon p35 overexpression (Fig. 6P). Beyond analysis of these misguided cells, we turned again to the muscle pattern in dissected and phalloidin-stained midguts (Fig. 6M–O). Quantification of LVM number (Fig. 6Q) and their thickness (Fig. 6R) revealed that inhibition of apoptosis could partially rescue LVM numbers but not significantly rescue fibre width. However, as the distribution and orientation of the LVMps and longitudinal muscles were still severely disrupted in the absence of side IV, it was difficult to determine whether these off-track cells were solely responsible for the lack of LVMs.
Beat II interacts with Side IV in trans
Based on the above findings, one hypothesis is that Beat IIs might work together with Side IV to guide CVM and LVMp cells properly and keep them on track. As Beat II and Side IV are expressed in different cell types, their recognition and interaction in trans could be the basis for their mutual recognition. To investigate whether Beat II is able to interact with Side IV in trans on a cellular level, we subjected suspensions of individual S2 cells to cell–cell aggregation assays.
S2 cells were first transiently transfected with Beat IIa–GFP, signal peptide (SP)-GFP–Beat IIb (the SP ensures targeting to the plasma membrane) or Side IV–Cherry in separate cell culture dishes. After a 2-day incubation period, transfected cells were mixed in all permutations and incubated on a shaker for 2 h to facilitate potential interactions. As Beat IIa and Beat IIb were co-expressed in CVM and LVMp cells, a batch of S2 cells co-transfected with expression constructs for both of these proteins were included in addition.
As shown in Fig. 7, two-colour cell aggregates expressing both GFP-tagged Beat proteins and Cherry-tagged Side IV (arrows in Fig. 7A‴,B‴,C‴) indicated that Beat IIa and Beat IIb individually interacted with Side IV in trans, and that co-transfected cells behaved similarly to singly-transfected cells. Note that untransfected S2 cells also formed small aggregates at high cell densities, but those aggregates did not contain, or only individual, fluorescent cells (arrowheads in Fig. 7A‴,B‴,C‴). Quantitative analysis of the size of these aggregates revealed that the three proteins did not form significant homophilic aggregates (Fig. 7D, see also Fig. S4). The combined expression of Beat IIa and IIb showed by far the strongest heterophilic interaction with Side IV, although aggregate size was quite variable, and the interaction of Beat IIb with Side IV did not reach significance (Fig. 7D). Aggregation frequencies, the percentage of a 500×500 µm^2^ area containing at least one cluster, confirmed this conclusion (Fig. 7E). Taken together, these results suggest that Beat IIa- and Beat IIb-expressing cells are able to recognize and interact with Side IV-expressing cells.
*Beat IIa- and Beat IIb-expressing cells interact with Side IV-expressing cells. (A–C‴) Confocal images (projections) showing transiently transfected S2 cells expressing Side-IV–Cherry (magenta) and Beat II–GFP (green) subjected to cell–cell aggregation assays. In these mixtures, transfected cells cluster in large cell aggregates showing two fluorescent colours, largely sorting-out themselves from untransfected cells, indicating in trans protein–protein interactions (A‴–C‴, arrows). The transmitted light channel (T-PMT) was recorded to show all cells. Untransfected S2 cells occasionally form small cell clusters at high cell densities but mostly lack transfected cells (A‴–C‴, arrowheads). Scale bar: 100 µm. (D) Quantification of aggregate size (µm2) from randomly chosen aggregates. Aggregates formed by Beat IIa-transfected or Beat IIa- and Beat IIb-cotransfected S2 cells in mixtures with Side IV-transfected S2 cells are significantly larger in size than those observed in the respective control groups. P-values were obtained using a two-tailed unpaired Mann–Whitney U test (test for normal distribution: Kolmogorov–Smirnov with Lilliefors correction). The box represents the 25–75th percentiles, and the median is indicated. The whiskers show the complete data range. (E) Beat IIa-transfected or Beat IIa- and Beat IIb-co-transfected S2 cells in mixtures with Side IV-transfected S2 cells formed aggregates (dark green bars) at significantly higher frequencies compared to controls. Light green bars, non-aggregated cells. P-values were obtained using a two-tailed Fisher's exact test. Abbreviations: B2a, Beat IIa–GFP; B2b, SP–GFP–Beat IIb; B2C, cotransfection of Beat IIa–GFP and SP–GFP–Beat IIb; S4: Side IV–mCherry; SP:, signal peptide. ns, not significant; *P<0.05; **P<0.01; **P<0.001. n values (technical replicates) in D: B2a: 20; B2aS4: 28; B2b: 14; B2bS4: 29; B2C: 4; B2CS4: 62; S4: 4; n values (technical replicates) in E: 79.
Side IV ectopically expressed in trachea misguides migrating LVMps
The interactions across cell membranes suggest that Side IV and Beat IIs might function as ligand–receptor pairs in vivo, too. To test whether Side IV is able to attract Beat II-expressing cells to ectopic tissues, breathless (btl)-GAL4 was used to force ectopic expression of Side IV in developing trachea. From embryonic stage 12 onward, visceral branches of the tracheal system extend towards the midgut to provide oxygen to the intestine. As filopodial activity in migrating LVMps during this period is highly dynamic (Frasch et al., 2023), these cells might detect Side IV in trachea.
To rule out possible overexpression effects, UAS-mCD8GFP were used as a control. Moreover, side IV mutant embryos were chosen for these experiments, as they are devoid of endogenous Side IV. As shown in Fig. 8A–B″, when we expressed mCD8GFP in side IV mutant controls, the migration pattern of CVM and LVMp cells was similar to that of side IV mutants alone (white asterisks indicate developing visceral branches extending toward the midgut). Although, some colocalizations were observed (whitish areas in Fig. 8A–B″), they were probably caused by random spatial overlaps, as no direct contacts could be detected during the analysis of individual focal planes in these Z-projections.
*Ectopic expression of Side IV in the trachea misguides migrating LVMp cells. (A–D″) Fluorescent confocal images (projections) of side IV mutant embryos (side IVΔ64/side IVΔ28) expressing either mCD8GFP [btl>mCD8GFP; depicted in green (anti-GFP)] or Side IV [btl>Side IV-V5; depicted in cyan (anti-V5)] in trachea. LVMp cells were marked in each background using HLH54Fb-cytoRFP [depicted in magenta (anti-RFP)]. Asterisks in A–D″ indicate visceral tracheal branches. Ectopic expression of Side IV leads to misguidance of LVMp cells toward the visceral trachea (arrows in C″ and D″). (E–G′) Confocal images (projections) of the endogenous fluorescence of LVM fibres in intact first-instar larvae expressing HLH54Fb–cytoRFP in wild-type (E-E′) and side IV mutant backgrounds (F–G′). Mutant larvae express in addition either mCD8GFP (F,F′) or Side IV–V5 (G,G′) in all tracheal branches using btl-GAL4 but are not co-stained here. Overexpression of Side IV–V5, but not mCD8GFP, results in ectopic attachment of LVMs (white arrows in G′) to the dorsal tracheal trunk (indicated by a green broken line in E–G′). (F″) Expression of mCD8GFP (green) in the tracheal system and cytoRFP (magenta) in LVMs in side IV mutants is shown for clarity in an intact L1 larva (white arrows mark the dorsal trunk). (H–J) Dissected midguts stained with phalloidin showing LVMs (arrows) on one side in third-instar wild-type (H) or side IV mutant larvae (I,J). Mutant backgrounds expressed additionally mCD8GFP (I) or Side IV–V5 (J) using the btl driver. Ectopic Side IV–V5 exacerbates LVM phenotypes. Scale bars: 20 µm (A–D″); 50 µm (H–J); 100 µm (E–G). (K) Quantitative analysis of LVM fibre number and width (in µm) in dissected midguts of third-instar larvae (both sides counted). In wild-type animals, the median number of LVM cells is 17 (range 13–20). In side IV mutant backgrounds, the median is reduced to 7 (range 5–10) in btl>mCD8GFP and 4 (range 1–7) in btl>Side IV-V5. The mean LVM cell width (±s.d.) in wild-type is 4.5±0.5 µm, which increases to 6.9±1.3 µm (btl>mCD8GFP) and 8.3±2.7 µm (btl>Side IV–V5) in side IV mutant backgrounds. In box plots, the box represents the 25–75th percentiles, and the median is indicated. The whiskers show the complete data range. ns, not significant; **P<0.001 (two-tailed unpaired Welch's t-test; test for normal distribution was Kolmogorov–Smirnov with Lilliefors correction). n value, number of biological replicates.
In contrast, when we expressed V5-tagged Side IV in side IV mutant embryos using the same driver line (Fig. 8C–D″, overexpression was verified by anti-V5 staining and depicted in cyan), we found that the dorsal-ventral distance between the two migrating domains of LVMp cells was much narrower (Fig. 8C–D″). Moreover, tight contacts between LVMps and the trachea was observed, indicating these cells were misrouted towards Side IV-expressing tracheal branches (Fig. 8C″,D″). This phenotype persisted until postembryonic stages, as whole-mount confocal microscopy of undissected first-instar larvae (Fig. 8E–G) revealed RFP-expressing LVMs tightly aligned along the main dorsal tracheal trunk in Side IV–V5- but not mCD8GFP-expressing side IV mutants (arrows in Fig. 8Gʹ). Ectopic attraction also aggravated the loss of LVM fibres in side IV mutant third-instar larvae (Fig. 8H–J). Quantitative analysis showed that the number of LVM fibres was further reduced, whereas their width did not significantly increase upon Side IV–V5 overexpression (Fig. 8K). It thus seems that ectopic Side IV provides chemoattractive cues to misdirect LVMps, suggesting that Side IV might play an important role in guiding the migration of CVM and LVMp cells.
DISCUSSION
In an expression pattern screen of members of the beat and side gene families, we found that beat IIb is expressed in CVM cells (Bae et al., 2017; Ismat et al., 2010). As Beat IIb and other family members, like Beat Ia and Beat VI, have been shown to function together with Side proteins to play a guidance role during neurogenesis (Carrier et al., 2024; Osaka et al., 2024; Siebert et al., 2009; Yoo et al., 2023), we supposed that Beat IIb, and its closely related paralog Beat IIa, together here called Beat IIs, might also exert a function in migratory mesodermal cells. To search for potential interaction partners, we focused on Side and Side IV, which have been previously reported to interact with Beat IIs (Li et al., 2017; Özkan et al., 2013). Interestingly, we found that Side IV is expressed in TVM cells, which serve as a substrate for migrating CVM and LVMp cells.
Although beat IIa or beat IIb single mutants showed only minor defects, beat II double mutants showed obvious CVM and LVMp migration defects, indicating that they function redundantly. Of note, beat II double mutant phenocopied side IV mutants, suggesting that Side IV, too, is involved in this process. To examine the terminal outcomes of the disrupted migration patterns, we isolated midguts from L3 larvae and compared LVM fibre numbers and widths to controls. Here, we found broader but fewer LVM fibres in both beat II and side IV mutants. Increased fibre width might result from an abnormal distribution of LVM cells during embryogenesis or a potential postembryonic function of Beat II and Side IV in the further growth of longitudinal fibres during larval stages. Regarding the decreased numbers, one possibility might be that the fusion of CVM founder cells with fusion-competent myoblasts is disrupted so that fewer differentiated LVM muscles develop. However, our results suggest that myoblast fusion does not seem to be affected, when Side IV is absent (Fig. S3).
Previously, reduced LVM fibre numbers (from 8–11 to 5–8 for one half of the midgut) have been observed with loss-of-function mutations in ligands of the fibroblast growth factor (FGF) family (Reim et al., 2012). In the absence of FGF signalling, CVM and LVMp cells detach from the substrate, and their death results in fewer LVM fibres (Reim et al., 2012). In collectively migrating cells, maintaining tissue homeostasis by eliminating abnormal or mis-migrating cells is crucial for embryonic development, as disordered collective migration is linked to chronic inflammation and metastatic cancer (Lou et al., 2021; Paluch et al., 2016). Interestingly, loss of substrate contact in epithelial cells leads to a special type of apoptosis called anoikis, which is an important control mechanism to maintain tissue homeostasis (Paoli et al., 2013). Macabenta and colleagues have convincingly demonstrated that collectively migrating CVM cells use both FGF signalling and bone morphogenetic proteins (BMPs) to eliminate mis-migrated cells via anoikis (Macabenta et al., 2022). Both FGF and BMP receptors are present in CVM cells. Although the BMP2- and BMP4-related ligand Decapentaplegic (Dpp) is broadly expressed throughout the embryo, the FGF ligand is restricted to the TVM. In this model, CVM cells migrating on track activate both FGF and BMP signalling pathways, which ensures cell survival. However, detachment from the substrate and exposure only to BMP ligands induces anoikis and thus cell death (Macabenta et al., 2022).
Although both FGF and BMP pathways act at earlier stages during anterior-posterior migration of CVM cells, Beat II–Side IV complexes might function further downstream during the dorsal-ventral migration of LVMps. In side IV mutants, we recognized an increase of LVMp cells that lost contact to their substrate. These cells often displayed abnormal morphologies with rounded and condensed cell shapes, a hallmark of cells undergoing apoptosis. TUNEL signals, which report on apoptotic DNA fragmentation, were present in a subset of these off-track cells. Additionally, overexpression of p35 improved the number of terminally differentiated LVM fibres. One possibility is that molecules with redundant functions compensate for the loss of side IV and further support the orderly development of these surviving cells. Taken together, in a working model, we propose that, in the absence of Side IV, LVMps lose close contact to their substrate, and this de-adhesion triggers the anoikis programme in those cells, which eventually results in fewer LVM fibres.
If Beat IIs in migrating cells functionally interact with Side IV in their substrate, the ability to recognize and interact with each other in trans would be crucial. Our results using S2 cells in cell–cell aggregation assays have provided the first evidence that similar interactions could take place during embryonic development. Most importantly, Osaka and colleagues have recently shown that Beat IIb interacts with Side IV in trans to induce the formation of layer-specific synapses in the Drosophila visual system (Osaka et al., 2024). Whereas Beat IIb is expressed in M1–M2 and M9–M10 layers of the medulla, Side IV-negative R7 photoreceptor axons never form synapses in these layers. However, when Side IV was ectopically expressed in R7, additional synapses formed in Beat IIb-positive layers (Osaka et al., 2024). In addition, overexpressed Side IV caused GFP-tagged Beat IIb to become punctate, a common phenomenon upon clustering of synapse organization transmembrane proteins. Most interestingly, ∼70% of Side IV and Beat IIb colocalized with ectopic synapses, strongly suggesting that Beat IIb-expressing neurons could attract Side IV-expressing axons to trigger synapse formation (Osaka et al., 2024). Thus, interaction of Beat IIs and Side IV might exert additional functions, beyond synaptogenesis and neurogenesis, and might also regulate the migration of CVM and LVMp cells.
Suppose a protein provides a chemoattractive signal for receptor-expressing migrating cells; in that case, it should be possible to reroute the migration path when it is ectopically expressed in a neighbouring tissue. Previous studies have shown that when Side is ectopically expressed in the tracheal system of side mutant embryos, that is, in the absence of endogenous Side, Beat Ia-positive motor axons are strongly attracted to tracheal branches and tightly attach to them (Siebert et al., 2009; Sink et al., 2001). In side mutants alone, that is, without any ectopic Side expression, motor axons do not establish any prolonged contacts with tracheal branches, indicating that Side-expressing substrates are attractive guides for Beat Ia-expressing motor axons (Siebert et al., 2009).
Similarly, if Side IV acts as an instructive molecule to guide the migration of Beat II-expressing CVM and LVMp cells, it should also be possible to re-route the migration path by creating ectopic attraction. During embryogenesis, the visceral branches migrate toward the midgut and distribute over the surface of the visceral mesoderm (Hayashi and Kondo, 2018). Meanwhile, at these stages, the filopodia activity of the LVMps is highly dynamic (Frasch et al., 2023), which means that when Side IV is overexpressed in the tracheal system, it should be detected by active filopodia of migrating LVMps. Although LVMp cell migration is disordered in side IV mutant embryos, these cells did not show any direct connections with mCD8GFP-expressing trachea. In contrast, when Side IV was overexpressed in tracheal branches, they attracted migrating LVMps, as many cells were observed migrating along tracheal tubes. At the same time, large spaces, especially in the anterior region of the midgut, were devoid of LVMps. As a result, most surviving LVMps were clustered along the visceral trachea, and only a few were dispensable for the formation of LVM fibres, indicating that Side IV plays a prominent role in the regulation of CVM and LVMp migration.
In conclusion, our study suggests that Beat IIs interact with Side IV to promote directional migration of longitudinal visceral muscle precursors over the substrate. In the absence of Beat II–Side IV interaction, these cells show abnormal migration patterns, resulting in fewer LVM fibres.
MATERIALS AND METHODS
Genetics and fly stocks
Fly stocks were raised and crossed at 25°C on a standard Drosophila cornmeal medium. The following stocks were used: *HLH54Fb-*GFP (Ismat et al., 2010); HLH54Fb-cytoRFP (also known as P{HLH54F.LVM-RFP}16c) (Hollfelder et al., 2014); HLH54Fb-LacZ (Ismat et al., 2010); Beat IIa::GFP (also known as beat-IIa::FlpTag-Constitutive) (Carrier et al., 2024); Beat IIb::GFP [also known as Beat IIb^MI03102-GFSTF.0^, Bloomington Drosophila Stock Center (BDSC) #59406] (Nagarkar-Jaiswal et al., 2015); Side IV::GFP (also known as Side IV-FsF-GFP; flip-out cassette was removed using hs-FLP, kindly provided by Takashi Suzuki, School of Life Science and Technology, Tokyo Institute of Technology, Japan) (Osaka et al., 2024); beat IIa^+2^ (Osaka et al., 2024); beat IIb^Δ14^ (Osaka et al., 2024); Df(3R)ED5780 (BDSC #8104) (kindly provided by Manfred Frasch, Institute of Developmental Biology, Friedrich-Alexander-University Erlangen-Nürnberg, Germany); Df(3R)Exel7320 (BDSC #7975); Sticks-and-stones::GFP (Sns::GFP), a C-terminal GFP insertion in the sns locus, also called nephrin-GFP (Lang et al., 2022); HLH54Fd-GAL4 (BDSC #91633); btl-GAL4 (Iordanou et al., 2014) (kindly provided by Mark Krasnow, Stanford University School of Medicine, Standford, USA); bap-GAL4, also called bap3-GAL4 (Zaffran et al., 2001) (kindly provided by Ingolf Reim, Institute of Developmental Biology, Friedrich-Alexander-University, Erlangen-Nürnberg, Germany); UAS-Histone-RFP (Emery et al., 2005); UAS-Side IV-V5 (Osaka et al., 2024); UAS-mCD8GFP (Lee and Luo, 1999); UAS-p35 (BDSC #5072) (kindly provided by Thomas Klein, Institute of Genetics, Heinrich Heine University, Düsseldorf, Germany).
Generation of CRISPR/Cas9 mutants for beat II and side IV
CRISPR/Cas9 (Jinek et al., 2012) was used to create beat IIa and beat IIb (beat II) double mutants in a beat IIa^+2^ background (Osaka et al., 2024). Briefly, a beat IIb gRNA was designed (5′-GGCACAGTAGCAGTAGCCAC-3′) using the CRISPR Optimal Target Finder (Gratz et al., 2014) and inserted into the pCFD3-dU6:3 vector (Port et al., 2014) (Addgene plasmid #49410) using the corresponding protocol and sent for injection into beat IIa^+2^ mutant background at FlyORF (FlyORF Injection Service, University of Zurich, Switzerland). The hatched flies were balanced over w^−^;; Dr/TM6C. Stable lines were established, and genomic DNA was amplified by PCR and sequenced for CRISPR/Cas9-induced mutations. A single stock carrying mutations in both beat IIa and beat IIb (beat IIa^+2^ beat IIb^Δ5+3^) was established. The Beat IIb protein sequence (isoform PA) is altered after isoleucine at position 79 to 74-YGSNVI-TATATVPFL* in this stock.
Mutations in side IV were generated by crossing a side IV-specific TRiP-KO line (BDSC #82064; Zirin et al., 2020) to Vasa-Cas9 (BDSC #51323). Male progenies were crossed to virgins of w;; Dr/TM6C, followed by backcrossing of individual offspring of the F2 generation to w;; Dr/TM6C. Homozygous larvae from the established stocks were used to isolate genomic DNA for PCR amplification and sequencing of the side IV locus. side IV^Δ28^ deletes 28 bp after alanine codon 131 in side IV mRNA isoform RC, which changes the reading frame to 125-RQFGQA-APAPTSAAPPIPPS*. side IV^Δ64^ deletes 64 bp after the same alanine codon, changing the reading frame to amino acid 125-RQFGQA-LS*.
Cloning of reporter constructs and generation of transgenic fly lines
UAS-Side IV-Cherry was a kind gift from Takashi Suzuki (Osaka et al., 2024). beat IIa and beat IIb (beat II) constructs were designed by amplifying defined fragments of full-length beat II cDNAs using regular PCR or overlap extension PCR (Nelson and Fitch, 2011) with Q5 proofreading polymerases (New England Bio Labs, Frankfurt, Germany). beat IIa cDNA was from the Drosophila Genetics Resource Center (RE17794). beat IIb cDNA (according to FlyBase beat IIb-RC transcript) was reverse transcribed from total RNA (Totally RNA Kit, Life Technologies, Carlsbad, CA, USA) extracted from ∼20 w^1118^ larvae using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific, Schwerte, Germany).
UAS-Beat IIa-Cherry and UAS-Beat IIa-GFP were generated as described previously for Beat Ia (Heymann et al., 2022). Briefly, full-length Beat IIa (isoform RA) lacking a stop codon [amino acids (aa) 1–431], was cloned into the Gateway Entry Vector pENTR D-Topo (Thermo Fisher Scientific, Schwerte, Germany) and sequenced from both ends. By using LR clonase (Thermo Fisher Scientific, Schwerte, Germany) and the destination vectors pUASTattB_rfA_mCherry or pUASTattB_rfA_eGFP (both provided by Floriano Rodrigues, Sven Bogdan and Christian Klämbt, Institute of Neuro- and Behavioral Biology, University of Münster, Germany), full-length Beat IIa was C-terminally fused to mCherry or eGFP, respectively. Owing to the LR recombination sites, linker sequences encoding additional amino acids (KGGRADPAFLYKVVISAS/R) were inserted between cDNAs and C-terminal tags. UAS-Beat IIa-Cherry was injected into the attP40 (located at 25C6 on chromosome II) (Bischof et al., 2013) to establish transgenic flies at FlyORF (FlyORF Injection Service, University of Zurich, Switzerland).
For generating the UAS-SP-GFP-Beat IIb construct, overlap extension PCR was used to insert the eGFP fragment (amplified from pUASTattB_rfA_eGFP) between the predicted signal peptide (SP) of Beat IIb (aa 1–20) and cloned into the Gateway entry vector. After sequencing, the SP-GFP–Beat IIb construct was recombined into the destination vector pUASTattB_rfA (provided by Floriano Rodrigues, Sven Bogdan and Christian Klämbt) as described above.
Immunofluorescence staining
Drosophila embryos were immunostained (Mahr and Aberle, 2006) and staged (Campos-Ortega and Hartenstein, 1985) as described in the given reference. For larval midgut stainings, midguts were dissected from third-instar larvae on dissection plates (Roland Vetter Laborbedarf OHG, Ammerbuch, Germany) using forceps (Dumont#5), microscissors (Vannas Spring Scissors, straight, 4 mm cutting edge) and 0.1 mm Minutien pins (equipments from Fine Science Tools, Heidelberg, Germany). Isolated midguts were fixed with 3.7% formaldehyde for 15 min, washed with PBS with 0.1% Triton X-100 (PTx), and blocked with PTx containing 5% normal goat serum (NGS) for 30 min. Stainings were developed using phalloidin conjugated with Texas Red (Thermo Fisher Scientific, Schwerte, Germany) for 2 h at room temperature. Midguts were washed with PTx, cleared in 70% glycerol in PBS, and mounted on microscope slides (coverslips 22×22 mm).
Dilutions of primary antibodies were as follows: rabbit anti-GFP 1:1000 (cat. no. TP401, Acris Antibodies, Herford, Germany), mouse anti-GFP 1:100 [clone 1D2, AB 2617419; Developmental Studies Hybridoma Bank (DSHB), Iowa City, IA, USA], rat anti-GFP 1:1000 (cat. no. 04404-26, Nacalai Tesque, Kyoto, Japan), rabbit anti-dsRed 1:500 (detects RFP, a monomeric form of dsRed; cat. no. 632496, Takara Bio**,** San Jose, CA, USA), mouse anti-βGal 1:100 (clone 40-1a, AB 528100; DSHB, Iowa City, IA, USA), mouse anti-V5 1:200 (cat. no. R960-50; Thermo Fisher Scientific, Schwerte, Germany), mouse anti-Fas III 1:40 (clone 7G10, AB 528238; DSHB, Iowa City, IA, USA). Secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) were diluted 1:500.
Fluorescence in situ hybridization
Embryos were fixed in 3.7% formaldehyde in PBS following a standard fluorescence in situ hybridization (FISH) protocol (Tautz and Pfeifle, 1989). The antisense digoxigenin (DIG)-labelled RNA probes targeting beat IIa, beat IIb, and side IV mRNAs were generated using a Dig-labelling mixture (#11277073910, Roche, Basel, Switzerland) with the corresponding plasmids beat IIa (DGRC #RE17794), beat IIb (DGRC #RE10028) and side IV (full-length side IV cDNA amplified from the UAS-Side IV-Cherry and inserted into pBluescript-SK). For detection of the endogenous transcripts, sheep anti-Digoxigenin-POD Fab fragments (#11207733910, Roche, Basel, Switzerland) were used in conjunction with Cy3-tyramide (AAT Bioquest, Pleasanton, CA, USA) according to the manufacturer's instructions. After tyramide staining, embryos were either mounted in 70% glycerol in PBS or subjected to standard antibody staining procedures.
TUNEL assay
Freshly fixed Drosophila embryos from overnight collections were subjected to the One-Step TUNEL assay kit according to the instructions of the manufacturer (Elabscience Biotechnology, Houston, TX, USA). Briefly, unspecific binding sites of dechorionized and permeabilized embryos were blocked using 500 μl PTx plus 5% NGS at room temperature (RT) for 30 min on a nutating shaker (VWR International, Leuven, Belgium). Primary antibodies were added, and embryos were incubated for 2 h at room temperature. After three washes with 500 μl PTx (10 min each), 100 μl TdT equilibration buffer was added. Embryos were incubated on a shaker at 37°C for 30 min, followed by adding the reaction mixture containing the secondary antibodies according to the instructions.
Cell–cell aggregation assay
The cell–cell aggregation assay using S2 cells (Thermo Fisher Scientific, Schwerte, Germany; cat. no. 51-4003) was performed as described previously (Heymann et al., 2022). Briefly, 2 ml S2 cells (2×10^6^–4×10^6^) at the fifth to tenth passage were seeded into six-well plates on the day before transfection. Cells were transiently co-transfected using specific UAS vectors and the common GAL4 vector (pBPGUw-Act5C-GAL4, provided by Sven Bogdan, Institute of Molecular Cell Physiology, Philipps-University Marburg, Germany) using Effectene Transfection Reagent (#1054250, Qiagen, Hilden, Germany). After incubating at 28°C for 3 days, a total of 500 μl transfected cells were seeded into a 35 mm glass bottom dish (ibidi GmbH, Martinsried, Germany) and incubated at room temperature under constant shaking at 80 r/min for 2 h before microscopy. Cells were tested monthly for mycoplasma and contaminations.
Microscopy
Confocal laser scanning microscopes (LSM710 and LSM880, Carl Zeiss MicroImaging, Jena, Germany) were used to acquire microscopic images. Fiji software (Schindelin et al., 2012) was used to process raw images [generally 1024×1024 pixels but 2320×2320 pixels for 40× oil objectives (Nyquist frequency); line averaging 2-4]. Z-stacks were compressed, unless otherwise noted, as maximum intensity projections and imported into Adobe Illustrator (Adobe Systems, Dublin, Republic of Ireland) for figure assembly. Microscopic images are assembled from at least three biological replicates (n>3). For imaging intact, undissected first-instar larvae, animals of a similar age were heat-shocked in a 65°C water bath for 1 s, mounted and immediately imaged. First-instar larvae and embryos were oriented dorsal side up and anterior left in all figures.
Protein structural prediction
Several programs were used to predict the domain structures of Beat II and Side IV proteins. SMART (Letunic and Bork, 2017), AlphaFold (Jumper et al., 2021) and ScanProsite (de Castro et al., 2006) were used to predict immunoglobulin domains. PredGPI (Pierleoni et al., 2008) and big PI-Predictor (Eisenhaber et al., 2003) were used to predict a possible GPI-modification site. DeepTMHMM (Hallgren et al., 2022 preprint) and InterPro (Blum et al., 2024) were used to analyse possible transmembrane domains, and Phobius was used to predict signal peptides (Käll et al., 2004).
Statistical analysis
The number of LVM fibres was evaluated in a central portion of the midgut, counting all fibres along its circumference (‘on both sides’). The width of the LVM fibres was measured using the ‘Straight selections’ and ‘Measure’ tools of Fiji software (Schindelin et al., 2012). Biological replicates are derived from different animals.
For quantitative analysis of cell–cell aggregation assays, aggregates were randomly chosen and evaluated using Fiji software. To analyse their size, their outlines were depicted using ‘Freehand selections’ followed by determining their area using the ‘Measure’ tool. The frequency of aggregate formation was quantified by evaluating 500×500 µm^2^ fields of the same dish (technical replicates; tile scan). Fields containing a minimum of one aggregate (minimum size 100 µm^2^) were counted positive and set in relation to all evaluated fields.
Data analysis was performed and presented using Prism 9 software (GraphPad Software, Boston, MA, USA). Statistical tests were performed as described in figure legends. Briefly, a Mann–Whitney U test, Fisher's exact test or Welch's t-test (normal distribution was tested using Kolmogorov–Smirnov test with Lilliefors correction) was used depending on the data sets.
Supplementary Material
10.1242/joces.264157_sup1Supplementary information
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