Hox Function Is Required for the Development and Maintenance of the ...

Article

Hox Function Is Required for the Development and Maintenance of the Drosophila Feeding Motor Unit Graphical Abstract

Authors Jana Friedrich, Sebastian Sorge, Fatmire Bujupi, Michael P. Eichenlaub, Natalie G. Schulz, Jochen Wittbrodt, Ingrid Lohmann

Correspondence [email protected]

In Brief By studying feeding behavior in Drosophila, Friedrich et al. show that the Hox transcription factor Deformed (Dfd) controls feeding motor unit formation. They show that Dfd controls wiring and maintenance of the feeding unit by stagespecifically regulating expression of target genes in motor neurons and in muscles.

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Hox gene Dfd is critical for feeding motor patterns in Drosophila

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Dfd instructs motor unit formation via stage- and tissuespecific target genes

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Synaptic specificity is dependent on actions of Dfd in motor neurons and muscles

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Maintenance of synapses is directly regulated by Dfd

Friedrich et al., 2016, Cell Reports 14, 1–11 February 2, 2016 ª2016 The Authors http://dx.doi.org/10.1016/j.celrep.2015.12.077

Please cite this article in press as: Friedrich et al., Hox Function Is Required for the Development and Maintenance of the Drosophila Feeding Motor Unit, Cell Reports (2016), http://dx.doi.org/10.1016/j.celrep.2015.12.077

Cell Reports

Article Hox Function Is Required for the Development and Maintenance of the Drosophila Feeding Motor Unit Jana Friedrich,1 Sebastian Sorge,1 Fatmire Bujupi,1 Michael P. Eichenlaub,2 Natalie G. Schulz,1 Jochen Wittbrodt,2 and Ingrid Lohmann1,* 1University

of Heidelberg, Centre for Organismal Studies (COS) Heidelberg, Department of Developmental Biology, 69120 Heidelberg, Germany 2University of Heidelberg, Centre for Organismal Studies (COS) Heidelberg, Department of Developmental Biology and Physiology, 69120 Heidelberg, Germany *Correspondence: [email protected] http://dx.doi.org/10.1016/j.celrep.2015.12.077 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

SUMMARY

Feeding is an evolutionarily conserved and integral behavior that depends on the rhythmic activity of feeding muscles stimulated by specific motoneurons. However, critical molecular determinants underlying the development of the neuromuscular feeding unit are largely unknown. Here, we identify the Hox transcription factor Deformed (Dfd) as essential for feeding unit formation, from initial specification to the establishment of active synapses, by controlling stage-specific sets of target genes. Importantly, we found Dfd to control the expression of functional components of synapses, such as Ankyrin2-XL, a protein known to be critical for synaptic stability and connectivity. Furthermore, we uncovered Dfd as a potential regulator of synaptic specificity, as it represses expression of the synaptic cell adhesion molecule Connectin (Con). These results demonstrate that Dfd is critical for the establishment and maintenance of the neuromuscular unit required for feeding behavior, which might be shared by other group 4 Hox genes.

INTRODUCTION Stereotypical motor behaviors are the primary means by which animals interact with their environment, forming the final output of most CNS activity. One such behavior is feeding, a crucial and highly conserved activity in all animals. The motor output consists of coordinated contractions of distinct head muscles in a rhythmic pattern required for chewing, sucking, and swallowing of food. Food uptake in adult flies has recently been shown to be controlled by a single pair of interneurons emanating from the subesophageal ganglion (SEG), an insect brain region primarily associated with taste and feeding (Flood et al., 2013). Although a substantial number of neurons are linked to different aspects of feeding behavior in flies (Cameron et al., 2010; Manzo et al., 2012), molecular factors critical for the estab-

lishment and development of feeding motor patterns have not been identified so far. The fruit fly Drosophila melanogaster is an excellent model to study the developmental aspect of feeding behavior for several reasons. First, Drosophila takes up food extensively during its larval stage, when the organism almost exclusively feeds to increase its body weight and size. Additionally, the anatomical framework and motor patterns critical for larval food uptake are well described. Feeding requires the rhythmic extension and retraction of the head skeleton, the cephalopharyngeal skeleton (CPS), coupled with coordinated elevation and depression of the mouth hooks (MHs), mandible-derived structures required for chopping up solid food, and subsequent food ingestion (Schoofs et al., 2009). The repetitive larval feeding movements are controlled by head muscles innervated by CNS nerves emerging from the SEG. CPS protraction and tilting are mediated by protractor muscles receiving input from the prothoracic nerve, while MH motor patterns are controlled by the mouth hook elevator (MHE) and depressor (MHD), which are innervated by the maxillary nerve. Food ingestion is achieved by the cibarial dilator muscle (CDM), which is connected to the CNS via the €ckesfeld et al., 2015; Schoofs et al., 2009). antennal nerve (Hu The cellular framework of Drosophila larval feeding is established during embryogenesis. Thus, molecular and genetic approaches can be used to identify and analyze factors controlling specification and communication of cell types critical for larval motor patterns. In contrast, neuromuscular units required for motor activities in adult flies develop from stem cell systems during larval and pupal stages in a process called metamorphosis. Due to the limited accessibility of this transitional phase, embryonic stages are better suited to study the development of neuromuscular units required for regional movements. The Hox family of transcription factors (TFs) have emerged as key regulators of motor behaviors (Dalla Torre di Sanguinetto et al., 2008; Guthrie, 2007; Philippidou and Dasen, 2013). One such behavior is locomotion, which Drosophila larvae perform by region-specific contractions of abdominal segments allowing them to crawl on substrate. Segment-specific changes of peristaltic movements in animals carrying mutations in the Hox genes Ultrabithorax (Ubx) and abdominal-A (abd-A) led to the assumption that Hox genes orchestrate the development of regional motor activities (Dixit et al., 2008). Recent studies Cell Reports 14, 1–11, February 2, 2016 ª2016 The Authors 1


(Baek et al., 2013; Philippidou et al., 2012) have now revealed that Hox proteins perform their task in a very refined manner and seem to have a direct transcriptional input on successive steps of motoneuronal development. As one such example, Hox5 function was shown to be required in motoneurons that control the contraction of breathing muscles in vertebrates: Hox5 deletion in mice leads to progressive death of phrenic motor column (PMC) neurons as well as to the inability of surviving PMC neurons to innervate the diaphragm muscle. However, despite the fact that blocking motoneuron apoptosis did rescue the decline in PMC neuron number, branching and innervation defects were still unchanged under these conditions. These findings imply that Hox5 proteins directly regulate early and late processes in the course of PMC neuron differentiation, a hypothesis still awaiting confirmation. Hox genes are segmentally expressed along the anteriorposterior body axis of animals (McGinnis and Krumlauf, 1992; Pearson et al., 2005), suggesting that members of this gene family expressed in the head region should control food uptake. Intriguingly, the Drosophila group 4 Hox gene Deformed (Dfd), which is known to specify the SEG (Hirth et al., 1998), has already been associated with feeding behavior before: animals carrying a hypomorphic Dfd allele starve to death as adult flies due to the inability to move their proboscis (Merrill et al., 1987; Restifo and Merrill, 1994) (Figures S1H, S1J, and S1K), an action crucial for food ingestion (Schoofs et al., 2009). Our work reveals that Dfd, which is expressed in many cell types, including a large €ckesfeld et al., 2015; Kuert et al., number of SEG neurons (Hu 2014; Schoofs et al., 2009), is functional in motoneurons and muscles that drive the movements critical for hatching and feeding. Most interestingly, we show that Dfd exerts its function via direct and stage-specific regulation of target genes. Importantly, we demonstrate that Ank2-XL, a microtubule organizing protein required for synaptic stability (Stephan et al., 2015), is under direct Dfd control throughout different stages in the animal’s life. Furthermore, we found synchronous expression of Dfd targets with critical function in synaptic target specificity, in particular, the cell adhesion molecule (CAM) Connectin (Con), in feeding neurons and muscles. This suggests that Dfd positively and/or negatively regulates different CAMs providing a specificity code required for the establishment of regional motor units. RESULTS The Hox Gene Dfd Is Required for Feeding and Hatching Motor Patterns To study the molecular basis of feeding motor patterns, we tested the Hox gene Dfd, which had been previously associated with adult feeding behavior in Drosophila (Merrill et al., 1987). Complete inactivation of the Dfd gene has severe consequences, since Dfd loss-of-function animals (Dfd16) die at the end of embryogenesis due to their inability to hatch from the eggshell (Figure S1G) (Merrill et al., 1987; Regulski et al., 1987). Embryonic death makes Dfd16 mutants poor candidates for the analysis of feeding motor patterns during larval stages. However, the same rhythmic movements that allow larvae to feed on solid food (Pereanu et al., 2007) are required earlier, when the vigorous elevation and depression of the MHs are used to tear open the 2 Cell Reports 14, 1–11, February 2, 2016 ª2016 The Authors

chorion during hatching (Pereanu et al., 2007). Due to the dependency of both hatching and feeding on the same motor unit, we used hatching behavior as a functional output of MH dependent motor activity in this study. At the same time, this setup allowed for the analysis of factors crucial for the establishment of feeding-associated neuromuscular units during embryonic stages, even if inactivation of these factors results in embryonic lethality. Rhythmic elevation and depression of the MHs is realized by two opposing muscles, the MHE and MHD, which receive synaptic input from the maxillary nerve (Figures 1A and 1E), a nerve €ckesfeld et al., bundle originating in the maxillary neuromere (Hu 2015; Schoofs et al., 2009, 2010). This part of the CNS, in combination with the labial and mandibular neuromeres, forms the feeding- and taste-related SEG (Figures 1C and 1E), expresses the Hox protein Dfd (Figures 1D, 1F, 1G, S1A, and S1B) (Hirth et al., 1998; Kuert et al., 2014), and depends on Dfd’s action for proper axogenesis (Figures S2A and S2B) (Hirth et al., 1998; Kuert et al., 2014). Our analysis revealed that in addition to severe axon guidance errors (Figures S2A and S2B), Dfd16 null mutants were unable to perform coordinated movements of the head at late embryonic stages (Movie S1) and, thus, were completely impaired in their hatching abilities in comparison to control animals (Figure S1G). In contrast, wild-type embryos of the same age intensely moved their MHs to free themselves from the eggshell (Movie S1). While loss of motor activity might explain the inability of Dfd16 null mutant animals to emerge from the eggshell, the absence of MHs in these animals (Figures S1D and S1E) (Merrill et al., 1987; Regulski et al., 1987) abolishes any kind of behavior associated with these structures. Thus, we analyzed head-specific motor patterns in animals with reduced Dfd levels (Dfd13/Df(3R)Scr) (Merrill et al., 1987), which are able to develop normal MHs (Figure S1I). However, despite their presence MH motility was severely impaired in Dfd13/Df(3R)Scr larvae (Movie S1) and 48.3% of mutant animals were unable to hatch (Figure S1G). These results demonstrate that rhythmic MH movements are dependent on the Hox gene Dfd and critical for head-associated behaviors. Dfd Is Expressed in a Few SEG Motoneurons that Innervate the MHE Motor control of behavior requires the precise wiring of motoneurons and the target muscles they innervate (Arber, 2012; Guthrie, 2007), indicating that Dfd is active in these cell types. Immuno-histochemical analysis revealed that Dfd protein is localized in OK371-GAL4 (Mahr and Aberle, 2006) labeled motoneurons of the SEG in late embryos (Figure 1G). Similar results on Dfd localization in feeding-associated neurons were obtained when using the Dfd neuronal autoregulatory enhancer (Figures 1F and S1B), termed DfdNAE667, known to be active exclusively in Dfd-expressing SEG neurons (Lou et al., 1995). In order to specifically label Dfd-expressing motoneurons, we restricted GAL4 expression patterns by applying the FLP-induced intersectional GAL80/GAL4 repression (FINGR) system (Bohm et al., 2010). Due to a recent report showing that all motoneurons in the SEG are generated exclusively during embryonic stages, unlike interneurons which also develop post-embryonically (Kuert et al., 2014), we were able to label and follow all Dfd-positive


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Figure 1. Dfd Is Expressed in SEG Motoneurons that Innervate Muscles Required for Head-Specific Motor Patterns

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motoneurons in DfdNAE667-Flp,tubP > GAL80 > ,OK371::mCD8GFP animals throughout their lifetime. mCD8-GFP expression starts in late embryos/first-instar larvae; strong and robust expression in third-instar larvae revealed that only two to three motoneurons per hemisegment within the SEG are Dfd positive and project axons via the maxillary nerve to the MH-associated muscles (Figure 1D). Importantly, Dfd-expressing SEG motoneurons exclusively innervate the MHE (Figures 1B and S1C), forming functional synapses as indicated by expression of the Drosophila vesicular glutamate transporter (DVGlut) (Figures 1B and S1C). Although the MHD is also innervated by motoneurons originating in the SEG and projecting their axons via the €ckesfeld et al., 2015), they do not express maxillary nerve (Hu Dfd. In sum, these results show that Dfd is expressed in a few motoneurons innervating the MHE and suggest that different upstream regulators are active in motoneurons controlling the counteracting parts of this motor unit. Dfd Neurons Control Hatching and Feeding Movements To test whether Dfd-expressing neurons and their neuromuscular connections are pivotal for the execution of MH motor activity, we blocked synaptic transmission in Dfd-positive SEG neurons by expressing the active form of tetanus toxin (TNT-R) known to inhibit neurotransmitter release at presynaptic endings

(A) Diagram of a third-instar (L3) larval head highlighting the structures critical for mouth hook movements: mouth hook (MH), cephalopharyngeal skeleton (CPS), mouth hook elevator (MHE), mouth hook depressor (MHD), maxillary nerve (MN). The cibarial dilator muscle (CDM), used in this study as control muscle, is indicated. (B) MHE and MHD of DfdNAE667-Flp,tubP > GAL80 > , OK371::mCD8-GFP L3 larvae stained with Myosin to label muscles, DVGlut to mark functional synapses and GFP. (C) Diagram of a L3 CNS with the brain lobes (BLs), the ventral nerve cord (VNC), and the MN exiting the subesophageal ganglion (SEG) highlighted. (D) SEG from a DfdNAE667-Flp,tubP > GAL80 > , OK371::mCD8-GFP L3 larval CNS stained with Dfd, GFP, and DAPI for the DNA, arrowheads mark two to three SEG motoneurons that also express Dfd, and inset shows 3D reconstruction of these neurons. (E) Diagram of the head of a stage 16 Drosophila embryo with the MHE and MHD muscle precursors, the MN, and the SEG highlighted. (F and G) Close up of the SEG of stage 16 embryos expressing mCD8-GFP in Dfd-positive neurons by means of the Dfd-specific neuronal driver DfdNAE667-GAL4 (F) or the motoneuronal driver OK371-GAL4 (G). Arrowheads highlight Dfdexpressing neurons, which project their axons into the MN. Scale bars, 50 mm in (B) and (D), 20 mm in (F) and (G). See also Figure S1.

(Sweeney et al., 1995) under the control of the DfdNAE667 enhancer. To exclude cholinergic sensory and interneuronal inputs, we introduced a choline-acetyltransferase (Cha)-GAL80 transgene, which restricts expression primarily to motoneurons (Pulver et al., 2009). The DfdNAE667 enhancer starts to be active during neurogenesis; thus, we monitored MH movements and screened hatching abilities at the end of embryogenesis. Due to the onset of strong GAL4-induced activity only in larval stages, we did not use DfdNAE667-Flp,tubP > GAL80 > ,OK371::mCD8GFP animals for the hatching assay. In comparison to control animals (Cha-tub-GAL80ts,DfdNAE667::IMPTNT-V1) (Movie S2), which express the inactive version of tetanus toxin (IMPTNTV1) (Sweeney et al., 1995), MH movements and hatching abilities were abolished in tub-GAL80ts,DfdNAE667::TNT-R animals, while peristalsis of more posterior body parts remained unaffected (Figure 2A; Movie S2). We next tested the requirement of Dfdpositive neurons during larval feeding. Here, we restricted TNT-R expression in tub-GAL80ts,DfdNAE667::TNT-R animals to larval stages by making use of the temperature-sensitive GAL80 transgene (McGuire et al., 2004). Motor activity of the MHs was examined by measuring the angle between the MHs and the H-piece during one feeding cycle in third-instar larvae (Figures 2B–2D). In control animals (tub-GAL80ts, DfdNAE667::IMPTNT-V1), this angle varied between 70 and Cell Reports 14, 1–11, February 2, 2016 ª2016 The Authors 3


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Figure 2. Dfd-Positive Neurons Control MH Movements during Embryonic and Larval Stages (A) Hatching rates of Cha-GAL80,DfdNAE667::IMPTNT(V1) and Cha-GAL80,DfdNAE667::TNT-R animals. (B) Measurements of the angle between the MHs and the H-piece during one feeding cycle in GAL80ts,DfdNAE667:: IMPTNT(V1), GAL80ts,DfdNAE667:: TNT-R and in DfdNAE667-Flp,tubP > GAL80 > ,OK371::IMPTNT(V1) and DfdNAE667-Flp,tubP > GAL80 > ,OK371::TNT-R larvae. Blue and red points indicate measurements of individual animals; blue and red lines represent the means of these measurements. (C and D) One cycle of MH movements, as indicated by cyan line, in GAL80ts,DfdNAE667::IMPTNT(V1) control (C) and GAL80ts,DfdNAE667::TNT-R (D) animals is shown in an exemplary larva and in the schematic drawing.

170 (Figures 2B and 2C), reflecting the depression and elevation of the MHs during feeding (Movie S3) (Schoofs et al., 2009). However, restricted expression of TNT-R in Dfd-positive neurons during larval stages almost completely abolished MH movements: the angle between the MHs and the H-piece varied only between 57 and 71 in tub-GAL80ts,DfdNAE667::TNT-R larvae (Figures 2B and 2D), which corresponds to the depression phase in control larvae (Figure 2B). The inability of these animals to elevate their MHs supports the exclusive innervation of the MHE by Dfd-expressing SEG motoneurons (Figures 1B and S1C). Similar results were obtained when studying MH movements in late first-instar larvae expressing TNT-R under the control of the DfdNAE667-Flp,tubP > GAL80 > ,OK371-GAL4 driver (Figure 2B), demonstrating that MH elevation movements are controlled more or less cell-autonomously by Dfd-expressing motoneurons. However, due to the activity of the OK371-GAL4 line also in very few non-motoneuronal glutamatergic neurons (Mahr and Aberle, 2006), we cannot completely exclude a minor cell non-autonomous contribution to the phenotypes observed. 4 Cell Reports 14, 1–11, February 2, 2016 ª2016 The Authors

Overall, these results show that Dfd is expressed in SEG motoneurons to control the MH-dependent motor program, which is required at the end of embryogenesis for hatching and during larval life for feeding. Dfd Is Crucial for Axon Outgrowth The loss of motor activity in Dfd mutant embryos could simply be a manifestation of the loss of feeding-associated SEG neurons. However, unlike in the epidermis (Lohmann et al., 2002), the number of apoptotic cells in the CNS was unchanged in Dfd mutant embryos (Figures S3B and S3C). Similarly, clonal inactivation of Dfd in the postembryonic SEG did not affect neuronal cell numbers (Kuert et al., 2014). Nonetheless, Dfd mutant cells are defective in their developmental program, since efferent, most likely motor projections of the maxillary nerve were not detectable in Dfd16 loss-of-function embryos (Figures 3E, 3G, and S2D) when compared to wild-type embryos (Figures 3D and S2C), while afferent projections from sensory organs were unchanged (Figures 3A and 3B). Efferent projections of the


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Figure 3. Dfd Is Crucial for Axon Outgrowth (A and D) Lateral view of stage 16 wild-type embryonic heads stained with the peripheral nervous system (PNS) axon marker Futsch/MAP1B (A), and the CNS axon marker FasII (D). The MN projecting from the CNS to the anterior part of the head is highlighted by closed arrowheads; the asterisks mark the exit point of the labial nerve from the CNS. (B and E) Lateral view of the heads of stage 16 Dfd16 null mutant embryos stained with Futsch/ MAP1B (B), and FasII (E). The presence or absence of CNS, most likely motoneuronal projections, are indicated by closed or open arrowheads. (C and F) Lateral view of the heads of stage 16 Dfd16 null mutant embryos, in which Dfd expression is restored in motoneurons using the OK371GAL4 driver. Axon projections from the CNS (maxillary neuromere) are partially restored (F), as highlighted by closed arrowheads. (G) Quantification of efferent maxillary nerve projections in wild-type, Dfd16, OK371::Dfd,Dfd16, Dfd13/Df(3R)Scr, Dfd16/Df(3R)Scr and embryos. The exit point of the labial nerve is marked by asterisks in all images. Lateral scale bars, 20 mm. See also Figure S2.

observation that efferent projections are completely missing in Dfd null mutant embryos indicates that Dfd also plays a non-cell-autonomous role in the development of other SEG motoneurons.

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maxillary nerve were also not compensated by ectopic projections originating from other neuromeres (Figures 3E and S2D). The effects of Dfd are specific, since loss of Dfd function did not affect other nerves, like the labial nerve that projects from the more posteriorly located labial neuromere (Figures 3E and S2D). Importantly, efferent projections of the maxillary nerve were reinforced when Dfd was transgenically activated in motoneurons of Dfd16 mutants using the OK371-GAL4 driver (Figures 3F and S2E), afferent projections from sensory organs were not influenced in this genetic background (Figure 3C). Due to the dependency of the DfdNAE667 autoregulatory enhancer on Dfd input (Lou et al., 1995), regionally restricted re-introduction of Dfd in maxillary neurons could not be performed. These results show that Dfd activity in motoneurons is necessary to instruct the proper developmental program in these cells. Interestingly, we only observed regional nerve outgrowth from the maxillary and in very rare cases from the labial neuromere, in the ‘‘rescue’’ situation, indicating that Dfd collaborates with additional regulatory proteins in SEG motoneurons to regulate their development, which it is not able to in other CNS motoneurons. Finally, our

Dfd Is Continuously Required during Feeding Unit Formation In line with a previously reported temporal requirement of Hox genes (Philippidou et al., 2012), we found efferent (motor) projections within the maxillary nerve, which are abolished in Dfd loss-of-function mutants (Figures 3E and S2D), to be normal in embryos with reduced Dfd levels (Dfd13/Df(3R)Scr) (Figure S2F). Nonetheless, the ability of these embryos to perform MH-dependent motor patterns was severely impaired (Movie S1), and their hatching abilities were significantly reduced (Figure S1G). These results indicated that Dfd is required not only during the early cell type specification phase, but also later when axons need to find and form functional synapses with their proper muscle targets. We used a Dfd temperature-sensitive loss-of-function allele (Dfd3) (Merrill et al., 1987), which allowed fast interference with Dfd activity at any time during development (Figure 4A), to support temporal requirement of Dfd. As expected, Dfd3 mutants raised at the permissive temperature (18 C) resembled wild-type embryos phenotypically and with respect to motor behavior (data not shown), while they exhibited morphological abnormalities and motor defects reminiscent of Dfd16 null mutants at the restrictive temperature (31 C) (Figure S1F) and died at the end of embryogenesis (Figure 4B). However, when Dfd3 embryos were raised at the permissive temperature up to the stage when synapses have Cell Reports 14, 1–11, February 2, 2016 ª2016 The Authors 5


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Figure 4. Dfd Controls Target Genes Required for Synaptic Stability and Connectivity (A) Diagram of temperature-shift regimens applied to Dfd3 mutants to stage-specifically interfere with Dfd function (for details, see Experimental Procedures). (B) Hatching rates of homozygous Dfd3 and wild-type/Dfd3 control animals subjected to shift A regimen, and of Dfd3 animals raised at the permissive (18 C) or restrictive temperature (31 C) throughout embryogenesis. Mean of three individual collections is shown. Error bars indicate 95% confidence interval. Unpaired t test, two-tailed, two-sample unequal variance was used. (C and D) Expression of Ank2-XL and the neural cell membrane marker HRP in MHE synapses of a L3 wild-type/Dfd3 control larva (C) and a Dfd3 larva subjected to shift B regimen (D). Closed arrowheads indicate granular pattern of Ank2-XL expression in synaptic boutons, open arrowheads indicate loss of Ank2-XL expression in synaptic boutons, and yellow arrowheads mark the areas of enlarged boutons. (E and F) Expression of Ank2-XL and HRP in MHE synapses of a L3 UAS-DfdVDRC50110 control larva (E) and a elav::dcr-2,DfdVDRC50110 larva. Ank2-XL is expressed in a granular pattern within synaptic boutons (closed arrowheads). Open arrowheads indicate loss of Ank2-XL expression in synaptic boutons; yellow arrowheads mark the areas of enlarged boutons. The same results were obtained with an independent Dfd RNAi line generated in the lab.

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formed (Prokop, 1999) and the first motor activity is initiated and shifted only then to the restrictive temperature during embryogenesis (shift A) (Figure 4A), a high number of Dfd3 embryos (40%) were unable to perform proper MH movements and did not hatch from the eggshell (Figure 4B; Movie S4) when compared to control animals (Figure 4B; Movie S4). This is surprising, since innervation of the MHE was comparable to control early first-instar larvae still enclosed in the eggshell at the end of embryogenesis (Figures S4A and S4B). Intriguingly, when we performed shift experiments to the restrictive temperature at early third-instar larval stages and analyzed head motor patterns at mid third-instar larval stages (shift B) (Figure 4A), we found MH movements to be impaired (Movie S4) in comparison to the control group (Movie S4). These results demonstrate that Dfd is required at different steps during neuromuscular network formation and indicates that, in addition to the establishment phase, Dfd also controls the system after synaptic connections are already functional. Dfd Controls Target Genes Required for Synaptic Stability and Connectivity Consistent with a temporal requirement of Dfd for motoneuronal development, we found an over-representation of neuronal genes among those genes associated with ChIP-seq identified Dfd binding regions (Sorge et al., 2012), which we classified as Dfd target genes (see Experimental Procedures). Importantly, grouping of these genes based on similar GO annotations (see Experimental Procedures and Table S1) showed that they operate at different time points in neuronal development: during neurogenesis and neuronal specification (32/182), when axon outgrowth and guidance decisions occur (86/182), and during synapse-related processes (85/182) (Figure S3A; Table S1). To test the temporal control of these genes by Dfd, we analyzed their expression when Dfd function was abolished at two different developmental stages. For early interference, we used Dfd16 loss-of-function embryos, while late interference was achieved using animals that carry the temperature-sensitive Dfd3 allele and were shifted to the restrictive temperature only during larval stages (shift B) (Figure 4A). We found early neurogenesis target genes, including prospero, a gene involved in asymmetric neuroblast division (Li et al., 1997), to be mis-localized in Dfd16 null mutant embryos (Figures S3D and S3E). Consequently the expression of genes required for subsequent processes in motoneuronal development, like the axon guidance genes capricious (caps) (Milań et al., 2001; Shishido et al., 1998), roundabout 2 (robo2) (Kidd et al., 1998; Rajagopalan et al., 2000), roundabout 3 (robo3) (Spitzweck et al., 2010), and Neural Lazarillo (NLaz) (Sańchez et al., 2000), were also affected (Figures S3F–S3M). Thus, Dfd16 null mutants are unable to form the neuromuscular unit required for MH movements due to the inability to activate the proper developmental program. In contrast, the MH-associated motor unit of Dfd3 animals shifted

to the restrictive temperature during larval stages was intact, with respect to outgrowth of maxillary nerve projecting motoneurons and MHE innervation (Figures 4C, 4D, S4E, and S4F). Accordingly, the expression of early Dfd neuronal targets was unchanged (data not shown). However, compared to the control group the expression of Dfd target genes critical for synapserelated processes was substantially altered in these late-shifted Dfd3 third-instar larvae (shift B) (Figures 4C, 4D, S4E, and S4F). This includes Ankyrin2 extra large (Ank2-XL), which is encoded in the ank2 locus (Koch et al., 2008). Ank2-XL, which is part of a membrane-associated microtubule-organizing complex, is known to be required for the establishment of appropriate synaptic dimensions and release properties (Stephan et al., 2015). We not only found Ank2 mRNA levels to be reduced in SEG neurons in late-shifted Dfd3 third-instar larvae (shift B) (Figures S4G and S4H), but we also observed decreased Ank2-XL protein expression in synaptic boutons, axons and their terminals on the MHE (Figures 4C, 4D, S4E, and S4F). Similar to a recent report (Stephan et al., 2015), we also found Futsch/MAP1B, a microtubule-associated protein known to form a membraneassociated complex with Ank2-XL, to be reduced in synaptic boutons of late-shifted Dfd3 third-instar larvae (Figures S4E and S4F). Concomitantly, the morphology of synaptic boutons on this muscle was also changed in late-shifted Dfd3 third-instar larvae (shift B): they were not of uniform size but appeared often dramatically increased compared to boutons of control animals (Figures 4C, 4D, 4I, S4E, and S4F). This is in line with the described phenotype of ank2-XL mutant animals (Stephan et al., 2015), which was suggested to reflect the failed separation of neighboring boutons. The effects observed are due to Dfd’s action in (moto)neurons, as tissue-specific knockdown of Dfd activity in neuronal cells only using the elav-GAL4 driver in combination with two independent UAS-DfdRNAi lines resulted in severe bouton phenotypes and Ank2-XL expression changes (Figures 4E, 4F, 4I), while the muscle architecture was completely normal (data not shown). Similar results on Ank2-XL expression and synapse morphology were obtained in Dfd13/Df(3R)Scr third-instar larvae that survived to this stage (Figures 4G, 4H, and 4I). The effect of Dfd on synapses on the MHE is specific, since neuromuscular junctions on control muscles, like the CDM, were completely normal with respect to their morphology and Ank2-XL expression in late-shifted Dfd3 third-instar animals (shift B) (Figures S4C and S4D). These results show that Dfd activity is continuously required during the formation of the feeding motor unit, from its specification to the establishment of synaptic connections, and that Dfd executes this function by directly regulating the transcription of phase-specific components. Intriguingly, our findings demonstrate that the Hox TF Dfd is one of the upstream regulators coordinating Ankyrin-dependent microtubule organization and synapse stability and provides evidence that Dfd function is required even after the initial establishment of the motor unit to control synapse-related processes

(G and H) Expression of Ank2-XL and the neural cell membrane marker HRP in MHE synapses of a L3 wild-type larva (G) and a Dfd13/Df(3R)Scr larva (H). Closed arrowheads indicate granular pattern of Ank2-XL expression in synaptic boutons, open arrowheads indicate loss of Ank2-XL expression in synaptic boutons, and yellow arrowheads mark the areas of enlarged boutons. Representative images are shown (n = 15 for C and D, n = 10 for E and F, n = 5 for G and H). (I) Quantification of bouton size from five neuromuscular junctions of each genotype (n = 88–207 boutons) are represented as Tukey boxplot. Scale bars, 10 mm. See also Figure S4.

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via its synaptic targets, like Ank2-XL. Finally, our results indicate that synaptic stability and plasticity is not only determined by the half-life of synaptic proteins, but is dependent on a robust transcriptional program that provides a continuous supply of essential synaptic components that maintain the system. Dfd Is Active in Muscles and Motoneurons that Form a Functional Neuromuscular Feeding Unit Our analysis has shown that Dfd is expressed in SEG motoneurons (Figure 1D). In addition, we found Dfd to be present in embryonic muscles, which later form the feeding/hatching motor unit (Figure 5A). We observed defects in the structure and number of the MH-associated muscles in the embryo when Dfd function was abolished (Figures 5B and 5G). As was the case in the CNS, Dfd seems to execute its muscle-specific function in an im8 Cell Reports 14, 1–11, February 2, 2016 ª2016 The Authors

Figure 5. Dfd Is Active in Muscles and Motoneurons Driving Head-Specific Motor Patterns (A) Lateral view of internal head muscles in a stage 16 wild-type embryo stained with Dfd, the muscle marker Myocyte enhancer factor 2 (Mef2), and Myosin. Closed arrowheads indicate head muscles neighboring the MHE and MHD that are devoid of Dfd protein. (B) Lateral view of internal head muscles in a stage 16 Dfd16 null mutant embryo stained with Dfd, Mef2, and Myosin. Open arrowheads indicate the reduced number of Mef2 positive nuclei. (C and D) Expression of the homophilic cell adhesion molecule Connectin (Con) in head muscles (C) and SEG neurons (D). (C) Lateral view of internal head muscles in a stage 16 wild-type embryo stained with Dfd, Con, and Myosin. Closed arrowheads indicate head muscles next to the MHE and MHD that express Con. (D) Lateral view of the CNS of a stage 16 wild-type embryo stained with Dfd, Con, and Elav. Closed arrowheads indicate neurons that express Con but not Dfd. (E and F) Expression of Con and Dfd mRNA in SEG neurons of stage 16 wild-type (E) and Dfd16 mutant (F) embryos. Arrowheads in (E) mark the few Con mRNA-expressing cells in the SEG devoid of Dfd mRNA. (G) Quantification of the muscle phenotypes in control (Dfd16/TM3) and Dfd16 homozygous mutants. In all cases, representative images are shown (n > 20). Scale bars, 10 mm.

mediate manner, since a substantial fraction (7.4%) of the genome-wide identified Dfd target genes (Sorge et al., 2012) is associated with mesoderm-related functions (Table S1). The innervation of the Dfd-expressing MHE by Dfd-positive motoneurons raised the intriguing possibility that the activity of the Hox protein Dfd provides a code on the functionally connected neurons and muscles crucial for the recognition and matching of the synaptic partners and, thus, the execution of rhythmic motor patterns. Consistent with this hypothesis, 27 of the ChiP-seq identified Dfd target genes encode factors with described functions in muscles and the nervous system (Table S1), and importantly nine of these genes play an important role in synaptic target recognition (Nose, 2012; Sanes and Yamagata, 2009; Winberg et al., 1998), like tartan (trn), Connectin (Con), or capricious (caps). Therefore, we analyzed the expression of the homophilic cell adhesion molecule Con and found it to be exclusively expressed in motoneurons and muscles devoid of Dfd protein in wildtype embryos (Figures 5C and 5D), suggesting that Dfd might function as a suppressor of Con expression. In order to provide vigorous proof for this hypothesis, we specifically labeled cells that were devoid of Dfd function in Dfd mutants and analyzed their ability to now express Con. Here, we made use of the fact that Dfd16 mutants that do not produce any functional protein


(protein-null mutants) still express Dfd mRNA. Consistent with our hypothesis, we found de-repression of Con mRNA expression in many Dfd mutant neuronal cells that were labeled by the presence of Dfd mRNA (Figures 5E and 5F). Due to the inability of Dfd mutant embryos to involute their heads (Merrill et al., 1987) (which reorganizes the order of the head muscles) and due to the high variance of the muscle phenotype, we do not show Con expression in this tissue in the Dfd loss-of-function situation. Taken together, these results demonstrate that Dfd is one of the critical upstream regulators, which coordinates the interdependent events of neuromuscular development and connectivity by positively or negatively regulating the expression of synaptic target selection molecules on the interacting motoneurons and muscles. Furthermore, it shows that the expression of synaptic cues is tightly regulated even in neurons located in close or direct proximity, allowing these cells to express different sets of synaptic recognition molecules thereby ensuring that they make the proper connections with their synaptic partners.

Hoxb4-GFP medaka embryos (Figure S5A). Intriguingly, a subset of neurons co-expressing GFP and Hoxb4 project their axons ventrally toward Hoxb4-positive cells within the pharyngeal region (Figure S5B). Both the branchial muscles and the pharyngeal jaw specialized for feeding in teleost fish develop from this area (Schilling and Kimmel, 1997). When medaka embryos have developed into hatchlings, axons emerging from GFPlabeled neurons innervate branchial muscles and the sternohyodeus (Figures S5C and S5D), muscle groups required for mouth opening and food swallowing (Schilling and Kimmel, 1997). Thus, the regulatory and transcriptional network dictating the formation of the respective feeding units in flies and fish could be conserved despite the fact that muscles and bones responsible for the execution of feeding movements are of different origin. In future, more functional studies are needed to validate the potentially conserved role of homology group 4 Hox genes in regulating rhythmic feeding movements throughout the animal kingdom.

DISCUSSION

EXPERIMENTAL PROCEDURES

Hox genes have been shown to control several motor activities along the anterior-posterior axis of animals (Arber, 2012; Dixit et al., 2008; Philippidou and Dasen, 2013; Philippidou et al., 2012); however, critical determinants regulating feeding movements had not been identified. In this study, we show that the Drosophila group 4 Hox gene Dfd controls multiple aspects in both the establishment and maintenance of the neural network controlling feeding behavior. A crucial finding from our study is that Hox TFs are required throughout the formation of regional motor units and mediate their effect not only through the induction of downstream TFs. In fact, we could show that Hox factors control distinct effector target genes, which realize stage-specific processes in a very immediate manner. This is true for Ankyrin2-XL, which, along with the MAP1B homolog Futsch, forms a membrane-associated microtubule-organizing complex that determines axonal diameter, supports axonal transport, and controls synaptic dimensions and stability (Stephan et al., 2015). Interestingly, we found Dfd was required for the maintenance of Ank2-XL expression, not only when the motor system is established but also when it is fully operational. In the light of recent findings showing that mis-regulation of Ankyrin 1 (ANK1) has an important role in the neurodegenerative Alzheimer disease (Lunnon et al., 2014), these results raise the intriguing possibility that Hox genes have a neuro-protective function. An important question arising from our study is whether the establishment of feeding-related motor patterns is one of the basic functions of group 4 Hox genes and thus conserved in the animal kingdom. Promisingly, it is known that tongue muscles critical for rhythmic feeding movements in mammals are innervated by the hypoglossal nerve (Guthrie, 2007). This nerve has its origin in rhombomere 8, which expresses several group 4 Hox genes, including Hoxb4 (Guthrie, 2007). Preliminary analysis using a previously identified fish Hoxb4 promoter (Hadrys et al., 2006) as a reporter in the teleost fish medaka (Oryzias latipes) shows that GFP is expressed in distinct neuronal subpopulations of the post-otic hindbrain and the spinal cord in stable

Fly Strains See Supplemental Experimental Procedures for a list and description of fly strains used in this study. Transgenics See Supplemental Experimental Procedures for a detailed description of the generation of transgenic flies. Immunohistochemistry and In Situ Hybridization Labeling of Drosophila embryos and larvae was performed as described previously (Tautz and Pfeifle, 1989) with minor modifications. Digoxigenin- and biotin-labeled antisense RNA probes were generated using the Roche RNA labeling system (Roche). Fluorescent duplex in situ hybridizations were done as described previously (Kosman et al., 2004). For probe detection, the TSA Plus Fluorescein and Cy3 Systems from PerkinElmer were used. For a list of antibodies used in this study and for details on image acquisition and processing, see Supplemental Experimental Procedures. Image Analysis and Statistics Images were analyzed with FIJI/ImageJ and Adobe Photoshop CS6. Synaptic bouton size was determined using horseradish peroxidase (HRP) staining on MHE NMJs using FIJI. Statistical analysis was performed using Microsoft Excel. Bar graph data are presented as mean values ± 95% confidence interval. Unpaired t test, two-tailed, two-sample unequal variance was used to calculate statistical significance. Boxplots were generated with BoxPlotR (http://boxplot.tyerslab. com) in Tukey-style. Central mark represents the median, the edges of the boxes the 25th and 75th percentiles and whiskers indicate 1.5 times interquartile range. Dots indicate outliers. Identification and Definition of Dfd Target Genes Dfd target genes were defined as genes located in the vicinity of Dfd binding regions (upstream, downstream, intronic) as previously defined by ChIP-seq experiments (Sorge et al., 2012). For this study, peak calling was done using Model-based Analysis of ChIP-seq (MACS [GEO: GSE73493]) (Feng et al., 2011) with a p value threshold of 0.001, which resulted in the identification of 3,897 enrichment peaks; the UCSC Genome Browser Database (Kent et al., 2002) was used to determine the location of all peaks. Genes associated with ChIP-seq peaks occurring in intergenic or intronic regions were subsequently classified according to their Gene Ontology (GO) annotations; genes with functions related to CNS development were further grouped into earlyphase, mid-phase, or late-phase neuronal genes based on specific annotation terms listed in Table S1.



TUNEL Labeling The in situ cell death detection kit, TMR red (Roche), was used to assay cell death in embryos according to the protocol (Krieser et al., 2007). Time-Lapse Movies See Supplemental Experimental Procedures for a detailed description. Tetanus Toxin Assay in Embryos To block synaptic transmission during embryogenesis, we used DfdNAE667GAL4;Cha-GAL80 flies crossed to UAS-TNT-R or UAS-IMPTNT(V1) (Sweeney et al., 1995) flies. Time-lapse movies were taken to analyze MH movements at late stages of embryogenesis before hatching. Hatching rates were determined 48 hr after egg laying (AEL). Tetanus Toxin Assay and the Documentation of MH Movements in First-Instar Larvae DfdNAE667-GAL4;tub-GAL80ts flies were crossed to UAS-TNT-R or UASIMPTNT(V1) (Sweeney et al., 1995) flies, respectively. Embryos of a 2-hr deposition at 25 C were kept at 18 C for the next 34 hr before they were shifted to 29 C. Six hours later, the hatched first-instar larvae were transferred to a piece of agar and placed on a microscope slide. In a second experimental setup, DfdNAE667-Flp;UAS-TNT-R or DfdNAE667Flp;UAS-IMPTNT(V1) flies were crossed to OK371-GAL4,5xUAS-mCD8GFP;tubP > GAL80 > flies. Embryos of a 2-hr deposition at 25 C were kept at 25 C for the next 40 hr until late first-instar larval stages. Larvae were transferred to a piece of agar and placed on a microscope slide. Time-lapse movies were taken from larvae using the Axio Zoom V16 microscope and AxioVision Release 4.7.2 software. The angles between the MHs and H-piece were measured using the ‘‘Angle tool’’ of the Fiji/ImageJ software as indicated by the dotted lines in the respective figures. Temperature-Shift Experiments We analyzed animals of the genotype Dfd3/Dfd3. Dfd3/wild-type was used as a control. For the first experimental setup (shift A), embryos of 1 hr egg depositions were raised at 18 C on yeast covered apple juice plates for 28 hr until embryonic stage 17b (Pereanu et al., 2007) and subsequently shifted to 31 C. Hatching rates were determined 48 hr AEL. Time-lapse movies were taken 5 hr after the temperature shift and shortly before hatching. For dissections and staining of the head apparatus and CNS, the vitelline membrane was removed manually from first-instar larvae before the time point of hatching. For late interference with Dfd function (shift B), Dfd3/Dfd3 and Dfd3/wild-type embryos of 1 hr egg depositions were raised at 18 C for 150 hr until they had reached early third-instar larval stage. Subsequently, the larvae were shifted to 31 C and kept for 20 hr (170 hr) before antibody staining. To document MH movements, movies were made from larvae using the Nikon SMZ18 microscope and Nikon DS-U3 camera. Dissections of the head apparatus and the CNS combined with antibody stainings were performed 20 hr after the temperature shift. RNA Interference Two independent Dfd-RNAi-lines, Vienna line 50110 and a Dfd siRNA line (this study), were crossed to elav-GAL4;UAS-dcr-2 flies. 1 hr egg depositions at 25 C were raised at 29 C until the third-instar larval stage, followed by dissection and antibody stainings. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, five figures, one table, and four movies and can be found with this article online at http://dx.doi.org/10.1016/j.celrep.2015.12.077.

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