Expression profiling reveals novel role of Hunchback in retinal glia cell development and blood-brain barrier integrity

3 Montserrat Torres-Oliva, Julia Schneider, Gordon Wiegleb, Felix Kaufholz, Nico Posnien 4 5 Universität Göttingen, Johann-Friedrich-Blumenbach-Institut für Zoologie und Anthropologie, 6 Abteilung für Entwicklungsbiologie, GZMB Ernst-Caspari-Haus, Justus-von-Liebig-Weg 11, 37077 7 Göttingen, Germany 8 9 Correspondence: nposnie@gwdg.de 10 11 MT-O: mtorres@gwdg.de 12 JS: schneider.julia86@gmail.com 13 GW: gordon.wiegleb@stud.uni-goettingen.de 14 FK: felix.kaufholz@uni-goettingen.de 15 NP: nposnie@gwdg.de 16 Abbreviations: after egg laying (AEL), first larval stage (L1), 2nd larval stage (L2), third larval 17 stage (L3), Hunchback (Hb), morphogenetic furrow (MF) 18


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The development of complex organs is often accompanied by extensive cell-and tissue 38 rearrangements. In some extreme cases, initially simple cells undergo profound morphological 39 changes such as extensive cell fusions of muscle precursor cells to form syncytial muscle fibers 40 (Rochlin et al. 2010). In the insect nervous system, for example, initially uniform neuroectodermal 41 cells first invaginate, divide following a very defined pattern and eventually undergo morphological 42 differentiation to give rise to highly polarized neurons with long axon projections and shorter 43 dendrites (Skeath and Thor 2003;Reichert 2011). Other cell types, such as germ cells first migrate 44 long distances before coming to rest in the developing gonads (Richardson and Lehmann 2010). 45 Although these cell-type specific processes need to be tightly controlled and coordinated with 46 those of other cell types of the same and neighboring organs, the molecular mechanisms involved The adult D. melanogaster head is composed of the compound eyes (the main visual system), the 53 three dorsal ocelli, the antennae, the ventral mouthparts and the head capsule that connects these 54 organs and encloses the brain (Snodgrass 1935). Most of these structures develop during larval 55 stages from eye-antennal imaginal discs, which originate from about 20 cells that are specified by 56 eyeless (ey) expression at embryonic stages (Cohen 1993; Garcia-Bellido and Merriam 1969; 57 Quiring et al. 1994). Throughout larval development, the eye-antennal discs grow extensively by 58 cell proliferation resulting in discs composed of more than 15,000 cells at the beginning of pupation 59 (Kenyon et al. 2003 represents the initiation of differentiation, at mid L3 stage (96h AEL) the morphogenetic furrow is 149 in the middle of the retinal field and the late L3 stage (120h AEL) represents the end of 150 morphogenetic furrow progression. Multidimensional scaling clustering clearly indicated that the 151 largest difference in gene expression (dimension 1) was between L2 eye-antennal discs (72h AEL) 152 and L3 eye-antennal discs (96h and 120h AEL) ( Figure S1). 153 After filtering out not expressed and very lowly expressed genes, we observed that 9,194 genes 154 were expressed at least in one of the three sequenced stages. As anticipated by the 155 multidimensional scaling plot ( Figure S1), the number of genes that changed their expression 156 between 72h AEL and 96h AEL was much larger than between 96h AEL and 120h AEL, (Table  157   S1). In only 24 hours, during the transition from L2 to L3, 60% of the expressed genes changed 158 their expression significantly. In the transition from mid L3 to late L3, in contrast, only 22% of the 159 genes underwent a change in their expression. 160 In order to better characterize the different expression dynamics of the expressed genes, we 161 performed a co-expression clustering analysis based on Poisson Mixture models (Rau et al. 2015). 162 Manual comparison of the different outputs showed that the 13 clusters predicted by one of the 163 models (Djump, (Baudry et al. 2012)) were non-redundant and sufficiently described all the 164 expression profiles present in the data. A total of 8,836 genes could be confidently placed in one 165 of these clusters (maximum a posteriori probability (MAP) > 99%). We ordered the predicted 13 166 clusters according to their expression profile (Figure 1): four clusters contained clearly early 167 expressed genes, two of them contained genes expressed only at 72h AEL (cluster 1 and 2) and 168 two contained genes predominantly expressed early, but also with low expression at 96h and/or 169 120h AEL (clusters 3 and 4); one cluster showed down-regulation at 96h AEL, but a peak of 170 expression again at 120h AEL (cluster 5); the genes in the largest clusters showed almost constant 171 expression throughout the three stages (clusters 6 and 7); one cluster showed constant 172 expression at 72h AEL and 96h AEL and down-regulation at 120h AEL (cluster 8); one cluster 173 showed a peak of expression at 96h AEL (cluster 9) and four clusters contained genes with 174 predominantly late expression, one with high and constant expression at 96h AEL and 120h AEL 175 (cluster 10), two with up-regulation in both transitions (cluster 11 and cluster 12) and one with 176 genes expressed only at 120h AEL (cluster 13). 177 A GO enrichment analysis for the genes in the individual clusters showed that the genome-wide 178 co-expression profiling and subsequent ordering of the clusters recapitulated the consecutive 179 biological processes that take place during eye-antennal disc development with a great resolution 180 ( Figure 1, Table S2). For instance, we found genes related to energy production mainly in clusters 181 2 and 3, while genes more specific for terms related to mitosis and cell cycle were found in clusters 182 4, 8 and 9, where genes have higher relative expression at 96h AEL than 72h AEL. Similarly, 183 cluster 10 contained the more general term "imaginal disc development", while cluster 12 showed 184 enrichment for "compound eye morphogenesis", and cluster 13 was the only with enriched terms 185 related to pupation processes and pigmentation. Although we sequenced the entire eye-antennal 186 discs, we found many GO terms related to eye development with high enrichment scores, while 187 very few GO terms specific for antenna and maxillary palps were observed (e.g. in cluster 12 "eye 188 development" appears with p=4.38e-24, "antennal development" with p =4.37e-08 and no GO 189 terms related to maxillary palps were found) (Table S2). However, many GO terms related to leg 190 formation and proximodistal pattern formation were highly enriched in the genes in cluster 9 191 ("proximaldistal pattern formation" with p=4.48e-05), cluster 10 ("leg disc development" with 192 p=7.85e-20) and cluster 12 ("leg disc development" with p=4.32e-12) ( Table S2). The assignment 193 of all expressed genes to their corresponding cluster is available along with the GEO submission 194 number GSE94915. 195 In summary, we showed that clusters with early expressed genes mainly represent metabolic and 196 energy related processes, while clusters with late expressed genes represent more organ specific 197 differentiation and morphogenetic processes. One of the most noticeable results of the statistical ranking analysis was that genes in 9 of the 13 208 clusters showed significant enrichment for Nejire binding sites ( Figure 1). Nejire (also known as 209 CREB-binding protein (CBP)) is a co-factor already known to be involved in many processes of 210 eye development and patterning (Justin P Kumar et al. 2004). Similarly, Pannier that has been 211 shown to play at least two important roles during eye-antennal disc development (Singh et al. 212 2005; Singh and Choi 2003; Oros et al. 2010) was found enriched to regulate the genes of many 213 clusters (clusters 2, 4, 6, 7, 8, 9 and 11). 214 Besides these highly abundant transcription factors, the clusters with genes predominantly 215 expressed at later stages were also enriched for transcription factors already known to play a role 216 in eye-antennal disc development. For instance, a significant number of Sloppy-paired 1 (Slp1) 217 target genes are up-regulated at L3 stage (cluster 12) and this transcription factor is known to play 218 a critical role in establishing dorsal-ventral patterning of the eye field in the eye-antennal disc (Sato 219 and Tomlinson 2007). A function of Daughterless (identified in cluster 13) is also described: it is 220 expressed in the morphogenetic furrow, it interacts with Atonal and is necessary for proper 221 photoreceptor differentiation (Brown et al. 1996). Finally, Snail (enriched in cluster 1 and 13) and 222 Twist (enriched in cluster 12) were previously identified as possible repressors of the retinal 223 determination gene dachshund (dac) (Anderson, Salzer, and Kumar 2006) and our results could 224 indicate that they regulate also other genes during eye-antennal disc development. 225 Cluster 5 contained genes that show a peak in expression at 72h AEL and 120h AEL stages, 226 which precede major stage transitions from L2 to L3 and from L3 to pupa stage, respectively. 227 These transitions are characterized by ecdysone hormone pulses before larval molting and 228 pupation (T. Li and Bender 2000). Intriguingly, the only potential transcription factor binding site 229 that was significantly enriched was that of the Ecdysone Receptor (EcR), that has been shown to 230 be expressed in the eye-antennal disc in the region of the progressing morphogenetic furrow 231 (Brennan et al. 1998). 232 The identification of many well-known transcription factors suggests that the applied clustering 233 approach indeed allows identifying key regulators of various processes taking place throughout 234 eye-antennal disc development. Interestingly, we identified a few generally well-known upstream 235 factors for which a potential role during eye-antennal disc development has not yet been 236 described. For instance, in clusters of very early expressed genes, we found an enrichment of 237 motifs for the transcription factor Caudal (Cad) (cluster 1 and 2) and the Hox protein Fushi tarazu 238 (Ftz) (cluster 1). The MADS-box transcription factor Myocyte enhancer factor 2 (DMef2) was 239 predicted to regulate genes found in clusters 2, 4 and 12 ( Figure 1). Using two independent 240 Dmef2-Gal4 lines to drive GFP expression, we confirmed expression of Dmef2 in lose cells 241 attached to the developing eye-antennal discs ( Figure S2). Eventually, we found an enrichment 242 of potential target genes of the C2H2 zinc-finger transcription factor Hunchback (Hb) in clusters 243 12 and 13, which are active mainly during mid and late L3 stages. Since GO terms enriched in 244 these two clusters suggested an involvement in retinal development or neurogenesis (Figure 1), 245 we examined a potential function of Hb in the eye-antennal disc in more detail. 246 hb is expressed in retinal subperineurial glia cells 247 Using in-situ hybridization we found hb expression in two cell nuclei at the base of the optic stalk 248 in the posterior region of late L3 eye-antennal discs (Figure 2A). With a Hb antibody we also 249 detected the Hb protein in these two basally located nuclei ( Figure 2B). DNA staining with DAPI 250 showed that the Hb-positive nuclei are bigger than those of surrounding cells, suggesting that they 251 are polyploid. Additionally, we tested two putative Gal4 driver lines obtained from the Vienna Tile 252 library (Pfeiffer et al. 2008) (VT038544; Figure 2C and VT038545; Figure S3). Both lines drove 253 reporter gene expression in the two polyploid nuclei as described above. Note that both lines also 254 drove the typical hb expression in the developing embryonic nervous system, but not the early 255 anterior expression (Jiménez and Campos-Ortega 1990; Kambadur et al. 1998) (not shown). The 256 regulatory region covered by the two Gal4 driver lines is located at the non-coding 3' end of the 257 hb locus accessible to DNA-binding proteins at embryonic stages 9 and 10 (X. Li et al. 2008) 258 ( Figure S4), a time when early-born neuroblasts express hb (Grosskortenhaus et al. 2005). The 259 lack of the early anterior expression may be explained by the fact that the DNA region covered by 260 the driver lines does not seem to be bound by Bicoid during early embryonic stages (X. Li et al. 261 2008) ( Figure S4). Based on these findings, we are confident that the regions covered by the two 262 Gal4 driver lines (VT038544 and VT038545) recapitulate native hb expression. 263 The basal location of the hb-positive cells suggests that they may be retinal glia cells. Co-264 expression of hb with the pan-glial marker Repo ( Figure 3A) further supported this suggestion. 265 Previous data has shown that two polyploid retinal subperineurial glia cells (also referred to as 266 carpet cells) cover the posterior region of the eye-antennal disc (Choi and Benzer 1994;Silies et 267 al. 2007). In order to test, whether hb may be expressed in carpet cells, we first investigated the 268 expression of the subperineurial glia marker Moody (Schwabe et al. 2005) and we found a clear 269 co-localization with Hb ( Figure 3B). 270 Carpet cells migrate through the optic stalk into the eye-antennal disc during larval development 271 (Choi and Benzer 1994;Silies et al. 2007). Therefore, we followed the expression of the hb driver 272 lines throughout late L2 and L3 larval stages (Figure 4). Already at the L2 stage, we could easily 273 recognize the hb-positive cell nuclei by their large size ( Figure 4A, A'). We could corroborate that 274 these cells indeed migrated through the optic stalk during late L2 and early L3 stages ( Figure 4A The most common phenotype observed in late L3 eye-antennal discs of RNAi and mutant flies 295 was the absence of one or both carpet cell nuclei ( Figure 5A-C). In wild type animals, we could 296 unambiguously identify two carpet cell nuclei in 72% of the eye-antennal discs. In 21% of the 297 analyzed discs, we found only one carpet cell nucleus ( Figure 5D). In contrast, in 35% to 40% of 298 the studied Hb loss of function discs only one carpet cell nucleus was observed ( Figure 5B and 299 D). In some cases, this single polyploid Repo-positive nucleus was located in the midline of the 300 retinal field ( Figure 5B). No carpet cell nuclei could be observed in 24% and 38% of the eye-301 antennal discs originating from moody>>hb dsRNA and Hb TS flies, respectively ( Figure 5C and D). 302 Note that we obtained comparable results when we expressed the hb dsRNA in all glia cells 303 (repo>>hb dsRNA ; not shown) or only in subperineurial glia cells (moody>>hb dsRNA ). 304 To identify larval stages at which Hb function is crucial for carpet cell development, we transferred 305 Hb TS flies to the restrictive temperature of 28ºC at 24h AEL (early L1 stage), at 48h AEL (late L1), 306 at 72h AEL (late L2) or 96h AEL (mid L3 stage) and assessed the presence of polyploid Repo-307 positive carpet cell nuclei in late L3 eye-antennal discs, respectively. In all cases, we found a 308 significant reduction of the number of carpet cell nuclei when compared to control discs ( Figure  309 5E). Although no clear significant differences in the number of carpet cells was detected between 310 . 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The copyright holder for this preprint (which   that remained in the optic stalk and did not span the entire retinal field of the eye-antennal disc 325 ( Figure 6B). In cases where one clear carpet cell nucleus was observed, the location of moody-326 positive cell membranes in eye-antennal discs depended on the location of the remaining nucleus. 327 If the nucleus was located on one side of the eye-antennal disc, we observed moody-positive 328 membranes more unilaterally ( Figure 6C), while the membrane was present in the center of the 329 disc if the polyploid nucleus was located centrally ( Figure 6D). 330 It has been shown that the extensive cell bodies of carpet cells provide a scaffold for other retinal 331 glia cells that migrate into the eye-antennal disc, pick up differentiating photoreceptor axons and 332 guide them through the optic stalk into the optic lobe (Choi and Benzer 1994; R Rangarajan, Gong, 333 and Gaul 1999). In accordance with this known function, we observed irregular and patchy 334 . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which . http://dx.doi.org/10.1101/114363 doi: bioRxiv preprint first posted online Mar. 8, 2017; patterns of Repo-positive cells in late L3 Hb loss of function eye-antennal discs, suggesting 335 impaired glia cell migration into the eye-antennal disc (compare Figure S6B to S6A). Additionally, 336 we used HRP staining to visualize axon projections in late L3 eye-antennal discs. While axonal 337 tracts were regular in control eye-antennal discs, we found unorganized axon projections upon 338 loss of hb expression (compare Figure S6B' to S6A'). 339

Loss of Hb function results in blood-brain barrier defects 340
Subperineurial glia cells cover the entire surface of the brain from larval stages onwards. They are 341 an integral part of the protective blood-brain barrier by establishing intercellular septate junctions 342 (Carlson et al. 2000). The blood-brain barrier prevents the substances that circulate in the 343 hemolymph to enter the brain and helps maintaining the proper homeostatic conditions of the 344 nervous system (J. S. Edwards, Swales, and Bate 1993). Since it has been shown that the carpet 345 cells migrate through the optic stalk towards the brain during pupal stages (T. N. Edwards et al. 346 2012), we tested, whether the loss of hb expression in developing carpet cells had an effect on 347 the integrity of the blood-brain barrier. 348 To this aim, we injected fluorescently labeled dextran into the abdomen of moody>>hb dsRNA adult 349 flies and scored the presence of this dye in the retina of the flies. Animals with a properly formed 350 blood-brain barrier showed a fluorescent signal in their body, but not in the retina ( Figure 7A). 351 However, in animals that had an incomplete blood-brain barrier, the dextran penetrated into the 352 retina and fluorescence was observed in the compound eyes ( Figure 7A'). Since it is known that 353 blood-brain barrier permeability can increase after exposure to stress conditions (H. S. Sharma 354 and Dey 1986; Skultétyová, Tokarev, and Jezová 1998), we only scored animals that survived 355 24h after the injection of dextran. In most cases, the two eyes of an individual presented different 356 fluorescent intensities, and even no fluorescence in one eye but strong signal in the other. 357 Therefore, we scored each eye separately. moody>>hb dsRNA flies had a significantly higher rate of 358 . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which . http://dx.doi.org/10.1101/114363 doi: bioRxiv preprint first posted online Mar. 8, 2017; fluorescent retinas (p = 8.08e-7, χ 2 test), indicating that their eyes were not properly isolated from 359 the hemolymph circulating in the body cavity ( Figure 7B). 360 In summary, our loss of function experiments further confirmed a central role of Hb in carpet cell 361 development. Besides impaired retinal glia cell migration and axon guidance, we showed that 362 upon loss of Hb function also the blood-brain barrier integrity is disrupted. 363

Expression of putative Hb target genes in eye-antennal discs 364
Since we have identified Hb because of an increase in expression of its target genes during 96h 365 and 120h AEL stages and hb itself is only expressed in carpet cells, we also investigated, whether 366 some of the targets were expressed in these cells. Using available ChIP-chip data for Hb from the 367 Berkeley Drosophila Transcription Network Project (BDTNP) (X. Li et al. 2008), we generated a 368 high confidence list of 847 putative Hb target genes (see Materials and Methods for details), of 369 which 585 were expressed in eye-antennal discs at least in one of the studied stages. More 370 precisely, we found that 267 of these genes were differentially expressed in the transition from 371 72h to 96h AEL and only 52 were differentially expressed between 96h and 120h AEL (Figure 8, 372 Table S4). In both cases, most of these genes were up-regulated, suggesting that Hb mainly 373 activates target gene expression in the eye-antennal disc. Focusing only on those target genes 374 that resulted in the identification of Hb in our clustering approach (see above), we found that 77 375 of the 585 expressed putative Hb targets were present in clusters 12 and 13. We searched the 376 GO terms for biological functions of these 77 genes and found that 17 code for transcription factors 377 and up to 25 code for proteins integral to the cell membrane. A number of GO terms were related 378 to neuronal development and eye development and to note is the presence of genes known to be 379 related to glia cell migration and endoreduplication (Table S5). 380 Based on their annotated GO terms, predicted or known cellular location and the availability of 381 driver lines and antibodies, we selected 13 of these target genes and tested if they were expressed 382 in carpet cells at 120h AEL. For 8 out of the 13 selected targets we found no clear expression 383 related to carpet cells (archipelago (ago), Delta (Dl), knirps (kni), rhomboid (rho), roundabout 3 384 (robo3), Sox21b, Src oncogene at 64B (Src64B) and thickveins (tkv), not shown). This could be 385 because they were false positives, but they could also be expressed at earlier stages than 386 analyzed here or the used driver constructs did not include the regulatory regions to drive 387 expression in carpet cells. In summary, we showed that 5 of the 13 computationally predicted Hb target genes that we tested, 406 were expressed in carpet cells, suggesting that our bioinformatic pipeline allows the identification 407 of new potential regulators of carpet cell development. 408 409 . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

Expression dynamics and clustering recapitulates developmental processes 411
Although compound eye development is one of the most extensively studied processes in D. 412 melanogaster, a comprehensive understanding of genome wide gene expression dynamics is still 413 missing. We performed a genome wide expression study of eye-antennal discs from three larval 414 stages representing late patterning processes and the onset of differentiation (late L2, 72h AEL), 415 differentiation progression (mid L3, 96h AEL) and the completion of the differentiation wave 416 (wandering L3, 120h AEL). 417 Our data showed that 9,194 of all annotated D. melanogaster genes are expressed in the 418 developing eye-antennal disc. We found extensive remodeling of the transcriptomic landscape 419 with 60% of all expressed genes significantly changing their expression profile during the transition 420 from late L2 stages to mid L3 stages. It has been shown that early eye-antennal disc stages are 421 mainly characterized by patterning processes that are necessary to subdivide the initially uniform 422 disc into the organ anlagen for the antennae, the maxillary palps, the compound eyes, the dorsal predominantly patterning and proliferation processes to the onset of differentiation. Accordingly, 432 the genes active at the late L2 stage were mostly involved in metabolic processes and generation 433 . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which . http://dx.doi.org/10.1101/114363 doi: bioRxiv preprint first posted online Mar. 8, 2017; of energy. At the end of L2 stages, the patterning processes are mostly concluded and 434 differentiation starts within each compartment. For instance, in the retinal field the progression of 435 the differentiation wave is accompanied by a reduction in cell proliferation (Wolff and Ready 1991;436 Jessica E. Treisman 2013). Therefore, mostly genes related to cell differentiation, nervous system 437 development, pattern specification and compound eye development were significantly up-438 regulated at the mid L3 stage. 439 On the level of transcriptome dynamics, the transition from the mid L3 stage to late L3 was less 440 pronounced, since only 22% of the expressed genes changed their expression. Interestingly, in 441 this transition again genes related to metabolism and energy production were down-regulated. 442 Treisman 2013). In the light of an ongoing differentiation, the GO terms of genes active at the late 447 L3 stage were also similar to those enriched in the transition from late L2 to mid L3. However, in 448 this case some terms related to later processes were obtained such as R7 cell differentiation or 449 pigment metabolic process, processes taking place late during eye-antennal disc development 450 (Jessica E. Treisman 2013). 451 The discrepancy between the number of differentially expressed genes in the two studied 452 transitions may in part also be because only female discs were analyzed between 96h and 120h 453 AEL, while we compared mixed males and females at 72h AEL with only females at 96h AEL was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which . http://dx.doi.org/10.1101/114363 doi: bioRxiv preprint first posted online Mar. 8, 2017; development because it has been shown that a strong sexual dimorphism in eye size and head 459 shape exists in D. melanogaster (Posnien et al. 2012). 460 Our clustering of expressed genes based on their dynamic expression profiles resulted in 13 non-461 redundant clusters (Figure 1), which represent a much more defined representation of the dynamic 462 expression changes during eye-antennal disc development. For example, cluster 7 grouped genes 463 that were similarly high expressed at 72h and 96h AEL, and their expression decreases at 120h 464 AEL. The known genes in this cluster have been described to be related to DNA replication and 465 cell cycle control (Table S2), which corresponds with the fact that active proliferation takes place 466 at these stages (Baonza and Freeman 2002). Thus, other genes that were grouped in this cluster, 467 but for which no previous knowledge is available, are likely also related to these biological 468 functions. Similarly, genes up-regulated in the later stages were separated in more specific 469 clusters, and most of the enriched GO terms are related to differentiation and neuron and eye 470 development. Members of well-known developmental signaling pathways such as EGFR, Notch 471 and cell cycle related genes (e.g. CycE) were present in cluster 9 that grouped genes with similarly 472 high expression at 96h AEL and 120h AEL (Figure 1). Among genes, which steadily increased in 473 expression throughout the three studied stages (cluster 11), we found for instance Delta  (Table S2). 481 Although, we dissected and sequenced full eye-antennal discs and this tissue contributes to the 482 formation of various organs, the GO enrichment analysis predominantly revealed terms related to 483 . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which . http://dx.doi.org/10.1101/114363 doi: bioRxiv preprint first posted online Mar. 8, 2017; general cellular and metabolic processes and retina development. The lack of terms related to 484 antennae or maxillary palp development may be a result of much more extensive research on eye 485 specific developmental processes in comparison to the other organs that develop from the same 486 imaginal disc. However, we revealed various clusters (e.g. clusters 9, 10 and 12) in which GO 487 terms related to leg formation and proximal-distal pattern formation are highly enriched (Table S2) clustering of dynamic expression profiles with potential transcription factor enrichment within each 507 cluster thus has the potential to reveal key regulators of eye-antennal disc development. 508 . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which . http://dx.doi.org/10.1101/114363 doi: bioRxiv preprint first posted online Mar. 8, 2017; Central and pleiotropic transcriptional regulators are expected to regulate target genes present in 509 different clusters. Accordingly, we found the CREB-binding protein (CBP), also known as Nejire, 510 enriched to regulate genes in nearly all clusters (Figure 1). This zinc-finger DNA binding protein is 511 a co-activator that can act as bridge for other transcription factors to bind specific enhancer 512 elements (Dai et al. 1996;Kwok et al. 1994 Besides the very central and general transcriptional regulators, we also identified one very specific 530 cluster that is enriched for genes predominantly regulated by the Ecdysone receptor (EcR) (cluster 531 5, Figure 1). The fact that this cluster contains mainly genes active at 72h AEL and 120h AEL 532 stages, which represent major stage transitions, confirms that dynamic expression profiling and 533 . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which . http://dx.doi.org/10.1101/114363 doi: bioRxiv preprint first posted online Mar. 8, 2017; subsequent clustering can yield highly process specific results. Interestingly, the interpretation of 534 ecdysone related hormonal control has been shown to regulate various aspects of eye-antennal 535 disc development. First, a very general role of ecdysone is to trigger stage transitions, which are 536 characterized by ecdysone hormone pulses before larval molting and pupation (T. Li & Bender, 537 2000). Second, ecdysone signaling has been shown to promote tissue growth in imaginal discs in 538 general and in the eye-antennal disc specifically (Herboso et al. 2015). Third, the progression of 539 the morphogenetic furrow during eye development is dependent on ecdysone signaling (Brennan 540 et al. 1998). Although the Ecdysone receptor is expressed in the region of the MF, it has later 541 been reported that the ecdysone response is transmitted by the Broad-complex (Brennan et al. 542 2001; Brennan et al. 1998). Our data provides a set of 282 potential target genes of the Ecdysone 543 receptor and thus represents an excellent starting point to further study the role of ecdysone 544 signaling during eye-antennal disc development. For instance, the target genes could be used to 545 reveal tissue specific genes to understand how a global signal, such as ecdysone can trigger a 546 tissue specific response. Furthermore, our data may be helpful in elucidating the role of the 547 Ecdysone receptor during eye development in D. melanogaster. 548

Identification of potential novel regulators of eye-antennal disc development 549
The identification of transcription factors with already well-described central roles during eye-550 antennal disc development suggests that also new important transcriptional regulators can be 551 identified. For instance, the transcription factor Caudal was found to putatively regulate many 552 genes in the first two clusters of very early expressed genes (Figure 1, cluster 1 and 2). It has 553 been described that Caudal is a downstream core promoter activator (Juven-Gershon, Hsu, and 554 Kadonaga 2008) and very recently it has been found that it cooperates with Nejire to promote the 555 expression of the homeobox gene fushi tarazu (ftz) (Shir-Shapira et al. 2015). Since Ftz was also 556 found enriched to regulate genes in a cluster of early expressed genes (cluster 1), our results 557 suggest that these three factors could also be acting together during early D. melanogaster eye-558 . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which Taken together, the combination of dynamic gene expression clustering and upstream factor 580 enrichment provides an excellent basis for the identification of potential new regulators involved 581 in a given biological process. Intriguingly, our approach seems to be successful, although the 582 ChIP-seq experiments that identified the direct interaction of a transcription factor with its target 583 . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which . http://dx.doi.org/10.1101/114363 doi: bioRxiv preprint first posted online Mar. 8, 2017; genes were not specifically performed in eye-antennal disc tissue at the stages we studied here. It has been described that in the absence of glia cells, projecting axons are not able to enter the 627 optic stalk or get directed to it (R Rangarajan, Gong, and Gaul 1999). Interestingly, our Hb target 628 gene analysis revealed many candidate target genes with GO terms related to axon guidance 629 (Table S5). A link between undifferentiated retinal glia cells and axon guidance has been 630 established as well. When perineurial glia cells contact newly forming photoreceptor axons, they 631 differentiate into wrapping glia cells and then they enwrap the axons to participate in their 632 projection to the brain lobes (Hummel et al. 2002). Hence, we could not only show impaired carpet 633 . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which not only the carpet cells, but also for instance all other subperineurial glia of the brain. Since hb 640 expression is very likely specific to carpet cells (see also below), the phenotype obtained here 641 may be more specific. It remains to be studied, however, how carpet cells and subperineurial glia 642 cells of the brain may interact to regulate perineurial glia cell migration in the eye-antennal discs. 643 In many cases, only one carpet cell could be observed in the eye-antennal disc, and this often had 644 a larger polyploid nucleus that was located in the midline of the eye field. In these cases, also no 645 perineurial glia cell over migration could be observed, which might indicate that a single carpet 646 cell could compensate the function of the other missing one. 647 Since subperineurial glia cells of the brain contribute to the blood-brain barrier (Bainton et al. conferring identity to retinal progenitor cells (Elliott et al. 2008). It is therefore tempting to 676 investigate a potential role of Ikaros in vertebrate blood-brain barrier development. 677

The molecular role of Hb during carpet cell development 678
The lack of ployploid large carpet cells during larval stages and the loss of blood-brain barrier 679 integrity could either indicate a central role of Hb in specifying carpet cell identity entirely or a more 680 specific role in defining aspects of carpet cell identity such as polyploidy and/or its migratory 681 behavior. The exact role of Hb, however, is still unclear and will require further in-depth analyses. 682 Based on our data presented here, we propose the following cellular functions: 683 . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which Second, Hb could be involved in establishing the migratory behavior of carpet cells. In the list of 695 putative Hb target genes, we found many genes with GO terms related to cell migration and, some 696 even specifically with the "glia cell migration" term. Additionally, many of the identified Hb target 697 genes are involved in the epidermal growth factor (EGF) pathway. This is a well-conserved 698 pathway that has received a lot of interest due to its various roles in development and cancer (Gao  pathway. Fas2 and sprouty are specifically expressed in carpet cells ( Figure 9C and 9D), 707 suggesting that Hb may actively influence the migratory behavior of carpet cells by activating 708 genes involved in EGFR signaling regulation. Another putative target gene of Hb is Ets98b. 709 . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which . http://dx.doi.org/10.1101/114363 doi: bioRxiv preprint first posted online Mar. 8, 2017; Intriguingly, it has recently been shown during early embryonic development in the common house 710 spider Parasteatoda tepidariorum that the ortholog Ets4/Ets98b induces ectopic cell migration 711 upon misexpression (Pechmann et al., submitted). We also performed preliminary misexpression 712 experiments and expressed hb ectopically in wrapping glia cells (not shown). In such 713 misexpression eye-antennal discs we observed cell nuclei between the axon bundles in the optic 714 stalk. These may be wrapping glia cells that over migrate into the stalk, although they normally 715 remain in the eye-antennal disc and only their extended cell membranes project to the brain lamina 716 or medulla to accompany the photoreceptor axons (Hummel et al. 2002). 717 In summary, our functional analyses in combination with computationally supported target gene 718 prediction suggests that Hb plays a central role in specifying key cellular features of carpet cells: 719 polyploidy and extensive migratory abilities. 720

Implication on the origin and nature of carpet cells 721
Although carpet cells fulfill fundamental functions, it is still unclear where these cells originate from. 722 Based on observations by Choi and Benzer (1994) using the enhancer trap line M1-126, these 723 cells originate in the optic stalk where they are present at late L2 stage (Choi and Benzer 1994). 724 It has also been proposed that carpet cells may originate from a pool of neuroblasts in the 725 neuroectoderm during embryogenesis (Homem and Knoblich 2012) or in the optic lobes (Apitz 726 and Salecker 2014). A clonal analysis using the FLP-out system suggests that various retinal glia 727 cell types, including the carpet cells, originate from at least one mother cell at L1 larval stage (R 728 Rangarajan, Gong, and Gaul 1999). Since only one polyploid cell nucleus seems to originate from 729 one clone (R Rangarajan, Gong, and Gaul 1999) and we show that in some loss of Hb imaginal 730 discs only one polyploid cell nucleus is present, we propose that the two carpet cells may originate 731 independently probably from two mother cells defined during L1 stages. Hb may be the key 732 transcription factor specifying carpet cell fate to distinguish them from other retinal glia cells. Our 733 observation that loss of Hb function resulted in loss of polyploid carpet cell nuclei when Hb TS 734 . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which Fas2 to be present in both datasets. All these data suggest that carpet cells are indeed a retina 745 specific subperineurial glia cell type that is molecularly very distinct from brain subperineurial glia 746

cells. 747
The use of the newly analyzed driver lines VT038544 and VT038545, which drive expression 748 specifically in the carpet cells in combination with the extensive list of potential Hb target genes, 749 of which many are likely to be expressed in this specific glia cell type, represents a valuable 750 resource to address the questions concerning the origin of these cells in more detail. 751

Conclusions 752
In this study, we identified a new role of Hb in retinal glia cell development. This finding has only 753 been possible because we studied the dynamic expression profiles of all genes expressed during 754 eye-antennal disc development. Since the RNA levels of hb in the entire eye-antennal disc are 755 negligible, we could identify Hb as central factor only through the expression profiles of its putative 756 target genes, which are steadily up regulated throughout development. This up regulation is very 757 likely due to the large cell bodies of the carpet cells, which need to produce high amounts cytosolic 758 . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

Quality control 800
Quality control was carried out using FastQC software (version 0.10.1, Babraham Bioinformatics). 801 All samples but one ("72hC" sample) had quality score >Q28 for all read positions. 12% of reads 802 in sample "72hC" had an "N" in position 45, probably due to an air bubble in the sequencer. 803 clusters that best describe the data (see (Rau et al. 2015)). Our previous experience with this 821 . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which . http://dx.doi.org/10.1101/114363 doi: bioRxiv preprint first posted online  package had shown that the BIC and ICL methods report always as many clusters as we have 822 allowed to test for (corresponding to the "gmax" parameter). Also for this analysis, both methods 823 reported 25 as the most likely number of clusters, which was the input "gmax" value. 824 Consequently, we discarded these results and we only analyzed the results of the methods that 825 use slope heuristics to calculate the best number of clusters, namely DDSE and Djump. The DDSE 826 method reported 19 clusters, with 8,626 genes having MAP > 99% while the Djump method 827 reported 13 clusters, with 8,836 genes having MAP > 99%. Careful inspection of the lambda 828 values of each of these clusters showed that the additional clusters predicted by the DDSE method 829 presented negligible variation to the 13 clusters predicted by Djump. Additionally, GO term 830 analysis (see below) of the genes in the 19 clusters predicted by DDSE showed redundant terms 831 for the very similar additional clusters, which was not the case with the 13 clusters predicted by 832 Djump. Therefore, we concluded that the additional clusters present in the DDSE prediction were 833 unlikely to represent significant biological differences and that the 13 clusters predicted by Djump 834 could sufficiently describe the profiles of the groups of co-expressed genes and we used them for 835 all following analyses. 836 Genes with predicted MAP < 99% were discarded. Cluster assignment results can be found at the 837 GEO repository (GSE94915). For the plots, the variance stabilizing transformation from DESeq2 838 (Love, Anders, and Huber 2014) library was used to normalize the background read count of the 839 genes belonging to each cluster. 840 The Gene Ontology terms enriched in each cluster of genes were obtained with the plugin BiNGO 841 was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

1511
. CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which . http://dx.doi.org/10.1101/114363 doi: bioRxiv preprint first posted online Mar. 8, 2017; . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

Figure 1. Co-expression clusters. 1512
All 13 profiles of co-expressed genes predicted by HTSCluster (see Materials and Methods). The 1513 number of genes assigned to a particular cluster are indicated below the cluster name. Blue dots 1514 represent relative expression levels (lambda value) of the genes of that cluster (y-axis on the right) 1515 at each stage. Background grey lines represent the normalized mean count of all genes belonging 1516 to a cluster (y-axis on the left). Below each cluster plot, the first four non-redundant GO terms 1517 enriched in the genes of that cluster are listed (see Table S2) and also the significantly enriched 1518 transcription factors (NES > 3, see Table S3). collection, see Figure S4 for details). Expression is localized in two large cells (C') (white arrows) 1530 in the same location as A and B, at the posterior end of the eye field, near the optic stalk (C'''). 1531 1532 . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

Figure 3. hb is expressed in subperineurial glia cells. 1533
A. hb expression (VT038544-Gal4 driving UAS-GFP, green) co-localizes (white arrows) with the 1534 pan-glial marker Repo (detected with rabbit α-Repo antibody, red). B. Hb (detected with rabbit α-1535 Hb antibody, red) co-localizes (white arrows) with the expression of the subperineurial glia cell 1536 marker moody (moody-Gal4 driving UAS-GFP, green). In all pictures, anterior is to the right. Eye 1537 disc (ed), optic stalk (os). Scale bar = 20 µm. 1538 1539 . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.  CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which . http://dx.doi.org/10.1101/114363 doi: bioRxiv preprint first posted online Mar. 8, 2017; Quantification of the number of polyploid nuclei observed in late L3 eye-antennal discs of flies that 1557 have been raised at 18°C until the indicated time points (24h AEL, 48h AEL, 72h AEL and 96h 1558 AEL), when they have been transferred to the restrictive temperature of 28°C. In D and E, the 1559 black bar indicates percentage of discs with two polyploid glia cell nuclei, grey indicates discs with 1560 . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which . http://dx.doi.org/10.1101/114363 doi: bioRxiv preprint first posted online Mar. 8, 2017; one polyploid glia cell nucleus and white indicates discs without polyploid glia cell nuclei. 1561 Pearson's Chi-squared test was performed to determine if the distribution of the different number 1562 of cells (0, 1 or 2) was equal between wild type and RNAi. *: p-val < 0.05, ***: p-val < 0.0005. 1563 1564 Figure 6. Carpet cell membranes after loss of Hb function. 1565 Membranes of carpet cells in late L3 eye-antennal discs are labelled with moody-Gal4 driven UAS-1566 mCD8::GFP expression (green). All glia cells are stained with rabbit α-Repo antibody (red). Carpet 1567 cell nuclei (white arrows) are recognized by their large size. In all pictures, anterior is to the right. 1568 Eye disc (ed), optic stalk (os). Scale bar = 20 µm. A. In wild type, the membranes of the two carpet 1569 . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which . http://dx.doi.org/10.1101/114363 doi: bioRxiv preprint first posted online  cells cover all the retinal field up to the edge of the most anteriorly located glia cells. B-D. 1570 Phenotypes observed after moody driven hb RNAi. In discs where carpet cell nuclei cannot be 1571 observed, GFP signal is detected only in the optic stalk (B). In discs where only one carpet cell 1572 nucleus can be observed on one side, the membrane signal is predominantly observed on that 1573 side (C). In discs where only one carpet cell can be observed in the disc midline, membrane extend 1574 to both sides (D), but do not extend so far anteriorly as in wild type (compare D to A). correctly formed blood-brain barrier present fluorescence in the body (not shown) but not in the 1579 compound eye. A'. In flies with incomplete blood-brain barrier, fluorescent dye can be observed 1580 in the compound eye as well as in the body. B. Quantification of eyes with (green) or without (red) 1581 dye penetration. hb knock-down flies have a significant increase in the penetrance of dye in the 1582 eye, indicating a defective blood-eye barrier. Pearson's Chi-squared test was performed to 1583 determine significance between the wild type results and the RNAi. ***: p-val < 0.0005. 1584 1585 . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which . http://dx.doi.org/10.1101/114363 doi: bioRxiv preprint first posted online Mar. 8, 2017; . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which . http://dx.doi.org/10.1101/114363 doi: bioRxiv preprint first posted online Mar. 8, 2017; . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which only observed between raising larvae at the restrictive temperature 48h AEL and 72h AEL. 1634 However, this difference is also significant in the wild type (WT). This can be due to the fact that 1635 more larvae die when transferred to the restrictive temperature too early (at 24h AEL or 48h AEL). 1636 B. Pearson's Chi-squared test was performed to determine if the distribution of the different 1637 number of cells (0, 1 or 2) was equal across the time points for the same conditions (WT or HB TS ). 1638 *: p-val < 0.05, ***: p-val < 0.0005. 1639 . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 72h AEL and 96h AEL and second sheet lists the differentially expressed genes between 96h AEL 1658 and 120h AEL. "Instances": number of Hb motifs found ±1000 bp from TTS. Right-side table shows 1659 how many of these genes belong to each cluster and the percentage over the total number of 1660 genes in that cluster. 1661

Supplementary Table 5. Putative Hb target genes in clusters 12 and 13. 1662
Table contains three sheets: first sheet contains the gene ID, name and symbol of the 77 genes, 1663 and the cluster they belong to; second sheet lists the GO terms associated to each of the 77 1664 genes; third sheet contains the number of times each GO term appears in the second sheet. 1665 1666 . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.