(C) PLOS One
This story was originally published by PLOS One and is unaltered.
 . . . . . . . . . .



Specification of the endocrine primordia controlling insect moulting and metamorphosis by the JAK/STAT signalling pathway [1]

['Mar García-Ferrés', 'Centro Andaluz De Biología Del Desarrollo', 'Cabd', 'Csic-Ja-Upo', 'Seville', 'Carlos Sánchez-Higueras', 'Jose Manuel Espinosa-Vázquez', 'James C-G Hombría']

Date: 2022-12 

The corpora allata and the prothoracic glands control moulting and metamorphosis in insects. These endocrine glands are specified in the maxillary and labial segments at positions homologous to those forming the trachea in more posterior segments. Glands and trachea can be homeotically transformed into each other suggesting that all three evolved from a metamerically repeated organ that diverged to form glands in the head and respiratory organs in the trunk. While much is known about tracheal specification, there is limited information about corpora allata and prothorathic gland specification. Here we show that the expression of a key regulator of early gland development, the snail gene, is controlled by the Dfd and Scr Hox genes and by the Hedgehog and Wnt signalling pathways that induce localised transcription of upd, the ligand of the JAK/STAT signalling pathway, which lies at the heart of gland specification. Our results show that the same upstream regulators are required for the early gland and tracheal primordia specification, reinforcing the hypothesis that they originated from a segmentally repeated organ present in an ancient arthropod.

The main endocrine organs controlling insect moulting and metamorphosis are the corpora allata and the prothoracic glands. Genetic experiments in Drosophila melanogaster suggested that, despite their extremely different morphology and function, the corpora allata and the prothoracic glands are homologous to the respiratory trachea. All three organs derive from a primordium arising at similar locations along the cephalic and trunk segments, they activate common developmental genes using the same cis-regulatory elements, and can be transformed into each other by modifying Hox expression. One key difference between glands and trachea is that the endocrine primordia activate the Epithelial to Mesenchymal inducer gene snail. Using the snail gland specific enhancer as a proxy for gland formation, we show that the glands are specified by the same inputs specifying the trachea. These include the JAK/STAT, the Hedgehog and Wingless signalling pathways as well as inputs from the Hox genes. These observations support the hypothesis that during arthropod evolution, a metamerically repeated organ diverged to give rise to endocrine glands in the head and respiratory organs in the trunk segments.

Funding: This work was supported by the Spanish Ministerio de Ciencia e Innovación (MICINN) and European Regional Development Fund (FEDER) grant PID2019-104656GB-I00 to MGF, CSH, JMEV and JCGH; grant BES-2017-081120 to MGF; the Consejería de Economía, Innovación, Ciencia y Empleo, Junta de Andalucía (Department of Economy, Innovation, Science and Employment, Government of Andalucia) grant P20-0003 to CSH and JCGH and the María de Maeztu Unit Excellence Grant CEX-2020-001088-M to MGF, CSH, JMEV and JCGH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

To find out what are the mechanisms inducing CA and PG specification we have analysed how snail expression is activated in the primordia of these organs. We show that the Wnt and Hh pathways determine the antero-posterior segmental location where the sna-rg enhancer is activated. This is achieved indirectly through the localised transcriptional activation of the upd gene, which encodes a ligand activating the JAK/STAT signalling pathway. We show STAT directly activates sna expression in the glands and propose that the Hox input required for activating sna expression is mediated indirectly.

In comparison to the extensive knowledge we have of the mechanisms specifying the Drosophila tracheae [ 10 – 20 ], little is known about CA and PG specification. The first signs of CA and PG specification are noticeable when these primordia start expressing the sna gene [ 5 ]. Snail is a zinc-finger transcription factor conserved in vertebrates where its function has also been associated to the induction of EMT [ 21 – 23 ]. Apart from its function in the endocrine primordia, Snail is also required for the formation of the mesoderm [ 24 , 25 ]. The sna-rg-GFP reporter gene, made with a 1.9 kb sna cis-regulatory element, is the earliest known specific marker for the CA and the PG primordia [ 5 ]. sna-rg-GFP expression is first activated at the beginning of organogenesis (st11), after the two gland primordia have just invaginated in the maxillary and labial segments, and its expression is maintained throughout embryonic gland development ( Fig 1G and 1H ). Thus, sna expression is a CA and PG specific marker comparable to what trh expression is for the trachea. Both genes encode transcription factors labelling the respective primordia at the earliest stages of development and both genes are required for the development of the organs where they are activated. Therefore, finding the upstream regulators of sna-rg expression should help uncovering the mechanisms required for gland specification. Moreover, the comparison of the gene network activating sna expression in the gland with that activating trh expression in the trachea will allow us to confirm if both organs share similar upstream regulators as would be expected if they shared a common evolutionary origin.

Despite their different morphology and function, the CA and the PG have several characteristics in common with the trachea. First, the CA and the PG are specified in the cephalic lateral ectoderm at homologous positions to those forming the tracheal primordia in the trunk segments. Second, all three organs express the gene encoding the transcription factor Ventral veinless (Vvl) activated through the same enhancer (vvl1+2). Third, ectopic expression of the Deformed (Dfd) or the Sex combs reduced (Scr) Hox genes can transform tracheal primordia cells into gland cells and, conversely, the ectopic activation of trunk Hox genes can transform the gland primordia into trachea. These observations led to the proposal that the CA, the PG and the trachea arose from a metamerically repeated ancient structure that evolved divergently in each segment giving rise to three completely different organs [ 5 ]. This hypothesis has been reinforced by functional studies performed in the Oncopeltus hemipteran insect [ 9 ].

(A) Scheme of a st16 embryo representing the CA and PG in green, the corpora cardiaca (CC) in red and the aorta and heart in blue. The migratory route followed by the three gland primordia towards their final position in the ring gland is represented by arrows starting from their approximate location at st11. (B) sna locus indicating the position of the transcription unit (black), the two mesoderm enhancers (brown), the ring gland enhancer (blue) and the sna ΔrgR2 deletion. (C-F) sna RNA expression in wild type embryos at st11 (C) and st13 (D), or sna ΔrgR2 embryos at st11 (E) and st13 (F). Arrows point to the CA and PG primordia, asterisks mark the absence of sna transcription. (G-H) sna-rg-GFP reporter in a st11 wild type embryo (G) before CA and PG coalescence, and at st13 (H) showing the coalesced CA/PG migrating towards the dorsal midline. (I-J) sna-rg-GFP sna ΔrgR2 homozygous embryos showing the CA and PG primordia at st11 (I) and at st13 (J) when degeneration is noticeable (asterisks). (G’-J’) Show DCP-1 co-expression (white) in the same embryos to reveal apoptosis. In control embryos (G’-H’) DCP-1 activation is restricted to ectodermal cells. In sna-rg-GFP sna ΔrgR2 homozygous embryos, gland cells show high levels of DCP-1 at st13 (J’) (yellow arrows). At st11 (I’), just after gland specification, DCP-1 starts being detectable before overt gland degeneration. (K-M) sna ΔrgR2 homozygous embryos carrying the sna-rg-mCherry reporter (green) and a sna-BAC rescue construct. The Sna protein in the BAC is tagged with GFP (red) revealing its expression in the gland primordia before coalescence (K, yellow because overlap with sna-rg expression), after coalescence (L, arrow), and after integrating in the ring gland (M). Apart from the gland primordia, the Snail BAC protein reveals other sites of expression: the oenocytes and the wing and haltere primordia (M, asterisks). Note that cell viability and migratory behaviour of the CA and PG are fully rescued by the BAC. All figures show lateral views with anterior left and dorsal up.

Analysis of development in Drosophila melanogaster showed that the CA and the PG primordia are specified in the lateral ectodermal cells of the maxillary and the labial segment respectively, at homologous locations to those giving rise in more posterior segments to the fly’s respiratory organs [ 5 ]. During early development, the CA and the PG primordia exhibit a similar behaviour to that of the tracheal primordia, with the epithelium invaginating to form small sacks of cells resembling tracheal pits. However, while the tracheal primordia maintain an epithelial organization throughout development, the gland cells soon experience an Epithelial to Mesenchymal Transition (EMT) induced by snail (sna) gene expression [ 5 ]. Following Snail activation, the CA and the PG coalesce into a single primordium that migrates across four segments until it reaches the dorsal part of the first abdominal segment (A1). This migration is guided by several intermediate landmarks that serve as “stepping stones” during their long-range migration ( Fig 1A ) [ 6 ]. Once in A1, the CA/PG primordium fuses ventrally to the corpora cardiaca, an independent endocrine organ of mesodermal origin [ 7 , 8 ], and dorsally to the contralateral primordium, giving rise to a ring structure encircling the anterior aorta. Therefore, the mature ring gland is a composite endocrine organ formed by three different glands, two of ectodermal origin (the CA and the PG) and one of mesodermal origin, the corpora cardiaca [ 1 , 6 ].

Arthropods are characterised by the presence of an external skeleton that protects them from injury but also constrains their growth during development. This problem is solved by a dedicated endocrine system controlling the periodic moulting of the exoskeleton. Two glands control the process of larval moulting and metamorphosis in insects: the corpora allata (CA), which secrete Juvenile Hormone; and the prothoracic glands (PG), which secrete Ecdysone [ 1 ]. In holometabolous insects, secretion of both of these hormones to the haemolymph induces the larva moulting into a larger larva, while secretion of Ecdysone alone induces metamorphosis [ 2 ]. Similar endocrine glands secreting hormones related to those produced by the CA and the PG have been identified in crustaceans, indicating that this system has an ancient evolutionary origin [ 1 , 3 , 4 ].

Results

sna expression in the CA and PG primordia is activated by a single cis-regulatory region Expression of the snail gene in the corpora allata (CA) and the prothoracic gland (PG) primordia is key for their specification and development [5]. To test if the sna-rg cis-regulatory region previously described is the only element activating snail expression in the CA and the PG primordia, we created snaΔrgR2, a deletion generated with the CRISPR-Cas9 system using specific single guide RNAs (Figs 1B and S1 and Materials and Methods). RNA in situ hybridization reveals snaΔrgR2 embryos lack sna expression in the CA and PG primordia while maintaining it in other organs (Fig 1C–1F). Embryos homozygous for snaΔrgR2 or heterozygous for this deletion over the sna1 null allele are not viable. These embryos develop a normal mesoderm with the only obvious phenotypic defect being the almost complete degeneration of the CA and the PG primordia (Fig 1G–1J). Embryo lethality and gland development are fully restored by a sna-GFP BAC construct [26], revealing that snaΔrgR2 lethality is due to the sna deletion (Fig 1K–1M). These results prove that the snaΔrgR2 deletion inactivates the only regulatory region driving sna expression in the CA and PG gland primordia, allowing us to use sna-rg-GFP reporter expression as a proxy to discover the upstream trans regulatory elements involved in sna transcription and CA and PG specification.

Requirement of the Wnt signalling pathway for gland specification The vvl and sna genes are co-expressed in the CA and the PG, but the expression of sna in the gland primordia does not depend on Vvl function [5], suggesting that both genes may respond to similar upstream regulatory cues in the gland region. As tracheal vvl expression expands in wingless (wg) mutants [20], we tested if sna-rg spatial activation is also restricted through the Wnt signalling pathway. In wgCX4 or in wgen11 homozygous mutant embryos sna-rg-GFP expression in the maxilla and the labium appears duplicated at st11 (Fig 2A and 2B). The duplicated primordia form in cells normally expressing Wg and are located at the same dorso-ventral position where the endogenous primordium of that segment forms. The ectopic and the normal sna-rg expressing cells become migratory coalescing into a single larger gland primordium, suggesting the ectopic cells form functional gland primordia, although this expanded primordium cannot reach the embryo’s dorsal side due to the general defects in wg mutants. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 2. Wnt pathway requirement for gland specification. (A-F) st11 gland primordia double labelled with sna-rg-GFP in green, and in red the wg expressing cells with a wgen11 reporter line (A-B) or the vvl expressing cells with a vvl1+2-mCherry reporter line (C-F). (A) Control sna-rg-GFP wgen11-lacZ/+ embryo. sna-rg labels specifically the CA and the PG cells invaginating at the anterior end of the maxillary and labial segments respectively. (B) In wgen11 homozygous mutants, ectopic patches of sna-rg expression appear on the wingless expressing cells (arrows) in the maxilla and labium at the same dorso-ventral positions as the endogenous ones. (C) Wg ectopic expression driven in the maxillary and labial segments with spalt-Gal4 results in the downregulation of sna-rg expression (asterisks). Downregulation of vvl1+2 cephalic expression is also observed. (D) Ectopic expression of activated Armadillo results in sna-rg downregulation (asterisks). (E) pan2(dTCF) null mutants present normal sna-rg expression and Engrailed (blue) stripe expression is maintained. (F) wgCX4 pan2 double mutant embryos do not present ectopic endocrine primordia. Embryo in panel F is also stained with anti-Wg to recognise the homozygous wg mutants. Scale bar 50 μm. https://doi.org/10.1371/journal.pgen.1010427.g002 Ectopic UAS-wg expression driven in the maxilla and labium with the sal-Gal4 driver eliminates sna-rg reporter expression (Fig 2C). This repression is mediated through the Wnt canonical pathway as sna-rg-GFP expression is also eliminated by ectopic expression of an activated form of Armadillo (UAS-ArmS10, Fig 2D) [27]. Surprisingly, we found that while sna-rg expression is normal in embryos homozygous for the pan2 zygotic null allele of dTCF [a.k.a. Pangolin [28,29]], the DNA binding protein downstream of the Wg signalling pathway (Fig 2E), double mutant wgCX4, pan2 embryos lack the ectopic gland primordia but not the endogenous ones (Fig 2F). These results suggest that Arm-dTCF can prevent sna-rg expression in Wg expressing cells but it does not affect the formation of the endogenous gland primordia which are out of Wg signalling range.

Requirement of the Hedgehog (Hh) signalling pathway for gland specification It has been reported that vvl expression in the tracheal primordia is strongly reduced in hh mutants [12]. The Hh and Wnt signalling pathways cross-regulate in the trunk epidermal cells where Hedgehog signalling is required for maintenance of wg expression in the adjacent ectodermal cells of the anterior compartment, and Wg signalling is required for the maintenance of hh and engrailed (en) expression in the posterior compartment [30]. As a result of this cross-regulation, wg, en and hh mutant embryos have similar phenotypes in the trunk ventral ectodermal segments [31]. However, in the cephalic region, where the glands are specified, such cross regulation does not occur, with Engrailed expression being maintained in the posterior segments of the maxilla and the labium in the absence of wg function [32]. To study the effect of Hh signalling on gland development, we analysed hhAC and enE homozygous mutant embryos and found an almost complete absence of sna-rg expression (Fig 3B and 3C). Engrailed activates hh expression in the posterior compartment, from where secreted Hh induces the pathway in neighbouring cells. The final target is the Cubitus interruptus (Ci) protein that can act either as a transcriptional activator or as a repressor depending on the pathway’s activation state. In the absence of Hh, Ci is cleaved giving rise to a protein repressing the transcription of its direct targets [33]. Conversely, in the presence of Hh, the pathway’s activation prevents Ci’s cleavage, giving rise to a transcriptional activator [34]. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 3. Hh pathway requirement for gland specification. (A-H) Embryos of various genotypes labelled with sna-rg-GFP (green) to mark the gland primordia. (A) Wild type embryo labelling the CA and PG location. (B) hhAC null and (C) enE null embryos show almost complete downregulation of the sna-rg enhancer (asterisks). (D) ci94 null embryos show normal sna-rg activation. (E) Double enE, ci94 mutants recover sna-rg expression in the CA and PG primordia. (F) Overexpression of the Ci repressor isoform with spalt-Gal4 downregulates sna-rg expression (asterisk marks the approximate position where stage 13 migrating primordia should be expected). (G) Overexpression of the Ci activator isoform with sal-Gal4 results in an expansion of sna-rg expression. (H) Double wgCX4, hhAC mutant embryos lack sna-rg expression (asterisks). (I) Model summarising regulatory interactions between the Wnt and Hh pathways and the sna-rg enhancer (The interactions represented are not assumed to be direct). Panels D-E were also stained with anti-Ci, panel E with anti-En and panel H with anti-Wg to identify the mutant embryos. All embryos are at st11 except F and G which are at st13 and st12. Scale bar 50 μm. https://doi.org/10.1371/journal.pgen.1010427.g003 We find that in ci94 null embryos sna-rg-GFP is expressed in its normal pattern (Fig 3D), indicating Ci is not a necessary activator of sna expression in the glands. We also found that in double enE, ci94 mutant embryos the sna-rg-GFP expression is recovered compared to enE embryos (Fig 3 compare panel C with E), indicating that the Ci repressor form prevents sna-rg activation. To confirm this, we expressed UAS-Ci76, the repressor isoform of Ci [33], with the sal-Gal4 line and found this causes an almost complete absence of sna-rg activity (Fig 3F). Although the above results indicate Ci is not absolutely required for sna-rg expression, we observed that overexpression of CiPKA, the active form of Ci, causes a non-fully penetrant expansion of sna-rg expression (Fig 3G) suggesting the possibility that sna-rg may be responsive to Ci and to a second activator. We also analysed double wg, hh (or wg, en) mutants and found that these embryos do not activate sna-rg, a phenotype similar to that of hh mutants (Fig 3H). These results indicate that Ci repression is epistatic over the derepression caused in wg mutants. The above data fit a model where sna-rg expression is under negative regulation, either directly or indirectly, mediated by the Wnt and Hh signalling pathways (Fig 3I). Although Ci repression of sna-rg activity should be relieved by Hh signalling anteriorly and posteriorly to the En expressing cells, the Wnt parallel repressive function prevents sna-rg activation in Wg expressing cells restricting the formation of the CA and PG primordia to the most anterior cells of the maxillary and labial segments.

Regulation of Upd ligand expression by the Wg and Hh pathways Previously we showed that JAK/STAT signalling is required for sna-rg expression [5]. To find out if the Wg and Hh signalling pathways regulate sna indirectly via JAK/STAT signalling, we reanalysed the spatio-temporal activation of upd in wild type and mutant embryos, paying special attention to the maxillary and labial segments where the gland primordia are specified. In st9 wild type embryos, upd is expressed in segmental stripes immediately posterior to the Engrailed expressing cells (S2 Fig). This pattern of transcription evolves to form a transient antero-posterior lateral stripe that rapidly resolves at early stage 11 into two patches of expression in the maxilla and labium corresponding to the sites where the CA and PG glands form (Figs 4C and S2E). Expression analysis of 10xSTAT-GFP, a reporter that is universally activated in cells where the JAK/STAT pathway is active [35,36] confirms JAK/STAT signalling activation at st10 and 11 in the CA and PG primordia (Fig 4A–4B). Although upd is transcribed in both primordia, we noticed that expression of both upd RNA and the 10XSTAT-GFP reporter is more transient in the CA than in the PG primordium (Fig 4A–4D). PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 4. JAK/STAT pathway requirement for gland specification. (A-B) Wild type embryos carrying the sna-rg-mCherry (red) and 10xSTAT-GFP (green) reporter genes. (A) At st10, embryos present patches of green staining in the maxillary and labial segments at the position where the gland primordia will become activated. Weak sna-rg expression starts to appear. (B) At st11, when sna-rg-mCherry is strongly activated, 10xSTAT-GFP expression has faded from the maxilla while is maintained in the labium. Note that 10xSTAT-GFP expression is also activated in the tracheal primordia. (C-I) upd mRNA in situ hybridization in st10-st11 embryos. (C) A wild type early st11 embryo showing upd mRNA in the maxillary and labial segments is restricted to two patches where the CA and the PG form. (D) At late st11, upd mRNA expression only remains in the PG (asterisk marks the position where the CA primordium is located). (E) hh null mutants lose maxillary and labial upd transcription (asterisks), which remains in the tracheal primordia. (F) In wg null mutants, upd transcription is extended in the maxillary and labial segments (arrows), as well as expanding around the tracheal primordia. (G) Dfd Scr mutants lose maxillary and labial upd transcription (asterisks). Embryos in (G) are also Abd-B homozygous mutant to allow distinguishing unambiguously the triple Dfd Scr Abd-B homozygous embryos by the absence of upd expression in the A8 posterior spiracle primordium. (H) upd expression in a control embryo at st10. (I) upd expression in an embryo expressing the activator isoform of Ci in a st10 embryo. Scale bars 50 μm. https://doi.org/10.1371/journal.pgen.1010427.g004 We next analysed if the Wnt and the Hh pathways affect upd transcription in the gland primordia. In hhAC null embryos, we find that the transient upd expression in the CA and PG primordia disappears (Fig 4E), while in wgCX4 mutants upd RNA expression expands (Fig 4F). We also found that ectopic expression of the activator Ci protein results in a non-fully penetrant expansion of upd expression in stage 10 embryos (Fig 4H–4I). These results suggest that the effects on sna-rg expression caused by mutations affecting the Wnt and Hh signalling pathways are mediated indirectly through the JAK/STAT signalling pathway.

Possible cross-regulation between Hox, wg, hh and upd in the maxillary and labial segments Development of the CA and PG and normal expression of the sna-rg reporter in the maxilla and the labium require Dfd and Scr function [5], therefore we studied if there are any cross-regulatory interactions among the genes involved in gland primordia specification. We first analysed wg and en mutant embryos and found that the expression of Dfd and Scr is not significantly affected (S3A–S3F Fig). Similarly, neither En nor Wg expression is affected in Dfd Scr mutant embryos (S3G–S3J Fig), discarding a possible interaction between Dfd and Scr and the Wnt/Hh signalling pathways. In contrast, we found that the transient expression of upd transcription in the CA and PG primordia almost disappears in Dfd Scr mutant embryos (Fig 4G), indicating that the Hox proteins can regulate JAK/STAT signalling as previously shown for Abd-B [37]. These results indicate that the Hox, and the Wnt/Hh pathways indirectly mediate the regulation of the sna-rg enhancer through their modulation of upd expression and JAK/STAT signalling activation. The sna-rg reporter is not expressed in Df(1)os1A embryos (Fig 5A and 5B). To test if generalised Upd expression in the maxilla and labium can activate sna-rg independently of other upstream positive or negative inputs, we induced UAS-upd with either the sal-Gal4 or the arm-Gal4 lines. We observe that these embryos have expanded sna-rg expression along the antero-posterior axis in the maxillary and labial segments (Fig 5C). Analysis of Sal expression, which labels the PG primordium [5] shows that Upd ectopic expression induces a moderate expansion of the CA primordia while resulting a much larger increase of the PG primordium (Fig 5D and 5E). This expansion occurs mostly in the antero-posterior axis from cells where the Hh and the Wnt pathways are normally blocking sna-rg expression, while expansion is less noticeable in the dorso-ventral axis. This indicates that most of the antero-posterior intrasegmental inputs provided by the segment polarity genes converge on Upd transcription but that the dorso-ventral information is registered downstream of Upd. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 5. Epistatic relationship between JAK/STAT, Wnt, Hh and Hox inputs over sna-rg regulation. (A-C) Embryos expressing sna-rg-GFP (green) and vvl1+2-mCherry (red) stained with anti-En (blue). (A) sna-rg and vvl1+2 expression in st11 control embryos. Panels (A’) and (A”) show each channel separately to appreciate the co-expression of both markers in the gland primordia. (B) Df(1)os1A embryos show an almost complete downregulation of sna-rg and vvl1+2 expression from the CA and PG (asterisks). (C) Ectopic Upd expression driven with sal-Gal4 induces ectopic sna-rg and vvl1+2 expression in the gnathal segments, which for sna-rg is more pronounced in the labium than in the maxilla. Note that in the maxillary segment Upd can induce ectopic dorsal vvl1+2 but not sna-rg expression, this is expected as Dfd only induces sna-rg ventrally in the maxilla. (D-E) sna-rg-GFP embryos stained with anti-GFP (green) and anti-Sal (red). In control embryos (D) Sal labels the PG primordium but not the CA. In arm-Gal4 embryos ectopically expressing Upd (E), the PG is more expanded than the CA as shown by the number of cells co-expressing Sal and GFP. (F-G) sna-rg-GFP expression (green) in st13 Dfd Scr mutant embryos (F), or Dfd Scr mutant embryos after ectopic Upd expression driven with the sal-Gal4 line (G) showing that Upd activation is not sufficient to rescue gland formation in Dfd Scr mutants. In Dfd Scr mutant embryos (F), although the gland primordia become apoptotic, residual GFP expression indicates that there must exist Hox independent inputs activating the sna-rg enhancer. Embryos in (F-G) are also stained with anti-Scr to recognise the homozygous mutants. Scale bars 50 μm. https://doi.org/10.1371/journal.pgen.1010427.g005 We finally tested if activation of UAS-upd with the sal-Gal4 driver line can rescue sna-rg activation in Dfd Scr mutant embryos. We found that the residual levels of GFP observed in sna-rg Dfd Scr mutant embryos are not increased in sal-Gal4 UAS-upd sna-rg Dfd Scr embryos (Fig 5F and 5G), indicating that besides regulating upd expression, the Hox input has further requirements for gland formation. Therefore, localised Upd expression defines the antero-posterior intrasegmental localisation of the CA and PG primordia, but other signals besides STAT must be controlling the dorso-ventral and the cephalic sna activation either directly at the sna-rg enhancer level or through unknown intermediate regulators.

Analysis of the direct regulation of sna-rg enhancer by STAT To find out if the Hox and STAT inputs regulate sna expression directly, we searched for putative binding sites in the cis-regulatory region of sna-rg. To facilitate the bioinformatic analysis we dissected the 1.9 kb sna-rg regulatory element down to a 681bp fragment we call R2P2 (S1A–S1E Fig). The sna-rg-R2P2-GFP reporter construct drives high levels of expression in the CA and PG and its expression is even more specific as it lacks the low levels of GFP expression observed in the haemocytes and neurons of the larger sna-rg-GFP reporter. Computational JASPAR analysis [38] of the 681bp R2P2 sequence identified three putative Hox-Exd-Hth and three putative STAT binding sites (Fig 6A). Further subdivision of sna-rg-R2P2 in two halves shows that neither the A1 nor the A2 half drive embryonic expression (Fig 6C and 6D). Reporters containing A1 fused to either the proximal part of A2 (the sna-rg A1+A2prox-GFP reporter containing a single STAT site) or to the distal part of the A2 element (the sna-rg A1+A2dist-GFP reporter containing two STAT sites) recovered ring gland expression (Fig 6E and 6F). The recovery of expression when A1 is fused to either fragment, both containing STAT binding sites, made us wonder if the lack of expression of the A1 fragment is due to the absence of STAT binding sites. To test this hypothesis, we added to A1 a 20bp fragment that contains a single functional STAT site taken from an unrelated gene [the vvl1+2 enhancer of the ventral veinless gene [20]] creating the sna-rg A1+STAT reporter. We find that A1+STAT drives expression in both the maxilla and the labium and that this depends on JAK/STAT signalling, as mutation of the STAT-binding site abolishes expression in the sna-rg A1+STATmut reporter (Fig 6G and 6H). Taken together, these experiments show that the presence of functional STAT binding sites is required for sna activation in the CA and PG primordia and that the 300bp A1 fragment can interpret the segmental cephalic positional information, suggesting that the Hox-Exd-Hth site located in A1 could mediate Dfd and Scr input to the enhancer. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 6. Direct regulation of sna-rg by STAT. (A) Representation of the minimal sna-rg R2P2 subfragments indicating the location of the putative STAT (pink crosses) and Hox-Exd-Hth DNA binding sites (black boxes). Mutated STAT binding sites are represented with a red X over the pink cross. (B) sna-rg R2P2-GFP expression. (C) No GFP expression is observed in A1 nor in A2 constructs (D). Gland expression is observed when A1 is joined to the A2 proximal half (E) or when A1 is joined to the A2 distal half (F). (G) A1 fused to a 20bp from fragment the vvl1+2 enhancer containing a functional STAT binding site. (H) A1 fused to the same 20bp fragment where the STAT binding site has been mutated. (I-K) Embryos carrying both the sna-rg-R2P2-GFP-PH (green) and the sna-rg-R2P2-STATmut Histone2B-RFP-GFP-PH (red and green) constructs. Red nuclear expression is not observed at early stage 11 (I) but can be detected later exclusively in the CA (J). Df(1)os1A embryos lacking all Upd ligands (K) do not express sna-rg-R2P2-STATmut mCherry-GFP-PH. Black arrows in (E-G) point to the CA/PG gland primordia, red arrows to ectopic expression outside the glands. Scale bars 50 μm. https://doi.org/10.1371/journal.pgen.1010427.g006 To confirm STAT’s binding sites requirement, we mutated all three putative sites on the R2P2 fragment generating the sna-rg-R2P2 STATmut construct expressing simultaneously the LifeActin-GFP and nuclear Histone-RFP reporter markers (Materials and Methods). Comparing its expression to the sna-rg-R2P2-eGFP-PH, we find that although mutating the three STAT binding sites completely abolishes the reporter’s expression in the PG, surprisingly, it does not eliminate its expression from the CA, where its activation is only slightly delayed (Fig 6I and 6J). The CA expression of the sna-rg-R2P2 STATmut construct still depends on upd activity as it disappears in Df(1)os1A embryos lacking all Upd ligands (Fig 6K). These results indicate that either there is a cryptic STAT site in sna-rg-R2P2 we did not mutate, or that in the CA the sna-rg-R2P2 enhancer can be activated both directly and indirectly by STAT through a site present in the A2 fragment (see discussion).

[END]
---
[1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1010427

Published and (C) by PLOS One
Content appears here under this condition or license: Creative Commons - Attribution BY 4.0.

via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/