Bab2 activates JNK signaling to reprogram Drosophila wing disc development

In response to cellular stress and damage, certain tissues are able to regenerate and to restore tissue homeostasis. In Drosophila imaginal wing discs, dying cells express mitogens that induce compensatory proliferation in the surrounding tissue. Here we report that high levels of the BTB/POZ transcription factor Bab2 in the posterior compartment of wing discs activates c-Jun N-terminal kinase (JNK) signaling and local, cell-autonomous apoptotic cell death. This in turn triggered the upregulation of the Dpp mitogen and cellular proliferation in the anterior compartment in a JNK-dependent manner. In the posterior compartment, however, dpp expression was suppressed, most likely by direct transcriptional repression by Bab2. This dual-mode of JNK-signaling, autocrine pro-apoptotic signaling and paracrine pro-proliferative signaling, led to opposite effects in the two compartments and reprogramming of the adult wing structure. We establish Bab2 as a regulator of wing disc development, with the capacity to reprogram development via JNK activation in a cell-autonomous and non-cell-autonomous manner. Summary statement Zhao et al. shows that the BTB/POZ transcription factor Bab2 is a potent activator of JNK signaling, apoptosis and compensatory proliferation, thereby driving both pro-tumorigenic and anti-tumorigenic processes.


Introduction 4 8
Regeneration refers to the organismal processes that ensure repair and replacement of lost 4 2 6 6 mis-expression and secretion of the mitogens Wg and Dpp, promoting compensatory 2 6 7 proliferation of neighboring cells (Perez-Garijo et al., 2004, Ryoo et al., 2004  that Wg signaling is compromised as a result of bab2 overexpression. However, we had 2 7 8 previously found that protein levels of Wg were reduced in parts of the wing disc upon 2 7 9 bab2 depletion via RNAi (Fig S1c,d). This suggests that indirect regulatory mechanisms The wing disc A/P compartment boundary constitutes a signaling center from which A-P 2 8 6 patterning originates and dpp is expressed in a narrow strip of cells in the AC along the 2 8 7 1 1 A/P boundary (Fig 1d) (Masucci et al., 1990). Secretion of Dpp from these cells creates an 2 8 8 activity gradient medially to laterally, which regulates patterning and growth of the wing 2 8 9 disc (Rogulja and Irvine, 2005, Schwank et al., 2008, Barrio and Milan, 2017 2017, Matsuda and Affolter, 2017, Barrio and Milan, 2020). To analyze whether dpp 2 9 1 expression was affected by bab2 overexpression, we again used the dpp-lacZ reporter.

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There was no overlap between the dpp-lacZ reporter and the mCherry-marked PC in 2 9 3 control discs (Fig. 5a), showing that dpp expression was confined to cells in the AC, in 2 9 4 accordance with previous findings (Masucci et al., 1990). However, dpp-lacZ strongly 2 9 5 increased in the AC in en-Gal4>bab2 discs (Fig. 5a,b), indicating that dpp was 2 9 6 upregulated in the AC in response to Bab2 expression in the PC. As described above, Bab2 expression in the PC also led to a decrease in the ratio between PC/AC areas (Fig. 4e). We further used the nub-Gal4 wing pouch driver to modulate dpp expression. In line was restricted to the AC compartment as it has previously been shown that the activation in the PC (Perez-Garijo et al., 2004, Ryoo et al., 2004.
To analyze if Bab2 binds directly to dpp and wg DNA sequence elements we performed Next, we investigated the effects of bab2 expression in the whole wing pouch on dpp-3 1 9 lacZ expression. Interestingly, the normal expression pattern of dpp-lacZ along the A/P 3 2 0 boundary was suppressed in most of the disc, and only a small patch of cells in the dorsal 3 2 1 part still expressed dpp-lacZ (Fig 5f,g), demonstrating that Bab2 represses dpp expression.

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Collectively, these results suggest that high-level expression of Bab2 directly repressed  To assess if expression of dpp-lacZ in the AC, in response to bab2 overexpression in the 3 2 6 PC, was dependent on the induction of apoptosis, we co-expressed P35 to block cell non-cell-autonomous manner. It is well established that JNK is involved in compensatory cell proliferation downstream al., 2011). We next examined whether the JNK pathway plays a direct role in bab2- induced non-cell-autonomous dpp upregulation in the wing disc. To this end, we reporter. Again, overexpression of Bab2 in the PC strongly upregulated dpp-lacZ in the 3 4 2 AC ( Fig. 6a,b). While downregulation of bsk itself did not affect the normal strip of dpp-  induced by high levels of Bab2 (Fig. 6a,b). This demonstrates that upregulation of dpp in  To test whether activation of the JNK signaling would be sufficient to promote non-cell- (Egr), a TNF superfamily ligand that activates the Drosophila JNK pathway.

5 0
Overexpression of egr in the wing pouch activated JNK signaling, as measured by non-cell-autonomously (yellow outlines in Fig 6c), comparable to what we have observed as well as non-cell-autonomous dpp expression. In this study, we show that the Drosophila BTB/POZ transcription factor Bab2 can  indicating that Bab2 is a transcriptional repressor of dpp. On the contrary, our results do bilaterally in the wing disc, regulating cell proliferation and tissue size (Fig. 6e).
Induction of apoptosis in the PC by Bab2 overexpression upregulated dpp non-cell- autonomously in the AC (Fig. 6f). Blocking bab2-induced cell death by silencing bsk or 3 9 2 expressing P35 abolished dpp upregulation, confirming that the proliferation is indeed resulted in wing disc reprogramming in the AC, which was, at least in part, mediated by 3 9 5 the JNK and Dpp signaling pathways (Fig. 6). The apoptotic stress in the PC caused a 3 9 6 decrease in the PC/AC ratio, and as a consequence, a reduction of the IV4 and IV5 3 9 7 territories of adult wings, while the IV2 territory increased as a result of over- proliferation. This indicates that apoptosis and JNK activation induced by Bab2 is 3 9 9 constantly active. This is in agreement with a recent study, showing that transient Importantly, unlike non-cell-autonomous proliferation driven by so-called "undead" cells 4 1 3 in AiP, bab2-promoted proliferation did not involve inhibition of apoptosis by P35.

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Rather, it was dependent on apoptosis and continuous JNK signaling, as dpp expression simultaneously.

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The non-cell-autonomous upregulation of dpp implies active crosstalk between the two 4 2 7 wing disc compartments, most likely by secreted molecules, but it may also be possible pouch region (Fig 6c). Thus, it is feasible that cells with upregulated JNK activity migrate  However, as dpp expression was repressed in the PC cells, it seems unlikely that those 4 3 7 cells would become dpp-expressing AC cells. Therefore, we suggest that secreted  were collected to build an en-Gal4, UAS-mCherry stock. image was saved in .tif format. Drosophila wing discs were dissected in phosphate-buffered saline pH 7.2 (PBS) and secondary antibodies for two hours at room temperature, followed by washing four times To generate a UAS vector for overexpression of a cDNA encoding an N-terminal Flag amplified, digested with EcoRI (ThermoFisher) and XbaI (ThermoFisher), and cloned 5 1 2 into the pUAST vector using the same restriction enzymes. The plasmid was extracted 5 3 8 primers was 300 nM.