GFP reporters detect the activation of the Drosophila JAK/STAT pathway in vivo

JAK/STAT signaling is essential for a wide range of developmental processes in Drosophila melanogaster. The mechanism by which the JAK/STAT pathway contributes to these processes has been the subject of recent investigation. However, a reporter that reflects activity of the JAK/STAT pathway in all Drosophila tissues has not yet been developed. By placing a fragment of the Stat92E target gene Socs36E, which contains at least two putative Stat92E binding sites, upstream of GFP, we generated three constructs that can be used to monitor JAK/STAT pathway activity in vivo. These constructs differ by the number of Stat92E binding sites and the stability of GFP. The 2XSTAT92E-GFP and 10XSTAT92E-GFP constructs contain 2 and 10 Stat92E binding sites, respectively, driving expression of enhanced GFP, while 10XSTAT92E-DGFP drives expression of destabilized GFP. We show that these reporters are expressed in the embryo in an overlapping pattern with Stat92E protein and in tissues where JAK/STAT signaling is required. In addition, these reporters accurately reflect JAK/STAT pathway activity at larval stages, as their expression pattern overlaps that of the activating ligand unpaired in imaginal discs. Moreover, the STAT92E-GFP reporters are activated by ectopic JAK/STAT signaling. STAT92E-GFP fluorescence is increased in response to ectopic upd in the larval eye disc and mis-expression of the JAK kinase hopscotch in the adult fat body. Lastly, these reporters are specifically activated by Stat92E, as STAT92E-GFP reporter expression is lost cell-autonomously in stat92E homozygous mutant tissue. In sum, we have generated in vivo GFP reporters that accurately reflect JAK/STAT pathway activation in a variety of tissues. These reporters are valuable tools to further investigate and understand the role of JAK/STAT signaling in Drosophila.     

otx2 expression in the ectoderm activates anterior neural determination and is required for Xenopus cement gland formation.

We previously showed that otx2 regulates Xenopus cement gland formation in the ectoderm. Here, we show that otx2 is sufficient to direct anterior neural gene expression, and that its activity is required for cement gland and anterior neural determination. otx2 activity at midgastrula activates anterior and prevents expression of posterior and ventral gene expression in whole embryos and ectodermal explants. These data suggest that part of the mechanism by which otx2 promotes anterior determination involves repression of posterior and ventral fates. A dominant negative otx2-engrailed repressor fusion protein (otx2-En) ablates endogenous cement gland formation, and inhibits expression of the mid/hindbrain boundary marker engrailed-2. Ectoderm expressing otx2-En is not able to respond to signals from the mesoderm to form cement gland, and is impaired in its ability to form anterior neural tissue. These results compliment analyses in otx2 mutant mice, indicating a role for otx2 in the ectoderm during anterior neural patterning.     

EGF Modulates Expression of STAT5 in Mammary Epithelial Cells

HC11 mouse mammary epithelial cells are capable of differentiatingin vitro.By growing cells in EGF-containing medium, and upon confluence withdrawing EGF, these cells become competent at responding to lactogenic hormone treatment and expressing milk proteins. We found that during proliferation and at confluence STAT5A and STAT5B proteins were expressed at equal levels or with STAT5B being predominant. In competent cells, expression levels of STAT5A and STAT5B increased markedly with STAT5A now being the predominant form, an expression pattern resembling the expression patterns of STAT5 proteins seen during mammary gland differentiationin vivo.This suggests that EGF has a suppressive effect on STAT5 expression, in particular, STAT5A, which we conclude to be mediated through ras/raf/MEK/MAPK pathway and to a lesser extent through a PI3-kinase-mediated pathway. Furthermore, we also found that EGF regulated a nuclear phosphatase capable of dephosphorylating tyrosine-phosphorylated STAT5. Our data show that HC11 cells have retained the expression patterns of STAT5 proteins seenin vivo.This makes HC11 cells useful for studying molecular mechanisms regulating expression of STAT factors and their participation in differentiation processes of mammary gland.     

Conserved and divergent expression of T-box genes Tbx2-Tbx5 in Xenopus

We report here the identification of four members of T-box family genes, Xltbx2-Xltbx5, in Xenopus. Two of them are probable pseudovariant genes of XTbx5 and ET, a putative Xenopus ortholog of Tbx3. We compared their expression patterns in both embryos and limbs. In embryos, expression of Xltbx2 and Xltbx3 showed novel diversities, such as Xltbx2 in the neural crest cells and Xltbx3 in the ventral spinal cord, together with mutual similarities in the following regions: dorsal retina, proctoderm, lateral line organ, cement gland and cranial ganglia. The patterns in limbs were highly conserved with mouse and chick orthologs, including the limb-type specific expression of Xltbx4 and Xltbx5. In addition, RT-PCR analysis showed that they are expressed weakly even in adult limbs as previously reported in the newt.     

SOCS3 promotes apoptosis of mammary differentiated cells

Growth and function of the mammary gland is regulated by cytokines and modulated by suppressor of cytokine signalling (SOCS) proteins. In vitro experiments demonstrated that SOCS3 can inhibit PRL induction of milk protein gene expression and STAT5 activation. We explored the SOCS3 expression pattern during mouse mammary development and its regulation by PRL and GH in wild-type and STAT5a-null mammary tissue. Our results suggest that, in vivo, PRL stimulates SOCS3 expression in stromal adipocytes, independently of STAT5a stimulation. In mammary epithelial cells, SOCS3 expression appears to be related to STAT3 activation. Together, our results are consistent with a role of SOCS3 in the mammary gland by promoting apoptosis of differentiated cells (adipocytes during gestation and epithelial cells during involution).     

Caveolin-1-deficient mice show accelerated mammary gland development during pregnancy,premature lactation,and hyperactivation of the Jak-2/STAT5a signaling cascade

It is well established that mammary gland development and lactation are tightly controlled by prolactin signaling. Binding of prolactin to its cognate receptor (Prl-R) leads to activation of the Jak-2 tyrosine kinase and the recruitment/tyrosine phosphorylation of STAT5a. However, the mechanisms for attenuating the Prl-R/Jak-2/STAT5a signaling cascade are just now being elucidated. Here, we present evidence that caveolin-1 functions as a novel suppressor of cytokine signaling in the mammary gland, akin to the SOCS family of proteins. Specifically, we show that caveolin-1 expression blocks prolactin-induced activation of a STAT5a-responsive luciferase reporter in mammary epithelial cells. Furthermore, caveolin-1 expression inhibited prolactin-induced STAT5a tyrosine phosphorylation and DNA binding activity, suggesting that caveolin-1 may negatively regulate the Jak-2 tyrosine kinase. Because the caveolin-scaffolding domain bears a striking resemblance to the SOCS pseudosubstrate domain, we examined whether Jak-2 associates with caveolin-1. In accordance with this homology, we demonstrate that Jak-2 cofractionates and coimmunoprecipitates with caveolin-1. We next tested the in vivo relevance of these findings using female Cav-1 (-/-) null mice. If caveolin-1 normally functions as a suppressor of cytokine signaling in the mammary gland, then Cav-1 null mice should show premature development of the lobuloalveolar compartment because of hyperactivation of the prolactin signaling cascade via disinhibition of Jak-2. In accordance with this prediction, Cav-1 null mice show accelerated development of the lobuloalveolar compartment, premature milk production, and hyperphosphorylation of STAT5a (pY694) at its Jak-2 phosphorylation site. In addition, the Ras-p42/44 MAPK cascade is hyper-activated. Because a similar premature lactation phenotype is observed in SOCS1 (-/-) null mice, we conclude that caveolin-1 is a novel suppressor of cytokine signaling.     

Xotx5b, a new member of the Otx gene family, may be involved in anterior and eye development in Xenopus laevis

We describe the cloning, expression pattern and functional overexpression analysis of Xotx5b, a new member of the Otx gene family in Xenopus laevis. Early expression of Xotx5b resembles that of Xotx2, being detected in the organizer region at early gastrula stage, and, shortly after, also in anterior neuroectoderm. During neurula stages Xotx5b exhibits a changing and dynamic pattern of expression. After neural tube closure, Xotx5b is expressed in the eye and pineal gland, both involved in photoreception. Overexpression of Xotx5b has a similar effect to that of Xotx2, producing posterior truncations and inducing ectopic cement gland and neural tissue in whole embryos. In animal cap assays, Xotx5b and Xotx2 are both able to activate XAG, to strongly suppress the expression of the epidermal marker XK81, and to reciprocally activate each other. Finally, in einsteck transplantation assays, Xotx5b is able to respecify a tail/trunk organizer to a head organizer.     

Forced activation of Stat5 subjects mammary epithelial cells to DNA damage and preferential induction of the cellular response mechanism during proliferation

Parity‐dependent adenocarcinoma tumors developed in postestropausal transgenic mice expressing a constitutively active Stat5 variant (STAT5ca) in their mammary gland. These tumors maintained elevated expression levels of genes regulating the cellular DNA damage response (DDR) mechanism, compared to the intact gland. No correlation with STAT5ca expression was observed for these genes in the established tumors. However, activated Stat5a in individual cells of the rarely and earlier developed hyperplasia was associated with induced Chk2 activity. Deregulated Stat5 may already cause DNA damage during the fertile period. This hypothesis and the specific vulnerable stage were further studied in mammary epithelial cells that were stably transfected with β‐lactoglobulin (BLG)/STAT5ca and exposed to a reproduced reproductive cycle. During the pregnancy‐like proliferative state, STAT5ca expression was induced by the added lactogenic hormones. Production of reactive oxygen species, rather than proliferation, served as the primary mediator of DNA damage and cellular DDR. Differentiated cells expressed higher levels of STAT5ca and retained the DNA nicks. However, the elevated expression of the genes involved in DDR was downregulated. Higher levels of DNA damage were also detected in the mammary gland of transgenic mice expressing the BLG/STAT5ca during pregnancy and lactation. However, the relative number of damaged cells was much lower than that in the reproduced in vitro stages and the insults were generally associated with apoptosis and DDR. This study implicates pregnancy as the vulnerable stage for deregulated Stat5 activity, and demonstrates that DNA insults in viable differentiated mammary epithelial cells are ignored by the DDR mechanism. J. Cell. Physiol. 226: 616–626, 2011. © 2010 Wiley‐Liss, Inc.     

Direct action of the nodal-related signal cyclops in induction of sonic hedgehog in the ventral midline of the CNS

The secreted molecule Sonic hedgehog (Shh) is crucial for floor plate and ventral brain development in amniote embryos. In zebrafish, mutations in cyclops (cyc), a gene that encodes a distinct signal related to the TGF(beta) family member Nodal, result in neural tube defects similar to those of shh null mice. cyc mutant embryos display cyclopia and lack floor plate and ventral brain regions, suggesting a role for Cyc in specification of these structures. cyc mutants express shh in the notochord but lack expression of shh in the ventral brain. Here we show that Cyc signalling can act directly on shh expression in neural tissue. Modulation of the Cyc signalling pathway by constitutive activation or inhibition of Smad2 leads to altered shh expression in zebrafish embryos. Ectopic activation of the shh promoter occurs in response to expression of Cyc signal transducers in the chick neural tube. Furthermore an enhancer of the shh gene, which controls ventral neural tube expression, is responsive to Cyc signal transducers. Our data imply that the Nodal related signal Cyc induces shh expression in the ventral neural tube. Based on the differential responsiveness of shh and other neural tube specific genes to Hedgehog and Cyc signalling, a two-step model for the establishment of the ventral midline of the CNS is proposed.     

Impaired differentiation and lactational failure of Erbb4-deficient mammary glands identify ERBB4 as an obligate mediator of STAT5

The ERBB family of type 1 receptor tyrosine kinases and their ligands have crucial functions during mammopoiesis, but the signaling networks that ultimately regulate ERBB activity in the breast have remained elusive. Here, we show that mice with Cre-lox mediated deletions of both Erbb4 alleles within the developing mammary gland (Erbb4(Flox/Flox)Wap-Cre) fail to accumulate lobuloalveoli or successfully engage lactation at parturition owing, in part, to impaired epithelial proliferation. Analysis of the mammary differentiation factor STAT5 by immunohistochemistry and western blot revealed a complete ablation of STAT5 activation in Erbb4(Flox/Flox)Wap-Cre mammary epithelium at parturition. Consistent with disrupted STAT5 function, Erbb4(Flox/Flox)Wap-Cre mammary glands at parturition failed to express the mammary epithelial differentiation marker NPT2B. Defects in epithelial functional differentiation at parturition were accompanied by a profound reduction in expression of the STAT5-regulated milk genes casein beta and whey acidic protein. We propose that ERBB4 functions as an essential mediator of STAT5 signaling, and that loss of STAT5 activity contributes to the impaired functional differentiation of mammary glands observed in mice containing conditional Erbb4 deletions.