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Transforming growth factor (TGF)-beta1 is a pluripotent cytokine that profoundly inhibits epithelial proliferation, induces apoptosis, and influences morphogenesis by mediating extracellular matrix deposition and remodeling. The physiologic roles of the action of TGF-beta in mammary gland, indeed in most tissues, are poorly understood. In order to understand the actions of TGF-beta, we need to take into account the complexity of its effects on different cell types and the influence of context on cellular responses. This task is further compounded by multiple mechanisms for regulating TGF-beta transcription, translation, and activity. One of the most significant factors that obscures the action of TGF-beta is that it is secreted as a stable latent complex, which consists of the 24-kDa cytokine and the 80-kDa dimer of its prepro region, called latency-associated peptide. Latency imposes a critical restraint on TGF-beta activity that is often overlooked. The extracellular process known as activation, in which TGF-beta is released from the latent complex, is emphasized in the present discussion of the role of TGF-beta in mammary gland development. Definition of the spatial and temporal patterns of latent TGF-beta activation in situ is essential for understanding the specific roles that TGF-beta plays during mammary gland development, proliferation, and morphogenesis.

Aberrant activation of the Wnt signalling pathway is required for tumour initiation and survival in the majority of colorectal cancers. The development of inhibitors of Wnt signalling has been the focus of multiple drug discovery programs targeting colorectal cancer and other malignancies associated with aberrant pathway activation. However, progression of new clinical entities targeting the Wnt pathway has been slow. One challenge lies with the limited predictive power of 2D cancer cell lines because they fail to fully recapitulate intratumoural phenotypic heterogeneity. In particular, the relationship between 2D cancer cell biology and cancer stem cell function is poorly understood. By contrast, 3D tumour organoids provide a platform in which complex cell-cell interactions can be studied. However, complex 3D models provide a challenging platform for the quantitative analysis of drug responses of therapies that have differential effects on tumour cell subpopulations. Here, we generated tumour organoids from colorectal cancer patients and tested their responses to inhibitors of Tankyrase (TNKSi) which are known to modulate Wnt signalling. Using compounds with 3 orders of magnitude difference in cellular mechanistic potency together with image-based assays, we demonstrate that morphometric analyses can capture subtle alterations in organoid responses to Wnt inhibitors that are consistent with activity against a cancer stem cell subpopulation. Overall our study highlights the value of phenotypic readouts as a quantitative method to asses drug-induced effects in a relevant preclinical model., Competing Interests: L.S.P is a founder and major shareholder of OcellO B.V, and C.E., H.-P.B and D.W are employees of Merck Healthcare KGaA. M.T and J.T are employees of Cellesce Ltd.T.C.D is a director of Cellesce Ltd. L.M.B and D.A.B are siblings. The funder provided support in the form of salaries for authors L.S.P, C.E.,H.-P.B, D.W, M.T and J.T but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Tcf7l2 mediates Wnt/β-Catenin signalling during development and is implicated in cancer and type-2 diabetes. The mechanisms by which Tcf7l2 and Wnt/β-Catenin signalling elicit such a diversity of biological outcomes are poorly understood. Here, we study the function of zebrafish tcf7l2 alternative splice variants and show that only variants that include exon five or an analogous human tcf7l2 variant can effectively provide compensatory repressor function to restore eye formation in embryos lacking tcf7l1a/tcf7l1b function. Knockdown of exon five specific tcf7l2 variants in tcf7l1a mutants also compromises eye formation, and these variants can effectively repress Wnt pathway activity in reporter assays using Wnt target gene promoters. We show that the repressive activities of exon5-coded variants are likely explained by their interaction with Tle co-repressors. Furthermore, phosphorylated residues in Tcf7l2 coded exon5 facilitate repressor activity. Our studies suggest that developmentally regulated splicing of tcf7l2 can influence the transcriptional output of the Wnt pathway., Competing Interests: RY, KE, VF, MA, JG, TD, SW No competing interests declared, (© 2019, Young et al.)

We have previously shown that deficiency of the methyl binding domain protein Mbd2 dramatically reduces adenoma burden on an Apc(Min/+) background. To investigate the mechanism underlying this phenomenon, we have determined the effect of Mbd2 deficiency upon the phenotypes imposed by the conditional deletion of Apc in the small intestine. Microarray analysis demonstrated a partial suppression of the Wnt pathway in the absence of Mbd2. Mbd2 deficiency also influenced one immediate cellular consequence of Apc loss, with normalization of Paneth cell positioning. From a mechanistic perspective, we show that deficiency of Mbd2 elevates levels of the known Wnt target Lect2, and we confirm here that Mbd2 binds the Lect2 promoter in association with NuRD. Furthermore, we show that Lect2 is capable of functioning as a Wnt pathway repressor. These results therefore provide a mechanistic basis for the epigenetic control of adenoma formation mediated through Mbd2.

There has been a surge of interest in the therapeutic targeting of the Wnt pathway following the demonstration that it is activated in a wide variety of tumors and that blocking aberrant signaling promoted tumor cell apoptosis or differentiation. This review describes recent therapeutic approaches and discusses potential opportunities for intervention at multiple levels within the Wnt pathway.

Transforming growth factor (TGF)-beta1 is a potent inhibitor of mammary epithelial proliferation. In human breast, estrogen receptor (ER)-alpha cells rarely co-localize with markers of proliferation, but their increased frequency correlates with breast cancer risk. To determine whether TGF-beta1 is necessary for the quiescence of ER-alpha-positive populations, we examined mouse mammary epithelial glands at estrus. Approximately 35% of epithelial cells showed TGF-beta1 activation, which co-localized with nuclear receptor-phosphorylated Smad 2/3, indicating that TGF-beta signaling is autocrine. Nuclear Smad co-localized with nuclear ER-alpha. To test whether TGF-beta inhibits proliferation, we examined genetically engineered mice with different levels of TGF-beta1. ER-alpha co-localization with markers of proliferation (ie, Ki-67 or bromodeoxyuridine) at estrus was significantly increased in the mammary glands of Tgf beta1 C57/bl/129SV heterozygote mice. This relationship was maintained after pregnancy but was absent at puberty. Conversely, mammary epithelial expression of constitutively active TGF-beta1 via the MMTV promoter suppressed proliferation of ER-alpha-positive cells. Thus, TGF-beta1 activation functionally restrains ER-alpha-positive cells from proliferating in adult mammary gland. Accordingly, we propose that TGF-beta1 dysregulation may promote proliferation of ER-alpha-positive cells associated with breast cancer risk in humans.

Transforming growth factor (TGF)-beta1 is rapidly activated after ionizing radiation, but its specific role in cellular responses to DNA damage is not known. Here we use Tgfbeta1 knockout mice to show that radiation-induced apoptotic response is TGF-beta1 dependent in the mammary epithelium, and that both apoptosis and inhibition of proliferation in response to DNA damage decrease as a function of TGF-beta1 gene dose in embryonic epithelial tissues. Because apoptosis in these tissues has been shown previously to be p53 dependent, we then examined p53 protein activation. TGF-beta1 depletion, by either gene knockout or by using TGF-beta neutralizing antibodies, resulted in decreased p53 Ser-18 phosphorylation in irradiated mammary gland. These data indicate that TGF-beta1 is essential for rapid p53-mediated cellular responses that mediate cell fate decisions in situ.

Transforming growth factor-beta1 (TGF-beta 1) is a pluripotent cytokine that can inhibit epithelial proliferation and induce apoptosis, but is also widely implicated in breast cancer progression. Understanding its biological action in mammary development is critical for understanding its role in cancer. TGF-beta 1 is produced as a latent complex that requires extracellular activation before receptor binding. To better understand the spatial and temporal regulation of its action during mammary gland development, we examined the pattern of activation in situ using antibodies selected to distinguish between latent and active TGF-beta. Activation was highly restricted. TGF-beta 1 activation was localized primarily to the epithelium, and within the epithelium it was restricted to luminal epithelial cells but absent from either cap or myoepithelial cells. Within the luminal epithelium, we noted a further restriction. During periods of proliferation (ie, puberty, estrus and pregnancy), which are stimulated by ovarian hormones, TGF-beta 1 activation decreased in some cells, consistent with preparation for proliferation. Paradoxically, other cells simultaneously increase TGF-beta 1 immunoreactivity, which suggests that TGF-beta 1 differentially restrains epithelial subpopulations from responding to hormonal signals to proliferate. These data suggest that endogenous TGF-beta 1 activation and thus activity are regulated by ovarian hormones. To determine the specific consequences of TGF-beta 1 activity, we manipulated TGF-beta 1 levels in vivo using Tgfbeta 1 knockout mice and undertook tissue recombination experiments with heterozygous tissue. In Tgfbeta 1 heterozygous mice, which have <10% wild-type levels of TGF-beta1, ductal development during puberty and alveolar development during pregnancy were accelerated, consistent with its role as a growth inhibitor. The proliferative index of Tgfbeta 1+/- epithelium was increased approximately twofold in quiescent tissue and fourfold in proliferating tissue but both ducts and alveoli were grossly and histologically normal. To test whether epithelial TGF-beta1 was critical to the proliferative phenotype, Tgfbeta 1+/+ and +/- epithelium were transplanted into +/+ mammary stroma. The outgrowth of Tgfbeta 1+/- epithelium was accelerated in wild-type hosts, indicating that the phenotype was intrinsic to the epithelium. Moreover, proliferation was 15-fold greater in Tgfbeta 1+/- than wild-type mice after ovariectomy and treatment with estrogen and progesterone, suggesting that TGF-beta 1 acts in an autocrine or juxtacrine manner to regulate epithelial proliferation. Together these data indicate that ovarian hormones regulate TGF-beta 1 activation, which in turn restricts proliferative response to hormone signaling.

Gender differences have been noted in the tissue distribution of mercury. We sought to determine if the uptake of low-dose inorganic mercury into motor neurons differs between male and female mice. Four male and four female mice were injected i.p. with 0.5 mg HgCl2/kg. In 50-microns sections of lumbar spinal cord stained with autometallography, six motor neuron cell bodies were selected for study. The volume percentage of mercury granules in the cell bodies was estimated using a confocal microscope. Mercury granules occupied more perikaryal volume in motor neurons from female mice (mean 3.7%) than from male mice (mean 2.2%) (p < or = 0.05). After the same dose, the amount of renal mercury measured by mass spectrometry was significantly less in six female than five male mice. In conclusion, female mice take up more inorganic mercury into their motor neurons than do male mice. This may be related to a smaller deposition of mercury in the female kidney, leaving more circulating mercury available to be taken up by motor axons.

Heavy metal toxicity has been implicated in the pathogenesis of motor neuron diseases. In an attempt to assess the efficacy of chelating agents to remove mercury from motor neurons, we quantitated the effect of the chelating agents meso-2,3-dimercaptosuccinic acid (DMSA) and 2,3- dimercaptopropane -1-sulphonate (DMPS) on the burden of inorganic mercury in mouse spinal motor neurons. Mice were injected intraperitoneally with 1.0 mg HgCl2/kg body weight and one week later with either 4,400 mg/kg DMPS, 3,600 mg/kg DMSA or 5% NaHCO3 (control) over 4 weeks. Mercury deposits in motor neurons of 50 micron frozen sections of lumbar spinal cord were visualised with an autometallographic technique. Optical sections of silver-enhanced deposits were acquired using a confocal microscope in reflective mode and the volume of the deposits within the perikaryon was estimated. Mercury deposits occupied significantly more volume in motor neurons after both DMPS (7.4%, SD +/- 0.7%) and DMSA (8.0% +/- SD 0.7%) treatment than in controls (4.3%, SD +/- 1.7%). The higher levels of neuronal inorganic mercury may be due to increased entry of mercury into motor axons across the neuromuscular junction as a result of chelator-induced elevated circulating mercury.

Our aim was to map the segmental distribution of somitic cells in the limb before and after the fusion of these cells into myotubes. Somitic cells of the brachial somites were labeled by injection of DiI and DiO into the somitoceles of embryonic Day 2 (E2) embryos. The quantitative distribution of dye-labeled cells from injected somites was examined in whole mounts of E4 wing buds and in sections of E5 wing buds using confocal microscopy. Dye derived from cells of anterior brachial somites 16 and 17 was highly concentrated in the anterior half of E4 wings, whereas dye from posterior brachial somites 20 and 21 was concentrated in the posterior half of the wing. Not more than 14% (on average) of the dye in the wing was found outside the preferred half. Dye was equally dispersed in the anterior and posterior halves of the wing when the middle two somites contributing to the wing musculature, numbers 18 and 19, were injected. The total amount of dye in the whole limb at E4 was not significantly different after injection of similar amounts of dye into either pair of brachial somites 16/17, 18/19, or 20/21. In E5 embryos the distribution of labeled cells in the newly formed muscle masses, identified with antibody 2H2 to myosin light chains, was examined in cryostat cross-sections of the wing. Dye was more widely distributed in the wing than at E4. As much as 32% (on average) of the dye was found in the muscle mass outside the preferred half after administration of dye into the anterior or posterior pair of somites. We conclude that the branchial somites contribute similar numbers of cells to the wing musculature and that the segmental origin of these cells is rigidly maintained during their lateral migration into the limb. Cells from adjacent segments then "mix" when they fuse to form myotubes at E5. In addition, we found a significantly greater amount of dye per unit of muscle mass in the proximal compared with distal parts of the limb. We argue that this finding is due to division of myogenic cells during the course of their migration in the wing.

We have examined the somitic cell contribution to the vertebral column of the chick by genetic labeling of sclerotomal cells in early development. Single somites of embryonic Day 2 embryos were filled with retroviral particles containing the lacZ transducing vector BAG. After a further 14 or 17 days of incubation the embryos were fixed and the vertebral column was sectioned and stained histochemically for the lacZ gene product beta-galactosidase. Cells staining for the enzyme were found exclusively on the injected side of two vertebral segments; the staining was largely restricted, however, to the caudal half of the more rostral segment and the rostral half of the next more caudal segment. No embryos were observed with labeling in less than two vertebral segments. Moreover, labeled cells were not uniformly distributed within the labeled region of each vertebra; the neural arch, for example, usually contained a higher proportion of labeled cells than did the centrum. These observations support the concept of resegmentation, whereby a vertebra forms from sclerotomal cells derived from two consecutive somites resulting in a vertebral column shifted by one half segment with respect to the segmented boundaries of the somites. The quantitative distribution of labeled cells in the vertebrae also suggests that sclerotomal cells populate the region of a future vertebral segment in an orderly fashion dependent on when the cells migrate from the somite.