Ubiquitin-Mediated Proteasome Degradation Regulates Optic Fissure Fusion

Optic fissure fusion is a critical event during retinal development. Failure of fusion leads to coloboma, a potentially blinding congenital disorder. Pax2a is an essential regulator of optic fissure fusion and the target of numerous morphogenetic pathways. In our current study, we examined the negative regulator of pax2a expression, Nz2, and the mechanism modulating Nlz2 activity during optic fissure fusion. Upregulation of Nlz2 in zebrafish embryos resulted in downregulation of pax2a expression and fissure fusion failure. Conversely, upregulation of pax2a expression also led to fissure fusion failure suggesting Pax2 levels require modulation to ensure proper fusion. Interestingly, we discovered Nlz2 is a target of the E3 ubiquitin ligase Siah. We show that zebrafish siah1 expression is regulated by Hedgehog signaling and that Siah1 can directly target Nlz2 for proteasomal degradation, in turn regulating the levels of pax2a mRNA. Finally, we show that both activation and inhibition of Siah activity leads to failure of optic fissure fusion dependent on ubiquitin-mediated proteasomal degradation of Nlz2. In conclusion, we outline a novel, proteasome-mediated degradation regulatory pathway involved in optic fissure fusion

Green tea as an antioxidant which protects against alcohol induced injury in rats - a histopathological examination

Our study with animal models was designed to test the hypothesis that green tea protects against chronic (over 4 weeks) alcohol induced liver injury in rats. The research was conducted on Wistar male rats divided into 4 research groups: I - received the Libera-De Carli control diet (L-DC), II - received (L-DC) and green tea, III - received (L-DC) and ethanol and IV - received (L-DC), green tea and ethanol. When comparing groups I and II we saw less intensive steatosis in group II than in group I, which can suggest that green tea may affect the accumulation of fat in the hepatocytes and protect them against steatosis and disruption. In III, the ethanol group, the steatosis of the liver increased considerably and the green tea which was given with ethanol in group IV did not halt this, as in group IV we also observed intensive steatosis in the liver. From this data we conclude that green tea has an important, although not fully understood role in preventing liver injury

Stable hZW10 kinetochore residency, mediated by hZwint-1 interaction, is essential for the mitotic checkpoint

The mitotic checkpoint is an essential surveillance mechanism that ensures high fidelity chromosome segregation during mitosis. Mitotic checkpoint function depends on numerous kinetochore proteins, including ZW10, ROD, and Zwilch (the ROD–ZW10–Zwilch complex). Through an extensive mutagenesis screen of hZW10, we have mapped the kinetochore localization domain of hZW10 as well as the hZwint-1 interaction domain. We find that hZwint-1–noninteracting mutants still localize to kinetochores. In addition, using fluorescence recovery after photobleaching, we have found that hZW10 residency at metaphase kinetochores is brief (half-time of 13 s). However, during prometaphase or at unattached kinetochores, enhanced green fluorescent protein–hZW10 becomes a stable component of the kinetochore. Moreover, we find that stable hZW10 kinetochore residency at prometaphase kinetochores is dependent on its interaction with hZwint-1, and is essential for mitotic checkpoint arrest

Intrakinetochore stretch is associated with changes in kinetochore phosphorylation and spindle assembly checkpoint activity

© 2009 Maresca and Salmon. This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 License. The definitive version was published in Journal of Cell Biology 184 (2009): 373-381, doi:10.1083/jcb.200808130.Cells have evolved a signaling pathway called the spindle assembly checkpoint (SAC) to increase the fidelity of chromosome segregation by generating a "wait anaphase" signal until all chromosomes are properly aligned within the mitotic spindle. It has been proposed that tension generated by the stretch of the centromeric chromatin of bioriented chromosomes stabilizes kinetochore microtubule attachments and turns off SAC activity. Although biorientation clearly causes stretching of the centromeric chromatin, it is unclear whether the kinetochore is also stretched. To test whether intrakinetochore stretch occurs and is involved in SAC regulation, we developed a Drosophila melanogaster S2 cell line expressing centromere identifier–mCherry and Ndc80–green fluorescent protein to mark the inner and outer kinetochore domains, respectively. We observed stretching within kinetochores of bioriented chromosomes by monitoring both inter- and intrakinetochore distances in live cell assays. This intrakinetochore stretch is largely independent of a 30-fold variation in centromere stretch. Furthermore, loss of intrakinetochore stretch is associated with enhancement of 3F3/2 phosphorylation and SAC activation.This work was supported by the American Cancer Society (grant PF0711401 to T.J. Maresca) and the National Institutes of Health (grant GM24364 to E.D. Salmon)

Hyaloid Vasculature and mmp2 Activity Play a Role during Optic Fissure Fusion in Zebrafish

Vertebrate retinal development requires timely and precise fusion of the optic fissure (OF). Failure of this event leads to congenital vision impairment in the form of coloboma. Recent studies have suggested hyaloid vasculature to be involved in OF fusion. In order to examine this link, we analyzed OF fusion and hyaloid vasculogenesis in the zebrafish pax2a noi mutant line. We first determined that pax2a−/− embryos fail to accumulate F-actin in the OF prior to basement membrane (BM) degradation. Furthermore, using 3D and live imaging we observed reduced OF hyaloid vascularization in pax2a−/− embryos. When examining the connection between pax2a loss of function and hyaloid vasculature, we observed significant reduction of talin1 expression, a regulator of hyaloid vasculature. In addition, cranial VEGF expression was found to be reduced in pax2a−/− embryos. Pharmacological inhibition of VEGF signaling phenocopied the pax2a−/− vasculature, F-actin and BM degradation phenotypes. Lastly, we determined that OF associated hyaloid vasculature is a source of mmp2, mmp14a and mmp14b expression and showed that mmp2 is functionally necessary for degradation of OF BM. Taken together we propose a pax2a driven mechanism that ensures proper and timely hyaloid vasculature invasion of the OF in order to facilitate availability of the BM remodeler mmp2

Proteasome-Mediated Regulation of Cdhr1a by Siah1 Modulates Photoreceptor Development and Survival in Zebrafish

Congenital retinal dystrophies are a major cause of unpreventable and incurable blindness worldwide. Mutations in CDHR1, a retina specific cadherin, are associated with cone-rod dystrophy. The ubiquitin proteasome system (UPS) is responsible for mediating orderly and precise targeting of protein degradation to maintain biological homeostasis and coordinate proper development, including retinal development. Recently, our lab uncovered that the seven in absentia (Siah) family of E3 ubiquitin ligases play a role in optic fissure fusion and identified Cdhr1a as a potential target of Siah. Using two-color whole mount in situ hybridization and immunohistochemistry, we detected siah1 and cdhr1a co-expression as well as protein localization in the retinal outer nuclear layer (ONL), and more precisely in the connecting cilium of rods and cones between 3–5 days post fertilization (dpf). We confirmed that Siah1 targets Cdhr1a for proteasomal degradation by co-transfection and co-immunoprecipitation in cell culture. To analyze the functional importance of this interaction, we created two transgenic zebrafish lines that express siah1 or an inactive siah1 (siah1ΔRING) under the control of the heat shock promoter to modulate Siah activity during photoreceptor development. Overexpression of siah1, but not siah1ΔRING, resulted in a decrease in the number of rods and cones at 72 h post fertilization (hpf). The number of retinal ganglion cells, amacrine and bipolar cells was not affected by Siah1 overexpression, and there was no significant reduction of proliferating cells in the Siah1 overexpressing retina. We did, however, detect increased cell death, confirmed by an increase in the number of TUNEL + cells in the ONL, which was proteasome-dependent, as proteasome inhibition rescued the cell death phenotype. Furthermore, reduction in rods and cones resulting from increased Siah1 expression was rescued by injection of cdhr1a mRNA, and to an even greater extent by injection of a Siah1-insensitive cdhr1a variant mRNA. Lastly, CRISPR induced loss of Cdhr1a function phenocopied Siah1 overexpression resulting in a significant reduction of rods and cones. Taken together, our work provides the first evidence that Cdhr1a plays a role during early photoreceptor development and that Cdhr1a is regulated by Siah1 via the UPS

Expression of connexin 43 in breast cancer in comparison with mammary dysplasia and the normal mammary gland

Gap junctional intercellular communication (GJIC) plays a critical role in tissue development and differentiation and probably in carcinogenesis. The purpose of the study was to evaluate the expression and localisation of Cx43 in 40 cases of mammary dysplasia and 29 cases of breast cancer (without primary chemotherapy). The tissue sections were investigated for Cx43 expression by immunohistochemistry. In the normal mammary gland there was an intercellular, punctate staining pattern, mainly between myoepithelial cells, characteristic of functional gap junctions. In dysplasias there was mainly mixed (cytoplasmic and intercellular) staining and in most cases of breast cancer we observed diffuse or granular, but cytoplasmic, staining of Cx43. Our results demonstrated that expression of Cx43 in dysplasias and breast cancer is changed and GJIC is probably impaired because of disruption of functional gap junction formation especially between breast cancer cells

Spatiotemporal Characterization of Anterior Segment Mesenchyme Heterogeneity during Zebrafish Ocular Anterior Segment Development

Assembly of the ocular anterior segment (AS) is a critical event during development of the vertebrate visual system. Failure in this process leads to anterior segment dysgenesis (ASD), which is characterized by congenital blindness and predisposition to glaucoma. The anterior segment is largely formed via a neural crest-derived population, the Periocular Mesenchyme (POM). In this study, we aimed to characterize POM behaviors and transcriptional identities during early establishment of the zebrafish AS. Two-color fluorescent in situ hybridization suggested that early AS associated POM comprise of a heterogenous population. In vivo and time-course imaging analysis of POM distribution and migratory dynamics analyzed using transgenic zebrafish embryos (Tg[foxc1b:GFP], Tg[foxd3:GFP], Tg[pitx2:GFP], Tg[lmx1b.1:GFP], and Tg[sox10:GFP]) revealed unique AS distribution and migratory behavior among the reporter lines. Based on fixed timepoint and real-time analysis of POM cell behavior a comprehensive model for colonization of the zebrafish AS was assembled. Furthermore, we generated single cell transcriptomic profiles (scRNA) from our POM reporter lines and characterized unique subpopulation expression patterns. Based on scRNA clustering analysis we observed cluster overlap between neural crest associated (sox10/foxd3), POM (pitx2) and finally AS specified cells (lmx1b, and foxc1b). scRNA clustering also revealed several novel markers potentially associated with AS development and/or function including lum, fmoda, adcyap1b, tgfbi, and hmng2. Taken together, our data indicates that AS-associated POM, or Anterior Segment Mesenchyme (ASM), is not homogeneous but rather comprised of several subpopulations with differing colonization patterns, migration behavior, and transcriptomic profiles

Expression of the apoptotic markers in normal breast epithelium, benign mammary dysplasia and in breast cancer

Apoptosis and proliferation are processes associated with the development and progression of breast cancer. The sensitivity of tumour cells to the induction of apoptosis depends on the balance between pro- and anti-apoptotic proteins. The expression of Bak and Bcl-2 was examined using an immunohistochemical method in 71 primary breast cancers. Furthermore, Bcl-2 and Bak were assessed in the normal mammary gland as well as in benign mammary dysplasia adjacent to breast cancer. Positive immunostaining for Bcl-2 was observed in 77.8% of cases of normal breast epithelium (NBE), 93% of benign dysplasia without intraductal proliferation (BBD) as well as in 94% of intraductal proliferative lesions of the breast (BIPL). Expression of Bak was detected in 39% of cases of NBE, 45% of BBD and in 67% of BIPL. In breast cancer Bcl-2 and Bak expression was found in 83% and 70% of the cases studied, respectively. Increased Bcl-2 expression in primary tumours significantly correlated with favourable prognostic factors, namely pT1, G2 and lack of metastases to the regional lymph nodes (p < 0.01, p < 0.03, p < 0.02, respectively). There were no relationships between Bak and the clinicopathological features studied, but our results indicate changes in the expression of Bak during breast cancer development and progression. It would appear to be important to assess and compare pro- and anti-apoptotic proteins between normal mammary gland, benign mammary dysplasia and the primary tumours of breast cancer. This knowledge should be helpful in understanding breast cancer development and progression