The study of regional trends in the rural-urban fertility gradient helps us to understand the pace of completion of the fertility transition and the geography of urban growth in the global South. We question whether the hypothesized inverted U-shaped evolution in rural excess fertility is confirmed in four developing regions, and investigate the underlying fertility dynamics by place of residence. Using multiple surveys for 60 developing countries, we analyze long-term rural and urban trends in cohort fertility. The regional comparison is controlled for the international heterogeneity in the stages attained in the fertility transition and the context of urbanization. We found a clearly inverted U-shaped trend in the rural-urban fertility gradient in Latin America, the Middle East and Northern Africa. In Asia, rural excess fertility remained limited. In sub-Saharan Africa it increased monotonically until the most recent cohorts. These differences stem from variations in the urban-to-rural diffusion of the onset of fertility transition and, in sub-Saharan Africa, from a slower pace of decline in rural areas. Starting in the 1960s, the fertility transition was almost complete in Asia and Latin America by 2010–15. However, women in sub-Saharan Africa were still having more than five children on average . The stuThe transformation from a predominantly rural to a predominantly urban society affects the cultural and socioeconomic environment of human reproduction. On the one hand, the earlier and faster pace of structural changes in urban societies leads to lower fertility compared to that of rural populations , 5. The According to spatial diffusion theory, the structural and ideational changes in society that motivate lower fertility spread from cities into peripheral areas during the process of regional development and integration , 12. A rRecent fertility levels are higher in rural than in urban areas in all developing regions , 17. TheWe question whether the inverted U-shaped evolution in rural excess fertility is confirmed in Asia, Latin America and the Caribbean (LACarr), the Middle East and Northern Africa (MENA), and sub-Saharan Africa (SSA). In the next section, we lay out the expectations about the regional differences, given the variations in the level of urbanization, the patterns of change in reproductive behaviors and the urban-to-rural diffusion of modern family planning. We then introduce the data and the methods used to estimate fertility trends over 80 birth cohorts in 60 countries. In the empirical section, we describe the regional trends in the rural-urban fertility ratio and the underlying dynamics by place of residence, while controlling for the international heterogeneity in the demographic and urbanization context. Finally, we summarize the results and discuss their implications for future fertility developments and the geography of urban growth in the global South.Since 1950, the percentage of the population living in urban areas has more than doubled in each of the four developing regions, but there are substantial differences in the levels. In 2010, 79% of the population in LACarr lived in urban areas, 60% in MENA, 43% in Asia , and 36% in SSA . InIn MENA, the progress in the diffusion of modern contraceptives was slower and concentrated in big cities. In rural areas, women used these methods primarily to space (rather than to limit) their births . This imUnlike the steady decrease in unwanted fertility in these three developing regions, the rate has stayed constant over the last 20 years in SSA . There, This contextual evidence motivates a number of hypotheses about the regional differentials in the evolution of the rural-urban fertility ratio over time. Given the fast diffusion of a stopping pattern of childbearing in urban LACarr, we would expect an early and sharp increase in rural excess fertility. The significant time-lag in the urban-to-rural diffusion of modern contraceptives should have led to a high peak in the fertility gradient by residence. As soon as family planning services became accessible in remote areas, the rural-urban fertility gradient should have started to decrease at a fast pace. The dominance of cities in the region’s population geography may accelerate the diffusion of the new behaviors in rural areas. A similar pattern can be expected for MENA, although rural (excess) fertility may have declined at a slower pace because of a slower spatial diffusion of behavioral changes in a society which was predominantly rural until the 1980s.The expectations for Asia are competing. On the one hand, the lower level of urbanization implies a slow diffusion of new reproductive behaviors to rural areas. The peak in the rural-urban fertility gradient may thus be high. On the other hand, governments’ commitment to restraining people’s freedom to have large families, and to spreading the use of modern contraceptives in rural areas early on in the fertility transition, may have put a ceiling on rural excess fertility.In SSA, the early decline in urban fertility may have been slower, when compared to other regions, because it was predominantly driven by birth spacing. This should have led to a slower increase in the rural-urban fertility gradient. However, this rise is likely to be more prolonged because of the important time lag in the urban-to-rural diffusion of modern contraceptives. This also reflects the minority status of the urban population from which behavioral innovation spreads. The subsequent acceleration of the urban fertility decline (due to the diffusion of birth limiting behaviors) should have led to a continuously widening rural-urban fertility gap, as rural fertility decline is likely to remain slow (due to the dominance of birth spacing).To uncover the regional differences in the trajectories of the urban-rural fertility gradient, we adopt an approach that controls for three major confounding factors in international comparisons . First, Third, we focus on cohort fertility. Rural and urban trends in period fertility are flawed, respectively, by temporary peaks and troughs of rural-to-urban migration–especially when migratory and fertility decisions are interlinked . Rural wWe used individual-level data on the number of children ever born (i.e. parity) and birth histories of women aged 30 to 49, drawn from 278 World Fertility Surveys (WFS) and Demographic and Health Surveys (DHS). In order to extend the observation windows into early or late stages of the fertility transition in a number of countries, we also relied on 18 Multiple Indicator Cluster Surveys  and Integrated Public Use Samples of the earliest or most recent population censuses . To incThis analysis covers 60 developing countries see , in whicAlthough SSA countries are overrepresented in the sample due to the greater availability of survey data, regional differences in the urbanization phenomenon are well reflected. The median percentages of the national populations living in urban areas increased from 6% in 1950 to 31% in 2000 among the SSA countries covered in this study, from 13 to 26% among the Asian, from 32 to 53% among the MENA, and from 33 to 61% in the LACarr countries see . The mosGiven the frequent absence of a clear definition of urban and rural type of residence location in the WFS and DHS, we assume that official definitions are relied upon (as in censuses). These national definitions may change over the years, and the city boundaries move out into formerly rural areas as the population sprawls. However, we can rule out the possibility of major analytical biases due to this rural-to-urban reclassification of populations (or vice-versa), as the fertility trends are cross-validated by successive surveys/census-samples see .International differences in the definition of urban areas pose bigger challenges for comparative research. In some countries, the non-inclusion of slum settlements in officially designated urban areas leads to an over-estimation of the extent of the urban fertility decline because the most fertile urban residents are not considered . In otheWe estimated urban and rural trends in average life-time parity, or total fertility of cohorts (TF), by country. We used cohort parity-progression ratios (PPR) which measure the proportion of women who have already had a given number of children and who go on to have an additional one. The TF is then estimated as a weighted average of the parities attained, with the weights being the parity distribution of women as implied by the chaining of the progression ratios from nulliparous to the first birth (PPR1) up to the progression from the fifth to the sixth birth . For the cohorts that have completed their childbearing career at the time of the interview , we directly estimated PPRs based on the distribution of women according to the reported parity at the survey date. Figures for cohorts with fewer than 60 women (in the unweighted sample) were discarded to avoid excessive noise in the estimation series due to sampling biases. In order to fill inter-cohort estimation gaps and extend the series with more recent cohorts, we estimated truncated PPRs of women aged 30–34 and 35–39 at the survey dates, and projected their completed PPRs.We applied the Brass-Juarez paired-cohort comparison procedure to project these PPRs , 51. TheAn internal and external quality assessment of our results confirms (1) the good quality of our data at older ages  and (2) the negligible impacts of the assumptions used to project completed parity progression ratios based on truncated estimates for younger women see . We thusUrban and rural fertility trends were analyzed according to cohort years relative to the onset of the national transition. We first defined the last calendar year in which the national TFR peaked before the first 10%-decline–according to the United Nations' World Population Prospects estimations  and the To estimate regional fertility trends, we first calculated the pace of change in urban and rural areas over cohorts, using linear spline regression models . Country fixed effects control for the unobserved heterogeneity in the national contexts of urbanization and in urban/rural definitions. The regression coefficients were then used to predict regional average within-country trends. Country-specific results illustrate the variations in the fertility developments within regions and illuminate atypical trends, but the differences are not directly interpretable due to the lack of comparability of the rural/urban definitions.No trend aligns on the red and dashed horizontal line at unity, meaning that urban and rural fertility did not decline at the same time and same pace. The majority of estimates are situated above this reference line, indicating a higher fertility in rural relative to urban subgroups of cohorts. The results tend to support the hypothesis of an inverted U-shaped trend in the rural-urban fertility ratio over time in all world regions, except in SSA.However, the form of this U-shape differed across regions. Among the first transition cohorts in LACarr, rural fertility was 25% higher than the urban level on average. Rural excess fertility increased sharply in the first twenty transition cohorts (to a ratio of 1.5 on average), and flattened out later on. The average rural-urban ratio started to drop after 30 transition cohorts. In the most recent cohorts, the ratio remained above 1.25. Our data allow us to observe a significant decline in rural excess fertility in eight out of the 12 countries in the region. In the MENA, the average trend was generally similar. Three out of the four countries in the sample revealed a decline of the rural-urban fertility ratio. A residual gap also persists in the youngest cohorts.The Asian region stands out with the lowest overall level of rural excess fertility. The inverted U-shaped trend over the cohorts is the flattest. Starting from an average ratio of 1.15 at the transition onset, it increased to slightly below 1.25 and declined thereafter. This drop is observed in four out of the 10 countries covered in the region.th transition cohort, the SSA rural fertility decline is not advanced enough to make a definitive statement . This explains its comparatively low peak in the rural-urban fertility ratio. The MENA follow behind with a 13 years lag. LACarr and SSA are characterized by the longest intervals, of 16 and 21 years respectively. In addition, LACarr and SSA reveal large interquartile ranges in the urban-rural transition lags. In SSA, the intercountry variation of 14 years represents approximately twice the variation observed within Asia and within the MENA. In eight SSA countries, rural areas have in fact not yet experienced advanced fertility decline. These long median lags between the urban and rural transition onsets account for the higher peak level of the rural-urban fertility ratio in LACarr and for its continuous increase in SSA.Once rural fertility has started to decline, the rural-urban fertility ratio is determined by the differential paces of the transition by type of residence location. To evaluate rural-urban differences in the pace of fertility decline by world region, we plotted in In general, the average urban and rural pace of fertility decline follows a similar U-shaped trend in all world regions once it has started: from an annual decline of about -0.075 children per woman in the first (two) transition decades, the pace tends to slow down to about -0.025 in the fourth decade. Once the rural transition had started, therefore, the rural pace of decline was significantly faster when compared to that observed in the urban areas, which was already more advanced. This explains the drop in the rural-urban fertility ratio from its peak level in all regions, except in SSA (see below). Moreover, the fertility decline slowed down earlier over the course of the urban when compared to the rural fertility transition in Asia, LACarr and the MENA. This intensified the shrinkage of the rural-urban fertility ratio after its peak.Unlike the other regions, the pace of fertility decline in SSA decelerated more rapidly in the countryside when compared to the cities. This implies that, even though fertility eventually started to drop in a number of rural areas, the slow rhythm (relative to the dynamics in urban areas) sustained the continuous increase in the rural-urban fertility ratio, instead of significantly inverting the trend.The analysis above is confounded by the demographic effect of rural-to-urban migrants, who are included in the urban estimates. Not only have these migrants been socialized to higher fertility norms in the countryside, but the process of adaptation to the lower urban fertility patterns is generally completed only among their descendants, who have been socialized in cities –56. TherMigrants are defined as women who have been socialized (in childhood) in another type of residence location when compared to that in which they have been interviewed, using 146 DHS and WFS with information on migration covering 55 countries see . The ferIn all world regions, rural-to-urban migrants constitute the large majority of movers. Their fertility levels were in between those of the non-migrants in the rural and the urban areas. However, the importance of the indirect effect of migration on urban fertility varies by world region, as it depends on both the difference in non-migrant versus migrant fertility and the level of urbanization.In LACarr, the fertility differential between urban in- and non-migrants was the largest and remained constant over cohorts (0.8 births on average). The in-migrants’ impact on urban fertility levels, however, was negligible in the first thirty transition cohorts because the level of urbanization in Latin America was already relatively high . Migrants started to inflate urban fertility only in the last transition stage (by up to 0.5 births) when the non-migrants’ fertility reached near replacement levels. In the MENA, the patterns were similar. The migrants’ impact in inflating urban fertility levels was more sustained and started earlier on in the transition because the level of urbanization was lower.In SSA, the fertility differential between rural-to-urban migrants and urban non-migrants was comparatively more limited (0.3 births on average). Yet the inflation of urban fertility due to in-migration started immediately after the transition onset because the city populations were small (i.e. the levels of urbanization were much lower than in other regions). Migrants were responsible for at least 0.4 births more per woman in the total relative to the non-migrant population in urban areas. The Asian region is characterized by the smallest differentials in the levels of childbearing by migrant status (0.2 births on average), which exerted negligible effects on the trend in urban fertility at advanced stages of the transition (inflating the level by 0.3 births at best).In sum, the indirect demographic effect of in-migrants (through their higher fertility) decelerated the urban fertility decline in the early stages of the transition in less urbanized regions (most notably in SSA), and in later stages of the fertility transition in all regions. Therefore, migration at least partially explains why the late urban fertility decline decelerated to a larger extent when compared to the rural trend, pushing the rural-urban fertility ratio closer towards unity. In SSA, the early and significant deceleration of the urban fertility decline caused by the arrival of in-migrants also accounts for the slower pace of the initial increase in the average rural-urban fertility ratio.Regional differences in the pace of urban fertility decline and the lagged dynamics in rural areas of developing countries are crucial for better understanding demographic change in an increasingly urbanized world. We examined whether the inverted U-shaped evolution in the rural-urban fertility ratio is confirmed from a longitudinal and long-term perspective of four developing regions, and we investigated the differences in the underlying dynamics by type of residence.The results corroborated an increase followed by a decrease in rural excess fertility over the course of the fertility transition in all developing regions. At the same time, the rise and peak level of this excess fertility, as well as its later decline, differed. We found a marked inverted U-shaped evolution in the rural-urban fertility gradient only in LACarr and the MENA. Rural excess fertility rose continuously over an extended number of cohorts in SSA and has not yet significantly decreased. In Asia, the rural-urban fertility ratio remained comparatively the smallest. This regional variation was not due to differential paces in the urban trends. Our results reveal a fast and continuous urban fertility decline once it has started in all developing regions, including SSA which is often believed to follow an exceptional pathway. This confirms the importance of the universal process of fast structural and ideational change in societies for the transformation of reproductive behaviors.Regional variations in the evolution of rural excess fertility are mainly attributable to the time lag until the fertility transition starts in rural areas. This confirms the major importance of the spatial diffusion of structural and ideational changes for national fertility decline. In Asia, the overall limited rural-urban gradient can be related to the fastest inter-sectoral diffusion of the onset of the fertility transition. By contrast, the rural transition onset lagged significantly behind the urban fertility decline in the MENA, LACarr and, especially, in SSA. This explains the stronger increase in rural excess fertility in these regions. In addition, the speed of fertility decline was less pronounced in rural than in urban areas of SSA–as opposed to other developing regions in which the two lagged transitions evolved at a similar pace. This peculiarity explains why rural excess fertility has not yet declined in SSA. The regional comparison is robust as we controlled for international variation in the demographic context and for intra-regional differences in the definition of urban/rural areas. Although we did not account for the inter-regional variations in the definitions, this is unlikely to challenge the main conclusions of this study. The rural-urban fertility gradient was limited in Asia, where urban environments are best identified. By contrast, the gradient in LACarr and, especially, SSA may be underestimated because officially designated urban areas tend to include agricultural communities.The descriptive approach of this study does not allow us to identify explanations for the atypical fertility trends by type of residence in SSA and Asia. But our contextual knowledge enables us to speculate about potential causes. Even though the low level of urbanization in Asia would imply a slower urban-to-rural diffusion of behavioral change, authoritarian family planning policies are probably key to understanding the near parallel decline in fertility by type of residence. Alternatively, the limited rural-urban fertility gradients may indicate a loose relationship between the transformations in the socioeconomic structures of urban and rural populations and their respective levels of fertility, especially in the formerly communist countries of the region. While the fertility transition was fast, the urbanization process was restrained by communist governments . Future The reasons for the late and slow diffusion of fertility decline from SSA’s cities to the countryside remain an open question with important demographic consequences. The region clearly cumulates structural characteristics that potentially impede the spread of fertility change across space: a low level of urbanization, a limited integration of the agricultural sector in the national development process and a weak commitment of governments to improve the provision of family planning in remote areas. Hence the rural fertility transition appears to be disconnected from that in urban areas. Even though education expanded in rural areas, its impact on fertility decline was also less important when compared to urban areas . Future The results of this study help to better understand the variations in the fertility transitions across developing regions. The fast fertility decline in LACarr and the MENA can be related, first, to the dominance of cities in the population geography when urban fertility started to fall. Second, the minority population remaining in rural areas caught up the decline at a similarly fast pace. Given the high level of urbanization in these regions, future national-level trends will be mainly determined by the fertility dynamics in urban areas. By contrast, the synchronized decline in urban and rural fertility in Asia explains why this region experienced the fastest fertility transition in worldwide comparison–despite a low level of urbanization. Given a persistent residual level of rural excess fertility, further progress in urbanization will significantly shape prospective fertility trends in the region.In SSA, the national-level trends of slow fertility decline in conjunction with rampant socio-economic development , 22 actuThe results also have implications for the future geography of urban growth. Given that fertility has declined continuously among women socialized in cities, migration eventually dominates the urban growth process through both its direct and its indirect demographic contributions. Urban growth can therefore be expected to be concentrated in Asia and SSA, where the pool of potential in-migrants remains large due to the low levels of urbanization. In SSA, the future urban-to-rural diffusion of fertility decline will be crucial to limiting demographic pressure for rural-to-urban migration and, consequently, the acute strains on urban infrastructure and labour markets, as well as the resulting pressure for secondary migrations to foreign countries.S1 File(DOCX)Click here for additional data file.S2 File(DTA)Click here for additional data file.


L and Mcl-1 have been identified to play a pivotal role in apoptosis resistance in ovarian cancer and constitute key targets for innovative therapeutic strategies. Although BH3-mimetics (i.e. ABT-737) potently inhibit Bcl-xL activity, targeting Mcl-1 remains a hurdle to the success of these strategies. Calcium signaling is profoundly remodeled during carcinogenesis and was reported to activate the signaling pathway controlling Mcl-1 expression. In this context, we investigated the effect of carboxyamidotriazole (CAI), a calcium channel inhibitor used in clinical trials, on Mcl-1 expression. CAI had an anti-proliferative effect on ovarian carcinoma cell lines and strongly down-regulated Mcl-1 expression. It inhibited store-operated calcium entry (SOCE) and Mcl-1 translation through mTORC1 deactivation. Moreover, it sensitized ovarian carcinoma cells to anti-Bcl-xL strategies as their combination elicited massive apoptosis. Its effect on mTORC1 and Mcl-1 was mimicked by the potent SOCE inhibitor, YM58483, which also triggered apoptosis when combined with ABT-737. As a whole, this study suggests that CAI sensitizes to anti-Bcl-xL strategies via its action on Mcl-1 translation and that modulation of SOCE could extend the therapeutic arsenal for treatment of ovarian carcinoma.The anti-apoptotic proteins Bcl-x L were able to cooperate to allow ovarian carcinoma cells to overcome apoptosis, as their concomitant inhibition was sufficient to induce cell death i reflecting calcium release from RE via the IP3 receptor [2 to the extracellular medium triggered a sustained increase in [Ca2+]i from baseline , which is consistent with a characteristic SOCE-mediated Ca2+ influx from the extracellular solution  or 48h and 72h (OVCAR3 and SKOV3) did not inhibit Mcl-1 expression at mRNA level Figure , suggestTo investigate whether the Mcl-1 decrease upon CAI treatment was a consequence of proteasomal degradation, we incubated ovarian carcinoma cells with CAI for 48h (IGROV1-R10) or 72h (OVCAR3 and SKOV3) but supplemented the medium with bortezomib, a proteasome inhibitor, for the last 24 hours before analysis. As depicted in Figure Therefore, we assessed the impact of CAI on the Akt/mTOR pathway, which is known to control Mcl-1 translation. Treatment with CAI dose-dependently inhibited mTORC1 activation in the three cell lines, as reflected by a decrease in P-mTORC1(Ser2448), P-4E-BP1 and P-p70S6K Figure . In agreWe then investigated the molecular link between SOCE inhibition and the decrease in mTORC1 phosphorylation. For this purpose, we evaluated the involvement of CamKII, a calcium/calmodulin-stimulated serine/threonine protein kinase acting as one of the major calcium sensors in the cell. CamKII was described to modulate several cellular processes such as carcinogenesis by controlling cancer cell survival including mTOR activation , 56. PhoMoreover, mTORC1 is known to be negatively controlled by phosphorylated AMP-activated protein kinase (p-AMPK) . As CAI 2+ re-addition” protocol was used in the three cell lines pre-treated or not with YM53483. YM53483 did not inhibit calcium release triggered by thapsigargin in Ca2+ depleted medium but, as expected, it strongly reduced calcium entry via SOCE upon calcium reintroduction  or 48h (OVCAR3 and SKOV3 cells). Then the medium was supplemented with 1μM ABT-737 for the last 24h of treatment. As illustrated in Figure L triggered massive apoptosis in the three cell lines, as suggested by caspase 3 and PARP cleavages  and the other to the cell plasma membrane (ORAI-1). Upon ER calcium depletion, these subunits interact with each other leading to a calcium influx from extracellular compartments in order to refill cytoplasm and ER stocks subsequently. Our finding is supported by other studies showing that CAI inhibits SOC in HEK293 , colon cCAI-induced Mcl-1 inhibition did not require transcriptional events and its effect was not reversed by proteasomal inhibition, implying that post-translational events were not involved in the Mcl-1 decrease. The Mcl-1 down-regulation we evidenced may be attributed to translational events, as CAI strongly repressed P-4E-BP1, which is known to control Mcl-1 protein expression , 12. ThiCalcium can control the PI3K-AKT-mTOR pathway at several levels”. Indeed, calcium/calmodulin(CaM) complex has been shown to activate P-Akt(Ser473), and subsequently mTORC1, through CaMKII activation in B lymphocytes . ConsistOur results strongly suggest that SOCE inhibition by CAI impaired PI3K/Akt/mTOR by inactivating mTORC1, as evidenced by the potent inhibition of mTORC1(Ser2448), p70S6K and 4E-BP1 protein phosphorylation. This effect was confirmed by the appearance of a transient upregulation of P-Akt(308) triggered by a compensatory feedback upon mTORC1 inhibition . It is nThe hypothesis that calcium could play an important role in the regulation of mTOR signaling was evidenced by the need for calcium to activate p70S6K –26. TherTo consolidate our findings with CAI, we then tested the effect of YM-58483, a well-known SOC inhibitor, on SOC channels and Akt/mTOR activation in ovarian carcinoma cells. As expected, YM58483 strongly down-regulated SOCE but had no impact on calcium release from IP3R, thereby confirming its action as an SOCE inhibitor. Moreover, its effect on the PI3K/Akt/mTOR pathway mimicked that obtained with CAI. YM85483 potently inhibited mTORC1 activation, leading to compensatory feedback loops on Akt and to inhibition of the mTORC1 downstream targets P-p70S6K and P-4E-BP1. Finally, YM-58483 also drastically inhibited Mcl-1 expression, so it is highly likely that Mcl-1 is a target for SOCE inhibitors. The link between SOCE and the expression of Bcl-2 family members had already been pointed out by a few studies but led to divergent conclusions. On the one hand, a constitutively active mutated STIM-1 led to induction of Mcl-1 transcription in HEK293; on the other hand, ORAI1 knock-down promoted Mcl-1 and inhibited Noxa expressions leading to survival of activated murine T cells , 72. TheSOC play a crucial role in tumor development and accumulating evidence suggests that their activation is involved in cancer progression. First, they are differentially expressed in malignant cells and STIM1 and ORAI1 were found to be more strongly expressed in hepatoma tissues than in pre-cancerous tissues or non-tumoral tissues from the same patients , 74. SecL strategies in the three cell lines tested. Because our experimental model is dependent on Mcl-1 and Bcl-xL to survive, the massive cell death we observed with SOC inhibitors and an inhibitor of Bcl-xL strongly suggests that Mcl-1 expression is regulated by SOC channels. Moreover, the anti-tumoral effect of this combination could explain the mild efficacy of CAI when it was used as a single agent in clinical trials, as the sole inhibition of Mcl-1 is not sufficient to induce apoptosis in ovarian cancer i) and was acquired every 5 seconds with a digital CMOS camera . The monochromator and the photometers allow emission and detection of fluorescence from ~12 cells in the field of view. Data analysis was performed using MM fluor software .Intracellular calcium concentration variations were analyzed by microspectrofluorimetry using the Caeviously . Brieflyt-test was used for statistical analysis of densitometry graphs and Wilcoxon test was used for analysis of percentage of viable cells. Differences were considered statistically different if p<0.05 (*); p<0.01 (**); p<0.001 (***).The values are presented as means ± S.E.M. for at least three independent experiments. Student's


Kidney development involves formation of nephrons intricately aligned with the vasculature and connected to a branched network of collecting ducts. Notch signaling plays multiple roles during kidney development involving the formation of nephrons composed of diverse epithelial cell types arranged into tubular segments, all the while maintaining a nephron progenitor niche. Here, we review the roles of Notch signaling identified from rodent kidney development and injury studies, while discussing human kidney diseases associated with aberrant Notch signaling. We also review Notch signaling requirement in maintenance of mature kidney epithelial cell states and speculate that Notch activity regulation mediates certain renal physiologic adaptations. The Notch signaling pathway is an evolutionarily conserved signaling pathway that has critical functions in embryonic development, tissue homeostasis, and disease. Unique features of the pathway include ligand and receptor expression in neighboring cells and a series of proteolytic cleavages of the receptor that lead to the expression of Notch target genes. After exit from the endoplasmic reticulum, the Notch receptors undergo processing in the Golgi where they are cleaved by furin-like convertases, resulting in a bipartite receptor that translocates to the plasma membrane . Notch rIn mammals, there are four Notch receptors (Notch1–Notch4) and five ligands [Delta-like  and Jagged (Jag1 and Jag2)]. These basic components, along with their downstream mediators orchestrate gene regulation in almost all tissues of multicellular organisms. Research conducted for over a century has contributed to our understanding of the mechanism of Notch regulated gene expression in a context dependent manner  signals from the metanephric mesenchyme to trigger ureteric budding from an epithelial tubular structure referred to as the Wolffian duct. The tip cells of the ureteric bud (UB) express high levels of Ret and GFRα1 receptors that are activated by GDNF , and facNephron formation occurs through a series of morphological changes that transform groups of cap mesenchymal cells that periodically exit the progenitor pool, into a tubular structure referred to as a nephron. Each nephron arises from an aggregate of cap mesenchymal cells, referred to as a pre-tubular aggregate (PTA) that forms below the UB tip. Mesenchymal aggregates epithelialize to form renal vesicles (RV) ,28, whicThe Notch pathway receptors and ligands have unique expression patterns within the developing kidney  35,38].,38.35,38RBPJ, or Notch1 and Notch2) sustain Six2 expression within the developing mouse kidney -S-phenylglycine t-butyl ester (DAPT) to inhibit Notch signaling  compartment of the RV closer to the UB occurs in Pax3-Cre;Notch2f/f kidneys just like in wild-type kidneys. However, the segment specific expression of Lhx1 is not maintained in the absence of Notch2 and the establishment of distinct nephron segments is defective from the RV stage onwards. Interestingly, Six2-Cre which drives Cre expression in the NPCs also reveals the requirement of Notch signaling in the establishment of the proximodistal axis within the RV and hence nephron segmentation, but only when both Notch1 and Notch2 are inactivated . Th. ThNotchelopment . A role d bodies  [42]. Th alleles .Notch1 and Notch2 along with a Rosa+/EYFP Cre reporter also results in defective nephrogenesis [Notch1 and Notch2 inactivation showed a deficiency in the expression of mature markers  of all nephron segments. The delayed inactivation of Notch1 and Notch2 using the Wnt4-Cre allowed for the initial segmentation of the RV along proximodistal axis but reduced the expression of these early stage nephron segment specific markers Lhx1 at later stages resulting in the failure to form normal SSBs and nephrons [Lhx1 in the distal RV compartment and its requirement for Dll1 expression in distal compartment of the RV is suggestive that both Notch signaling and Lhx1 are critical for development of distinct cell types within the RV [Interestingly, delaying Cre expression to the pre-tubular aggregate stage just prior to RV formation, using the Wnt4-Cre to inactivate both ogenesis . Mutant nephrons . Downregnephrons . The expn the RV .Wnt4-Cre;Notch1f/f;Notch2f/f kidneys clearly implicates a critical requirement for Notch signaling subsequent to suppressing Six2 expression and allowing NPCs to exit the progenitor state. This later function involves the proper conversion of RVs to SSBs. At the cellular level this conversion step requires the sufficient proliferation of Jag1+ cells within the early stage nephrogenic bodies [Lhx1 and Hnf1b [The failure to form SSBs in the c bodies , and at nd Hnf1b .Notch1 alone or Notch2 alone within Six2 expressing cells allows for normal nephron formation but results in proximal tubular cysts  yields a variety of phenotypes, including hypoplastic kidneys, tubular cysts, and unilateral aplasia [In addition to patterning cell types within the collecting duct, altering Notch signaling affects ureteric bud branching. Overexpression of  aplasia . All the aplasia . HoweverWhile the role of Notch signaling in kidney development is well established (as reviewed above), the requirement for ongoing Notch signaling in the adult kidney is only beginning to be unraveled. Knowing the adult kidney functions of Notch signaling is important as therapies that target Notch signaling have been considered for treating various diseases including end stage kidney disease . Notch sNotch signaling is essential for podocyte development , but theDiseases such as focal segmental glomerulosclerosis (FSGS) and diabetic nephropathy are caused by podocyte loss and dysfunction. In human biopsy samples and rodent models of type 1 and type 2 diabetes and of albuminuria and FSGS, Notch signaling is re-activated in the podocytes . This stAdditional studies support a role for injury induced increase in Notch signaling in adult kidney tubules promoting tubulointerstitial fibrosis (TIF). Injury induced by either Folic acid injection or unilateral ureteral obstruction (UUO) increased transcripts of some genes of the Notch signaling pathway. Pretreatment of mice with the gamma secretase inhibitor DBZ before initial insult with either folic acid or UUO reduced TIF progression. Further evidence of the involvement or requirement of Notch signaling came from reduction of TIF in tubular epithelial cell-specific knockout of RBPj, and from induction of ICN1 by a doxycycline inducible model that led to TIF. Four-week-old mice administered doxycycline in food for four weeks had an increase in Notch pathway genes and showed signs of fibrosis . Going fPpargc1a as a direct target of Hes1. A more recent study [Tfam) to be both differentially expressed in NICD1-overexpressing kidney tubules and a direct transcriptional target of RBPj. Similar to PGC-1α, Tfam was also down-regulated in NICD1-overexpressing tubules and in multiple models of kidney fibrosis. Tubule-specific knockout of Tfam led to altered mitochondrial structure and increased expression of profibrotic factors. In cultured renal epithelial cells, higher Notch signaling led to decrease in fatty acid oxidation, and this effect could be reversed by increasing Tfam levels. Taken together, the findings of these two studies indicate a role of Notch in perturbing metabolic pathways associated with cellular energy production to promote fibrosis.The downstream targets of Notch signaling in promoting kidney fibrosis are largely unknown. Recently, Han et al.  reportednt study  identifiAcute kidney injury (AKI) is triggered by a brief duration of injury that rapidly, within 1–2 days, results in loss of kidney function and decrease of urine output. AKI can be induced by a host of factors, including but not limited to surgery-related injuries, and other physiological events such trauma, stress, infection, and drug toxicity . One in de novo Sox9+ expression in response to injury. The Susztak group showed that these highly proliferative Sox9+ cells do not express any transcription factors commonly seen in the developing kidney, such as Lgr5, Pax2, and Six2, suggesting their progenitor nature. In vivo lineage tracing showed these Sox9-positive cells could give rise to all epithelia of the kidney except collecting duct and glomeruli. This cell population showed increased proliferation in the adult kidney after injury by either folic acid or IRI. Eventually, these cells led to tubule generation in different segments of the kidney [While many studies suggest a pathological role for increased Notch signaling following AKI as discussed above, recent studies reveal beneficial roles for Notch signaling activation following AKI. Following AKI-induced tubular epithelial damage, repair mechanisms are initiated to restore nephron structure and function. The repair is mediated by the neighboring less damaged tubular epithelial cells , but thee kidney . The expe kidney  and invoe kidney  make Note kidney . More ree kidney .The collecting duct segments of the kidney consist of two broad cell types, the principal cells (PCs) and the intercalated cells (ICs) intermingled with each other. These two cell types arise from a common progenitor, and proper functioning of the collecting duct system requires the relative ratio of these cell types be maintained throughout the tubule. These cell types are characterized by the presence of specific marker proteins. Differentiation of the ICs require the presence of the forkhead transcription factor Foxi1 , and sevOther studies point to a role for increased Notch signaling activity in the epithelial cells of the distal nephron segments to remodel the cell types in response to inactivation of the sodium chloride cotransporter (NCC). The distal nephron remodeling process is observed to involve induction of Jagged1/NOTCH signaling, and increased Hes1 levels. This then results in an increase in PCs within the cortical connecting tubule and replacement of acid-secreting alpha-ICs with pendrin expressing-ICs . Hence, Several developmentally important signaling pathways, such as Wnt signaling and hedgehog signaling are re-activated after injury (reviewed in ). Similade novo in genes coding for Notch signaling pathway components are associated with renal diseases  is an autosomal dominant disorder that can affect multiple organs of the body including the liver, heart, skeleton, eyes, and kidneys. ALGS is caused by heterozygous mutations in function ,101. Howfunction . Due to function . Accordifunction . To dateescribed . The majed [JAG1  and anotCH2 gene ,91. The JAG1. The ALGS associated renal anomalies include multicystic dysplastic kidneys, renal tubular acidosis, vesicoureteral reflux, obstruction, chronic renal failure, end-stage renal disease, acute kidney injury, renal lipidosis, renal artery stenosis, focal segmental glomerulosclerosis, duplex collecting system, and other renal diseases in a minor proportion [Renal anomalies are found in around 40% of the ALGS patients with mutations in oportion ,94. Curroportion , althougNOTCH2 gene, that results in the truncation of NOTCH2 protein and the absence of the c-terminal pest domain, which contains sequences necessary for the ubiquitinylation and degradation of NOTCH2 following association of the intracellular domain with RBPJ and Mastermind. The absence of the PEST domain is predicted to increase the half-life of NOTCH2 resulting in gain-of-function effects [Hajdu-Cheney syndrome  is a rare genetic disorder with prominent features including bone abnormalities and to a lesser extent renal and cardiac abnormalities among others . The dis effects . So far  effects ,107, and effects ,107. Sim effects . Brennan effects . Conside effects .JAG1 or NOTCH2 are associated with CAKUT. What is perhaps surprising is that mutations in other Notch pathways genes have so far not been associated with CAKUT. However, the genetic basis of CAKUT in most patients remains to be determined.Congenital anomalies of the kidney and urinary tract (CAKUT) includes a spectrum of anomalies affecting the kidney, the urinary tract, or both . CAKUT oDiabetic nephropathy is also called diabetic kidney disease (DKD), and is the leading cause of end-stage kidney disease and occurs in approximately 40% of patients who are diabetic. Diabetic nephropathy occurs due to hyperglycemia observed in diabetic patients, although the symptoms are quite variable between type I versus type II diabetes . Among tRenal carcinoma is one of the most commonly occurring cancers with nearly 270,000 cases diagnosed yearly and accounting for 116,000 deaths annually . Notch sRenal cell carcinoma can be further divided into at least three subtypes: Clear cell renal cell carcinoma, papillary renal cell carcinoma, and chromophobe renal cell carcinoma. Some of Notch signaling components are uniquely associated with each of these subtypes based on mRNASeq data of the patient samples . Low expNOTCH2 mutation may have no renal defects, or congenital cystic kidney disease or late adult onset kidney disease. This is similar to mouse lines with a sensitized level of Notch signaling during kidney development some of which develop small kidneys while others develop normal sized kidneys. The sensitized level of Notch signaling likely allows for normally insignificant genetic variations, or dietary changes, or environmental factors to affect the severity of the kidney disease. For example, normally a null mutation in one allele of Notch1 does not cause kidney disease in mice, however when we combine this sensitized level of Notch signaling with the conditional inactivation of Notch2 in the developing kidney nephron progenitors this results in development of multicystic hypoplastic kidneys. These mouse studies suggest that additional variations in the Notch signaling pathway genes could be present in some ALGS patients with severe kidney disease in addition to a mutation in JAG1 or NOTCH2. Additional genetic modifiers may also be present in genes involved in unique functions that Notch signaling mediates in the kidney of ALGS patients with severe kidney disease. The role of Notch signaling in the maintenance of adult kidney cell types, or in the distal tubule cell type remodeling to adapt to chronic dietary changes, or in renal epithelial repair following injury is suggestive that a secondary event in adults with reduced Notch signaling could trigger late onset of kidney disease in some ALGS patients.Mouse studies have revealed many functions for Notch signaling in kidney development, in the maintenance of adult kidney cell types and in the response to kidney injury. The myriad functions of Notch signaling may in part explain the high degree of variability in the onset and severity of kidney disease observed among individuals with the same genetic defect in the Notch signaling pathway. ALGS patients with the same


Osteoarthritis (OA) is the most common joint disease worldwide and a leading cause of disability. Characterized by degradation of articular cartilage, synovial inflammation, and changes in periarticular and subchondral bone, OA can negatively impact an individual’s physical and mental well-being. Recent studies have reported several critical signaling pathways as key regulators and activators of cellular and molecular processes during OA development. Wnt signaling is one such pathway whose signaling molecules and regulators were shown to be abnormally activated or suppressed. As such, agonists and antagonists of those molecules are potential candidates for OA treatment. Notably, a recent phase I clinical trial (NCT02095548) demonstrated the potential of SM04690, a small-molecule inhibitor of the Wnt signaling pathway, as a disease-modifying oseoarthritis drug (DMOAD). This review summarizes the role and mechanism of Wnt signaling and related molecules in regulating OA progression, with a view to accelerating the translation of such evidence into the development of strategies for OA treatment, particularly with respect to potential applications of molecules targeting the Wnt signaling pathway. Osteoarthritis (OA) is a degenerative joint disease typically characterized by articular cartilage degeneration, abnormal bone remodeling with osteophyte formation and subchondral bone sclerosis, and fibrosis and hyperplasia of the synovial membrane . GloballWnt is an extracellularly secreted glycoprotein whose signaling involves 19 Wnt genes and various Wnt receptors that regulate canonical β-catenin-dependent and non-canonical β-catenin-independent signaling pathways. Both downstream pathways are associated with numerous biological processes such as cell proliferation, differentiation, polarization, and fate determination during embryogenesis and late stages of development ; as wellWith regard to the canonical β-catenin-dependent Wnt signaling pathway, in the absence of Wnt proteins, β-catenin is degraded n the cytoplasm by the enzyme glycogen synthase kinase 3β (GSK3β) in a “destruction complex” that includes Axin1/Axin2, adenomatous polyposis coli (APC), Dishevelled (Dvl), and casein kinase 1(CK1) in a phosphorylation-dependent manner , 5. HoweProtein levels of Wnt3a and β-catenin were increased, and collagen II was reduced in rat models of normal exercise-induced OA and injured exercise-induced OA groups . FurtherIn view of previous studies, moderate WNT activity is essential for chondrocyte proliferation and maintenance of their typical characteristics . HoweverWnt1-inducible-signaling pathway protein 1 (WISP1), which positively controls canonical Wnt signaling and aggravates OA pathology , is a feWnt inhibitory factor 1 (Wif-1) blocks Wnt3a-dependent activation of the canonical Wnt signaling pathway in chondrogenic cells . MoreoveFibulin-4, an extracellular matrix (ECM) protein reported to be abnormally elevated in human OA chondrocytes , augmentVarious members of the Wnt signaling pathway are overexpressed in the synovium during experimental OA . Indeed,Abnormal remodeling of subchondral bone and osteophyte formation are hallmarks of OA progression . In miceBased on the data presented above, we propose that excessively activated canonical Wnt signaling in cartilage, synovium, and subchondral bone plays a critical role in OA development, in both an independent and interacting manner. Accordingly, the Wnt signaling-mediated network that functionally regulates chondrocytes, synovial cells, and osteoblasts/osteoclasts should be highlighted, as the mechanisms underlying these events remain unclear. The degree to which these events share similarities or differ with regard to signaling is not yet fully resolved, nor are interactions between different cell populations Fig. .Fig. 1T2+ and planar cell polarity (PCP) pathways. Through the activation of calcium signaling by phospholipase C/protein kinase C (PKC)/Ca2+ and calmodulin-sensitive protein kinase II (CaMKII), the Wnt/Ca2+/CaMKII pathway activates the transcription factor nuclear factor associated with T cells (NFAT) to regulate cytoskeletal rearrangements, cell adhesion, and migration. In the PCP pathway, Wnt binds to Fzd receptors, which activates Dvl to trigger Rho/Rho-associated kinase and Rac/c-Jun N-terminal kinase (JNK) signaling, and actin polymerization in stimulated cells. These complex signaling events are integrated to mediate cytoskeletal changes, cell polarization, and motility during gastrulation. Moreover, recent evidence supports the involvement of Wnt5a in inflammatory responses, innate immunity, [Key molecules and cascades in the non-canonical Wnt signaling pathway have been previously summarized , 36. Brimmunity, , 38 and −/− and Col2a1-Wnt5b mice, but decreased expression in Col2a1-Wnt5a transgenic mice; therefore, it is possible that Wnt5a promotes chondrocyte hypertrophy in part by decreasing the transcriptional activity of Sox9 [Expressions of Wnt5a in articular cartilage has been positively correlated to progressive damage of knee OA joints . In addi of Sox9 .An in vitro study found that Wnt5a reduced ACAN while promoting MMP1 and MMP13 expression via activating β-catenin independent signaling including JNK and CaMKII in human OA cartilage . ConditiExosomes, small extracellular microvesicles of endosomal origin, are gaining increasing recognition for their important roles in mediating cell-cell communication . ExosomaTaken together, activation of the non-canonical Wnt signaling pathway, predominantly Wnt5a in view of recent data, likely enhances cartilage degradation by stimulating catabolic metabolism of cartilage by upregulating MMP expression and decreasing collagen type II production. Mechanisms underlying this process involve interactions between Wnt5a/CaMKII and key molecules from signaling pathways including JNK, c-Myc, cyclin D1 and Sox9.With regard to the synovium, Lambert et al. compared the gene expression patterns of synovial cells from inflamed and normal/reactive areas of synovial membrane obtained from the same OA patient; 896 differentially expressed genes were identified, of which Wnt5a and LRP5 were upregulated . In isol−/− mice showed impaired mineralization even treated with BMP2 [Cxcl12 and Rankl gene expression induced by JNK and Ca2+/NFAT signaling pathways led to activation of osteoclast differentiation and enhanced subchondral bone turnover [As the dominant cells in bone remodelling, osteoblasts and osteoclasts are functionally regulated by Wnt5a . Osteoblith BMP2 . These fith BMP2 . Osteoblith BMP2 . Increasturnover .Collectively, these data indicate Wnt5a signaling and its downstream cascades may lead to an abnormal balance between osteoblasts and osteoclasts, which can increase subchondral bone remodeling and participate in excessive mineralization or even osteophyte formation. Cell-specific modulation of Wnt5a expression and its receptor in osteoblasts or osteoclasts would provide direct evidence to elucidate the precise role of this signaling pathway in OA Fig. .Fig. 2TWith respect to the mechanisms by which the Wnt signaling pathway participates in OA development, a series in-depth studies indicated that the pathogenesis of OA is, at least in part, the result of interactions between Wnt and multiple signaling pathways. In normal chondrocytes, the network formed by signaling pathways such as Wnt, BMP, Hedgehog, etc. are needed to maintain their normal phenotype . Based o2+-dependent membrane-binding proteins) interacted with p65 resulted in increased nuclear translocation and retention of the active p50/p65 NF-kB complex, whereas plasma membrane-associated AnxA6 interfered with the membrane-association of the Wnt signalosome complex required for the activation of Wnt/β-catenin signaling in human cartilage degradation during OA pathology [NF-κB signaling is important for several biological and pathological processes , such asathology . Similarathology . In addiathology . This inathology . Thus, sCrosstalk between Wnt and BMP pathways not only participates in chondrocyte hypertrophy and matrix degradation, but also stimulates bone formation via chondrogenic or osteogenic differentiation. In vitro, BMP2-induced Wnt/β-catenin signaling pathway activation through increased β-catenin nuclear translocation and LRP-5 expression and that the BMP-2-induced LRP-5 upregulation is mediated through Smad1/5/8 binding on LRP-5 promoter, resulted in MMPs and ADAMTS-5 expression, and hypertrophic maturation of chondrocytes by stimulating collagen X expression . In addiNemoto et al. demonstrated an association between BMP2-mediated osteoblastic differentiation and increased Wnt5a and Ror2 expression in vivo and in vitro, and silencing gene expression of Wnt5a and Ror2 resulted in suppression of BMP2-induced expression of ALP and osteocalcin (OCN), suggesting that Wnt5a and Ror2 signaling form a substantial component of BMP2-mediated osteoblastic differentiation in a Smad independent pathway . ComparePrevious studies suggest that the TGF-β/Smad pathway plays a critical role in regulation of articular chondrocyte hypertrophy and maturation during OA development –66. OverInterestingly, Wnt/β-catenin signaling regulates initiation of chondrocyte hypertrophy by antagonizing PTHrP signaling, whereas it acts independently of PTHrP signaling to control the final maturation of hypertrophic chondrocytes . Ma et aRockel et al. reported that in adult chondrocytes, activated hedgehog signal induction expression of dominant negative equivalent TCF7L2 (dnTCF7L2) isoforms, and that increased expression of TCF7L2 protein isoforms limited signaling by β-catenin, resulting in an inhibition of expression of FGF18, leading to cartilage degeneration via induction of expression ADAMTS4 and MMP13, which are involved in cartilage degeneration as part of OA . TherefoNotch is actively involved in various life processes including osteogenesis, and that Hes1, an essential mediator of Notch signaling, generally mediates Notch signaling by repressing expression of target genes , 73. CaMchon mice with OA that were intra-articularly injected with PKF118–310, an inhibitor of the TCF4/β-catenin interaction, exhibited reduced cartilage degradation and MMP13 expression [HIF1α is a crucial hypoxic factor for chondrocyte growth and survival during development . HIF1α ipression .The Hippo/YAP signaling pathway is important for mediating organ size and tissue homeostasis, and inhibition of YAP using YAP siRNA is a promising way to prevent cartilage degration in OA . Wnt5a aAn in vitro study found that Wnt5a reduced ACAN while promoting MMP1 and MMP13 expression via activated β-catenin independent signaling including p38, extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K) in human OA cartilage .Management of the pathogenesis of OA has become central for treatment and alleviation of related clinical symptoms. Osteoarthritis Research Society International recommendations cover the use of non-pharmacological , pharmacological modalities  and surgical modalities  . AlthougIn recent years, pluripotent stem cells and regenerative medicine strategies have been considered promising to repair cartilage damage in OA. However, their long-term effects remain uncertain based on limited clinical data. In view of the important role of Wnt signaling pathways and cascades molecules play in OA, they might be potential target for the treatment of OA.Here we summarize emerging therapies that target or act via Wnt signaling pathways, in terms of small molecule antagonists or agonists, herbs, enzymes, and tissue engineering and so on.SOX9 and ACAN gene expression, and decreased COL10A1 expression [First and most importantly, SM04690 was shown to elicit protective effects on cartilage during joint destruction in a preclinical model of knee OA . A furthpression .LRP5 plays an essential role in Wnt/β-catenin signaling mediated OA cartilage destruction by upregulating catabolic factors  and downregulating the anabolic factor type II collagen . These eLorecivivint inhibited CDC-like kinase 2-mediated (CLK2) phosphorylation of 61 serine/arginine-rich (SR) proteins and DYRK1A-mediated  phosphorylation of SIRT1 and FOXO1, suggested a novel mechanism for Wnt pathway inhibition, enhancing chondrogenesis, inhibited expression of cartilage catabolic enzymes, and anti-inflammation . It is aPsoralea corylifolia L.), reportedly promotes chondrocyte proliferation by activating the Wnt/β-catenin signaling pathway, and may play an important role in OA treatment [Psoralen, the active ingredient of Fructus Psoraleae (dried ripe fruit of reatment . Additioreatment  and berbreatment  were shoArtemisinin (ART) inhibition of OA progression and cartilage degradation through upregulation of FRZB in IL-1β-induce chondrocytes and downregulation of the expression of β-catenin, which suggests that it may act as a Wnt/β-catenin antagonist to reduce the release of inflammatory mediators and enhance cell proliferation, glycosaminoglycan deposition, and prevent cartilage apoptosis and degeneration .Ma et al. reported that knockdown of peroxiredoxin 5 (Prdx5) by RNA interference activated apoptosis of OA chondrocytes, which was mediated through decreased scavenging of endogenous reactive oxygen species and promotes the nuclear translocation of β-catenin, decreases GSK-3β activity, and enhances β-catenin/TCF-dependent transcription in osteoarthritic chondrocytes . TherefoKim et al. reported that the tri-butanoylated N-acetyl-D-galactosamine analog , a multifunctional carbohydrate-based drug candidate, improved cartilage tissue production in 3D cultures in vitro by inhibiting Wnt/β-catenin signaling , suggestPraxenthaler et al. shows a correlation of WNT/β-catenin activity with de- and re-differentiation and ECM deposition in human articular chondrocytes with time in 3D culture, thus establishing that the mechanical loading response of chondrocytes is modulated by WNT/β-catenin activity levels . TherefoMianserin suppresses R-spondin 2-induced activation of Wnt/β- catenin signaling in chondrocytes and prevents cartilage degradation in a rat model of osteoarthritis . FluoxetMiR-320c suppress the Wnt/β-catenin signaling pathway through the downregulation of β-catenin protein level in nucleus, inhibiting chondrogenic degeneration in osteoarthritis . OverexpAfter stimulation with IL 1β or fibronectin fragments, and blockage of Wnt signaling by DKK1, Selene et al. showed that ERK inhibition decreased Runx2 activation, ADAMTS 7, 12 expression and cartilage oligomeric matrix protein (COMP) degradation in OA synovial fibroblasts (SF) .Osteoclast activity plays a significant role in the interaction between articular cartilage and subchondral bone, and knee loading may suppress osteoclastogenesis through the Wnt signaling pathway to serve as a treatment for OA mice . TherefoBurt et al. reported that a fibroblast growth factor 23 (FGF23) neutralizing antibody was able to partly ameliorate OA abnormalities in subchondral bone and reduce degradative/hypertrophic chondrogenic marker expression in high-molecular-weight joints in vivo . Moreove2+ signaling in bone marrow-derived MSCs [Pulsed electromagnetic field (PEMF) treatment prevented subchondral trabecular bone loss and preserved subchondral trabecular microarchitecture in a low-dose monoiodoacetate rat model, at least partially via Wnt/β-catenin signaling . In addived MSCs .Dkk1 competitively binds to Wnt co-receptors LRP5/6 to inhibit Wnt signal transduction , 105. SeThe pathogenesis of OA is the result of multiple interacting factors. Although the mechanism is unclear, Wnt signaling has been shown to contribute to whole joint disease. Recent studies have demonstrated interactions between multiple signaling pathways in OA, including canonical and non-canonical Wnt, Hedgehog, TGF-β, and NF-ĸB signaling pathways, which represent potential targets for the treatment of OA.Studies have shown that palmatine  may delay the progression of OA, as determined by assessments of articular cartilage in a rabbit OA model and cultured rabbit chondrocytes stimulated with IL-1β , 110, poSnelling et al. provided evidence that Dkk3, a non-canonical member of the Dkk family of Wnt antagonists, was upregulated in OA, whereby it mediated protective effects on cartilage partially through upregulation of TGF-β signaling and inhibition of Wnt signaling . Thus, DLigustrum lucidum [SPN is an extracted agent of the fruit of  lucidum . Ma et a lucidum . Similar lucidum .The anti-osteoarthritic effects of emodin by inhibiting the expression of MMPS and ADAMTS via the suppression of IκB-α degradation, the down-regulation of IKK-β and NF-κB p65 levels, as well the reduction of Wnt/β-catenin activity by inhibiting β-catenin in-vitro and in-vivo . In vivoHou et al. reported that the cationic amphipathic peptide designated as p5RHH is capable of siRNA transfection without significant cytotoxicity at all tested doses . Yan et Both canonical and non-canonical Wnt signaling pathways are excessively activated during OA development. A series of studies have revealed that cell-specific modulation of Wnt or its downstream effectors can lead to or prevent aberrant changes in OA tissues. The underlying molecular mechanisms involve other signaling pathways such as Hedgehog, NF/κB, BMP/TGF-β/Smad, PTH, Notch and HIF1α, − more specifically, the network they construct. Further studies using various knockdown or knock-in strategies to test functional roles of these components are necessary to better understand the nature of these events. Although the details of cell-specific molecular events, especially the cascades by which Wnt signaling interacts with the other signaling pathways described above, remain to be elucidated, key points of the network composed by these interacting pathways are promising potential targets for OA treatment. Notably, a recent phase I clinical trial (NCT02095548) demonstrated that the Wnt signaling pathway inhibitor SM04690 has potential as a DMOAD The development of additional emerging genetic therapies or small molecules targeting the Wnt signaling pathway, though only currently supported by evidence from animal studies, should be encouraged. Moreover, in vivo experiments and clinical data are needed to clarify mechanisms of action and clinical value of the signaling networks affected by natural antioxidants Prdx5 and Traditional Chinese Medicines containing ART and psoralen, as they have been shown to delay the process of OA via Wnt signaling. In addition, we should develop a more in-depth understanding of the beneficial mechanisms of PEMF and ESWT physiotherapy to provide more powerful evidence for clinical treatment of OA. Moreover, new therapeutic strategies such as engineered cartilage, pluripotent stem cells, and regenerative technologies should be explored to seek more effective therapies for OA. However, before seeking new agents for OA treatment that target the Wnt signaling pathway, further study is needed to test the efficiency and safety of SM04690 and its derivates before they are prescribed as agents for OA treatment.


Neovascular age-related macular degeneration (nAMD) is one of the leading causes of blindness among the aging population. The current treatment options for nAMD include intravitreal injections of anti-vascular endothelial growth factor (anti-VEGF). However, standardized frequent administration of anti-VEGF injections only improves vision in approximately 30–40% of nAMD patients. Current therapies targeting nAMD pose a significant risk of retinal fibrosis and geographic atrophy (GA) development in nAMD patients. A need exists to develop new therapies to treat nAMD with effective and long-term anti-angiogenic effects. Recent research on nAMD has identified novel therapeutic targets and angiogenic signaling mechanisms involved in its pathogenesis. For example, tissue factor, human intravenous immune globulin, interferon-β signaling, cyclooxygenase-2 (COX-2) and cytochrome P450 monooxygenase lipid metabolites have been identified as key players in the development of angiogenesis in AMD disease models. Furthermore, novel therapies such as NACHT, LRR and PYD domains containing protein 3 (NLRP3) inflammasome inhibition, inhibitors of integrins and tissue factor are currently being tested at the level of clinical trials to treat nAMD. The aim of this review is to discuss the scope for alternative therapies proposed as anti-VEGFs for the treatment of nAMD. Age related macular degeneration (AMD) is the most common cause of irreversible blindness among the elderly population ,2. CurreThe formation of neovascularization in AMD is a complex process involving multiple signaling pathways mediated by vascular endothelial growth factor (VEGF), platelet-derived growth factor, fibroblast growth factor, transforming growth factor, the Wnt pathway, the NLRP3 inflammasome, mitogen-activated protein kinase (MAPK) signaling, interleukins and chemokines ,7,8,9,10Injection of VEGF inhibitors into the vitreous is the current standard for nAMD treatment . HoweverPhotodynamic therapy (PDT) for nAMD involves intravenous injection of verteporfin, an approved FDA agent ,29. InjeCurrent treatment strategies for nAMD require repeated, frequent intravitreal injections . Long-teSemaphorins were initially discovered as molecules that contribute to the development of the embryonic nervous system . SemaphoTissue factor (TF) is a transmembrane receptor for plasma coagulation factor VII ,41. StudCytochrome P450 (CYP) is a class of enzymes that can synthesize fatty acid metabolites . CYP monDespite the reported protective role of the ω3, ω6-LCPUFAs , in reducing nAMD in animal models, results from the Age-Related Eye Disease Study 2 (AREDS2) a multi-center, randomized, double masked phase III human clinical trial, report conflicting results . AREDS2 Interleukin-33 (IL-33), a pro-inflammatory cytokine, is a member of the type-2 IL-1 family . Once acFlt23k intraceptors, (2) PLGA-biodegradable nanoparticles, and (3) the tripeptide adhesion motif Arg-Gly-Asp (RGD) .,99.98,99Cyclooxygenases (COXs) are a group of enzymes that are involved in inflammatory immune responses required for the conversion of arachidonic acid to prostaglandins . Out of CCR3  is a cell surface chemokine receptor that is expressed by eosinophils, Th2 cells, basophils, and mast cells . CCR3 exSeveral other pathways and novel agents that involve VEGF signaling were recently implicated in the pathogenesis of nAMD. Such mechanisms/agents include but are not limited to the complement pathway, BMP9/ALK1 signaling, erythropoietin signaling, long non-coding RNAs, STAT3 activation, neuropilin 1, platelet activating factor, mTOR signaling, and Yes-associated protein (YAP) inhibitors. Some of these pathways act in parallel , downstream (neuropilin 1) or upstream of VEGF signaling . Since the primary aim of this review is to focus on the pathways independent to VEGF, other signaling pathways that inhibit VEGF directly or indirectly with possible therapeutic benefits in nAMD are not discussed in detail, but are listed in form of table , brieflyThe current review highlights a broad range of non-VEGF pathway targets that offer potential new treatments for nAMD. Intravitreal injection of anti-VEGFs has been the standard treatment of nAMD for many years. Despite the success of anti-VEGFs, there is no improvement in vision for one-third of nAMD patients, and the long-term use of anti-VEGF therapy is associated with adverse events such as the development of GA and retinal fibrosis, to name a few. Therefore, a need exists to develop improved strategies that can reduce or eliminate ocular injections and improve clinical outcomes. Recent research has identified many molecular targets other than anti-VEGFs, as well as alternative drug delivery routes, which are currently being tested at the level of clinical trials, offering potential new avenues for treating nAMD. It could also be postulated that employing multiple targeted approaches to treat nAMD could yield better results than single pathway targeting, especially for simultaneous treatment of nAMD and subretinal fibrosis.


Total Nrf2 protein level was positively correlated with AHI and TSat90 and negatively correlated with minimum SaO2 (LSaO2), while nuclear Nrf2 protein and HO-1 protein were positively correlated with LSaO2. Moreover, significant positive correlations were found between maze scores and expression of nuclear Nrf2 protein, HO-1 protein, and SOD and Trx levels. Furthermore, inverse relationships between total Nrf2 protein in PBMCs and HVLT-R and maze scores were found. Multiple linear regression showed plasma Trx concentration as a potential predictor of maze and BVMT-R scores. In conclusion, the expression of Nrf2-ARE molecules and related antioxidases is significantly decreased in patients with OSAHS and is correlated with neurocognitive dysfunction. The Nrf2-ARE signaling pathway may play a crucial role in neurocognitive impairment in patients with moderate to severe OSAHS. Further studies are needed to explore the exact mechanisms and potential treatment interventions.The present study investigated the nuclear factor erythroid 2-related factor 2- (Nrf2-) antioxidant response element (ARE) signaling pathway in patients with moderate to severe obstructive sleep apnea-hypopnea syndrome (OSAHS). Their correlation with neurocognitive impairment metrics was investigated to explore potential pathogenesis in OSAHS. Forty-eight patients with OSAHS and 28 controls underwent testing with the Epworth Sleep Scale (ESS), MATRICS Consensus Cognitive Battery (MCCB), Stroop Color and Word Test, polysomnography (PSG), and measurements of the concentration of plasma superoxide dismutase (SOD) and thioredoxin (Trx). Further, 20 pairs of matched patients with OSAHS and controls were selected for measurement of the expression (protein and mRNA) of Nrf2 and of its downstream antioxidase, heme oxygenase-1 (HO-1), in peripheral mononuclear cells (PBMCs). Finally, correlations between neurocognitive impairment and the above metrics were analyzed. Expression of Nrf2 and HO-1 mRNA and protein in the PBMCs, as well as plasma SOD and Trx levels, were significantly reduced in patients with OSAHS. After adjusting for education, sex, age, and smoking index, the expression of Nrf2-ARE signaling pathway proteins (or mRNA) was closely correlated with sleep respiratory parameters. An inverse relationship was demonstrated between the expression of nuclear Nrf2 in PBMCs, concentration of plasma SOD and Trx, and apnea-hypopnea index (AHI) in patients with OSAHS. Trx, nuclear Nrf2 protein, and HO-1 protein were also negatively correlated with the percent of time that SaO Obstructive sleep apnea-hypopnea syndrome (OSAHS) is characterized by nocturnal intermittent hypoxia/reoxygenation and sleep disturbances and is caused by repeated upper respiratory tract collapse during sleep. OSAHS has a roughly 22% prevalence in men and 17% in women, and this is higher in obese people and the elderly . FurtherForty-eight patients with moderate to severe OSAHS diagnosed using polysomnography (PSG) in the Second Xiangya Hospital of Central South University from September 2015 to March 2017 were included (41 men). Among them, 15 cases were moderate OSAHS and 33 were severe OSAHS. The control group consisted of 28 healthy volunteers (20 men) with no statistical differences from the OSAHS group in age, sex, body mass index (BMI), or years of education. The basic clinical characteristics of these two groups are listed in 2; (3) history of brain, kidney, liver, blood, endocrine, or reproductive system diseases, malignant tumors, coronary heart disease, and so on; (4) current pregnancy; (5) use of hormones, hypnotics, sedatives, psychotropic substances, or tobacco; (6) infection, trauma, or major surgery in the previous 2 weeks; (7) history of any other respiratory diseases; (8) color blindness or color weakness; (9) previously received a similar or the same neuropsychological assessment; and (10) history of mental illnesses such as anxiety or depression. Where appropriate, these criteria applied to the healthy controls as well, except that they had no sleep disorder.Patients were included if they (1) had moderate to severe OSAHS, determined based on the guidelines recommended by the American Academy of Sleep Medicine  , and (2)Participants were provided with an Epworth Sleepiness Scale (ESS) evaluation and neuropsychological cognitive assessment . The monitoring was performed in a specialized sleep center.A 10 ml sample of fasted venous blood was collected from every participant and centrifuged at 3000 rpm for 5 min at 4°C. Plasma was removed and stored in a −80°C freezer. The PBMCs were separated from the remaining blood components via density gradient centrifugation and stored in three tubes. Two tubes were directly stored at −80°C for Western blot analysis, and 1 ml TRIzol  was added to the other tube for real-time quantitative PCR analysis, and the tube was stored at −80°C.Blood samples were analyzed using a commercially available sandwich enzyme-linked immunosorbent assay kit. The SOD testing kits were purchased from Abcam  and the Trx testing kit was from Cusabio Biotech .GAPDH) was used as an internal control gene. The sequence of each primer was as follows:Total PBMC RNA was extracted using TRIzol. The RNA concentration was measured using a spectrophotometer , and first strand cDNA was generated using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Relative expression levels of Nrf2 and HO-1 were measured using All-in-One qPCR Mix . Glyceraldehyde 3-phosphate dehydrogenase .β-actin , or proliferating cell nuclear antigen  overnight at 4°C. Membranes were then incubated with secondary antibodies labeled with horseradish peroxidase (HRP) for 1 h at room temperature. Finally, the membranes were imaged using chemiluminescence. β-Actin and PCNA were used as the internal control proteins to normalize slight variations in protein loading. The ratio of the optical density of the target protein and the internal control protein was taken as the relative expression of the protein.PBMCs were washed twice in ice-cold 1x phosphate buffered saline (PBS). Total protein was extracted with a protein lysate buffer , and nuclear proteins were extracted using a nuclear lysate buffer (Thermo Fisher Scientific). The protein concentration was determined using the bicinchoninic acid assay (BCA). After adjusting the concentrations, cell lysates were boiled at 100°C for 5 min. After cooling, each protein sample was separated on 10% Bis-Tris polyacrylamide gels and then electrotransferred to polyvinylidene difluoride (PVDF) membranes  via SDS-PAGE electrophoresis. The electrotransferred PVDF membranes were blocked in 5% skim milk dissolved in tris-buffered saline with 20% Tween-20 (TBS-T) for 1 h at room temperature and then incubated with specific antibodies against Nrf2 , HO-1 , t-tests. Nonnormally distributed data were analyzed using nonparametric tests. Pearson's correlation coefficient was used for the correlation analysis of normally distributed data, and Spearman's rho was used for correlation analysis of nonnormally distributed data. Partial correlation analysis was used to analyze the correlation between levels of Nrf2-ARE signaling pathway, PSG parameters, and tests of neurocognitive function. Multiple linear regression was used to further evaluate the potential independent factors to predict the scores of neurocognitive function tests. A p value less than 0.05 was considered statistically significant.SPSS statistical software  was used for statistical analysis. Normally distributed data determined by a Kolmogorov-Smirnov test were analyzed using independent sample p < 0.05) than healthy controls on all neurocognitive function tests included in MCCB and Stroop Color and Word Test (SCWT).Differences in neurocognitive function between patients with OSAHS and healthy controls are shown in p < 0.01), as shown in Tables Plasma SOD and Trx levels as well as Nrf2 and HO-1 mRNA and protein expression levels in PBMCs were significantly lower in the OSAHS group than in the control group (2 < 90% (TSat90), and ESS scores, and positively correlated with LSaO2 and MSaO2 after adjusting for the influence of education, sex, age, and smoking index  = 3.367, p < 0.002, R2 = 0.563; F = 3.660, p < 0.002, R2 = 0.528)  . For theCIH in patients with OSAHS during sleep can produce large amounts of ROS, which might lead to oxidation/anti-oxidation imbalances, increased oxidative stress, and decreased antioxidant capacity—an important factor in the dysfunction of patients with OSAHS , 4. ReseThe Nrf2-ARE signaling pathway is dysfunctional in the mouse model of intermittent hypoxia (IH) –20. The 2, MSaO2, ODI, and TSat90. Furthermore, REM% showed no significant correlation with the above antioxidation indicators. Above results indicated that the impaired Nrf2-ARE signaling pathway may be more closely correlated to CIH, an important characteristic of OSAHS, rather than to disturbed sleep architecture. Furthermore, the expression of nuclear Nrf2 protein was negatively correlated with AHI and TSat90, while the expression of total Nrf2 protein was positively correlated with AHI and TSat90. This indicates that the balance of total Nrf2 protein and nuclear Nrf2 protein may be an important regulator of antioxidative capacity in OSAHS. In addition, levels of total Nrf2 protein, nuclear Nrf2 protein, SOD, and Trx were negatively correlated with AHI, which suggests that the Nrf2-ARE signaling pathway might also be closely associated with the severity of the disease.In the present study, compared to those in the healthy control group, the mRNA and protein expression of Nrf2 and HO-1 in PBMCs as well as plasma levels of SOD and Trx in patients with OSAHS were all significantly reduced, meaning that antioxidant capabilities are systematically impaired in OSAHS. After adjusting for the influence of age, education, sex, and smoking index, the expression of Nrf2-ARE signaling pathway components in patients with OSAHS, including total Nrf2 protein, nuclear Nrf2 protein in PBMCs, and plasma Trx levels, were all correlated with TSat90 and AHI, and HO-1 protein levels were correlated with LSaONeurocognitive impairment, including declines in memory, attention, and executive function, is very common among patients with OSAHS. These impairments reduce the work efficiency and quality of life of patients with OSAHS to different degrees, in addition to negatively influencing social, public safety, and economic development on a societal level . DisturbThe Nrf2-ARE signaling pathway is one of the most important anti-oxidative signaling pathways in vivo. Studies have confirmed that Nrf2-ARE dysfunction plays a vital role in the development of cognitive dysfunction, especially in neurodegenerative diseases such as Alzheimer's disease (AD), amyotrophic lateral sclerosis, and Parkinson's disease . AggravaAnimal experiments have shown that sulforaphane, an Nrf2 activator, can reduce cerebral oxidative stress and inflammation, thereby improving cognitive impairment in an AD mouse model . Both mRThis study, however, is not without limitations. First, a low number of women were involved in this study due to the lower prevalence of OSAHS among the female population in China. In our sleep center, women account for fewer than 20% of patients with moderate to severe OSAHS. Moreover, obesity, a strong risk factor for OSAHS, is more common for middle-aged men in China. However, many studies have demonstrated a sex bias in physiological adaptations to oxidative stress in the healthy population as well as in patients with chronic neurodegenerative diseases, immune dysfunction, and cardiovascular disease, among others –40. Men The present study is the first to demonstrate that the expression of components of the Nrf2-ARE signaling pathway, an extremely crucial antioxidant pathway in vivo, and related key antioxidases in peripheral blood are all significantly decreased in patients with moderate and severe OSAHS. More importantly, the reduced expression of Nrf2-ARE proteins is significantly correlated with dysfunction of memory and executive function. Besides, we adopted MCCB, an internationally recognized battery of neurocognitive tests evaluating commonly impaired cognitive domains to evaluate cognitive impairment in patients with OSAHS. The results revealed that MCCB is likely to be a potentially effective neurocognitive test battery in assessing neurocognitive function in these patients. Moreover, we demonstrate that dysfunction of the Nrf2-ARE signaling pathway may be an important pathophysiological mechanism in the development of cognitive impairment in patients with moderate to severe OSAHS. The present study provides an important base for further research in vivo and in vitro to explore the specific mechanisms behind this dysfunction and potential treatments for improving neurocognitive function in patients with OSAHS.


Drosophila melanogaster have proved invaluable for identifying new conserved RTK pathway components, delineating their contributions, and for the discovery of conserved mechanisms that control RTK-signaling events. Here we provide a brief overview of the RTK superfamily and the general mechanisms used in their regulation. We further highlight the functions of several RTKs that govern distinct cell-fate decisions in Drosophila and explore how their activities are developmentally controlled.Cell-to-cell communication mediates a plethora of cellular decisions and behaviors that are crucial for the correct and robust development of multicellular organisms. Many of these signals are encoded in secreted hormones or growth factors that bind to and activate cell surface receptors, to transmit the cue intracellularly. One of the major superfamilies of cell surface receptors are the receptor tyrosine kinases (RTKs). For nearly half a century RTKs have been the focus of intensive study due to their ability to alter fundamental aspects of cell biology, such as cell proliferation, growth, and shape, and because of their central importance in diseases such as cancer. Studies in model organisms such a  Receptor tyrosine kinases (RTKs) play essential roles in the cellular communication network that orchestrates the development of metazoans. They are a major class of enzyme-coupled cell surface receptors activated when bound by extracellular signals from the environment such as growth factors, cytokines, and hormones. Initially, RTKs were discovered in the 1970s as the key factors responsible for transducing several potent growth and proliferative signals, including nerve growth factor (NGF), epidermal growth factor (EGF), and insulin  or phosphotyrosine-binding (PDB) domains [Ligand-binding triggers the juxtaposition of the cytoplasmic TKDs, which in turn results in autophosphorylation in  domains . For exa domains .The most common cascades employed to transmit the signal from RTKs are the mitogen-activated protein kinase (MAPK) cascades, PLCgamma, phosphatidylinositol 3-kinase (PI3K), and Janus Kinase and Signal Transducer and Activator of Transcription (JAK/STAT) signaling pathways . The MAPDrosophila EGFR [Drosophila Torso [In addition to the MAPK pathway, several RTKs including the Insulin receptor family use the PI3K-Akt-TOR signaling axis to modulate general protein synthesis, cell cycle progression, and inhibit apoptosis via S6K and/or the forkhead transcription factor FOXO ,34. Otheila EGFR ,38, PDGFila EGFR , and Drola Torso . The events described above are programmed to take place at precisely controlled locations and times both during development and in adult tissue homeostasis. Indeed, the spatiotemporal regulation of receptor abundance, as well as the availability and density at the membrane are all major determinants of RTK output . ReceptoLigand location and abundance must also be coordinated with that of the receptor to ensure that the desired output is both achieved and robust to perturbation . Over foDrosophila RTKs and highlight the general mechanisms by which they are controlled.A great deal of these mechanistic insights have been gained through studies of RTKs and their ligands in both cell culture systems and in model organism. In particular, genetic and functional analyses of RTKs in models, including the mouse, fruit fly, and nematode worm have provided the crucial contextual links for understanding how these proteins serve to control processes that underlie development. Studies of these organisms have revealed novel insights at all levels of RTK regulation and identified many of the components that underpin these mechanisms ,54,55,56Drosophila melanogaster have contributed substantially to our present understanding of RTK function in the context of development. This is largely owing to its genetic tractability, speed of life-cycle, and the strong conservation that exists between Drosophila and humans, which extends to most of the RTK families and the downstream intracellular signaling pathways [Drosophila for studying RTKs is the low level of within family complexity; the human genome encodes ~58 RTKs across 20 families, whereas the Drosophila genome encodes 20 known RTKs with single representatives for 11 of the 20 mammalian families  is the only view see , Table 2Drosophila genome encodes four ligands for EGFR; three that are produced as transmembrane precursors, Spitz, Keren, and Gurken, and one that is constitutively secreted called Vein [EGFR signals via the canonical Ras/MAPK to activate the transcription of target genes in a context-dependent manner via the Pointed ETS transcription factor . Its expled Vein . Spitz hrhomboid (rho) phenocopy loss of spitz, and the highly dynamic expression pattern of rho closely mirrors that of EGFR-induced MAPK activation, suggesting that rho expression is a critical localizing determinant of EGFR signaling [Surprisingly, like EGFR, Spitz expression is also broad. This is because its activity (and that of Keren and Gurken) further requires proteolytic processing for membrane release and secreted ligand activity ,119. At ignaling . rho is also a transcriptional target of EGFR signaling. Induction of rho in signal receiving cells therefore converts them into a signal source. Since EGFR ligands predominantly act at close range (one to two cells away), this mechanism permits expansion of the signal and can generate complex tissue patterning such as during compound eye development [In several contexts, elopment . During elopment ,122.Drosophila EGFR activity have also described the use of negative feedback circuits to produce binary switch-like outputs. The most well studied of these involves the transcriptional target argos, which encodes a secreted EGFR mimetic that acts to sequester and inhibit secreted Spitz [argos results in phenotypes resembling Egfr gain-of-function mutants [Drosophila, some operating only in particular developmental contexts [Studies of ed Spitz ,123,124. mutants . Argos icontexts , the EGFDrosophila insulin-like receptor (InR) is a major player in the control of cell, organ, and body growth [Like its mammalian counterparts, IR and IGF1R, the y growth . InR is y growth , and is y growth ,127. Losy growth ) and faiy growth . More wey growth . chico nulls, however, suggests that other InR signaling substrates may also exist [Consistent with their role as the predominant downstream transducers (and regulators) of the InR signal, reductions in Chico , PI3K, PTEN, Akt, TOR, and FOXO function all profoundly affect cell size and number ,130. Theso exist . This isso exist . Drosophila genome encodes eight insulin-like peptides (DILP1–8) that share structural similarity to preproinsulin , IGF1 , and thDILP1–5, ,135. TheDILP1–5, . Of partd growth ,138. Ilp independently, as well as their translation and secretion into the lymph. For example, adipokinetic hormone (glucagon-like) signaling in the IPCs has been shown to trigger selective release of DILP3 [The IPCs, like pancreatic beta-cells in mammals, are under tight control. IPCs sense an expanding list of different neurotransmitters and peptides from other neurons, as well as factors from the gut and other tissues that all converge on DILP regulation (for review see ). These of DILP3 . Intriguof DILP3 . Thus, sPvr encodes the only known Drosophila ortholog of the platelet derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) receptor families. Akin to its mammalian relatives, Pvr comprises an ectodomain with seven immunoglobulin-like repeats and a split intracellular TKD [Drosophila Pvr. For instance, PDGFR and VEGFR families are critical for hematopoiesis and blood vessel formation, respectively in mammals  causes greatly reduced hemocyte numbers and defective dispersal patterns [Unlike emocytes . Hemocytemocytes ,145. Heremocytes ,153. Exppatterns ,144. Inipatterns . Howeverpatterns . Another informative migratory event involving Pvr permits a small cluster of ovarian follicle cells, the border cells, to migrate to the anterior end of the oocyte during oogenesis . This prtor and other terminal class genes revealed that terminal patterning is the result of RTK signaling. Torso (Tor) was identified in the seminal mutagenesis screens of the 1980s as one of a handful of maternal factors critical for specifying cell fate at the embryonic termini ,65. Losstrunk (trk), a member of the cysteine-knot family of growth factors and cytokines, and whose expression mirrors that of Tor [Like other maternal gene products, the mRNA that encodes Tor is deposited into the developing oocyte during oogenesis and translated upon fertilization where it is then thought to be present ubiquitously on the early embryo plasma membrane . The ligt of Tor . Despitet of Tor , Figure tailless and huckebein [tsl causes terminal patterning defects identical to loss of trk and tor, and when tsl is ectopically expressed beyond the termini, ectopic Tor activity is observed [Tsl is localized to the inside of the innermost layer of the eggshell coinciding with the domain of Tor activation as measured by activation of ERK and its zygotic transcriptional targets uckebein ,158. Losobserved . Early wobserved ,160. Theobserved ,161,162.Based on protein sequence Tsl is not predicted to have a signaling function. Instead, Tsl is a member of the membrane attack complex/perforin (MACPF) protein superfamily known for their roles as pore-forming effectors in vertebrate immunity ,164. We If Trk matures into the Tor ligand prior to secretion, it would presumably require physical separation from Tor during its trafficking to the membrane to avoid Tsl-independent, and therefore unrestricted, activation. Alternatively, Trk may require other factors  or post-translational modifications before being capable of binding Tor. Whether such events are linked to the function of Tsl remain to be known. Interestingly, there are MACPF proteins in vertebrates that are also involved in developmental events (for review see ). UndersTor has a second function during late larval development in the major endocrine gland, the prothoracic gland, that when activated acts as an instructional cue for the initiation of metamorphosis . Tor sigDrosophila have and continue to yield important insights into the components and mechanisms that control them. Importantly, this work has provided an appreciation for how decisions of cell fate are determined in the context of development and how disease arises when such processes fail. However, there is still much to learn. For example, we know very little regarding how the dynamics of RTK activation (and deactivation) contributes to cell-fate decisions and the role that their ligands play in this. How important is it that RTK signaling occurs at a particular time or place, or at a level or duration sufficient to ensure a cell-fate decision is made? Answers to these questions requires advances in quantitative in vivo approaches that permit precise measurements of signaling activity and the ability to modulate RTK signaling in native developmental contexts.Studies of RTKs and their pathways in model organisms such as Still, the most widely employed approach to measuring RTK signaling output in vivo uses sample fixation and antibodies raised against phosphorylated (active) signaling substrates, such as ERK and Akt e.g., ,174,175),175174,1Drosophila