We also examine the associated inequality within each country in the proximate determinants of fertility–marriage, contraception, and breastfeeding. Applying Bongaarts’ proximate determinants of fertility framework, for 14/21 countries we analyze, we find that those in the richest wealth quintiles have had a more rapid decline in fertility rates than those in the poorest wealth quintiles. The rapid decline for those in the richest wealth quintiles is attributable to delayed marriage and modest increases in contraceptive use. Although the poorest lag in fertility decline, postpartum abstinence and breastfeeding are the most important factors for them for fertility regulation. Further encouraging maternal health programs that focus on natural methods of fertility regulation will work in favor of the poorest in sub-Saharan Africa in moving through the demographic transition.In this paper, we examine the inequality in the dynamics of the total fertility rate  Examination of the fertility rates at the sub-national level can inform us of inequalities that may hinder the decline in the national-level fertility rates. For example, the richest declining rapidly in their fertility rates, but the poorest remaining stagnant or increasing in their fertility rates. At the aggregate level, reflected in the national-level fertility rates, it would appear that fertility decline is slow for everyone. Rather, there is great variation across the wealth spectrum within a country in the rate and timing of fertility decline. Examination at the sub-national level reveals these within-country inequalities in the total fertility rate dynamics. Furthermore, inequalities in the total fertility rate reflect inequalities in the proximate determinants of fertility: child marriage, access to contraception and safe abortion, and postpartum breastfeeding or abstinence, can vary within countries across wealth quintiles. Examination of these proximate determinants of fertility can reveal underling drivers of inequality in the total fertility rates across subgroups of the national population.Globally, countries have seen a decline in their national-level total fertility rates over the past 100 years, although this decline has begun in different years and evolved at different rates. The variation across countries in the rate and timing of fertility decline may be a reflection of variations The total fertility rate as a calculation is the number of children born per woman if she were to age through her childbearing years and have children according to the current schedule of age-specific fertility rates. It is not the number of children a woman gives birth to, but a hypothetical projection of what she would give birth to if her own age-specific fertility patterns across her life course matched the population average age-specific patterns in the present.An inequality in the total fertility rate refers to an observable and measurable difference between or among individuals, subgroups of the population, or groups occupying unequal positions in society .consequences of inequality in the total fertility rates. In this paper, however, we examine the existence of inequality in the total fertility rate within countries, and the proximate determinants of this inequality in the total fertility rates. We leave the examination of the consequences of inequality in fertility rate on inequality in economic development for another paper.A decline in the total fertility rate to the replacement rate of 2.1 children per women has been shown to have a positive impact on a nation’s economic prosperity (the demographic dividend ). The coOur examination of the inequality in the total fertility rate at the sub-national level, and investigation of the drivers of this inequality in the total fertility rates, enables us to observe the underlying pathways of how inequality in age of marriage, contraceptive use, and breastfeeding affects inequality in the total fertility rate.At the sub-national level, the examination of inequality in the total fertility rate has been observed within countries by education levels . EloundoAs a result, in this paper we conduct a descriptive data analysis on individual-level data to illustrate inequality in changes in the total fertility rates within subgroups  of a country’s population. We use Bongaarts’  model ofBongaarts’  model ofWhile Bongaarts’ model has received its share of citicism , 13, we The social determinants of fertility are education, wealth, and urban/rural living. In this paper, we consider the role of wealth as a moderator of the relationship between the proximate determinants of fertility and the total fertlity rate. We hypothesize that we will observe differences in the roles of the proximate determinants of fertility across wealth quintiles within a country, which will illustrate the underlying reason for differences in the total fertility rate across wealth quintiles within a country.In this paper, we aim to show: 1) the total fertility rate by wealth quintiles over time, 2) inequality in the total fertility rates over time, 3) the proximate determinants of fertility by wealth quintile over time; and 4) inequality in the proximate determinants of fertility over time.This decomposition of the data enables us to see which countries are experiencing inequality in fertility decline and what is driving this inequality. The decomposition of the proximate determinates enables us to observe a side-by-side comparison where policy can be most effective. For example, we can see how policies ending child marriage, increasing contraceptive use, or promoting natural methods of fertility regulation could be promoted, and which groups within a country would benefit most from such policies.In this paper we focus on the sub-Saharan African continent, as their demographic transition is beginning, but is unfolding slowly  and its and breastfeeding. Thus, there may be differences in our reported numbers to the fertility rates reported by others, as our sample is restricted to women who respond to all these questions.It is worth noting that a large-scale secondary source dataset such as the Demographic and Health Surveys, which interviews across different time points or different countries, have varied in quality. Furthermore, the methods for calculating the total fertility rate published by the national statistical office or the United Nations, or total fertility rates by wealth quintile published in DHS Final Reports may differ from our estimation methods. Moreover, our sample may differ from the sample used in the DHS Final Reports, as we only include women who respond to questions regarding wealth, birth history, sexual union, contraceptive use, postpartum abstinence In this paper we used data from the Demographic and Health Surveys (DHS) , 17, whiThe 21 countries included were Benin, Burkina Faso, Cameroon, Cote d’Ivoire, Ethiopia, Ghana, Guinea, Kenya, Madagascar, Malawi, Mali, Mozambique, Namibia, Niger, Nigeria, Rwanda, Senegal, Tanzania, Uganda, Zambia, and Zimbabwe. See http://data.princeton.edu/eco572/asfr.html) applying birth histories from the DHS to calculate the age-specific fertility rate in the three-year period before the survey. The second method for calculating the total fertility rate is an application of the Bongaarts’ proximate determinants of fertility model [The outcome of interest is the total fertility rate. This is estimated using two methods. The first uses the Rodriguez’s Stata code .We examine the total fertility rate and the proximate determinants of fertility across the 21 sub-Saharan African countries and by wealth quintile within each country, over multiple surveys (time points). The wealth quintiles calculated by the DHS  are a coIn this study, we followed Bongaarts’  model ofDelaying marriage or delayed initiation of sexual activity will reduce the years that a woman of reproductive age is exposed to the risk of pregnancy. Contraceptive use decreases the risk of pregnancy. Abortion reduces the number of live births. Postpartum infecundability is a combination of postpartum abstinence and postpartum amenorrhea. The latter is a result of breastfeeding following a birth.The proximate determinant variables point directly to policy instruments that are relevant in the maternal health space: child marriage, low contraceptive use, high rates of unsafe abortions, and the need for support in the post-partum period. All are issues that plague women, particularly in low-resource settings. Bongaarts’ papers , 9, 20, Each of the proximate determinants work to reduce fertility from the biological maximum, which we set at 15.3 following Bongaarts’ earlier paper . If the We examine trends in the total fertility rate, and the proximate determinants, by wealth quintile, following the approach of Majumder and Ram  who focuIn this paper, we calculate the total fertility rate by wealth quintile using Rodriguez’s code for age-specific fertility rates. decline. This decline ranged from a drop of 1.9 children per woman for the poorest in Rwanda between 1992 and 2010, to a 0.1 drop in the number of children per woman in Ethiopia (7.8 in 2000 to 7.7 in 2010) and Senegal . In the case of Rwanda, Rutayisire et al [no change in the total fertility rate of the poorest wealth quintile over time. For six countries, Cameroon, Guinea, Mozambique, Nigeria, Uganda, and Zambia, the total fertility rate of the poorest increased from the earliest to the latest survey. The poorest in Mozambique had the largest absolute increase in the total fertility rate, from 6.6 children per woman in 1997 to 8.1 children per woman in 2011.Over time, for the poorest quintile , 19/21 countries saw a decrease in their total fertility rate. Benin saw no change in the total fertility rate of the richest over time. Tanzania saw an increase of 0.1 children per woman for the richest quintile between 1996 and 2009.gap in total fertility rate of the poorest minus the total fertility rate of the richest. We found that for 14 of the 21 countries, the gap between the poorest and richest total fertility rate increased. That is, inequality in the total fertility rate increased for these 14 countries: Burkina Faso, Cameroon, Cote d’Ivoire, Ethiopia, Ghana, Guinea, Kenya, Malawi, Mali, Mozambique, Nigeria, Senegal, Uganda, and Zambia.The gap between poorest and richest was constant in Namibia. For the remaining six countries in the sample, the gap between poorest and richest declined .Of the 14 countries that saw an increase in inequality in fertility decline, for six countries this was due to more rapid declines for the richest compared to the slower declines of the poorest quintiles over time . For another 6/14 countries that saw an increase in fertility inequality, this rise in inequality was due to the poorest experiencing an increase in the total fertility rate while the richest experienced a decline in the total fertility rate over time . For Kenya and Malawi, there was an increase in inequality of the fertility rate due to a decline in the total fertility rate over time for the richest, but no change in the total fertility rate over time for the poorest.For Namibia, the total fertility rate decreased by the same amount for the richest and poorest,  and so the gap between rich and poor remained the same over time. The poorest had a total fertility rate of 7.2 in the earliest survey, and the richest had a total fertility rate of 4.1. By the time of the most recent Namibian survey, the total fertility rate was 5.9 for the poorest (down 1.3) and 2.7 (down 1.3) for the richest.For the six countries that saw a closing of the gap between the richest and poorest total fertility rates over time, four countries did so due to the decline in fertility of the poorest being greater than the decline in fertility of the richest . For Benin, the poorest saw a decline in the total fertility rate over time, while the richest saw no change. For Tanzania, the poorest saw a decline in the total fertility rate over time, while the richest saw an increase in their total fertility rate over time.Thus far, we have examined how the gap between rich and poor in the total fertility rate has widened  or narrowed  over time. Furthermore, we examined whether this widening inequality is due to the poor lagging behind or the rich accelerating ahead.We now turn to examine how the proximate determinants of fertility–marriage, contraception, and postpartum abstinence/amenorrhea, play a role in shaping the widening or narrowing fertility inequality.Tables We take the example of Mozambique in That is, for the poorest in Mozambique in the earliest survey, postpartum infecundability and delayed marriage were the most effective methods in bringing fertility rates down from the biological maximum. Contraceptive use played a minor role.For the richest wealth quintile in the earliest survey in Mozambique, the observed total fertility rate was 4.9. For these women, postpartum abstinence and amenorrhea reduced the total fertility rate by 4.6 from the biological maximum of 15.3. Abortion reduced the total fertility rate by 1.2. Contraceptive use reduced the total fertility rate by 1.5. Non-exposure  brought fertility down by a further 1.2. The unexplained residual brought fertility down by 1.9.In the earliest survey in Mozambique, the rich (4.6) relied on postpartum infecundability more than the poor (4.5) in reducing the total fertility rate from the biological maximum. The rich (1.5) used contraception more than the poor (0.1) to reduce fertility from the biological maximum. The rich (1.2) also saw non-exposure to sex as a determinant of their fertility rates more than the poor (1.0). The unexplained residual brought fertility down from the biological maximum for both the rich and poor, but it was larger for the poor (2.3) than the rich (1.9).In the latest survey for Mozambique, For Mozambique, not only did the total fertility rate of the richest and poorest change over time, but so too did the proximate determinants of fertility.Postpartum infecundability became an increasingly important factor to bring down the total fertility rate from the biological maximum for both the rich (4.6 to 5.1) and the poor (4.5 to 5.1). The influence of contraceptive use in reducing fertility from the biological maximum increased for the rich from 1.5 to 2.4 and for the poor from 0.1 to 0.2. The influence of delayed marriage did not change for the richest, remaining at 1.2 over time, but increased for the poorest (1.0 to 1.3).To summarize, postpartum infecundability became important to both the rich and the poor in Mozambique. The rich used contraception much more than the poor. There was little change over time in the forces of delayed marriage for the rich, but slightly positive for the poor.gap between rich and poor total fertility rate (Rodriguez) increased for Mozambique between the earliest and latest surveys. The poor relied on delayed marriage more than the rich did. However, the rich utilized contraception more than the poor did.In changes in the gap in total fertility rate and changes in gap in the proximate determinants of fertility. In the first column, for the total fertility rate (Rodriguez), a negative value indicates that the gap between richest and poorest total fertility rate is closing–declining inequality in the total fertility rate between richest and poorest. A positive value indicates that the inequality in fertility between richest and poorest is increasing. Mozambique saw an increase in the gap between richest and poorest total fertility rate over time of 2.5 children per woman–increasing inequality.In For the residual R, a negative value indicates that the proximate determinant has a bigger impact on observed fertility rates for the richest compared to the poorest. A positive value indicates that the proximate determinant has a bigger impact over time on observed fertility rates for the poorest compared to the richest.Of the 21 countries, 14 countries saw the richest rely on delayed marriage at an increasing rate compared to the poorest in reducing fertility from the biological maximum.Of the 21 countries, 13 countries saw the rich use contraception at an increasing rate compared to the poor in reducing fertility from the biological maximum.Of the 21 countries, 16 countries saw the poor rely on postpartum infecundability at an increasing rate relative to the rich in reducing fertility from the biological maximum. .Of the 21 countries, 11 countries saw the rich unexplained residual reducing fertility at a greater rate than the poorest.For 14/21 countries, the gap between richest and poorest total fertility increased . For these countries, the richest saw increases in contraceptive use and age of exposure (delayed marriage) to a greater extent than the poorest. This puts more downward pressure on the total fertility rate of the richest than the poorest through these two proximate determinants. Moreover, for this group on average, both the poorest and richest decreased their reliance on postpartum infecundability . However, this decreased reliance on postpartum infecundability did not offset the role of delayed marriage and contraction in decreasing the total fertility rate. The poor also reduced their reliance on postpartum infecundability by less than the richest.For the group of countries that saw an increase in fertility inequality due to the richest decreasing in the total fertility rate while the poorest increasing their total fertility rate , delayed marriage and contraceptive use both increased in their role for the richest and poorest putting downward pressure on total fertility rate for both wealth groups. The upward pressure on total fertility rate created by increased use of postpartum infecundability for the richest and poorest, did not sufficiently offset the role of contraception and delayed marriage to cause the increase in total fertility rate for the poorest. In this case, it was the role of the residual, which saw total fertility rate of the poorest increase, highlighting the need to discover what this residual represents.For Kenya and Malawi, these counties also saw an increase in the inequality between richest and poorest for the total fertility rates–driven by the richest decreasing in total fertility rate while the poorest remained unchanged  in their total fertility rate over time. For these two countries, the richest increased in their reliance on delayed marriage. The poorest in Malawi saw a decrease in age of marriage or sexual debut between the latest and earliest surveys. The richest and poorest in both countries saw an increased role of contraceptive use; this was stronger for the richest in Malawi. In Kenya, the poorest decreasingly relied on postpartum infecundability, while the poorest and richest in Malawi increased in their reliance on postpartum infecundability.For Namibia, where the richest and poorest declined equally in their total fertility rate, age of marriage/exposure actually declined for both groups (poorest more than richest) putting upward pressure on total fertility rate. However, in Namibia, contraceptive use of the poorest increased as a rate greater than the increase for the richest. This catch-up in contraceptive use of the poorest meant that both the richest and poorest saw equal decline in their total fertility rates.on average there was no change in age of marriage, equal increase in contraceptive use by richest and poorest, and a stronger role of postpartum infecundability for the poorest than the richest. Postpartum infecundability played the role of closing the gap between richest and poorest total fertility rate.For six countries, the gap between richest and poorest total fertility rate decreased. For Madagascar, Niger, Rwanda, and Zimbabwe, For Benin, the richest saw no decline in their total fertility rate while the poorest saw a decline in total fertility rate. The gap between rich and poor total fertility rate narrowed. For the poorest there was increased reliance on delayed marriage, contraceptive use, and postpartum infecundability , and thus all the proximate determinants worked to decrease total fertility rate of the poorest. However, the richest saw no change in their contraceptive use, a declining dependence on postpartum infecundability . Age of marriage/exposure of the richest increased .For Tanzania, where the poorest saw a decrease in their total fertility rate and the richest an increase in their total fertility rate , this was driven by the unknown residual of the richest.In summary, we could say that for those countries that saw an increase in inequality of fertility rates, the poorest were declining in their use of postpartum infecundability and did not compensate or keep up with the richest in terms of contraceptive use. Trends in increasing the age of exposure/marriage also lagged for the poorest.For those countries that saw a decrease in the gap between richest and poorest in terms of the total fertility rate, there was an increased role of postpartum infecundability in reducing fertility rates for the poorest. Moreover, contraceptive use of the poorest for these countries increased and kept pace with the richest. Delayed marriage played a minor (or non-existent) role.To investigate why fertility inequality changed differently for the richest and poorest countries, we used the Bongaarts’ proximate determinants of fertility model, in which these proximate determinants of fertility directly determine the fertility rate. In this paper, we found that there was increasing inequality in the fertility rate across wealth quintiles for 14/21 SSA countries. We assume that the proximate determinants of the levels of fertility are also the proximate determinants of inequality in fertility. Such that, inequality in sexual exposure, inequality in contraceptive use, and inequality in postpartum infecundability lead to inequality in fertility rates.We found that increasing inequality in the total fertility rate was driven by positive trends in delayed marriage for the richest wealth quintile and modest gains in contraceptive use by the richest. However, the poorest did not see these gains, hence the rising inequality the total fertility rate between the richest and poorest. Rossier et al  have notPostpartum infecundability has played a very large role in reducing fertility rates for the poorest wealth quintile . Without this important mechanism of postpartum infecundability, there would have been a much greater divergence of the fertility rate between the richest and poorest. As Rossier and Corker  pointed Research typically focuses on the role of early marriage , contraceptive access and use, and programs designed to foster these fertility regulating instruments . HoweverMoreover, through the descriptive analysis in this study, we have found that the residual proximate determinant—which is yet unknown—has played an increasingly important role in how the rich move away from the poor in terms of fertility decline. Further work is needed to fully understand what the residual is.Following from the results of this study, the implications for policy are: 1) programs targeting delayed marriage need to be inclusive of the poorest wealth quintile to ensure that fertility decline through this mechanism is experienced across the wealth spectrum; 2) programs promoting contraceptive use for the poorest will help with an equal decline in the total fertility rate; and, 3) postpartum infecundability is employed very effectively by the poor, and when it is promoted for the poor, it helps close the gap in total fertility rate. In fact, when postpartum infecundability is de-emphasized, inequality in total fertility rate increases.While some previous research does emphasize the role of postpartum infecundability in fertility regulation in poor resource settings , 27, polIn addition to the higher-level limitations addressed in the introduction, there are limitations to this analysis that should be recognized so that interpreting the results can be made with caution. The residual was often high, and we do not fully understand its composition. Misreporting and potentially under-reporting of contraceptive use would downplay the contribution of contraception to the determination of the observed total fertility rate. In addition, the abortion rate we used was for the entire region and was not country and age specific, as the other components were calculated. This means that our interpretation of abortion rates was not specific to countries or wealth quintiles.Despite these limitations, this paper has shown that inequality in fertility is increasing in 14 out of the 21 sample countries. This divergence in fertility rates between the richest and poorest wealth quintiles is due to the richest wealth quintile’s fertility decline outpacing the fertility decline of the poorest in 6/14 countries, and the poorest increasing in their total fertility rate compared to declines in the richest quintile in 6/14 countries. The fertility decline of the richest is driven by delayed marriage and modest increases in contraceptive use. Likewise, for the richest, postpartum abstinence and postpartum amenorrhea played a decreasing role. However, for the poorest, postpartum abstinence and postpartum amenorrhea played an increasingly important role in reducing fertility rates .Without Bongaarts’ proximate determinants framework, the policy focus is on child marriage and contraceptive access in completely different fields. This paper, however, shows that natural methods of fertility regulation are important in reducing fertility rates–especially for the poor. Any programmatic or policy shift away from these natural methods will see an increase in inequality in fertility rates.


Wnt signaling pathway. Wnt/beta-catenin signaling pathway controls a myriad of cellular processes that are essential for normal cell functioning. Cell cycle progression, differentiation, fate determination, and migration are generally orchestrated by canonical Wnt signaling. Altered Wnt/beta-catenin signaling has been considered a promoting event for different types of cancers and the oncogenic potential of Wnt signaling have been discussed in many cancer types, including breast, colon, pancreatic as well as head and neck. Furthermore, Wnt signaling is critical for the maintenance and stemness of both the normal as well as cancer stem cells. This review sheds new light on Wnt signaling and explains how it can regulate normal physiological processes and curtail the development of cancer. It depicts the vital functions of Wnt signaling in the stem cell growth and differentiation by focusing on current druggable targets that have been ascribed by recent studies. Thus, Wnt signaling pathway retains a tremendous potential in eradicating head and neck squamous cell carcinoma.Cellular maintenance and development are two fundamental mechanisms regulated by the canonical  Head and neck squamous cell carcinoma (HNSCC) are one of the most aggressive cancer types with high fatality rate due to late diagnosis and high recurrence. It arises from the mucosal surfaces of various organs with epithelial linings. Common organs include sinuses, oral cavity, tongue, pharynx, and larynx . The incidence rate of HNSCC overall in the United States is 11 per 100,000 individuals and is more prevalent in black population compared to white people, and it is the most prevalent type of tumor after cervical cancer . Head and neck squamous cell carcinoma accounts for more than 330,000 death worldwide with more than 650,000 cases of HNSCCs reported annually . In the US alone it has been reported that HNSCCs was responsible for 10,800 deaths and more than 53,000 American population developed the disease on an annual basis . In addition to this HNSCCs incidence comprises 3% of all malignancies in the US and 4% of all cancer-related malignancies in Europe respectively. However, in Europe, nearly 250,000 cases of HNSSCs are recorded each year, and death tally is 63,500 per year. HNSCCs is more prevalent in males as compared to females with ratios between 2:1 to 4:1 respectively. The incidence rate of HNSCCs is 20 per 100,000 in different regions of the world moreover, HNSCCs is most common type cancer In Asian countries  Risk of recurrence, unavailability of suitable therapies and low efficacy of currently used drugs are the major setbacks that have hampered the efforts towards a valid therapeutic approach for the treatment of HNSCC .Wnt signaling pathway has been reported to be a Promoting event for different types cancers , such as colon, breast and lung and even reported to implicate decisive role in safeguarding the CSC, as well as support the fundamental machinery that promotes HNSCC progression . This review aims to discuss the involvement of Wnt signaling in maintenance of stemness in CSC with particular reference to HNSCC and different therapeutic and diagnostic options for HNSCCs furthermore it shed light on the use of a natural compound as a promising therapeutic agent for the HNSCC treatment. Like any other tumor, it has become really difficult to treat HNSCCs. Overall it is the sixth most prevalent type of cancer with survival rate less than 60% , like other cancers, physicians can hardly treat HNSCC patients as they are facing several hindrances such as the rapid growth of tumor, in availability of early medical care, reluctance of people towards early treatment and the presence of cancer stem cells (CSC) worsen the tumor progression . Indeed these are a sub-population of cells which are resistant to chemo and radiotherapy and frequently promote recurrence by halting or escaping clinical efforts also in HNSCC . CSC show coherent features, i.e. similar to normal stem cells such as self-renewal, development and differentiation properties. Understanding the mechanisms involved in the maintenance of CSC holds promising benefits for delineating the causes of tumorigenesis, metastasis, self-renewal . Moreover, understanding cell-cell interaction can aid in the development of appropriate druggable targets for HNSCC and consequently potential treatment. Aberrant Cancer Stem Cells in HNSCCThe ability of self-renewal and differentiation are the two main features of stem cells (SC). Stem cells only divide to give rise to the new progeny of cells as they lack any tissue-specific structure . Depending upon specific cellular signaling SC can undergo cell division to produce either cells with retained pluripotency or progenitor cells for the synthesis of more mature and functionally active cells which can differentiate into more specific cell or tissue. In the definition of SC also falls embryonic stem cells (ESC) which originate from the blastocyst of the embryo. ESC possesses a fundamental ability, pluripotency, a feature that guarantee the development of any tissue or organ. Differently adult stem cells (ASC) are cells characterized as unipotent or multipotent, they reside in the specific tissue and can produce a variety of cells, but not all cells from which the organ was built .The origin of CSC came into limelight by the experiments conducted by Bonnet and Dick in 1997 when they successfully transplanted immune-deficient mice with stem-like cells termed as severe combined immunodeficiency disease leukemia-initiating cells (SL-IC). These cells retain the properties of SC in such a way that they can proliferate and differentiate and can produce leukemia. Furthermore, these cells maintain the capacity of self-renewal and have the potential to establish disease in other mice injected with SL-IC . Later other studies provided substantial evidence for the existence of CSC in different forms of cancers such as breast, lung, prostate, pancreatic and head and neck . Altogether these findings established the existence of CSC and further underlined their ability of pluripotency and self-renewal. Involvement of CSCs in HNSCCs came from Prince and colleagues . Their findings revealed that expression of CD44+ a lineage marker was indispensable for persistent tumor heterogeneity in HNSCCs. Using limiting dilution assay they confirmed the high prevalence of CSCs in HNSCCs after breast cancer, indicating the fact that cancer stem cells are pivotal for epithelial cells mediated HNSCCs . CD44+ separated by the aldehyde dehydrogenase (ALDH) has been reported to have more tumorigenic properties and self-renewal capacity compared to normal CD44+ not separated by this enzyme. ALDH has been linked to confer therapy resistance and tumor prevalence via modulating the expression of PI3K (phosphoinositide 3-kinase) and SOX2 (SRY-box 2) signaling cascades. ALDH concentrations have been directly associated with increased tumor size and symptoms of cancer in cell lines of HNSCCs cancer  Apart from CD44 a broad of range of markers have been investigated in CSCs and specifically HNSCCs. CD133 is a transmembrane glycoprotein whose presence have been linked to increase drug resistance against cisplatin in vitro . This marker has been studied extensively for its role in modulating the expression of various tumors such as lung cancer, HNSCCs and other solid tumors . CD24 another glycoprotein has been reported to hinder the efficacy of drugs like cisplatin gemcitabine by promoting drug resistance in CSCs of HNSCCs . Several other factors regulates the expression of CSCs in HNSCCs. Enzymes such as ALDH and proteins such as BMI and transcription factors such as the Nanog and SOX2 all have been implicated to play a decisive role in proliferation and drug resistance of CSCs. Furthermore, CSC has also been reported to have a longer life span and negate the process of apoptosis due to the presence of drug resistance proteins such as ATP-binding cassettes family transporters. Cancer stem cells trigger tumorigenesis in a variety of cancers such as lung cancer and HNSCCs as they confer drug resistance and disease relapse in these tumors. .Wnt Signaling PathwayWnt signaling is vital for a plethora of cellular function ranging from homeostasis to the development of mature tissue. Embryonic development also requires Wnt-mediated canonical signaling . Moreover, Wnt signaling is inevitable for regulating cellular proliferation, apoptosis, metastasis, and migration of cells . Of note, Wnt operates through either canonical or non-canonical pathways which are differentiated by beta-catenin involvement . Wnt protein initiates the classical Wnt signaling to Frizzled transmembrane receptors , non-canonical signaling requires other molecules for downstream activation such as co-receptors Receptor tyrosine Kinase-like orphan receptor 2 (ROR2) or receptor-like tyrosine Kinase (RYK) . Together these signal transducers activate the calcium-dependent signaling cascade via activation of scaffolding proteins disheveled (DvL). Thus, the initiated signaling aids in the cellular movement and cytoskeleton remodeling by activation of Rho family of GTPases that in turn activates RhoA and Rac. Cellular polarity is mediated by the commencement of c-Jun N-terminal kinase (JNK) signaling cascade through triggered Rac. DvL mediated activation of Wnt signaling induces the launch of heterotrimeric G protein which promotes cellular export of calcium to extracellular environment . This transportation activates several kinases which include JNK, nemo like Kinase (NLK) and nuclear factor of activated T-cell (NFAT) through activation of calcium responsive kinase C and calcium/calmodulin-dependent Kinase II (CaMKII) . NFAT via its activation through calcineurin plays a decisive role in the gene expression and embryogenesis . On the other hand, non-canonical signaling mediates the activation of canonical Wnt signaling through phosphorylation of T-cell factor (TCF) via NLK . This indicates that both canonical and non-canonical signaling are antagonized one each other and calcium modulates such counteract by either activation of one the signaling   .Wnt signaling is commenced by the binding of Wnt to the cytoplasmic beta-catenin signaling cascade . In the absence of Wnt, beta-catenin is normally degraded by the E3 ligase. Cytoplasmic beta-catenin usually exists freely or attached to APC, but a trimeric complex composed by Axin, CK1 and GSK3 phosphorylate cytoplasmic beta-catenin at serine residues via phosphorylation of Axin and APC making beta-catenin vulnerable for the E3 ubiquitination and degradation  the β-catenin is targeted for degradation (2) (Phosphorylation) by the complex. The complex consists of a tumor suppressor gene (APC), scaffold protein (Axin) and kinase  (3). Activation of Axin-CK1-GSK3 complex initiates the phosphorylation of APC that promotes beta-catenin degradation. When there is the activation of canonical Wnt signaling, Wnt can attach to Fzd along with lipoprotein receptor-related protein (LRP) to form a trimeric complex . This trimeric complex helps in the phosphorylation of DvL. Phosphorylation of DvL recruits Axin-CK1-GSK3 complex to the cell membrane preventing it from phosphorylating APC which is attached to beta-catenin, thus allowing cytoplasmic beta-catenin accumulation and translocation into the nucleus where it can activate Wnt target genes   .Wnt signaling has been implicated in a broad range of cellular processes such as cell growth, differentiation, and migration: at the same time, it inhibits cell death through apoptosis. Transcription and signaling of molecules such as Cyclin D1, c-Myc, cyclooxygenase 2 (COX2), bone morphogenetic protein 4 (BMP4), matrix metalloproteinases7 (MMP7), Axin2, multi-drug resistance 1 (MDR1), b-TRCP and endothelial growth factor (EGF) have been reported to be under the influence of the Wnt-mediated canonical signaling, suggesting that Wnt has the key authority in sustaining cell development, migration and early embryogenesis .Wnt Signaling in HNSCC Wnt/Beta-catenin signaling has been a hallmark in tumor biology of both HPV+ as well as HPV- types HNSCCs . Many factors play a crucial role in the development of the tumor. Epigenetic modifications in (surfactin exporter in surfactin self-resistance) SRFP, (Wnt inhibitory factor) WIF and  DKK3 that inhibit the Wnt signaling cascade. However, mutation prompts them to activate the Wnt Signaling in the basal cells of HNSCCs . Overexpression of Beta-catenin has been strictly related to the increased transcriptional activity in HNSCCs . Derailed expression of the Beta-catenin can, in turn, promotes poor prognosis and increased differentiation in HNSCCs . Epigenetic changes that promote growth and regulation of cancerous stem cells has been affiliated with the Wnt/Beta-catenin axis . Although mutations in the Wnt/Beta-catenin are not so common in the HNSCCs yet other signaling cascades such NOTCH, FAT1 and AJUBA do interplay with Wnt/Beta-catenin to intricate changes in its activity . Epithelial differentiation program of HPV- HNSCCs has been orchestrated by signaling cascades such as the (Ajuba LIM Protein) AJUBA,  FAT1 and Notch drosophila homolog) NOTCH. Viral oncoproteins E6/E7 of HPV has been implemented to alter the prognosis of HPV positive HNSCCs . In the case of oropharyngeal squamous cell carcinoma (OPSCCs), viral oncoprotein E6 has been involved in nuclear localization of beta-catenin via activation of  EGFR pathway. Inhibition of E6 with small interfering RNAs resulted in the decreased expression of the EGFR receptor activity in OPSCCs via de-mobilization of the beta-catenin into the nucleus in vitro . It has come to limelight less lately that certain microRNAs have certain implications in attenuation of HPV+/HPV- HNSCCs. Histone modifications and acetylation results in modulation of the Wnt/Beta-catenin downstream signaling. Microarray analysis conducted by the Sannigrahi et al. showed that miR-132-5p overexpression targeted Wnt/Beta-catenin mediated signal transducer . Further studies are required to elaborate on the role of the E6/E7 in the modulation of the HPV+ HNSCCs. Mesenchymal-Epithelial transition factor c-Met or Hepatocyte growth factor Receptor (HGFR) have been reported to interplay with the Wnt/Beta-catenin Pathway in HNSCCs . Involvement of c-Met and Wnt/ Beta-catenin has been studied well in Colon cancer cells where their activity harness the cell fate determination in cancer stem cells. However, in HNSCCs, induction of c-Met inhibitor resulted in the elimination of CSCs in the presence of Beta-catenin. Frizzled class receptor 8 (FZD8) a modulator of Wnt/Beta-catenin pathway was reported to increase the expression of CSCs in HNSCCs via reactivation of (extracellular regulated MAP kinase) ERK/c-Fos signaling axis . These findings suggest the fact that various cellular cascades modulate Wnt/Beta-catenin signaling. HNSCCs involves the spread of malignant cells at multiple sites. It is a malignancy characterized by distinctive heterogeneity between inter and intra-tumor environment  . HNSCCs are divided into Human papillomavirus-positive (HPV+) and HPV- tumors. Each type is bearing its characteristic features of clinical, pathological and epidemiological significance . Abrogated Wnt signaling has been well understood in diverse cancer types, the role of Wnt in the HNSCC first came to light by the study conducted by Leethanakul et al. that observed the effects of Wnt using microarray technology. Also involvement of (Frizzled) Fzd and (Dishevelled) DvL homologs were compared with normal tissue samples. Their findings revealed that both of these target genes were over-expressed suggesting the intrusion of Wnt-mediated canonical signaling in the development of HNSCCs . To further support this hypothesis, experiments conducted by Lee et al. showed that the Wnt homologs (such as Wnt1 and Wnt10 b) were quite common in HNSCC tissues and expressed at higher values. Conclusively, Fzd and DvL are co-related the modulated Wnt signaling in the development of HNSCCs .Even though modulated Wnt-mediated signaling is its involvement in the CSC differentiation and development. Aberrant Wnt signaling has been directly associated with the over-expression and differentiation of CSC. Indeed CSC survival and differentiation and Wnt seem strictly coupled: activated Wnt signaling is indispensable for cancer development . More recent advances in the Wnt signaling in the development of breast cancer via CSC. Their findings suggest that aberrant Wnt signaling promotes the accumulation of several downstream targets such as Axin2 that promotes stable beta-catenin necessary for cell survival. Furthermore, these downstream targets block the efficacy of Wnt signaling inhibitors such as DKK1 resulting in the persistent survival of CSC. Altogether this suggests that Wnt signaling is differentially expressed in normal and CSC .A study conducted by Semenov et al. reported the crucial role of Wnt and Drug Resistance in HNSCCWnt in conferring resistance to chemotherapy by up-regulating the CSC activity in HNSCC. Warrier et al. using (secreted frizzled-related protein 4) SFRP4, an antagonist of Fzd/Wnt, demonstrated that SFRP4 increased drug sensitivity by 25% in HNSCCs. They showed that SFRP4 directly competed with the Wnt and greatly enhanced the effects of cisplatin-induced apoptosis and reduced tumor cell viability. Moreover, the antagonist did not affect the normal CS indicating the fact that Wnt signaling has a mighty role in the development and differentiation of CSC related to HNSCC .Disease relapse is a major hallmark in HNSCC primarily due to the presence of drug-resistant CSC. These cells foster the development of more resilient phenotypes that negate any possible way of apoptosis and thus produce more aggressive tumors . Tones of evidence have indicated the role of Wnt mediates the activation of induced CSC is still a dilemma. However, compelling evidence has shed light on a set of transporter proteins such as the ABC family. These cell transporters have been strongly associated with an astounding chemoresistance in a broad range of tumor cell lines. Transporter proteins affect the drug efflux of the cell resulting in a massive anti-apoptotic effect. The most suitable candidate involved in disrupting the normal drug efflux in HNSCC is ABCG2 (ATP-binding cassette sub-family G member 2). This transporter protein seems to be the sole cause of chemoresistance as it is highly upregulated in HNSCC {Guan, 2015 #83}. Five types of ABC transporters namely ABCC1 to ABCC5 have been considered to be prime regulators in the classical activation hyper-activation of Wnt signaling pathway in spheroid cells of HNSCC. Furthermore, the ability of spheroid cells to produce chemoresistance triggered by CSC was completely exhausted by knocking out the genes responsible for beta-catenin synthesis. This knock-out also contributed to the loss of SC markers necessary for self-renewal . Despite these evidence the actual cross talk that underlies drug resistance regulated by the interplay between Wnt Pathway and ATP binding cassette family (ABC) proteins still needs to be explained. However, further developments in unraveling the interaction between Wnt and ABC proteins will show the potential of how ABC transporter Protein channel regulates the overexpression of CSCs in HNSCCs .The mechanisms underlying the up-regulation of chemoresistance in CSC are still poorly understood and how Wnt and Druggable TargetsWnt signaling dysregulation in cancer . Some of these tiny molecules have been observed to restrain Wnt signaling cascade at diverse steps including activation of target proteins and modulation of the enzymatic activity responsible for Wnt-mediated cell growth. Thus, they hold promising benefits in devising strategies to understand Wnt signaling. It is the need of the hour to explore the potential of these promising biosynthetic molecules for promising therapy against Wnt-mediated cancer . In recent years, preclinical trials have determined a broad class of synthetic as well as natural compounds that have been implicated in Wnt signaling, a variety of approaches have been utilized: one of them is a blockade of Wnt activity by definite inhibitors. PORCN also was known as a porcupine is an enzyme mainly restrict the activation of Wnt signaling at serine residues. PORCN activity promotes the palmitoylation of the Wnt . Proffitt et al. demonstrated the sudden decrease in the expression of Wnt signaling by using small inhibitors of PORCN such as ETC-159 and Wnt-C59. Their findings shed light on how PORCN deficiency mediates the interplay between Wnt and Fzd proteins. PORCN directly stalls the production of excessive Wnt and consequently inhibits the interaction between Wnt and Fzd proteins. Therefore Wnt downstream signaling is inhibited, and cancer growth can be potentially hindered. The efficacy of Wnt inhibition by PORCN has been established in various animal models and cell lines .To suppress Wnt signaling in the rodent model of breast cancer. The effectiveness of these small molecules as inhibitors of Wnt has been established in almost all formats of cancer in various mammalian models. Moreover, PORCN inhibitors have been demonstrated to play a pivotal role in the down-regulation of Wnt target genes with minimal toxicity to the normal tissues.Furthermore, Liu et al., (2013) evidenced the synergy between inhibitors of PORCN such as LG974 and Wnt-mediated mice tumors of mammary glands, Kabiri et al., (2015) demonstrated that using Wnt-C59 (Wnt inhibitor) had no significant effect on the normal blood cell count suggesting the fact these inhibitors can block “cancerous” Wnt signaling with precision and have almost zero toxicity in normal hematopoietic SC. Furthermore, the authors speculated that other possible mechanisms for self-renewal of normal SC than the abrogated Wnt signaling could be present. They also suggested that there might be a competition between lower levels of Wnt and the adaptability of SC and this could force SC to proliferate using a different set of mechanisms. Interestingly, compelling evidences have suggested the involvement of these inhibitors in tumor regression having little or almost no side effects.In Wnt signaling biosynthetic inhibitor, Huang et al. demonstrated that XAV939 has greater efficacy installing Wnt signaling by targeting Tankyrases (TNKS). TNKS have been reported to be essential mediators for the synthesis of Axin. Indeed inhibition of Axin promotes degradation of beta-catenin resulting in the delayed or no activation of Wnt signaling .In seeking for Wnt signaling involve protein-protein interactions. Disrupting the interaction between proteins can help to promote the down-regulation of Wnt signaling. PNU74654 has been considered a promising drug for Wnt-mediated tumors . It has the potential to block the interplay between the transcription of T-cell receptor factor 4 (TCF4) and beta-catenin. It inhibits their interaction resulting in blocking of the Wnt signaling . Acetylsalicylic acid (ASA) and other anti-inflammatory and antipyretic drugs have been implicated in Wnt signaling inhibition in various cancers. Dihlmann et al., (2003) demonstrated the involvement of ASA in mediating initiation of phosphorylation at serine residues of beta-catenin. This promotes inhibition of Wnt signaling in the colorectal carcinoma. Furthermore, ASA accelerates the phosphorylation at Tyr307 in PP2A enzyme a member of Heat repeat protein family. This phosphorylation promotes the interaction and phosphorylation of the beta-catenin, resulting in the suppression of the canonical Wnt signaling . Other pharmacological approaches for limiting Wnt signaling, curcumin has been investigated extensively. It plays a vital role in the suppression of the Wnt canonical signaling, through a dose-dependent manner . Curcumin inhibits the nuclear localization of beta-catenin and also blocks the interaction between Wnt and TCF4. Furthermore, other natural compounds as extracts from green tea have been implicated in lung cancer growth inhibition. Extracts directly interact and promote the unmethylation of early methylated genes such as WIF1. WIF1 is an antagonist of Wnt signaling and thus can inhibit the downstream signaling of the Wnt signaling  1,8-cineol a terpenoid is another biologically active natural compound that has been reported to inhibit Wnt/Beta-catenin signaling cascade in HNSCCs via targeting GSK-3 an essential regulatory protein of the Wnt pathway. Glycogen synthetase Kinase 3 (GSK-3) interacts with APC that in turn destroys the active Beta-catenin complex. Using HNSCCs Cell lines Roettger et al. demonstrated that 1,8-cineol was responsible for reducing WNT11 mediated cellular proliferation in HNSCCs . Despite these powerful improvements in the search for Wnt signaling modulators, fewer drugs have been entered in the clinical use CSCs is one stumbling block that has hampered suitable drug formulation for HNSCCs. Diverse cell-population, high rate of metastasis, migration and invasion properties of the CSCs might be the major setbacks in development of an effective drug for the Wnt-mediated HNSCCs. Tumor recurrence is another hindrance in the development of a drug. Intrinsic factors such as the transmembrane proteins, incubation stages and mutations in the apoptotic machinery are also chief culprits responsible for the resistance to cytotoxic agents and radiation. Another dilemma for the development of effective drug therapy is the self-renewal capacity of the cancer stem cells. Radiation therapy has little or no influence on the CSCs as they can produce new stem cells instantly . OMP-54F28, a chimeric product of human IgG1 and frizzled protein Fzd8 have been implicated in reducing growth in pancreatic cancer cells. Interestingly, this drug has been entered in different clinical trials, but results are still to be reported. Only one work, a phase I clinical trials , underlined as OMP-54F28 was well tolerated in patients with solid tumors. Indubitably more research is needed to address the role of Wnt signaling modulators in cancer.Among natural inhibitors of Wnt signaling in tumors. Different preclinical trials have opened new horizons, searching for new therapeutic interventions are still required for Wnt-mediated cancer. Furthermore, strategies and techniques are required to cope with CSC produced through aberrant Wnt-mediated signaling. As these cells share the same characteristics  as the normal human SC, it is necessary to devise methodologies that will be more effective and precise in treating such CSC. Wnt signaling is the main mediator for the production of CSC concerning HNSCC. Evaluating the various aspects of signaling can diversify insight into this crucial pathway and will help in understanding the cross-talk between different cellular signaling pathways. This will enable to develop broad therapeutic interventions for both eradicating as well as coping with the disease relapse in HNSCCs. In conclusion, a massive improvement is still required in developing an effective therapeutic intervention for targeting Cancer is a multifactorial disease and targeting a specific pathway in such a complex pathology is hindered by several obstacles. Toxicity, allergic reaction, immune suppression, and chemo-resistance, non-specific off-target binding are some of the several factors that impede the tailored treatment of cancer. Overcoming such hurdles can enhance the chances of therapy to maximum potential. Moreover, improving drug development and efficacy by choosing the right target or using a combination of drugs can narrow the possible side effects and guarantee the best therapeutic value. Complex and profound studies on the efficacy, safety, and toxicity of candidate drugs are fundamental to discover novel agents that potentially have low side effects and great medical benefits overcoming health risks for cancer patients.The authors declare no conflict of interest.


Precise control of inflammatory gene expression is critical for effective host defense without excessive tissue damage. The principal regulator of inflammatory gene expression is nuclear factor kappa B (NFκB), a transcription factor. Nuclear NFκB activity is controlled by IκB proteins, whose stimulus-responsive degradation and re-synthesis provide for transient or dynamic regulation. The IκB-NFκB signaling module receives input signals from a variety of pathogen sensors, such as toll-like receptors (TLRs). The molecular components and mechanisms of NFκB signaling are well-understood and have been reviewed elsewhere in detail. Here we review the molecular mechanisms that mediate cross-regulation of TLR-IκB-NFκB signal transduction by signaling pathways that do not activate NFκB themselves, such as interferon signaling pathways. We distinguish between potential regulatory crosstalk mechanisms that (i) occur proximal to TLRs and thus may have stimulus-specific effects, (ii) affect the core IκB-NFκB signaling module to modulate NFκB activation in response to several stimuli. We review some well-documented examples of molecular crosstalk mechanisms and indicate other potential mechanisms whose physiological roles require further study. By. By20). In addition to upregulating TLR expression, IFNγ also upregulates expression of TLR accessory proteins , such assocs1−/− mice, which suggests that the primary role of SOCS1 is to restrain IFNγ-dependent inflammation and pathology.While IFNγ upregulates the expression of TLRs and accessory proteins that promote inflammatory responses, it also upregulates negative feedback regulators to maintain homeostasis . To enabmyd88 mRNA is IFNγ-inducible  to propagate signals downstream , 55. Thenducible . Furthernducible . HoweverSignal transduction from TLRs to NFκB involves recruitment of several enzymes to the TLR signaling complex . The recPeli1) is dependent on IFN-regulatory factor 3 (IRF3), evidence suggests Peli1 is also a target gene of ISGF3, which is induced by type I IFNs (Similar to TNF receptor 1 (TNFR1), TRIF engages the adaptor protein tumor necrosis TNFR1-associated death domain protein (TRADD) and the kinase receptor-interacting protein (RIP)1 , 67. NFκe I IFNs . Whethere I IFNs . HoweverThe primary E3 ubiquitin ligase that transduces signals from MyD88 to IKK is TRAF6 –73. DownA20 is a highly inducible NFκB target gene that attenuates cytokine- and pathogen-mediated inflammatory signaling , 77. Lostnip1−/−) may lead to embryonic lethality  is a TNF-inducible binding partner of A20 –86. ABINethality . Tnip1−/aneously , 90. ABIaneously , 91. TheZc3h12a−/− mice develop lymphadenopathy, splenomegaly, growth retardation, and chronic autoimmunity and die prematurely  is a TNF-, IL1β-, and IL4-inducible deubiquitinase that negatively regulates NFκB activity –94. In tmaturely , 93.nfkbia mRNA, which encodes IκBα, can mediate cross-regulation of NFκB activity (nfkbia (nfkbia (nfkbia is controlled by eukaryotic initiation factor (elF)2α and eIF4E [J. , PERK (pancreatic eIF2α kinase/RNA-dependent-protein-kinase-like endoplasmic-reticulum kinase), and GCN2   , 99, 101sible-2) , 101.nfkbia translation and enhance NFκB activity by inhibiting the phosphorylation and activation of eIF4E  permits multiple layers of inflammatory regulation , 107. ItMaintaining a delicate balance between effective host defense and deleterious inflammatory responses requires precise control of NFκB signaling . Multiplin silico that may be prohibitive in vitro or ex vivo  that control NFκB-dependent gene expression , 121. ThAA conducted the literature review, prepared figures, and wrote the manuscript. AH provided supervision, outlined the scope, and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The segregation of cells with distinct regional identity underlies formation of a sharp border, which in some tissues serves to organise a boundary signaling centre. It is unclear whether or how border sharpness is coordinated with induction of boundary-specific gene expression. We show that forward signaling of EphA4 is required for border sharpening and induction of boundary cells in the zebrafish hindbrain, which we find both require kinase-dependent signaling, with a lesser input of PDZ domain-dependent signaling. We find that boundary-specific gene expression is regulated by myosin II phosphorylation, which increases actomyosin contraction downstream of EphA4 signaling. Myosin phosphorylation leads to nuclear translocation of Taz, which together with Tead1a is required for boundary marker expression. Since actomyosin contraction maintains sharp borders, there is direct coupling of border sharpness to boundary cell induction that ensures correct organisation of signaling centres. During embryo development, sharp borders form at the interface of adjacent tissues and between domains within tissues that have a different regional identity. These borders are generated by cell segregation mechanisms that establish and maintain a precise organisation of tissues . At someEph receptor and ephrin signaling has major role in cell segregation and border sharpening in many tissues in vertebrates . Eph recEph receptors and ephrins that have a high affinity for each other are expressed in complementary domains in many tissues , such thrfng, which by inhibiting neurogenesis promotes the maintenance of boundary cells , each with a distinct anteroposterior identity and demarcated by borders across which cell intermingling is restricted . These bry cells . Boundarry cells , and expry cells . In the ry cells . Clues t markers . These fepha4-expressing cells and are up-regulated by forward signaling. By creating a series of truncation and point mutants in epha4, we show that kinase-dependent and PDZ domain-dependent signaling both contribute to regulation of border sharpening and boundary-specific gene expression. We find that boundary marker expression is regulated by myosin II phosphorylation that occurs downstream of EphA4 activation and increases mechanical tension at segment borders. Mechanotransduction that induces boundary marker expression is mediated by nuclear translocation of Taz. The regulation of actomysosin contraction by Eph signaling thus couples the maintenance of sharp borders and induction of a boundary signaling centre.We set out to dissect mechanisms of signaling that underlie border sharpening and boundary cell specification in the zebrafish hindbrain. EphA4 and ephrinB3 act as a signaling pair since knockdown of either component disrupts the same segment boundaries . We findepha4 (epha4a) is expressed in r3 and r5 (ephrinb3 (efnb3b) in r2, r4 and r6  which enables sensitive fluorescent detection of multiple transcripts , deleting most of the tyrosine kinase domain and all C-terminal domains, and thus completely lacks forward signaling but potentially still activates reverse signaling. The kinase-dead mutant (KDephA4) replaces a lysine residue essential for kinase function with methionine. The ΔPDZBDepha4 mutant is truncated at residue 994 which removes the five C-terminal amino acids containing the PDZ binding domain (PDZBD) motif.Knockdown of interact : r2/r3, interact ; there i5 border . To test5 border . The nulepha4 has the same phenotype described previously for epha4 knockdown, with loss of rfng expression at the r2/r3, r3/r4 and r5/r6 borders  and actomyosin contraction  were injected into one-cell-stage embryos. All injections were done in pathways . The ants  and processed for immunostaining or in situ hybridization.ephrinb3b mutants were generated using TALENs designed and constructed as previously outlined  containing three silent mutations at the gRNA target site, the K658M mutation and an additional silent mutation that generated an RsaI restriction site. Mutations were identified by amplicon restriction using restriction enzymes or T7 endonuclease I (#M0302L NEB) and verified by sequencing. A fish was identified carrying the K658M mutation together with a 6 bp deletion affecting three additional residues (649-651) in the kinase domain.mypt1, yap1, taz, tead1a and tead3a, 3 to 5 crRNAs targeting the same gene (Key resources table) were obtained from Integrated DNA Technologies Inc . crRNAs were annealed with equimolar amounts of tracRNA and 100 to 150 pg of each gRNA were co-injected with Cas9 protein. The generation of deletions was validated by PCR and in some cases by detection of mismatches by T7 endonuclease I digestion . Anti-EphA4 was described previously . Actin wegr2b  using reagents obtained from Molecular Instruments (In Situ HCR v3.0) and the method described by For in situ hybridization, embryos were fixed in 4% PFA overnight at 4°C and kept in methanol at −20°C prior to processing. The probes used have been previously described: egr2b , rfng ; Robb Krumlauf (Reviewer #2).Thank you for submitting your article "Actomyosin regulation by Eph receptor signaling couples boundary cell formation to border sharpness" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:This interesting study by Cayuso et al. addresses ae long standing question of how border sharpening and boundary cell formation are coupled. By examining these events in the developing zebrafish hindbrain, they show that boundary cell markers are induced in EphA4-expressing cells and are up-regulated by forward signaling in rhombomeres 3 and 5. They show that myosin II phosphorylation downstream of EphA4 activation increases mechanical tension at segment borders, in turn inducing boundary cell markers via nuclear translocation of Taz.Their work couples for the first time boundary sharpening by mechanical forces with the induction of boundary-specific genes, via EphA4 forward signaling. The findings are highly significant and novel. While the study is very well done, a few changes described below would improve the paper and render it ready for publication. The detailed reviews are included below for additional clarification.Essential revisions:-/-epha4 at 16hpf presented in Figure 6D is currently not very convincing. The authors' case would be strengthened by presenting the Taz phenotype in -/-epha4 at 18hpf.1) The Taz phenotype in 2) Genotyping data for CRISPants should be added as well as validation of the effects of the MO on protein levels should be added.3) Regarding whether EphA4 forward signaling drives all the downstream actin rearrangement/Taz localization, the data suggests that this occurs via forward signaling, but are not conclusive. While adding data from a forward signaling mutant would be optimal, this is not essential but would ask that you soften this point by modifying text.Reviewer #1:epha4-expressing cells and are up-regulated by forward signaling. Moreover, they demonstrate that myosin II phosphorylation downstream of epha4 activation increases mechanical tension at segment borders, which in turn results in induction of boundary cell markers via nuclear translocation of Taz. Thus, Eph signaling couples border sharpening with boundary cell formation through mechanotransduction and Taz/Tead nuclear regulation of gene expression. These findings provide very important novel insights and will be of great interest to the readers of eLife.This elegant study addresses one long standing and elusive key question concerning rhombomere (r) compartment formation in the developing hindbrain, namely how border sharpening and boundary cell formation are coupled. Jordi Cayuso and colleagues now beautifully identify the underlying mechanism in the hindbrain of the zebrafish embryo. They show that, in r3 and r5, boundary cell markers are induced in epha4 signaling to regulation of border gene expression by Taz-mediated mechanotransduction is provided in Figure 6. However, the presentation of data in Figure 6D (-/-epha4) must be improved. First, the stage of Figure 6D is not mentioned on the panel nor in the figure legend. I assume is 16phf, as in Figure 6B. If so, then the distribution/placement of the yellow arrowheads when comparing Figure 6B and D makes difficult to appreciate the described reduction in nuclear Taz at the affected boundaries. Maybe using asterisks in addition to yellow arrowheads could help. Also, as it might be difficult to fully appreciate the defect in Taz localization at the 16hpf stage, the authors may add a quantification of the results as they did for MO-mypt1 in Figure 6I. Nonetheless, it appears that the nuclear localisation of Taz at boundaries at 16hpf is still not fully refined and may suffer from a certain variability, even among wild type embryos. In contrast, Taz expression pattern at boundaries is very clear at 18hpf, as in Figure 6C. I believe therefore the analysis would very much benefit from presenting the Taz phenotype in -/-epha4 at 18hpf, which would further strengthen the authors' case. Lastly, the stage in Figure 6E should be mentioned as well – i.e. 18hpf, and Figure 6G, G', H, H' are not 14hpf but 18hpf.A critical demonstration linking Reviewer #2:This is a very interesting and comprehensive study which investigates the fundamental question of whether morphogenetic processes, associated with sharpening of inter-rhombomeric boundaries in the zebrafish hindbrain, are coupled with cell fate determination events that create unique boundary cells. They utilize gene editing to create an allelic series of EphA4 genes to clearly demonstrate that forward signaling from EphA4 is required to establish boundary cell identities and this process involves dual inputs of kinase and PDZ signaling. These experiments indicate that there is a tight coupling of boundary sharpening and formation of boundary cell identities regulated by EphA4.While published studies from these authors and other have previously analyzed events and processes in boundary sharpening this study provides novel insight into mechanisms of cell fate determination modulated by EphA4 signaling that govern formation of boundary cell identity. They show that mechanical tension gated by phosphorylation of MLC directly impacts the regulation of boundary cell identity. This involves actomyosin dependent nuclear localization of Taz that regulates boundary cell. identity markers.This a very strong paper because the authors elegantly show through genetic manipulations and cellular analyses precisely how the opposing domains of EphA4 and Efnb3 trigger a series of events that coordinates morphogenetic process of cell sharpening directly to cell differentiation. This is important because it provides a means for regulating the neural progenitor stem population at rhombomere boundaries. These findings have significant relevance beyond the formation hindbrain boundaries as this type of signaling is important in other contexts where cell sharpening and differentiation programs appear to correlate with EphA4 signaling.The figures and data supporting the conclusions are very clear and beautifully presented. I believe this is very high quality work, with novel insight and broad general interest to cell and developmental biologists. My only comment is that I would have liked to have seen a summary/model that illustrates the steps in these processes. I believe it would help tie this together and reinforce the findings with the reader.Reviewer #3:eLife. Yet, there are some weaknesses in the data that should be addressed prior to its acceptance.In this manuscript, Cayuso et al. report that forward activation of EphA4 via the kinase and PDZ domains leads to actomyosin phosphorylation and actin contraction at segment borders, which in turn increases nuclear localization of Taz that induces the expression of boundary markers by interacting with Tead1a. This is a very interesting work, which couples for the first time boundary sharpening by mechanical forces with the induction of boundary-specific genes, via EphA4 forward signaling. These findings are significant and novel. Therefore, in principal I fully support its publication in - The study uses many types of KO and KD techniques to eliminate the expression of multiple genes. However, no demonstration of the genotypes of the Crisp-Cas lines/Crispr-Cas transiently injected embryos as well as the loss of protein expression in the MO-injected embryos is provided to support the genetic and/or expression loss of the desired sequence/protein. These validations are necessary in the manuscript.rfng and wnt is induced by the forward signaling of EphA4, these data are not sufficient to conclude that the entire pathway upstream to rfng expression is mainly mediated through the forward signaling. Only by using of the different EphA4 mutants in additional levels the pathway the authors can argue that the mechanical stabilization and Taz nuclear localization in the borders is mediated via the forward signaling.- A general EphA4 KO led to loss of pMLC in r2/r3, r3/r4 r5/r6 borders, as well as prevented the nuclear localization of Taz. However, there is no confirmation that these effects occur via the forward signaling only. Although the authors showed that the border expression of mypt1 KO. This is necessary in order to fully support the suggested model. Moreover, also in the rescues it would be nice to show not only the rfng expression rescue but the actual rescue if the actin cables/pMLC, as these are the actual readouts of the mechanical forces induced by EphA4.- The authors don't show the pattern of the actin cables/pMLC in the rfng in all boundaries, even in those not affected by epha4 KO. Moreover, Tead1a KO leads to loss of rfng in a much milder way than Taz KO. These findings have to be explained in view of the suggested model of activation.- Taz KO/KD leads to loss of - The quality of the data in Figure 6 is poor. The nuclear localization data of Taz upon the different manipulations are hard to evaluate by the DAPI+ immunostaining. This is a very significant part of the study, and the authors have to present the cytoplasm-to-nuclear localization in a better way. Essential revisions:-/- at 16hpf presented in Figure 6D is currently not very convincing. The authors' case would be strengthened by presenting the Taz phenotype in epha4-/- at 18hpf.1) The Taz phenotype in epha4We have repeated the immunostainings at 18 hpf and present better data. We now also show examples of nuclear localisation in different boundaries in Figure 6—figure supplement 1. We have clarified that there is an increase in Taz levels at boundaries, consistent with evidence that the actin cytoskeleton can regulate the stability as well as nuclear localisation of Taz . Not all boundary cells have nuclear Taz, which may reflect dynamic nuclear localisation.2) Genotyping data for CRISPants should be added as well as validation of the effects of the MO on protein levels should be added.We now present these data in Figure 2—figure supplement 1 and describe our and previous validations of MOs in Materials and methods (subsection “Generation of mutants”). All phenotypes from MO knockdowns have been validated by Crispr-mediated knockout. We discuss this in more detail in the response to reviewer 3.3) Regarding whether EphA4 forward signaling drives all the downstream actin rearrangement/Taz localization, the data suggests that this occurs via forward signaling, but are not conclusive. While adding data from a forward signaling mutant would be optimal, this is not essential but would ask that you soften this point by modifying text.This is a good point, as analysing Taz localisation in the different mutants would test whether it correlates with boundary cell formation. We have now analysed Taz localisation in the forward signaling mutants (truncated and kinase dead EphA4) and find that the same boundaries are disrupted as in the null mutant.Reviewer #1:-/-) must be improved. First, the stage of Figure 6D is not mentioned on the panel nor in the figure legend. I assume is 16phf, as in Figure 6B. If so, then the distribution/placement of the yellow arrowheads when comparing Figure 6B and D makes difficult to appreciate the described reduction in nuclear Taz at the affected boundaries. Maybe using asterisks in addition to yellow arrowheads could help. Also, as it might be difficult to fully appreciate the defect in Taz localization at the 16hpf stage, the authors may add a quantification of the results as they did for MO-mypt1 in Figure 6I. Nonetheless, it appears that the nuclear localisation of Taz at boundaries at 16hpf is still not fully refined and may suffer from a certain variability, even among wild type embryos. In contrast, Taz expression pattern at boundaries is very clear at 18hpf, as in Figure 6C. I believe therefore the analysis would very much benefit from presenting the Taz phenotype in epha4-/- at 18hpf, which would further strengthen the authors' case. Lastly, the stage in Figure 6E should be mentioned as well – i.e. 18hpf, and Figure 6G, G', H, H' are not 14hpf but 18hpf.[…] A critical demonstration linking epha4 signaling to regulation of border gene expression by Taz-mediated mechanotransduction is provided in Figure 6. However, the presentation of data in Figure 6D  that there is an increase in Taz levels at boundaries, consistent with evidence that the actin cytoskeleton can regulate the stability as well as nuclear localisation of Taz. Not all boundary cells have nuclear Taz, which may reflect dynamic nuclear localisation. We now mention the embryo stage for each panel on the figure and in the legend.Reviewer #2:[…] My only comment is that I would have liked to have seen a summary/model that illustrates the steps in these processes. I believe it would help tie this together and reinforce the findings with the reader.Thank you for these helpful ideas. We now include a summary model as Figure 7. We tried to provide a broader context on possible relevance to other tissues in the subsection “Concluding Perspectives”, and we have now improved this section.Reviewer #3:[…] There are some weaknesses in the data that should be addressed prior to its acceptance.- The study uses many types of KO and KD techniques to eliminate the expression of multiple genes. However, no demonstration of the genotypes of the Crisp-Cas lines/Crispr-Cas transiently injected embryos as well as the loss of protein expression in the MO-injected embryos is provided to support the genetic and/or expression loss of the desired sequence/protein. These validations are necessary in the manuscript.mypt1 MO, there is to our knowledge no antibody available to detect the zebrafish protein. mypt1 MO and mutant embryos were previously shown  to alter cell shape and to increase phosphorylation of MLC, which is a target of Mypt1. Likewise, we find that mypt1 morphant and mutant embryos have the same phenotype (increased boundary marker expression). The yap1 MO has been validated by Fukui et al. (2014) by rescue and by analysis of splicing, and also used by Cebola et al., Nat Cell Biol 17, 615-626, 2015; Agarwala et al., eLife e08201, 2015; Kozlovskaja-Gumbriene et al., eLife e21049, 2017.We now include our data for the deletions in transient gene knockouts and the relevant sequence information for mutants . The MOs used have previously been described and validated by others – we now make this clearer in the Materials and methods (subsection “Generation of mutants”). For all MO-mediated knockdowns, we also carried out transient Crispr-mediated knockouts to ascertain whether this leads to the same phenotype. When possible, we validated the effect of MO on protein level. We show that the Taz MO  leads to a decreased level of protein expression . For the - A general EphA4 KO led to loss of pMLC in r2/r3, r3/r4 r5/r6 borders, as well as prevented the nuclear localization of Taz. However, there is no confirmation that these effects occur via the forward signaling only. Although the authors showed that the border expression of rfng and wnt is induced by the forward signaling of EphA4, these data are not sufficient to conclude that the entire pathway upstream to rfng expression is mainly mediated through the forward signaling. Only by using of the different EphA4 mutants in additional levels the pathway the authors can argue that the mechanical stabilization and Taz nuclear localization in the borders is mediated via the forward signaling.mypt1 knockdown.We have now analysed Taz localisation in the forward signaling mutants (truncated and kinase dead EphA4) and find that the same boundaries are disrupted as in the null mutant. As we have not analysed pMLC in the forward signaling mutants, we have softened our discussion of this point . A number of studies have shown that forward signaling is the main driver of segregation and border sharpening, and acts by increasing actomyosin tension . However, we have shown in a cell culture model that reverse signaling leads to weaker repulsion that can contribute to cell segregation . Nevertheless, if reverse signaling does lead to some increase in pMLC at boundaries, this is not sufficient to induce boundary gene expression in forward signaling mutants, even after amplifying tension by - The authors don't show the pattern of the actin cables/pMLC in the mypt1 KO. This is necessary in order to fully support the suggested model. Moreover, also in the rescues it would be nice to show not only the rfng expression rescue but the actual rescue if the actin cables/pMLC, as these are the actual readouts of the mechanical forces induced by EphA4.mypt1 knockdown as this had been shown by Gutzman and Sive (2010) by Western blot analysis and immunostaining of the hindbrain. They were not able to detect pMLC at hindbrain boundaries in wild type embryos, but did when pMLC increases in the mypt1 mutant. Their Western blot analysis revealed a five-fold increase in pMLC after mypt1 knockdown. By optimising the immunostaining we were able to detect pMLC at hindbrain boundaries in wild type embryos. This extends the previous findings of myosin and actin cables at boundaries, which Calzolari et al. (2014) showed are EphA4-dependent. We agree that it would be nice to show that mypt1 knockdown increases pMLC at boundaries in the EphA4 mutant, but feel that our findings together with the published work give compelling evidence for the pathway from EphA4 –> pMLC –> Taz –> boundary gene expression.We do not show the increase in MLC phosphorylation after - Taz KO/KD leads to loss of rfng in all boundaries, even in those not affected by epha4 KO. Moreover, Tead1a KO leads to loss of rfng in a much milder way than Taz KO. These findings have to be explained in view of the suggested model of activation.taz and tead1a are required for boundary marker expression”). That Taz knockout affects all hindbrain boundaries is consistent with increased pMLC and Taz nuclear localisation at all segment borders. EphB4 and ephrinB2 are segmentally expressed and drive cell segregation, and likely contribute to pMLC and boundary marker expression at the r1/r2, r5/r6 and r6/r7 borders that are not affected in the EphA4 mutants (mentioned in the Introduction and Results). However, we have found that there is some redundancy at these borders, and that at least one further Eph-ephrin pair is segmentally expressed. We now mention (see the aforementioned subsection) that the milder phenotype of tead1a knockout may be due to partial redundancy with family members that have not yet been analysed.We have clarified these points in the revised manuscript . We also clarified  that there is an increase in Taz levels at boundaries, consistent with evidence that the actin cytoskeleton can regulate the stability of Taz. Not all boundary cells have nuclear Taz, which may reflect dynamic nuclear localisation. We now show examples of nuclear localisation in different boundaries .We agree that the quality of the staining is not great, which unfortunately reflects that the Taz antibody is tricky to use. For example, Voltes et al. (2019) analysed later stages and detected increased Taz protein but not its nuclear localisation at boundaries (they obtained indirect evidence for pathway activation by using a Tead reporter gene). We tested different conditions and found that it is important to carry out antigen retrieval (now mentioned in the subsection “Immunohistochemistry and in situ hybridization”). We have carried out stainings for


Giardia duodenalis is an important intestinal protozoan infecting both humans and animals, causing significant public health concern and immeasurable economic losses to animal husbandry. Sheep and goats have been reported as common reservoirs of G. duodenalis, but only a limited amount of information is available for native breeds of these small ruminants in China. The present study investigated the prevalence and multilocus genotypes of G. duodenalis in black-boned sheep and goats, two important native breeds in Yunnan Province, southwestern China.G. duodenalis was determined by the nested PCR targeting the β-giardin (bg) gene. The assemblages and multilocus genotypes (MLGs) were investigated based on analyses of three genetic loci, i.e. bg, glutamate dehydrogenase (gdh) and triosephosphate isomerase (tpi).Fecal samples were collected from 336 black-boned goats and 325 black-boned sheep from five counties  of Yunnan Province and the genomic DNA was extracted from these feces. The prevalence of Giardia duodenalis infection was detected in both black-boned sheep and goats, and the prevalence of G. duodenalis in black-boned sheep  was significantly higher  than that in black-boned goats . Significant differences in prevalence were also observed in goats and sheep from different counties  and age groups . Zoonotic assemblage A and animal-specific assemblage E were identified in both black-boned sheep and goats with the latter as the predominant assemblage. Based on sequences obtained from the three genetic loci , 16 MLGs were obtained in black-boned sheep and goats, including 15 MLGs in assemblage E and one MLG in assemblage A.G. duodenalis, but also revealed high genetic variations in G. duodenalis assemblages. The findings of the present study also provide baseline data for preventing and controlling G. duodenalis infection in black-boned sheep and goats in Yunnan Province.Our results not only extended the host range of  Giardia duodenalis  is one of the most common parasites that causes intestinal infections of humans and various animals worldwide  . We also found that the occurrence of G. duodenalis decreased as age increased in these animals, with the highest prevalence found in animals < 2 months , followed by 3–6 months , 7–12 months  and > 12 months  .In the present study, of the 661 animal fecal specimens examined, 87 were G. duodenalis were found among different age groups of both black-boned sheep  and goats . Of the 325 fecal samples collected from the four age groups of black-boned sheep, the highest prevalence of G. duodenalis was found for the 0–2-month group ; however, G. duodenalis was not found in this age group of black-boned goats (0/3) due to the number of samples being too small. The highest prevalence of G. duodenalis was found in 3–6 month black-boned goats . In black-boned sheep, the lowest prevalence was found in sheep aged more than 12 months , while only one of 200 (0.5%) black-boned goats in this age group was found with G. duodenalis infection  of G. duodenalis in both black-boned sheep and goats. Of the 71 bg-positive black-boned sheep samples, 70 isolates from three counties and four age groups belonged to the animal-specific assemblage E, while only one isolate from a 3-month sheep in Shilin county was identified as the zoonotic assemblage A. Ten isolates of assemblage E were detected in 16 bg-positive black-boned goat samples and all 6 assemblage A isolates were detected in Meishui County  and five novel subtypes    and three novel subtypes  , two previously published sequence with no subtype name (named as E32 and E33) were identified (Table tpi locus two known assemblage A subtypes (A1 and A4), one known assemblage E subtype (E3) and one previously published sequence with no subtype name (named as E28) were identified , and two samples were mixed infection with assemblages E and A  and 4.8% (16/336). The prevalence in black-boned sheep was higher than in other kinds of sheep previously reported in China (1.82–13.11%) [G. duodenalis prevalence tends to decrease, with a significant difference being observed among different age groups. Our results were consistent with previous reports [G. duodenalis than adult sheep.In this investigation, –13.11%) –24, seve–13.11%) , 41–47, –13.11%) –56. As b reports , 56, 57,G. duodenalis prevalence in black-boned goats was slightly higher than that reported in some provinces of China [G. duodenalis among different age groups, and the prevalence in black-boned goat kids (< 6 months)  was significantly higher than in black-boned goats over 6 months . A higher G. duodenalis prevalence in goat kids has also been observed in other studies [G. duodenalis was detected in black-boned goats of this age group due to the limited number of sampled animals (n = 3).Similarly, the of China , 25–27 aof China . Howeverof China , 59–63.  3.0%, 9/4. A high studies , 59, 64.G. duodenalis assemblage A. Interestingly, genomic analysis of assemblages A, B and E showed that the nucleotide similarity between assemblages A and E is much higher than that between assemblages A and B [G. duodenalis to humans.Assemblage E was the most prevalent genotype in both black-boned sheep 70/71) and goats (10/16) in our study. Assemblage E was also reported as the predominate genotype in sheep and goats in other studies , 61, 62./71 and g A and B . These rgdh and tpi) of 87 bg-positive samples were analyzed by the multilocus genotyping tool with high resolution [gdh locus, four assemblage E subtypes and one assemblage A subtype at the tpi locus were identified in black-boned sheep. Two assemblage E subtypes and one assemblage A subtype at the gdh locus, and two assemblage E subtypes and two assemblage A subtypes at the tpi locus were observed in black-boned goats  and the presence of the assemblage A MLGs suggest that the black-boned goats has higher potential in transmitting zoonotic G. duodenalis than the black-boned sheep.To further reveal the genetic variations in assemblages A and E, another two genetic loci  in black-boned goats. These results provide baseline data for preventing and controlling G. duodenalis infection in black-boned sheep and goats as well as humans.The present study revealed the presence and identified assemblages of