Ischemic heart disease (IHD) is a multifactorial pathological condition strictly related to genetic, dietary, and lifestyle factors. Its morbidity and mortality rate represent one of the most important pathological issues that today involve younger people in a stronger way than in the past. IHD clinical outcomes are difficult to treat and have a high economic impact on health care. So prevention of this pathological condition through cardioprotective maneuvers represents the first line of intervention, as already underlined by several animal and human studies. Even if the time of intervention is important to prevent severe outcomes, many studies highlight that sex-dependent responses are crucial for the result of cardioprotective procedures. In this scenario sexual hormones have revealed an important role in cardioprotective approach, as women seem to be more protected toward cardiac insults when compared to male counterparts. The aim of this mini review is to show the molecular pathways involved in cardioprotective protocols and to elucidate how sexual hormones can contribute in ameliorating or worsening the physiological responses to IHD. Ischemic heart disease (IHD) is a pathological condition characterized by reduced or absent blood flow in coronary arteries due to total or partial occlusion of these vessels by atherosclerotic plaque or blood clots formation. This condition causes improper supply of oxygen and nutrients to myocardial tissue, and, depending on ischemia duration, myocardial cell death can occur. Among all cardiovascular diseases (CVD), IHD is the leading single cause of death in Europe: IHD mortality represents 19% of deaths among men and 20% of deaths among women . Emergin2+ impairment as already reviewed elsewhere  is characterized by detrimental effects of both ischemia and reperfusion. From a clinical perspective, even if restoration of blood flow could be dangerous, it represents the only possible intervention to have better survival chance when coronary occlusion occurs, and researches are moving toward new insights to reduce IRI and improve cardiac outcomes after prolonged ischemia.Experimental and clinical studies have underlined sex differences in response to IRI, with women showing most detrimental outcomes, probably due to improper diagnosis and poorer prevention procedures than male counterparts . IHD shoNowadays it is largely accepted that there are different IRI manifestations in male and female subjects, among all ages and outcome of cardiac injury. Differences studied and results obtained suggest a possible role of sexual hormones in several animal models.Despite the traditional dualism of estrogens and androgens in conditioning the cardioprotective responses, mainly highlighted by experiments from animal models, human studies and clinical data show a more complex scenario. In this section, cardioprotective pathways activated by both estrogens and androgens are presented, with the aim to underline the weakness of sex-adapted cardioprotective strategies only focused on these mechanisms.2+ channels with a reduction of Ca2+ loading, and it regulates Ca2+ uptake in mitochondria through the extracellular signal-regulated kinases (ERK1/2)  (Estrogen binds to different receptors: estrogen receptor-α (ER-α), estrogen receptor-β (ER-β), or G-protein coupled estrogen receptor (GPR30 or GPER). Even if ER-β has a strategical role in cardioprotection, the involvement of ER-α against IRI effects is still controversial; nevertheless, consistent data suggest that estrogen-mediated protective response may rely on both ERs . Several(ERK1/2) . Despite (TNFR2) . Further (TNFR2) .Cardioprotection in female hearts involves also endogenous antioxidant systems: catalase, superoxide dismutase (SOD), glutathione (GSH), and GSH peroxidase (GPx) are highly expressed if compared to male counterparts . MoreoveOther animal studies centered their work in the evaluation of the effect of testosterone in cardioprotection, and like estrogen, androgens can activate both genomic and non-genomic responses by binding to androgen receptors (AR) .2+ release channels (SERCA) and activation of L-type calcium channels, and it has been observed that in severe ischemic condition it may contribute to Ca2+ overload suggesting that specific context may modulate the response to this sexual hormone  before the infarcting ischemia; it could be performed through mechanical or pharmacological stimulation or with exercise . AccordiGiven the effect of PreC, and the implications of sex hormones in cardioprotection, it could be interesting to evaluate if there are different responses to PreC in male and female subjects.In order to assess any difference between sexes in PreC followed by ischemia, it has been evidenced in animal models that not only sex but also age influences the resulting cardioprotection: in fact, young female animals show no response to PreC presumably due to incomplete sexual maturation, while male subjects show cardioprotection after PreC at any age .ATP channels and heat shock proteins (HSP) that are constitutively more activated in females  is considered among cardioprotective maneuvers; it consists of brief cycles of ischemia and reperfusion in a peripheral organ or tissue (even in arms or legs), remote from the heart . It can In this brief report, we have outlined different responses in female and male hearts to IRI, and in particular we focused on a primary role of estrogens in cardioprotection. Some of the most studied cardioprotective maneuvers, in order to reduce IRI, have been described with a particular focus on females and males’ different responses. The overview outlined here shows that differences between sexes in cardioprotective interventions could be linked, but not exclusively, to the physiological role of sexual hormones that change throughout the lifespan highlighting a complex relationship toward age and sex in response to cardioprotective maneuvers. In conclusion, sex and age differences have to be considered in cardioprotection in order to optimize the clinical application of these procedures. As GQ, MG, SA, and CP conceived the study and its design, and revised the manuscript for important intellectual content. GQ, MG, and CP wrote the manuscript. FG created the figure and revised the manuscript. All authors read, edited, and approved the final 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.


IDH-wildtype Glioblastoma Multiforme (GBM) using combination therapy has been shown to be synergistic  reports that an estimated 9.6 million people died from cancer in 2018  and poor patient survival. They utilized the Oncomine database, Gene Expression Profiling Interactive Analysis (GEPIA), and Integrative Molecular Database of Hepatocellular Carcinoma (HCCDB) to analyze the expression of hepatocellular CSC (HCSC) markers in 364 liver cancer samples. The correlation of HCSC markers to tumor-infiltrating immune cells was tested by Tumor Immune Estimation Resource (TIMER). Out of 10 differentially deregulated HCSC markers, 3  were highly expressed and had a positive correlation with poor prognosis. In contrast, the expression of CD13, CD34, and ALDH1A1 was associated with prolonged overall survival. The authors noted that SOX12 in particular might constitute a therapeutic target for hepatocellular carcinoma. Complementary to that work is the Li and Zhu manuscript, which reviewed recent advances in experimental studies on liver CSCs. They showed an update on the latest advances in experimental studies on non-coding RNAs (ncRNAs), oncogenes, and oncoproteins, with a particular focus on three pathways: the Wnt/β-catenin signaling pathway, phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway, and interleukin 6/Janus kinase 2/signal transducer and activator of transcription 3 (IL6/JAK2/STAT3) signaling pathway. Known associated roles for more than 30 CSC-related genes were discussed in detail. In particular, they conclude that octamer 4 (OCT4) and NANOG are important functional genes that play a pivotal role in liver CSC regulation and HCC prognosis.Using bioinformatics tools, Tian et al., applied a weighted gene co-expression network analysis on gene expression data sets from head and neck squamous cell carcinomas (HNSCC) to define an mRNA expression-based stemness index consisting of genes that served as prognostic markers. Raw data for 643 samples were downloaded from the Cancer Genome Atlas (TCGA) database and the Gene Expression Omnibus (GEO) website. The study showed that the combined deregulated expression of eight stem-cell-related markers  has a powerful capacity for overall survival prediction. They support their bioinformatics data by detecting the level of expression in HNSCC cell lines. The authors concluded that their proposed model could contribute to a better understanding of the role of HNSCC stem cells in developing targeted therapy.Another bioinformatics-based paper, published by Li et al. This work investigated the association of SET Domain Containing 2 (SETD2) gene mutations/variants with clinical features and prognosis in patients with Myelodysplastic syndrome (MDS). SETD2 is a transcriptional regulator and has been previously shown to be required for the self-renewal of hematopoietic stem cells (HSCs), and SETD2-deficient HSCs were shown to contribute to the development of MDS. Using targeted next-generation sequencing, the results indicated that out of 203 patients with MDS, 37 patients had SETD2 gene mutations/variants, and these patients exhibited a significantly increased frequency of TP53 mutations. Low expression of SETD2 in patient tumor cells was identified as a risk factor for progression-free survival (PFS). The study concluded that SETD2 deficiency contributes to genomic instability and is associated with unfavorable prognosis in patients with myelodysplastic syndrome.An experimentally based approach was presented in the manuscript authored by Azzarelli, which discussed the emerging 3D models of glioblastoma that overcome certain limitations of monolayer cultures. The author concluded that glioblastoma brain organoids provide the opportunity to study CSC lineages and serve as tools to predict tumor progression and treatment response. In a second review, Xu et al. discussed the role of N6-methyladenosine (m6A) in the differentiation of CSCs. The authors highlighted that targeting m6A modification of CSCs constitutes a yet not fully explored option for drug treatment of cancer. The third review was presented by Alhabbab, and it described how CSCs employ various mechanisms to modulate the immune system response. The review outlined the recent knowledge for the interactions between CSCs' common markers, including CD133, CD90, EpCAM, CD44, ALDH, and EGFRVIII, and the immune system. Current information on CAR T cell genetic engineering and signaling, CAR T cells, and the barriers in using CAR T cells as immunotherapy to treat solid cancers in the context of targeting CSCs were detailed.Three more review articles were published in this collection. The first was a mini review by Manzo investigated the nature of tumor growth within a mathematical model, which assumes tumors encompass CSCs that behave similarly to para-embryonic stem cells and divide into a hierarchic sequence of CSCs and non-CSCs. Tabulating theoretical data using this model, the author identified defined mathematical relationships between CSCs and non-CSCs that were similar to experimental data. The model explains tumor progression in a modular way that recalls the propagation of tumor spheres in vitro. Furthermore, the author discussed similar features, including nature form, dimension, cell distribution, and layer compartmentation for avascular tumors, tumor spheres, and preimplantation blastocysts. The author concluded that the presented mathematical model provides further support for the para-embryonic nature of the cancer process.Finally, in a theory-based article, The research on CSCs is ongoing, and several concepts still remain to be addressed or fully explained. For example, what combinations of markers define different types of CSCs, and how does the “combined markers identity tag” correlate with therapeutic prognosis? Perhaps next-generation single-cell sequencing in combination with multiplex protein array technology could shed more light on the characteristics of CSCs and CSC markers. A unified CSC-specific interactive database for the mutational signatures and genomic instability of CSCs is likely to improve cancer research. Some questions remain: How do CSCs contribute to metastasis, and what are the therapeutics that can be given to combat CSCs movement and colonization? How can the gene expression profile of CSCs be stabilized and prevented from shifting in response to the microenvironment? What are the clinically relevant CSCs models that provide highly efficient translational protocols that can be utilized in a clinical setting?Taken together, the variety of the authors' topic contributions, either by focused reviews, theoretical considerations, or research articles, has shed light on current advances in CSC biology and support further approaches for integrative CSC research.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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 COVID-19 pandemic has drastically affected healthcare systems throughout the world. Though all domains of healthcare are busy battling this deadly pandemic, oncology care has taken a drastic hit due to cancer patients being immunocompromised and predisposed to acquire COVID-19 infection. Patients suffering from acute myeloid leukemia are at greater risk of acquiring Severe Acute Respiratory Syndrome Coronavirus 2(SARS CoV 2) infection along with developing complications related to COVID-19 due to the immunosuppression caused by the malignancy, as well as the high-intensity chemotherapy provided in acute myeloid leukemia. We report a case of 28-year-old male who was a known case of acute myeloid leukemia diagnosed three months ago, presented with high-grade fever with cough and breathlessness. Nasopharyngeal swab of the patient for SARS CoV2 by reverse transcriptase-polymerase chain reaction turned out to be positive. The patient was managed successfully with steroids, remdesavir, granulocyte colony-stimulating factor, and other supportive measures, and was discharged in a stable condition. Ever since the beginning of the COVID19 pandemic there have been emerging challenges for healthcare professionals around the globe. Patients who are immunocompromised due to malignancies, HIV, chemotherapy, or history of a solid organ transplant have emerged as the weak ring of the lot with increased risk of contracting COVID-19 and increased risk of developing serious complications such as acute respiratory distress syndrome . Acute m2 was 91 percent on room air. On systemic examination, trachea was centrally placed, there were fine crackles heard in the inframammary region of the chest, a systolic murmur was heard in the left second intercostal space which was functional, the abdomen was soft and non-tender with no hepatosplenomegaly and the patient was conscious and oriented. The patient was admitted for further evaluation and his nasopharyngeal swab for COVID-19 came positive by reverse transcriptase-polymerase chain reaction method. Blood investigations of the patient revealed pancytopenia and raised inflammatory markers with normal renal and liver function test (Table A 28-year-old male presented with the chief complaint of fever with chills along with cough and breathlessness for five days. He had a history of being diagnosed with acute myeloid leukemia three months ago, for which he had received chemotherapy cycle of 7+3 with daunorubicin and cytarabine. He had no history of hypertension, diabetes mellitus, tuberculosis or bronchial asthma. On general examination, the patient was febrile with a temperature of 101 °F, pulse of 106 beats per minute, blood pressure of 110/70 mm Hg in right arm supine position; pallor was present and SpOAcute myeloid leukemia is one of the commonest leukemia in India . In acutA detailed understanding of granulocyte colony stimulating factor and leukemia is essential in tackling a patient with acute myeloid leukemia with COVID-19. A thorough assessment of risk versus benefit ratio may help the treating physician in choosing to give granulocyte colony stimulating factor which increases alveolar macrophages and innate immunity along with increasing lymphocytes, thus helping in preventing morbidity and mortality.


Fruits are vital food resources as they are loaded with bioactive compounds varying with different stages of ripening. As the fruit ripens, a dynamic color change is observed from green to yellow to red due to the biosynthesis of pigments like chlorophyll, carotenoids, and anthocyanins. Apart from making the fruit attractive and being a visual indicator of the ripening status, pigments add value to a ripened fruit by making them a source of nutraceuticals and industrial products. As the fruit matures, it undergoes biochemical changes which alter the pigment composition of fruits.The synthesis, degradation and retention pathways of fruit pigments are mediated by hormonal, genetic, and environmental factors. Manipulation of the underlying regulatory mechanisms during fruit ripening suggests ways to enhance the desired pigments in fruits by biotechnological interventions. Here we report, in-depth insight into the dynamics of a pigment change in ripening and the regulatory mechanisms in action.This review emphasizes the role of pigments as an asset to a ripened fruit as they augment the nutritive value, antioxidant levels and the net carbon gain of fruits; pigments are a source for fruit biofortification have tremendous industrial value along with being a tool to predict the harvest. This report will be of great utility to the harvesters, traders, consumers, and natural product divisions to extract the leading nutraceutical and industrial potential of preferred pigments biosynthesized at different fruit ripening stages. Fruits are important and health-promoting food resources and are a part of our diet as a potential source of nutrients . With thFruit color is an indicator of its stage of maturity, freshness and quality and serves as an important parameter in their classification . As the Fruit color signifies a genetic trait with ecological and nutritional worth. Plants mainly make use of color for seed dispersion and to attract animals. The other adaptive challenges were entrusted on fruit color by environmental factors and domestication further resulting in diversification. The unprompted mutations occurring repeatedly in pigment biosynthetic pathways lead to variations in fruit color which were often propagated. In the recent decades, the pigmentation pattern was further enriched by introgression breeding coupled with the unravelling of genetic determinants underneath fruit pigments .The emergence of these pigments as a result of fruit ripening has multifold prospects for the growing plant. The chlorophyll content and development of chloroplasts at mature green stage affects the net carbon yield of a ripened fruit .Chlorophyll and carotenoids are the main photosynthetic pigments in plants and play a vital role in enhancing the net carbon yield of the plant . These pa and b are present throughout the photosynthetic machinery whereas carotenoids like β-carotene, violaxanthin, antheraxanthin, zeaxanthin and lutein are mainly present in antenna systems of light harvesting complexes [Chlorophyll omplexes , 16. Duromplexes  and prevomplexes . Also, aApart from their crucial role in photosynthesis, pigments like carotenoids and anthocyanins enhance the antioxidant status and therapeutic potential Table  of the fCarotenoids like β-carotene, lycopene, zeaxanthin, and lutein are the major antioxidants in fruits and act as scavengers of free radicals. Lycopene, lutein and astaxanthin and the colorless carotenoid precursors phytoene, phytofluene have also been associated with a decreased risk of cancers including prostate cancer –41, coloBixa orellana [Among the carotenoids and their cleavage products, the highest oxidation potential of 0.94 V has been reported for bixin, an apocarotenoid, obtained from the seed arils of orellana  and therorellana  as well orellana .Fig. 1InBerries have also been found to be richest source of anthocyanins among fruits, and blue berries have been reported to be the richest source of antioxidants with a TEAC  value of 14.98 mM Trolox/100 g of dry weight due to high levels of proanthocyanidins and anthocyanidin .Apart from their health benefits, pigments extracted from fruits have been found to have numerous applications as food colorants, in cosmetics, textiles & pharmaceutical industry  Fig. 1)1). ConsiAmong the several pigments present in fruits the major pigments such as chlorophyll, carotenoids, and anthocyanins possess diverse pigment functionality as they are essential for photosynthesis, possess multiple bioactivities, potent antioxidants, therapeutic properties, and industrial use, therefore an in-depth analysis of the factors regulating their biosynthesis, metabolism and storage during ripening is inevitable. This will further aid in designing biotechnological tools to enhance the production of these highly beneficial pigments in fruits.Pigment change during fruit ripening is a tightly controlled phenomenon signaled by plant growth hormone, several transcription factors, gene families, enzymes of the pigment biosynthetic pathways, and environmental stimuli . Also, ss)  involved in chlorophyll degradation [s could lead to a stay-green phenotype, which has recently been reviewed by Zhu et al. [Nr (never ripe) mutants in tomato. The variations of color in tomato clarifies that regulation of carotenoid biogenesis is unique for each species. Recently, Solanum habrochaites  was studied to decipher the molecular reasoning for green color retention. i) In SH a shift towards the β-carotene branch of carotenoid biosynthesis was found missing and both α- and β (carotene) branches were found to make equal contributions, ii) SH fruits were found to be insensitive to ethylene induced carotenogenesis, as in spite of emitting high levels of ethylene, they remained green, iii) SH fruits were found to retain the proteins related to photosynthesis and were lacking in proteins involved in conversion of chloroplasts to chromoplasts, iv) lack of carotenoid accumulation in spite of uncompromised expression of chromoplast specific genes such as PSY 1(phytoene synthase1) and LCYB (lycopene β-cyclase) in SH due to probable blocking by SNP’ (single nucleotide polymorphisms) resulting in lack of abundance of key proteins PSY1 and LCYB, v) diminished levels of homologues of fibrillin (FIB) like PAP3 (plastid lipid-associated protein) and CHRC (chromoplast-specific carotenoid-associated protein) involved in sequestration [As the fruit begins to ripen, the degradation of the green pigment chlorophyll, is initiated to promote remobilization of nutrient and promote biosynthesis of vitamins. The de-greening of fruits is important to promote detoxification of chlorophyll released from its binding proteins . Light, see Fig. , which aradation , 76. Howu et al. . Howeveru et al. . Severalu et al. , 78–81 au et al. . Besidesu et al.  elevatedu et al. , melatonu et al. , and chlu et al.  resultinu et al.  and insestration  However,stration .Fig. 2ReFIB gene was assessed and it was found that it delayed thylakoid loss in differentiating chromoplast and resulted in an increase in plastoglobuli number and thereby increased the concentrations of carotenoids like β-carotene and lycopene [The degradation of chlorophyll in fruits like tomato and pepper is accompanied by a well-regulated conversion of chloroplast into chromoplast see Fig. , accumullycopene .Fig. 3Res GLK1 and GLK2 play a crucial role in chloroplast development and accumulation of chlorophyll [APRR2-LIKE genes is carried out by TKN2 and TKN4, the two KNOX (Class I KNOTTED1-LIKE HOMEOBOX) proteins [The chlorophyll content of an unripe fruit such as tomato has been found to be directly linked to the sugar content of the ripened fruit, thereby affecting the nutritional quality and taste of a ripened fruit. Therefore, the regulatory mechanism involved in chloroplast development and accumulation of chlorophyll are crucial. DET1 (De-etiolated 1) and DDB1 (UV-damaged DNA-binding protein1) control chloroplast number, size and development as negative regulators , Also GOorophyll  along wiorophyll  , Physalis peruviana L. (orange), and Physalis alkekengi L. (red) wherein the yellow and orange fruit of Physalis showed gradual accumulation of β-carotene and lutein at varied concentration, justifying their different hues on ripening. However in the red fruit of Physalis (on being fully ripe) traces of β-carotene, and high levels of β-cryptoxanthin and zeaxanthin were reported [With the development of chromoplast the carotenoids which would determine the final fruit color are accumulated and the predominant carotenoid shape up the final form of its deposition which is crucial for color, bioavailability, and stability of the carotenoid. This phenomenon has been demonstrated for fruits of reported . Thereforeported . The regreported ). TherefIt has been shown on several occasions that secondary metabolism and, particularly, plastid metabolism is not only transcriptionally but also post-transcriptionally regulated. Recent developments in technologies based on mass-spectrometry has paved the way for a deeper coverage of protein expression. This has shifted the focus on translational and post translational protein expression in tomato and other fruits, which is currently limited to transcription, thereby adding a novel dimension to the already existing data on transcriptomic, metabolomic and genomic resources . The rols and growth hormones such as ethylene and ABA play a critical role in activation of ripening genes. Together they regulate accumulation of carotenoids (in chromoplast) and anthocyanins (in vacuoles). Among the MADS BOX family genes positively regulating ethylene signaling and carotenoid biosynthesis ripening inhibitor (RIN) is reported as the master regulator. Studies on mutants ripening-inhibitor (rin), non-ripening (nor), and Colorless-nonripening (Cnr) have led to novel findings in the ripening process of climacteric fruits like tomato [RIN (generated by CRISPR/Cas9) led to partial ripening with only 10% concentration of ethylene and carotenoids in comparison to the wild type [, which regulates the quantity and quality of carotenoid in fruit ripening [PSY1, and the other enzymes like carotene isomerase (CRTISO) and LCYE were reported to be regulated negatively by PYL9 [. In contrast, LCYB was positively regulated by PYL9, thereby limiting the entry of metabolites in the lycopene pathway and channeling the carbon towards β-carotene and ABA  [PSY1 in fruits, PSY2 in leaf tissues and PSY3 in roots under stress [PSY3 expression was induced in periods of stress like drought leading to an increase in the production of ABA and escalation of the carotenoids [recent findings it has been suggested that the PSY gene family compensates for the loss of PSY1 as reported in a yellow pepper variety, Micro Pep Yellow [The expression of carotenoid pigment is regulated at three important stages: biosynthesis, degradation, and lipoprotein sequestering structures, respectively. The regulation of carotenoid biosynthetic pathway in fruit ripening see Fig.  is multie tomato . It has ild type . Other tild type . As per ripening . These rripening . Further by PYL9 . In contorted by ). These ell wall . Also, clycopene . Along wlycopene  and similycopene . The PSYPoaceae)  and fruir stress , 119. Inotenoids . In addip Yellow .Fig. 4BiPSY genes are positively regulated by transcription factor RIN and negatively regulated by SGR1, PIF1 and RAP2.2 [PSY gene promoter site, thereby leading to an altered expression of the pigment [CRTISO gene is carried out by SDG8 (histone methyl transferase enzyme), responsible for methylation of chromatin linked with CRTISO gene. The absence of SDG8 leads to reduced gene expression and resultant decline in the biosynthesis of lutein and strigolactones [CRTISO mutant ccr1, has been reported to downregulate more than 80 genes along with CRTISO gene. Another CRTISO mutant ccr2 was found to stimulate increased production of cis-carotenes in chromoplasts [PSY) and LCYE (for CRTISO) respectively. This phenomenon has been observed in etiolated Arabidopsis seedlings where an elevated expression of PSY increased carotenoid levels due to DXS accumulation, which indicates metabolic feedback regulation triggered through PSY, which in turn enhanced the availability of MEP substrates [The d RAP2.2 . A light pigment . The genlactones . A CRTISmoplasts . In addibstrates .PSY while CRY (cryptochromes) are sensitive to blue light and enhance the levels of pigments like chlorophyll and anthocyanin in leaves, as well as lycopene in fruits along with a decline in LCYB expression [phyAB1B2 (phytochrome silenced plants) demonstrated that the biosynthesis of the major carotenoid lycopene was found to be sensitive to PHY-temperature perception [Apart from enzymes, there are several environmental factors which are involved in regulation of carotenoid biosynthesis, among them, one of the crucial factors is light. Photoreceptors like PHY (phytochromes) are sensitive to red light and enhance the activity of pression . In tomarception .The light signaling is regulated positively by HY5 (elongated hypocotyls) and negatively by DDB1, DET1 and COP1 (constitutive photomorphogenic1) see Fig. . The PIFIn addition to the natural regulatory mechanism of the plant, selected carotenoid targeted regulation has been made possible by genetic engineering models to enhance the content of a specific carotenoid in the fruit. The biotechnological interventions for transgenic metabolic engineering, multigene engineering (gene scissor: CRISPR/cas9) and genetic breeding, in the last two decades have made it possible to enhance the productivity, nutritional adequacy and economic value of fruits  with enhIn horticultural crops, carotenoids accumulate in chromoplasts, which could vary from crystalline , globular (mango), fibrillary (pepper)  membranoSeveral complex biochemical changes take place in the chromoplast leading to transformation of previously synthesized pigments and de novo biosynthesis of new pigments. In fruits, carotenoids are subjected to many chemical reactions like oxidation, epoxidation, (cis-trans) isomerization and cleavage of polyene chains. To increase the carotenoid accumulation in plant cells esterification is essential. Recently, several esterases XES (xanthophyll esterase) have been identified in plants , 139. ThCarotenoids undergo esterification with fatty acids which makes them fat soluble and also facilitate their build up in chromoplast . The yelIn contrast to yellow xanthophylls, the red xanthophylls like capsanthin and capsorubin, found in ripe pepper fruit, demonstrate more stability owing to their esterification by lauric and palmitic acid which are short chain fatty acids. The augmented carotenoid stability by esterification could aid in biofortification and thus be explored in depth by future research projects . In addiThe chemical changes in the chloroplast lead to the accumulation of keto-carotenoids with changes in metabolism of lipids as galactolipids levels have been found to diminish while phospholipids have been reported to accumulate in chromoplasts. Oxygenated configurations of carotenoids having acylcyclopentanol end groups like capsanthin, capsanthin-5,6-epoxide and capsorubin have been observed to control the intensity of red color in chili pepper fruits . In ripeThe ripening of the tomato fruit has been investigated recently for the coordinated regulation of the genes involved, chromatin, epigenetics, transcriptomics, post translational and at the level of protein expression. These investigators have reported that the non-coding RNAs play a crucial regulatory role in fruit ripening at transcriptional and post transcriptional levels (for details see review ).CCD genes namely CCD1, CCD4, CCD7 and CCD8 and five NCEDs (9-cis epoxy carotenoid dioxygenases) namely NCED2, NCED3, NCED5, NCED6 and NCED9 have been identified [CCD1 and CCD4 are inversely proportional to each other [CCD1 is closely related to production of volatile compounds like aroma in fruits, CCD7 and CCD8 play a vital role in strigolactones production [NCED family plays a major role in production of the growth hormone ABA [The final concentration of carotenoids in plant tissues is dependent upon the enzymes; lipoxygenases, and CCDs (carotenoid cleavage dioxygenases) involved in degradation and cleavage. In Arabidopsis four entified , 149. Mach other . Also, woduction –153 and mone ABA .PpCCD4 attenuates its levels resulting in yellow flesh variety due to truncated protein with reduced carotenoid degradation activity [CCDs cleave the carotenoids into apocarotenoids and majority of them like, bixin, saffron, crocin, apocarotenals, apolycopenoides, ionones , peridinin and many more have been reported as bioactive compounds with therapeutic efficacy and industrial applications , 155–159activity , 165.Citrus unshiu), cleavage of β-cryptoxanthin and zeaxanthin by CitCCD4 results in the formation of β-citraurin, which is responsible for the reddish color in the peel. The production of C30 apocarotenoid in orange has been linked to CCD4b, thereby influencing the carotenoid accumulation in citrus fruits [In citrus fruits  gene has been reported to initiate biogenesis of chromoplasts, as its mutants have been reported to initiate non colored plastid differentiation into chromoplasts with an augmented ability to amass β-carotene in potato, cauliflower [Or gene resulting in augmented chromoplast size in very young fruits, promoted flower and fruit development, enhanced ethylene production, expression of ripening associated genes a [Or gene has been a much sought-after target for nutritional biofortification and alteration of horticultural traits in agricultural products [Or gene by genome editing has been proposed to manipulate carotenoid biosynthetic pathway, thereby making use of its interaction with PSY resulting in enhanced sequestering, protection from degradation and improved plants tolerance to abiotic stress [Carotenoids are produced in various plastids but accumulate in chromoplast at high levels resulting in attractive hues of fruits thereby the regulation of chromoplast biogenesis has a strong impact on the carotenoid biosynthesis and accumulation. At the molecular level the liflower  and meloliflower . Recent  genes a . Thus, Oproducts , 174. ThOr and Hsp70 in liaison with the Clp protease complex in the stroma, thereby suggesting that different chaperone families target distinct processes with regard to carotenoid accumulation and metabolism in fruit ripening [Furthermore, the activity and accumulation of biosynthetic enzymes such as PSY was found to be regulated by chaperones ripening . Severalripening , 82). Horipening  and a 2-ripening , 177. Suripening . The overipening . These aripening .Furthermore, the transfer of carotenoid biosynthesis to the cytosol can be brought about by the activity of crtB in plastids and its combination with cytosolic bacterial enzymes to produce carotenoids in extraplastidial sites , 179 andPunica granatum L.) peel were cyanidin 3,5-diglucoside and cyanidin-3-O-glucoside respectively [Persea americana Mill.), color changes from green to purple to black have been attributed to a fall in chlorophyll and an increase in glycosylated anthocyanin; cyanidin 3-O-glucoside [Prunus avium) [s regulating the structural genes of anthocyanin biosynthesis at an individual level and as a team as MBW complex. In a recent analysis RrMyb10 was reported as the anthocyanin inducer in Ribes species, and its role in manipulating anthocyanin biosynthesis in heterologous systems has been highlighted [s in two varieties of banana, Musa acuminata and Musa balbisiana would enable future prospects for functional studies [SEPALLATA and SQUAMOSA class MADS BOX genes also regulate anthocyanin accumulation in fruits. In addition, anthocyanin biosynthesis can also be triggered by sucrose signaling by inducing production of PAP1 (anthocyanin pigment1) [s positively involved in regulation of MYB-bHLH-WD40 and increasing the expression of MYBL2 (a negative R3-MYB regulator) along with down regulation of SUC1 (sucrose transporter 1), PAP1, TT8 and GL3 [. In addition, auxins have also been reported to regulate anthocyanin biosynthesis in a negative manner [FaNCED1 and LbNCED1 resulted in minimal anthocyanin production in strawberries and lyceum plants [Vaccinium myrtillus L.) (non-climacteric fruit) demonstrated that treatment of unripe bilberry fruits (attached/detached from the plant) with ABA resulted in anthocyanin accumulation and cell wall modification while the sucrose treatment of unripe bilberry fruits did not promote anthocyanin biosynthesis. Therefore, these results indicate that regulation of fruit ripening, is fruit specific and would differ from one fruit to the other [There are numerous examples of fruits and vegetables like pomegranate, cherry, plums, turnip, blackberries, blueberries, strawberries, red pears, onions, red cabbage, red apples, prunes, eggplant, cranberries and grapes which accumulate anthocyanin when ripe and change from green to purple or red . Fruits ectively . Also, ilucoside . The ants avium) . The biohlighted . Also, r studies . Apart figment1)  along wiigment1) . Though igment1) , 191, ho and GL3 . In addie manner , 194. The manner . Silencim plants . Among the other .Fig. 5Res involved in regulating anthocyanin biosynthesis have been found to enhance the flavonoid content. The silencing of DET1 gene has resulted in reformed developmental processes mediated by light and an augmented flavonoid content [chalcone isomerase (CHI) gene in tomatoes [Ros1 and Del which encode MYB and bHLH resulted in the most substantial increase in anthocyanin content leading to development of fully purple hue on tomato fruits [Del/Ros1 lines with overexpressing lines of AtMYB12 resulted in enhanced accumulation of anthocyanin [Del and Ros1 were recently transferred from transgenic Micro-tom to Moneymaker tomato cultivar via traditional breeding. The anthocyanin content in the inbred fruit enriched with anthocyanin was escalated to about 131% of the parent level, phenolic compounds upsurged by 51% coupled with an augmented antioxidant activity and reduced growth of bacteria [s could engineer anthocyanin accumulation in a ripened fruit and thereby enhance its nutraceutical and industrial potential.Manipulation of both the functional genes and the TF content . Also, ttomatoes . Howevero fruits . In addihocyanin . Furtherbacteria . Recentlbacteria . TherefoMYB genes  related to anthocyanin biosynthesis. The maximum anthocyanin levels were observed at 15 °C in light treatment as compared to a fall in anthocyanin at 35 °C in dark [Litchi Chinensis which identified an R2R3-MYBTF encoding gene; LCMYB1 whose expression enhanced on exposure to ABA and sunlight. The expression of this gene also correlated with tissue anthocyanin content and expression of LCUFGT gene [s which promote photomorphogenesis by Ub- proteasome system. Moreover, in apples higher levels of MdMYB1 accumulate on exposure to light following which it interacts with MdCOP1 [s like HY5 and it regulates their breakdown and degradation via the 26S proteasome pathway [CHS  gene and promote flavonoid build up as a response to stimulus like light and UV-B radiation. Recent reports suggest the role of UV-B induced accretion of anthocyanins through the COP1 regulated signaling in binding of HY5 to MYB gene promoters [VvANR, VvLAR1, VvLAR2 and regulatory genes MYB5a, MYB5b, MYBPA1 in grapes [Reaumuria soongorica, the expression of flavanone-3-hydroxylase, an enzyme involved in anthocyanin biosynthesis increased under the influence of UV radiations and stress due to drought [Fragaria vesca) with white color fruits, a mutant RAP (reduced anthocyanin in petioles) has been reported. This mutation was a stop codon in the synthesis of GST (glutathione s-transferase) gene. Among the eight genes in GST family, RAP is present abundantly in ripening fruits and acts as a transporter of anthocyanin. However, in cultivated strawberry, RAP mutant acts downstream to FvMYB10 which is the fruit specific TFs and results in a reduction in fruit color [s involved in anthocyanin biosynthesis could be utilized to engineer anthocyanin production and enhance its accumulation as an essential nutraceutical in a ripened fruit.Anthocyanins have been termed as “chameleons” or the “color diversity hub” due to frequent changes in expression of color related to biotic or abiotic factors like a drop-in temperature, brininess, dearth of nitrogen and minerals like phosphorous which are generally expressed as purple coloration on stem, leaves and other parts of the plant as response to low light stress. Anthocyanins display changes in tones due to environmental factors . The soi in dark . A positFGT gene . The molh MdCOP1 . The pro pathway . In Arabromoters . The expromoters . UV radin grapes . In the  drought . The effit color . TherefoSeveral anthocyanins have been isolated from plant species based on a single basic flavonoid framework of carbon atoms as C6-C3-C6. The basic flavonoid structure contains one integrated aromatic ring as A ring, the B ring which is a phenyl component and the C ring comprises of a heterocyclic benzopyran . The staAnthocyanin synthesis takes place at endoplasmic reticulum following which they are transported to the anthocyanic vacuolar inclusions for storage. Electron microscopy of plant cells exhibiting anthocyanin pigments depict anthocyanic vacuolar inclusions (AVI) which are formed because of hydrogen bonding of anthocyanin to the protein matrix. The concentration of anthocyanins have been reported to be intensified in areas rich in AVI . These vAnthocyanins exhibit a property of co-pigmentation which leads to stabilization of color in plants. The co-pigments can be of a wide range from alkaloids, other flavonoids, nucleotides, organic acids, and metals. The complexes thus formed result in increased absorption intensity and change in wavelength leading to increased hue intensity of anthocyanins . Many faConsidering the importance of pigments in nutraceuticals and varied industries and their accumulation across different developmental stages of ripening, decisions regarding the optimum time for harvest could be made based on the pigments desired. Harvesting of un-ripened fruits could be a potential source of nutraceuticals like chlorophylls’ and xanthophylls’, the breaker stage would contribute to a supply of both chlorophyll and carotenoids, while fully ripened fruits will be a reservoir of pigments like anthocyanins and carotenoids along with their cleavage products, many of which have important clinical functions , 233–235− 1) is used to treat mild to moderate depression with no side effects [Also, extensive research on pigment biosynthetic pathways, stability, degradation, storage, and the underlying regulatory mechanisms highlights ways to engineer pigment content with the aid of biotechnological advances and genome editing . Denovo  effects . These dSome of these compounds have also been used as biopesticides and bioherbicides making them a potential source of alternate farming compounds that could be used to reduce our needs on chemicals that may be harmful to the environment, wildlife, and insect populations. Increasing their content and marketable yield either through breeding, selective harvesting or genetic engineering could reduce the overall costs, making them more attractive alternatives to current use chemicals.Another opportunity to potentially manipulate fruit metabolite content is to manipulate primary metabolism , 247 dirThis review generates new hypothesis for future research as different stages of ripening induce several structural changes in pigments resulting in the formulation of novel unexplored pigments, which may prove to be a boost to the existing era of green technology.


Epigenetic mechanisms allow the establishment and maintenance of multiple cellular phenotypesfrom a single genomic code. At the initiation of development, the oocyte and spermatozoa providetheir fully differentiated chromatin that soon after fertilization undergo extensive remodeling,resulting in a totipotent state that can then drive cellular differentiation towards allcell types. These remodeling involves different epigenetic modifications, including DNAmethylation, post-translational modifications of histones, non-coding RNAs, and large-scalechromatin conformation changes. Moreover, epigenetic remodeling is responsible for reprogrammingsomatic cells to totipotency upon somatic cell nuclear transfer/cloning, which is oftenincomplete and inefficient. Given that environmental factors, such as assisted reproductivetechniques (ARTs), can affect epigenetic remodeling, there is interest in understandingthe mechanisms driving these changes. We describe and discuss our current understandingof mechanisms responsible for the epigenetic remodeling that ensues during preimplantationdevelopment of mammals, presenting findings from studies of mouse embryos and when availablecomparing them to what is known for human and cattle embryos. The simplicity of the morphological changes that occur during early embryo development, mostlycleavage division at initial stages, masks the molecular events that underlie the profoundand dynamic remodeling of the embryonic transcriptome and epigenome during this period. Pre-implantationdevelopment in all animal species encompasses unique features, such as drastic transcriptionaland epigenetic remodeling . EpigenThe early stages of pre-implantation development occur in the absence of transcription anddevelopment relies on maternal proteins and mRNAs stored in the cytoplasm of the oocyte duringoocyte growth and maturation . The trThe epigenetic information of sperm and oocytes is extensively remodeled with formation oftotipotent blastomeres . This rin vitro embryo production, SCNT, and epigenetic selection and editingfor improved phenotypes.At the molecular level, epigenetic information is represented mainly by DNA methylation andposttranslational histone modifications. While global changes in epigenetic informationduring preimplantation development and after SCNT have been studied, much less is known aboutthe locus-specific changes of these epigenetic marks across the genome. The current availabledata is mostly for mice, and while informative, some differences in development between miceand livestock species indicate that it will be important to gather species-specific knowledgeif a clear understanding of early development is desired, which could fuel applications suchas Although there are more research resources and tools for mouse than for other mammalian species,substantial advances in current genomic technologies have effectively leveled the playingfield for many other species, such as cattle. The advent of sequencing technologies to determinetranscriptomic and epigenomic features have demonstrated that similar information can bereadily collected in any species for which a high quality and well annotated genome exists, e.g.,cattle, sheep, pigs. Furthermore, siRNA and gene editing technologies like CRISPR/Cas9 nowallow generation of knockdown (KD) and knockout (KO) embryos/animals, respectively, in almostany species. For modeling human development, cattle preimplantation embryos have similaritiesto human in areas in which mice differ, such as a similar timing for genome activation and reprogramminggene expression, and a more similar genome sequence and organization . For exIt is quite possible that species differences in timing of the major EGA, when the dramatic reprogrammingin gene expression occurs and is essential for further development, could reflect differencesin epigenetic remodeling leading to EGA. In mice, EGA occurs during first cell cycle , althouAlso, differences between mouse and human embryos are apparent during the first embryo differentiationevents. Single-cell analysis in human embryos revealed marked differences between human andmouse embryos with respect to lineage specification in the early embryos and X-chromosome inactivation(XCI) ,the function of protecting the female genome from TET3 activity is conserved across these species. DPPA3 is one of the most abundant transcripts in oocytesand protects 5-mC from TET3-mediated conversion to 5-hmC by binding to chromatin containingH3K9me2, which is abundant in the oocyte and but mostly lacking in sperm, with some H3K9me2 observedin paternally imprinted genes of the mature sperm , resultingin recruitment of DNMT1 to the imprinted control region and therefore maintaining the methylationstatus at the imprinted control region after DNA replication (Recent studies suggest a big role for DNA replication dependent (passive) demethylation, eitherfrom a native (5mC) or oxidized (5hmC) form; however, a small contribution for active demethylationcannot be excluded. Such active demethylation, if present would only be minor. Importantly,the role of Thymine DNA Glycosylase (TDG) in active demethylation in the zygote was discardedby studying mutant mice, implying that other enzymatic activity could be responsible .DNA demethylation is necessary for epigenetic reprogramming of the somatic nuclei ; and isin vitroembryo development, have been suggested as the main reason for the appearance of the Large OffspringSyndrome (LOS)  .In bovine, the presence of DNMT3A, DNMT3B, and DNMT3L during oocyte growth is related to the establishmentof imprinted genes .In Eukaryotes, the DNA is packaged in chromatin inside the nucleus. The nucleosome constitutesthe basic unit of chromatin and consists of a segment of DNA (~147bp), wrapped twice around anoctamer of histone core proteins  . The amSperm chromatin is unique in that most histone proteins are replaced by protamines . Upon fwriters are EHMT1/2, SUV39H1/H2, SETDB1 and EZH2, which are responsible for the methylationof H3K9me2, H3K9me3, and H3K927me3, respectively  activity.Down-regulation of KDM6B in cattle oocytes, which prevents the decrease in H3K27me3, resultsin impaired EGA and reduced development to blastocyst, in both parthenogenetic  , and during re-differentiation (early lineage commitment).Recently, the derivation of bovine embryonic stem cells . IntereSimilarly, H3K4me3 was localized to a large set of genes  common to bovine, mouse andhuman ESCs, with a larger proportion of genes shared between human and bovine ESCs than betweenhuman and mouse ESCs . This methylation level is overshadowedby the demethylation level observed in PGCs of mice and humans, which achieve 3-6% methylation.Therefore, the greatest remodeling of epigenetic information seems to occur during germ cellformation, rather than after fertilization. The level of methylation reached after fertilization, together with the dynamics of demethylation, suggest that most ofthe methylation reduction could result from one round of DNA replication (maybe during the firstcycle in PN-stage embryos) without maintenance of DNA methylation activity. At the male PN,replication dependent demethylation is facilitated by conversion of 5-mC to 5-hmC by TET proteins.Importantly, TET-dependent hydroxymethylation of 5-mC is also present in the female genome,although at a much lower level than the male one. And, some evidence for active removal of DNA methylationstill exists, while at very low levels and the mechanism remains unclear. Demethylation of preimplantationdevelopment seems to be conserved between mouse and human embryos, while these two species differin timing of EGA, and therefore suggest that DNA methylation remodeling may play a minor rolein EGA or that human and mouse embryos may have different mechanisms in place that lead to EGA.Bovine embryos have an EGA timing similar to that of human embryos, but comparable informationin terms of DNA methylation at base-specific level is not available. It will be interesting todetermine the extent to which DNA methylation remodeling is conserved across other mammalianspecies.Regarding histone modification remodeling, only recently some information about locus-specificdynamics of a few epigenetic marks has been produced in mouse embryos. This information producedunexpected interesting data that has not yet been fully understood, including the localizationof H3K4me3 to broad domains, and lack of association of H3K27me3 marking and gene expression.Most other information about histone modifications is limited to overall levels determinedby immunostaining studies and the overall role of some modifications and enzymes involved intheir deposition/removal.In view of the fact that genomic and epigenetic functional resources are getting better and morewidely available, it is likely that a more complete and detailed picture of the molecular mechanismsof epigenetic remodeling during the preimplantation embryo development will arise. Such knowledgewill likely result in our better ability to assess the impact of ART on embryos and progeny andto provide a basis for the “modification” of epigenetic information from animalembryos for improved production characteristics, as well as helping devise strategies forimproved SCNT results.


In this review, we focus on the tremendous progress attained with the use of engineered mouse models together with high throughput genome-scale analysis in disclosing key signals, pathways and networks that are required for normal endometrial responses to progesterone during the periimplantation period. Many molecular mediators and modifiers of the progesterone response are implicated in cross talk signaling between epithelial and stromal cells of the endometrium, an intercellular communication system that is critical for the ordered spatiotemporal control of embryo invasion within the maternal compartment. Accordingly, derailment of these signaling systems is causally linked with infertility, early embryo miscarriage and gestational complications that symptomatically manifest later in pregnancy. Such aberrant progesterone molecular responses also contribute to endometrial pathologies such as endometriosis, endometrial hyperplasia and cancer. Therefore, our review makes the case that further identification and functional analysis of key molecular mediators and modifiers of the endometrial response to progesterone will not only provide much-needed molecular insight into the early endometrial cellular changes that promote pregnancy establishment but lend credible hope for the development of more effective mechanism-based molecular diagnostics and precision therapies in the clinical management of female infertility, subfertility and a subset of gynecological morbidities.Synchrony between progesterone-driven endometrial receptivity and the arrival of a euploid blastocyst is essential for embryo implantation, a prerequisite event in the establishment of a successful pregnancy. Advancement of embryo implantation within the uterus also requires stromal fibroblasts of the endometrium to transform into epithelioid decidual cells, a progesterone-dependent cellular transformation process termed decidualization. Although progesterone is indispensable for these cellular processes, the molecular underpinnings are not fully understood. Because human studies are restricted, much of our fundamental understanding of progesterone signaling in endometrial periimplantation biology comes from  Estimated to reach eight billion by 2024 , the proin vitro fertilization followed by embryo transfer (IVF-ET); reviewed in The relatively low MFR in humans also sets limits for success when using assisted reproductive technology (ART), which includes Apart from deficiencies in embryo quality, defects in endometrial function are now considered important etiologic factors in implantation failure and early miscarriage following a natural or assisted conception . Moreovein vitro approaches, which are indispensable for progesterone-dependent endometrial receptivity and decidualization, cellular processes that ensure successful embryo implantation in members of the eutherian mammalian class, which includes the human and mouse.Therefore, if we are to increase pregnancy success rates currently achieved by natural or assisted conception as well as improve outcomes for those women at high-risk for early pregnancy loss, significant expansion not only in our cellular but also molecular understanding of endometrial function during the periimplantation period is required. With the aforementioned as background, this review will profile a selection of pivotal molecular mechanisms, primarily revealed by mouse studies in conjunction with in vivo. Despite being a distant evolutionary relative, the mouse shares with the human many important endometrial cellular responses to hormones during early gestation. While the initial cellular events of embryo implantation in the human endometrium are interstitial  as opposed to eccentric  in the mouse  at the whole organism level. Specifically, mouse engineering in general and conditional genetic technology in particular have proven invaluable interrogative methodologies in accelerating our functional understanding of progesterone-dependent endometrial cellular processes during the periimplantation window. However, a mouse study is not complete without follow-up translational validation, usually with e system ].Before proceeding to the review’s main focus, an overview of the salient stages of the implantation process in the murine endometrium—with relevant cross comparisons to the human—is instructive to provide physiological and cellular context to the described associated progesterone-dependent molecular mechanisms that follow.Following oocyte fertilization in the oviduct , the single-celled zygote—through a series of cell divisions—develops to the advanced morula stage upon entry into the uterine cavity, a developmental journey of 3–4 and 5–7 days in the mouse and human, respectively . On the In the human, the putative attachment reaction is considered to occur within the uterine fundus during the mid-secretory phase of the cycle (or ∼7–10 days after ovulation) . Unlike From GD 3 onwards in the mouse, a synchronous postovulatory rise in systemic progesterone levels is critical for the estrogen-primed endometrium to enter the transient receptive state that is permissive to embryo attachment, adhesion and ultimately invasion . ProduceOn the afternoon of murine GD 5, subepithelial stromal cells encircling the nidating blastocyst within the epithelial implantation chamber undergo rapid progesterone-dependent proliferation. Toward the anti-mesometrial (AM) pole, proliferating stromal cells differentiate to form the primary decidual zone (PDZ). The transformation of stromal fibroblasts into densely packed epithelioid decidual cells of the PDZ is termed decidualization . By actiDecidualization spreads throughout the anti-mesometrial region forming the anti-mesometrial decidua before advancing to the mesometrial region, which eventually will form the decidua basalis . After Gin vitro, requiring only cAMP, estradiol, and a progestin in the culture medium , decidualization is not triggered by an implanting conceptus . Insteade medium . This cemilieu to prevent rejection of the hemiallogeneic conceptus  cells [termed decidual NK (dNK) cells in the human ], macropCollectively, investigations of uterine periimplantation biology demonstrate that progesterone-dependent endometrial receptivity and decidualization are crucial reproductive cellular processes that comprise a tightly synchronized continuum of endometrial cellular changes in which the successful conclusion of one cellular process relies on the successful completion of preceding cellular events . ImportaAs with any biology that can succumb to pathology, advancing our understanding of the critical molecular mechanisms that drive normal progesterone-dependent endometrial responses during early pregnancy is a prerequisite to developing novel mechanism-based diagnostics, prognostics and therapeutics not only for implantation failure but also for later gestational complications that occur due to derailment of normal endometrial receptivity and decidualization.For the remainder of the review, a brief synopsis of the progesterone receptor (PGR) is provided before describing a signaling network that typifies the quintessential mediator role of the early progesterone response, which is required for development of the endometrial receptive and decidualized state. Finally, the review concludes by showcasing one example each of an epithelial and stromal modifier of the progesterone response that is critical for early pregnancy establishment.The majority of tissue and cellular responses to systemic progesterone are mediated by the PGR, which is a group C member of nuclear receptor subfamily 3 (NR3C), a subfamily of 3-ketosteroid binding receptors of the nuclear receptor superfamily of transcription factors . The PGRvia its nuclear receptor  in the endometrial stroma . A pivotlization ; endometlization . Apart flization , angiogelization , and loclization , all of Hand2)  increases stromal Hand2 levels; however, HAND2 suppresses stromal fibroblast growth factor (Fgf) family members  in the endometrium  , which iometrium . Accordiablation .Through paracrine signaling within the normal endometrium, stromal FGFs bind their cognate transmembrane tyrosine kinase receptors (FGFRs with ancillary docking factors) located in epithelial cells. Engagement of stromal-derived FGF ligand with its epithelial-derived receptor elicits phosphorylation (and activation) of extracellular signal regulated kinases 1 and 2 (ERK1/2). In turn, activated ERK1/2 phosphorylate ESR1 to activate and stabilize ESR1 in the epithelium . During Therefore, perturbation in the normal levels of any component of the progesterone-PGR-IHH-COUP-TFII-HAND2 signaling axis is predicted to result in unwarranted activation of epithelial ESR1 .Bmp2) (Bmp2 levels are associated with increased levels of wingless-type MMTV integration site (WNT) family member 4 (Wnt 4) in the stroma (Egfr) signaling is linked to increased levels of Bmp2 and Wnt4 in the murine uterus during early pregnancy (Msx) family of transcription factors (Msx 1 and 2)  , which ie stroma . Both cle stroma . Also, eregnancy . Increas1 and 2) , which a1 and 2) .Molecular modifiers of PGR activity modify its regulation of target gene transcriptional output through various mechanisms: from controlling PGR stability and ligand binding affinity to coregulating PGR-dependent gene expression programs. An increasing number of epithelial and stromal modifiers of PGR action has recently been characterized in the mouse, many of which have strong translational significance.GATA DNA sequence, the six member family of GATA-binding transcription factors control a broad spectrum of biological processes, ranging from hematopoiesis, adipocyte development to pituitary gland function  a NR3C subfamily member and close relative of PGR; in vitro  is a member of the p160/SRC family of coactivators, which also comprises SRC-1 and SRC-3 . As pleiin vitro . Numerouin vitro . Althougin vitro —stronglyStudies on the SRC-2 knockout (SRC-2KO) mouse were the first to demonstrate a critical role for this coactivator in both male and female reproductive function . In the Of translational importance, SRC-2 is also required for progesterone-driven decidualization of primary human endometrial stromal cells in culture , furnishvia the PFK-1 checkpoint, anabolic pathways  can accelerate endometrial stromal cell proliferation to ensure timely and complete decidualization. Both in cultured human endometrial stromal cells and in the mouse, inhibiting PFKFB3 activity blocks decidualization  Does stromal SRC-2 control other metabolic programs that promote endometrial stromal cell decidualization? The question is raised because SRC-2  is an established coregulator of carbohydrate, lipid, and amino acid metabolism in other physiological systems . Furtherde facto model for interrogating endometrial function in a living eutherian mammal. Due to its genetic and experimental malleability, the mouse has shed new light into the “black box” of endometrial periimplantation biology (Even after 75 million years of evolutionary divergence , the ute biology , particuThis review profiled just a small selection of endometrial molecular mediators and modifiers of the progesterone response that are essential for embryo implantation. While many of these factors are causally linked to the development of the decidual cell from the stromal fibroblast, their role in the manifold functions of the decidual cell following its development is not fully understood. As described previously, many of these functions include angiogenesis, local immunosuppression, immune cell influx and prevention of pre-mature senescence. Exploration of these aspects of decidual cell function in the mouse using cre-dependent gene ablation technologies can only occur with the development of new cre drivers that are selectively expressed in the stromal cell following decidualization.in vivo importance of these enhancer-promoter pairs to murine endometrial periimplantation biology is now a reality.With the advent of CRISPR/Cas9 gene editing methodologies , structuSingle cell or cell atlas technologies are recently responsible for stunning advances in our molecular understanding of the interactome that operates between cells derived from the human trophoblast and the endometrial stroma . Elegantin vivo model that matches the mouse in terms of interrogating the molecular mechanisms by which progesterone governs endometrial function at the whole organism level. Therefore, the engineered mouse will continue to enrich our understanding of endometrial progesterone responsiveness as it relates to pregnancy establishment. Looking forward, we predict that molecular phenotyping of the murine endometrium at high resolution—in concert with translational studies—will provide a more comprehensive molecular understanding of progesterone’s role not only in endometrial receptivity, decidualization and implantation success but also in gynecological morbidities suffered by women who are diagnosed with an endometrium with a compromised progesterone response.At present there is no Far from hype, the aforementioned provides genuine hope that we are entering a propitious time for the accelerated development of personalized mechanism-based diagnostic and targeted therapeutics for endometrium-based infertility and associated progesterone-responsive disorders. The ultimate hope is that such developments will be actionable in the clinic sooner rather than later to maintain, restore or optimize endometrial function in otherwise healthy women.All authors contributed equally to researching, writing, and reviewing 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.


Satb2, Pbx1/2, Fgf8, and Foxg1 as exemplars, we examine the role of apoptosis in the elaboration of jaw modules, the evolution and elaboration of the lambdoidal junction, the developmental integration at the mandibular arch hinge, and the control of upper jaw identity, patterning and development. Lastly, we posit that apoptosis uniquely acts during craniofacial development to control patterning cues emanating from core organizing centres.Coordination of craniofacial development involves an complex, intricate, genetically controlled and tightly regulated spatiotemporal series of reciprocal inductive and responsive interactions among the embryonic cephalic epithelia  and the cephalic mesenchyme — particularly the cranial neural crest (CNC). The coordinated regulation of these interactions is critical both ontogenetically and evolutionarily, and the clinical importance and mechanistic sensitivity to perturbation of this developmental system is reflected by the fact that one-third of all human congenital malformations affect the head and face. Here, we focus on one element of this elaborate process, apoptotic cell death, and its role in normal and abnormal craniofacial development. We highlight four themes in the temporospatial elaboration of craniofacial apoptosis during development, namely its occurrence at (1) positions of epithelial-epithelial apposition, (2) within intra-epithelial morphogenesis, (3) during epithelial compartmentalization, and (4) with CNC metameric organization. Using the genetic perturbation of  Craniogenesis, the development of a patterned and functionally integrated head, is an especially elaborate process, one under intense developmental and evolutionary pressures . This apSatb2, Pbx, Fgf8, and Foxg1), aberrant CFD and morphogenesis, and the alteration of apoptotic profiles during CFD. And secondly, we utilize what has been presented from our first aim to posit a potentially unique role for developmentally programmed apoptosis in idiosyncratic aspects of early CDF and patterning.Here, we focus on one element of this elaborate process — apoptotic programmed cell death (PCD) — and its role in normal and abnormal craniofacial development (CFD). Rather than intending an exhaustive catalogue, our aims are rather more modest. Firstly, we aim to provide a sufficient correlative overview of the mechanics and normal topography of apoptosis during CFD to promote the notion of apoptosis as an active biologic tool during the elaboration of CFD and morphogenesis, one acting more as a scalpel than as a sledgehammer. In this context, we present basic primers of both embryonic CFD  and the mechanics of apoptosis that together provide reference points as we discuss apoptosis in normal and abnormal CFD. These sections are intended to inform those new to the topics and can easily be skipped by those previously well-versed. We then describe features of the normal topography of apoptosis during early CFD, focusing on: (1) positions of embryonic epithelial-epithelial apposition and fusion; (2) intra-epithelial morphogenesis; (3) embryonic epithelial compartmentalization; and (4) early CNC organization. In addressing apoptosis in abnormal CFD we take two approaches. First, we turn to the molecular mechanics of apoptosis to underscore how CFD is (and is not) affected by mutations in the genes encoding components of the apoptosis machinery. In the other, we utilize examples from our own work that we believe exemplify the correlations between targeted mutations of craniofacial patterning genes  that migrates into BA1, surrounds the forebrain and eye, and migrates to colonize the frontonasal region  endoderm (PE) is crucial to overall understanding of CFD. While the CNC is migrating from the NF, a signaling cascade establishes an iterative, cranio-caudal sequence of invaginations within the nascent PE that creates the pharyngeal pouches  the hinge organizing centre likewise plays a significant role in instructing the CNC to either adopt a mxBA1-upper jaw identity or a mdBA1-lower jaw identity  establishing upper jaw versus lower jaw positional identity within the CNC, (2) generating and maintaining the point of articulation between the upper and lower jaw arcades, and (3) keeping the arcades in appropriate functional registration during subsequent development and morphogenesis. Actualization of these tasks involves elaboration of a “hinge and caps” system of establishing positional information in the CNC through several regional signaling centres . BA1 is Crucially, BA1 primordia and the jaws that they give rise to are both fundamentally polarized . This chThough much less complex structurally and developmentally, the SCE of the distal-most midline of mdBA1 also secretes many of the same signaling molecules as λ — thereby creating the corresponding lower-jaw “cap” organizing centre. In sum, the CNC are patterned by the integration of the hinge organizing signals with countering epithelially derived patterning signals posteroproximally propagated through mdBA1 from the mandibular organizing “cap” and patterning signals posterodistally emanating through mxBA1 from the maxillary “cap” centred at λ .Apoptosis, recognized as a specific subcategory of PCD, is a fundamental component of both animal development and adult homeostasis . Among tunwanted cells, in particular during such developmental and morphogenic processes topographically correlated with apoptosis as those briefly enumerated above  caspases, in particular Caspases-3, -6, and/or -7. This activates executioner caspases to cleave - and so to either activate or inhibit - a multitude of other proteins. Among these newly activated proteins are endonucleases that drive DNA fragmentation and inhibitors of flippases which then unbalance the constituent components of the bi-layered organization of the plasma membrane. Subsequently, these latter events reveal “find me” and “eat me” signals leading to the eventual phagocytic engulfment and degradation of apoptotic debris  family ligands, in particular of FAS ligand (FASL) to FAS receptor, which then potentiates two distinct series of events. When present, procaspase-8 is activated (through a TRADD-RIPK1-FADD pathway) to cleave and thereby activate effector Caspases-3, -6, and/or -7 which can effectuate the proteolytic conversion of inactive BID into activated tBID — which then activates BAK and BAX with predicable consequences. Absent facient Caspase-8, the necroptotic cell death regimen  can be initiated; notably, however, this leads to changes in cellular morphology that are distinct from those encountered with apoptosis and the process is thought to rarely occur during development .Axiomatically, expression of the components of the apoptosis machinery are under the disparate regulation by numerous transcription factors, including p53/63/73, AP-1, NF-κB, IRF-1, Myc, Foxo, Hox, and Snail . ExpressIn his compendium 70 years ago on cell death in normal vertebrate ontogeny, Craniofacial apoptosis is predictably and consistently found where cranial epithelia of one source grows to meet, in apposition, other epithelia — especially where the meeting, fusion, and subsequent degeneration is fundamental to further growth and development . As we pTypical of apical-apical apoptotic appositions is the growth and convergence of the mammalian secondary palate, a multi-step process that begins with specification of CNC within mxBA1 . This isNotably, apical-apical apoptotic apposition is also seen at λ where the epithelium of the inferolateral mFNP converges (beginning around E10.5 in a mouse) to meet mxBA1 and the inferolateral lFNP at the ventral intranasal line  and subsequently fuse to ensure the buccal organ is continuous laterally and the obicularis oris muscle can completely encircle the oral opening . FailureFormation, migration, closure and subsequent re-opening of the eyelids provides yet another example of complex craniofacial apical-apical epithelial appositions for which apoptosis appears as a part of the normal ontogenic progression. The optic and periocular SCE undergoes repeated rounds of coordinated epithelial morphogenesis and migration during development — to first generate a lens placode, and then to invaginate the placode to form a lens vesicle, pinch off completely as a vesicle from the SCE, generate a distinct corneal layer, build and properly position corneal  furrows, and then undergo focal glandular branching morphogenesis and tubulogenesis . Importantly, the SCE-derived epithelium must also establish a migratory wave of cells, encased in an epithelial periderm layer, to build eyelids that will grow as migratory waves across the sclera and then appose one another over the cornea, fuse and eventually split open again — all without becoming physically attached to the corneal or scleral surfaces . Once thAs noted above, basal-basal epithelial apoptosis-associated appositions likewise occur during normal craniofacial development, and includes both SCE-SCE and SCE-PE epithelial contacts. In addition to the primary and secondary palates and the upper lip, morphogenesis at λ includes the formation of the primary chonae, the internal oronasal aperture . ExternaSCE-PE basal-basal epithelial apoptosis-associated appositions also characterize the development and rupture of both the buccopharyngeal membrane and the PPts . The bucEpithelial folding is a fundamental morphogenic event as it acts to transform simpler two-dimensional epithelial sheets into complex three dimensional structures that are often essential for tissue or organ function as well as for organismal life . Indeed,in situ, their own specialized neurosensory cells. Strong evidence indicates that topographically regulated apoptosis normally seen within the amniote SCE assists the eventual individuation of the trigeminal and, within the PPR, the physical compartmentalization of the epibranchial and otic placodes , unique to this surface ectoderm, that has been shown to represent a specialized competence for placodal induction . Experimplacodes . Notablyplacodes . Thus, iHox– population includes cells that delaminate from R1 and R2, the midbrain, and the posterior forebrain. The Hox+, post-trigeminal CNC of BA2 and BA3 mostly emigrates from the even numbered posterior R such that R4 CNC enters BA2 while R6 CNC enters BA3. This coordinated segmentation is key to generating positional information between the BAs. Notably, this mechanism for pattern control is actively maintained by the early apoptosis-induced elimination of CNC at the third and fifth rhombomeres  follows mesodermal positional cues . The crambomeres .Apaf1 in mice resulted in significant rostral neural tube closure defects, mid-facial clefting, failure of palatal shelf fusion, and jaw dysmorphology  the role of Pbx genes in controlling apoptosis in the nasal fin at λ and the plausible significance of this for CFD and evolution, (3) how attenuation of Fgf8 expression leads to increased apoptosis in the CNC at the hinge as well as decouples jaw integration, and (4) how loss of Foxg1 leads to drastically altered apoptotic topography in the SCE and its signaling centres that results in the loss of suppression of lower jaw molecular identity in the upper jaw primordia.Analysis of both naturally occurring and targeted mutations in vertebrates has frequently included correlations between changes in the spatiotemporal topography of apoptosis and the larger phenotypic outcome of the mutation. Assessing the immense breadth of all such analyses lies well beyond our here intended scope; rather, to facilitate the notion of craniofacial apoptosis as chisel rather than sledge-hammer, we highlight four instances for which alteration of apoptosis has been invoked as integral to the phenotypic outcome of a mutation associated with craniofacially expressed genes. To aid the discussion, and to underscore the rationale of the hypotheses at the core of our second aim, we focus our examples around apoptosis and the molecular dynamics of jaw patterning. Specifically, we discuss (1) how the absence of Satb2  positive CNC population defines an integrated set of coordinated modules, topographically peri-sagittal to the midline, between the upper and lower jaws , a condition characterized by distinct craniofacial abnormalities, developmental delay, and intellectual disability  homeodomain transcription factors known for their interactions with Hox and Meis encoded proteins, and they govern disparate developmental processes including apoptosis in the seam of the nasal fin at λ  on a Pbx2 or Pbx3 heterozygous background likewise leads to CL and 1CP  through evolutionary transitions among amniotes  of the mouse is accompanied by the catastrophic loss of the hinge region of both the upper and lower jaws. This included of all the developmental modules within BA1 except those furthest from the hinge and centred at the caps  and null murine Fgf8 alleles. This allows for a modulation of Fgf8 signaling by reducing functional expression levels to approximately 20% (Fgf8null/Neo), 40% (Fgf8Neo/Neo), 50% (+/nullFgf8), or 70% (+/NeoFgf8) of normal (wild-type) levels  and the functional upper jaw articulation  levels . These gcapsules . The higcapsules . In Fgf8and Dlx6 . Notablyand Dlx6 .Foxg1 is an evolutionarily conserved member of the Fox/Forkhead family of winged-helix transcription factors  syndrome  and hinge . Notably, Foxg1 acts to suppress the induction of lower jaw identity in the upper jaw primordia as without Foxg1 mxBA1 partially acquire deep mdBA1 molecular identities , as well as associated with the recently closed anterior neuropore  and the medial olfactory field . In –/–Foxg1 littermates these fields are all altered: apoptosis is undetected in the medial olfactory field and it is expanded in the middle SCE of BA2. Most noticeably, extensive ectopic apoptosis is found along the aboral border of BA1 and bridging the OE and PC1 at the hinge  as well as within craiofacial epithelia that are undergoing obvious and significant morphogenesis . Additionally, we have seen that normally observable focal patches of apoptotic cells correlate with the placodal compartmentalization within the SCE.Using the genetic perturbation of Apoptotic cells are, however, also notably detectable during CFD in topographies that do not readily lend themselves either to an aspect of the epithelial morphogenesis or apposition or to structural compartmentalization. This is exemplified, for instance, by the patch of apoptotic cells found within mxBA1 ectoderm at E10.5 in mice see , 6F. MorFgf8-generated developmental cues and aboral, PPt1-derived Fgf8-generated developmental cues. We know that Foxg1, restricted in its expression to the upper jaw-associated SCE as it is, is part of solution to the first task: in its absence, normal structural development across the hinge is lost and a large domain of mxBA ectopically acquires a mdBA1 molecular identity. Thus Foxg1 makes the epithelial environment of the upper jaw distinct from that of the lower jaw. Included in this environment is control of the foci of apoptotic cells in the SCE. Though admittedly full understanding of the correlation of morphogen level and morphogenic outcome has yet to be achieved, we also know that thresholds of morphogens such as Fgf8 are necessary for normal patterning across the hinge. Perturbation of morphogen levels leads to alteration of gene expression, morphology and the topography of apoptosis. These two things — control of epithelial environment and control of morphogen concentration — are likely linked on numerous levels.It is patent that developmental mechanisms must be employed that properly instruct an equipotent, fungible population of CNC to (1) discriminate instructions for upper jaw pattern and development from those for lower jaw pattern and development and (2) then discriminate and integrate both OE-derived, Fgf8-producing cells of the anterior neural ridge, and the epithelial patterning centres of the enamel knot of developing teeth (Apoptotic control of a morphogen producing cell population is not an undocumented occurrence during development. Cell death has previously been proposed as a mechanism for regulating the effective potency of a secreted morphogen by regulating the numbers of cells producing the factor. Such a mechanism for patterning has been suggested for the PCD associated with Shh-producing cells in the limb bud, ng teeth . In eachWith regard to the SCE, we suggest that another mechanism to regulate the effective elaboration of patterning signals is to program apoptotic foci as a means of micromanaging epithelial environments that inform the CNC. It is possible that the mxBA1 apoptotic foci, for example, acts to restrict the spread of a hinge signal that could otherwise easily be interpreted by regionally subjacent mxBA1 CNC to make them act as though they were mdBA1 CNC. Either physical presentation of factors from the apoptotic cells or the topographic restrictions of the effective spread of a signal through the epithelium could both subtly but effectively micromanage CNC interpretations of local patterning cues either specific to mxBA1-hinge signals or for integrated OE and PPt1 signaling (schmatized in Similar roles may well be posited for other patches of apoptosis detected during CFD, including those found in the SCE between the anterior neuropore and the olfactory placode. However, while we have some regard for the topography of the normal apoptotic profile during CFD, understanding of the total picture of the spatiotemporal topography of apoptosis during vertebrate CFD — let alone all of its plausible functions — has yet to be fully realized. With regard to roles for apoptosis in normal CFD and patterning, moreover, we would suggest that investigative optics are better set at higher magnification and evidence of a scalpel’s touch rather than a sledgehammer’s punch be sought as we address the goal of understanding the roles that apoptosis plays during CFD.MD and CC conceptualized the themes presented and prepared the manuscript. All authors participated in data collection and interpretation, contributed to the article, and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Members of this family control gene expression to promote self-renewal of progenitor cell populations and govern mechanisms of cell differentiation. When the function of SIX genes becomes disrupted, distinct congenital defects develops both in animal models and humans. In addition to the embryonic setting, members of the SIX family have been found to be critical regulators of tumorigenesis, promoting cell proliferation, epithelial-to-mesenchymal transition, and metastasis. Research in both the fields of developmental biology and cancer research have provided an extensive understanding of SIX family transcription factor functions. Here we review recent progress in elucidating the role of SIX family genes in congenital disease as well as in the promotion of cancer. Common themes arise when comparing SIX transcription factor function during embryonic and cancer development. We highlight the complementary nature of these two fields and how knowledge in one area can open new aspects of experimentation in the other.The  The founding member, sine oculis (so), was first identified in Drosophila melanogaster where it was discovered to be required for compound eye formation , the optix subfamily (Six3 and Six6), and the DSix4 subfamily (Six4 and Six5). The vertebrate orthologs contain the same SIX and HD domains as the ancestral Drosophila proteins with significant amino acid sequence identity between the functional domains of family members. For example, the mouse SIX domain amino acid identity ranges from 63 to 93% while sequence identity in the HD ranges from 59 to 98% between family members, with Six3, Six4, and Six5 showing the most divergence. For a more complete review of SIX family protein structure and sequence comparisons see The ormation . SubsequSIX genes play critical roles in tissue formation and organogenesis, such as for the head, ear, retina, nose, brain, skeletal muscle, and kidney , renal dysplasia, hearing loss, and frontonasal dysplasia syndrome , exhibited defects of the cranial base and cleft palate , a heterogeneous collection of forebrain malformations, with incomplete penetrance due to SIX3 haploinsufficiency or hypomorphic function (Sonic Hedgehog (SHH) and other members of this signaling pathway . DM1 is dominantly inherited and presents with myotonia, muscle wasting, cardiac conduction defects, fertility defects, and cataracts , particularly SIX1, have been frequently implicated in the promotion, invasion, and survival of a variety of cancers   , non-smama (HCC)  and coloma (HCC) . As one ms tumor  as well ms tumor  and nephms tumor . SpecifiSIX6 has been found to be associated with T-cell acute lymphoblastic leukemia (T-ALL)  , though rognosis . Increast cancer . In start cancer , astrocyt cancer , glioblat cancer , and lunt cancer .SIX4 expression has been detected in non-small cell lung  promoter region . Studies in several cancer types have shown a relationship between SIX gene expression and increased EMT, cell migration, and tumor invasion (Cadherin-1 (CDH1), which encodes for the epithelial marker, E-cadherin. Increased expression of either SIX1 or SIX2 in several cancer types reduced the level of CDH1 through either activating known repressors of CDH1, such as Zeb proteins, or by CDH1 promoter methylation  which in turn upregulated Wnt1 . Differential methylation patterns have been identified during embryonic development and in cancerous tissues for Six2, Six3, Six5, and Six6 (Six2 and Six6, hypomethylation of promoter and genic regions correlated with increased expression (SIX6 with cancer types, however, expression levels of SIX6 were not addressed and it remains unclear the significance of gene methylation in these instances (SIX5 expression was associated with increased DNA and repressive-associated histone methylation linked to trinucleotide expansion in DM1, suggesting a similar epigenetic mechanism to control SIX gene expression (SIX3, the tumor suppressor appeared to be hypermethylated in lung cancer and glioblastoma accounting for decreased expression in these cancer types (Experimental evidence has suggested a significant role for epigenetic regulation in controlling and Six6 . For propression . Two repnstances . Reducedpression . For SIXer types .SIX expression (SIX mRNA transcripts were downregulated, allowing for SIX transcription factor induced cell proliferation and EMT. Several miRNAs have been identified that target Six1, Six2, and Six4 in a diverse array of cancers (SIX gene expression have been detected during embryonic development. Six1 was found to be directly regulated by miR30a during zebrafish skeletal muscle development (Six2 has been shown to be regulated by miR-181b in cultured metanephric mesenchyme cells and the interaction may function to control cell differentiation (Six3 may potentially be regulated by miRNAs during eye development, although these results need to be further investigated to confirm this mechanism (In addition to DNA methylation, several miRNAs have been identified as epigenetic regulators that down regulate pression . Much of cancers . In addielopment . The relntiation . Finallyechanism .SIX gene family in vertebrates. However, many questions remain including how the gene family is regulated, the regulation of activation or repression complexes, and whether SIX proteins are viable therapeutic targets. Efforts have been made to identify transcriptional regulators and characterize the promoters of SIX genes. For example, complex ChIP-seq analysis identified regulatory regions of Six2 in kidney progenitor cells and how Six2 may function in complex transcriptional regulatory networks (Six2 regulatory sequences and how they function in the balance between kidney progenitor cell maintenance and differentiation remains to be determined. It is also unclear whether any of the identified transcription factor binding sites upstream of Six2 or other SIX family members become re-engaged in cancer. In addition to the specific proteins that regulate SIX family enhancers and promoters, it is of interest to further identify upstream signal transduction pathways that can induce SIX gene expression. Evidence has shown a role for Notch, Wnt, and TGF-β pathways to not only be targets of SIX proteins but also to feedback onto SIX genes (SIX gene haploinsufficiency and compensation by wild-type alleles could reduce disease severity. Upstream activation pathways of SIX expression would also be of interest in cancer where they could provide new opportunities to reduce SIX induced tumorogenesis. Another tumorogenic target to reduce SIX function would be miRNAs, which have been found to be reduced in several SIX family associated cancers (The fields of developmental biology and cancer research together have provided great insight into the important functions of the networks . The funIX genes . Identif cancers . MicroRN cancers .A second area to further investigate is the regulation of activator vs. repressor complexes involving SIX proteins and other conserved factors such as Eya and Dach proteins. During development, these three protein families are commonly co-expressed to maintain a balance between progenitor cell proliferation and differentiation. Does the presence of Eya proteins always promote transcriptional activation even in the presence of repressors such as Dach  or is thTraditionally, transcription factors have been seen as poor drug targets despite the central role they can play in disease such as cancer . Past diLM, LF, and TC conceived and developed the manuscript outline, and wrote and edited the manuscript. LM, LF, BB, and TC researched the literature and developed the tables. LM and TC created the figure. All the authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.