Phosphatidylinositol-specific phospholipase Cγ2 (PLCγ2) is a critical signaling molecule activated downstream from a variety of cell surface receptors that contain an intracellular immunoreceptor tyrosine-based activation motif. These receptors recruit kinases such as Syk, BTK, and BLNK to phosphorylate and activate PLCγ2, which then generates 1D-myo-inositol 1,4,5-trisphosphate and diacylglycerol. These well-known second messengers are required for diverse membrane functionality including cellular proliferation, endocytosis, and calcium flux. As a result, PLCγ2 dysfunction is associated with a variety of diseases including cancer, neurodegeneration, and immune disorders. The diverse pathologies associated with PLCγ2 are exemplified by distinct genetic variants. Inherited mutations at this locus cause PLCγ2-associated antibody deficiency and immune dysregulation, in some cases with autoinflammation. Acquired mutations at this locus, which often arise as a result of BTK inhibition to treat chronic lymphocytic leukemia, result in constitutive downstream signaling and lymphocyte proliferation. Finally, a third group of PLCγ2 variants actually has a protective effect in a variety of neurodegenerative disorders, presumably by increased uptake and degradation of deleterious neurological aggregates. Therefore, manipulating PLCγ2 activity either up or down could have therapeutic benefit; however, we require a better understanding of the signaling pathways propagated by these variants before such clinical utility can be realized. Here, we review the signaling roles of PLCγ2 in hematopoietic cells to help understand the effect of mutations driving immune disorders and cancer and extrapolate from this to roles which may relate to protection against neurodegeneration. IP3 and DAG then facilitate secondary signaling for membrane-bound immunological and growth factor receptors (Phosphatidylinositol-specific phospholipase Cγ2 (PLCγ2) belongs to a group of intracellular enzymes that cleave the membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PIPte IP3) A. IP3 an A. IP3 aB). Interaction between the cSH2 domain and residues surrounding the catalytic active site results in autoinhibition of PLCγ2. Upon Y759 phosphorylation, the cSH2 domain is removed from the catalytic active site and interacts with phosphorylated residue, allowing the substrate PIP2 access to the active site (B) is essential and sufficient for activation by Rac2, with the crucial amino acids at the PLCγ2–Rac2 interface determined by NMR spectroscopy  domain, and SH3 domain B. Interaive site . Biochemive site B is essetroscopy . Rac2-mePLCγ2 is also a known risk factor and an important driver in a multitude of diverse disease circumstances including those with an immunological basis such as inflammation, autoimmunity, immunodeficiency, and allergy, as well as in hematological malignancies . Moreove3, DAG, and calcium flux, PLCγ2 activation results in activation of phospholipase D and cell spreading  is stimulated by its cognate antigen. Early studies of PLCγ2 function, typically conducted in the chicken B cell line DT40, revealed that BCR signaling through PLCγ2 required its association with other signal transduction molecules including Syk and Btk in a complex located in a lipid raft, or signalosome. PLCγ2 would then be phosphorylated at four tyrosines . Mopreading , 16, 17,preading . Studiespreading . Upon BCmembrane .in vitro for PLCγ2 phosphorylation and resultant calcium flux after BCR stimulation  with a neomycin cassette . Unlike ult mice . Indeed,antigens , 27, 28.−/−PLCγ2−/− mice where floxed enzymatic PIP2 domains of PLCγ2 were inducibly deleted  associating with the BCR and via binding its known ligand, herpes virus entry mediator and activating SHP-1 -mediated proliferation were normal in the absence of PLCγ2 , 26, witIn summary, PLCγ2 plays a fundamental role in B cell development affecting the survival of mature B cells and antibody production, as well as being an absolutely necessary and immediately proximal component of the BCR signaling cascade. While PLCγ1 plays a greater role in T cells, PLCγ2 is still important in terms of TCR signaling and the calpain-calpastatin system and when combined with PLCγ1 deletion. Considering these vital roles for PLCγ2 in the adaptive immune system, it is of little surprise its dysregulation or malfunction is also then of crucial importance in a number of disease settings of an immunological and hematological nature.PLCG2 gene  and common variable immunodeficiency (CVID). PLAID is specifically driven by a gain-of-function amino acid mutation in the PLCγ2 gene , 64, wheCG2 gene . PatientCG2 gene , 66. TheCG2 gene . Chemotain vitro . Patientin vitro .In vitro studies revealed that after BCR signaling, the cSH2 domain of PLCγ2, coupled with poor phosphorylation of Syk, Btk, and BLNK, stabilized the early signaling complex , and mast cells often because of its utility in facilitating downstream signaling after FcR engagement. Moreover, PLCγ2 is reported to be involved in essential signaling processes associated with integrin activation and signaling downstream of critical myeloid receptors including cytokine receptors and TLRs. This signaling drives subsequent antibacterial functions including degranulation and production of reactive oxygen species (ROS) and inflammatory responses.3 production, degranulation, and inflammatory cytokine release  receptor, essential for macrophage development, also associates with PLCγ2, leading to its activation, resulting in PI3K activation, thus showing a potential role in myeloid cell differentiation , 77. Bloin vivo ) in the macrophage cell lines  is a fundamental cytokine governing bone formation and destruction nd NF-κB . In contnd NF-κB . They alnd NF-κB . Moreovend NF-κB , 100. Thnd NF-κB . Anothernd NF-κB . Ephrin nd NF-κB . These nvia engagement of their antigen-specific IgE receptors that in turn release their proinflammatory payload. PLCγ2 is implicated in allergic responses, specifically with regard to the function of mast cells and their crosslinking of FcεRI (IgE-binding receptor) and FcγR and subsequent degranulation  in PLCγ2 resulting in spontaneous inflammation  and innate system  as well as various forms of inflammation , as evident by an upregulation of the PLCγ2 gene signature of patient peripheral blood mononuclear cells . Indeed,oduction . Pathogeoduction .PLCG2 also link the enzyme to type 1 diabetes (Lyn tyrosine kinase autoactivation (gain-of-function) mice, after BCR stimulation, showed constitutive phosphorylation of Syk/PLCγ2, with a resultant increased calcium flux, observed in their resting B cells . These cdiabetes  and venodiabetes , which cOverall, PLCγ2 clearly exhibits an important role in the manifestation of autoinflammatory disease with hypermorphic PLCγ2 signaling resulting in spontaneous autoinflammatory disease. These PLCγ2 signaling-induced autoimmune diseases produce a range of inflammatory phenotypes in both patients and disease models, typically driven by autoantibodies (with the exception of APLAID) and broad autoimmune cell expansion. These disease situations highlight the potential to develop PLCγ2-modulating therapies that treat immune disease but must be tailored so as to avoid effects on other disease relevant processes such as cancer and neurodegeneration. Next, we explore recent insights and advances in these fields.The crucial nature of PLCγ2 in both the innate and adaptive immune system is further underscored by its vital roles in hematological malignancy. This is perhaps most evident in chronic lymphocytic leukemia (CLL), but PLCγ2 is also observed to play a role in diffuse large B cell lymphoma (DLBCL), the most common form of non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, myelodysplastic syndrome, endemic Burkitt lymphoma (EBL), MALT-associated gastric lymphoma and multiple myeloma. Finally, PLCγ2 has also been shown to be a relevant part of Epstein–Barr virus (EBV) transformation of B cells.A powerful tool for validating the roles of oncogenes and tumor suppressors is the Eμ-Myc mice lymphoma model where all mice will succumb to lymphoma. Using this Eμ-Myc lymphomagenesis mouse model in the absence of PLCγ2 demonstrated that PLCγ2 was indeed vital to early B cell development, resulting in an accumulation of large pre-B cells . PLCγ2 dWhile the absence of PLCγ2 can further enable lymphogenesis in certain murine lymphomas, in humans, PLCγ2 signaling can drive leukemogenesis in some cases. Ibrutinib, which inhibits Btk and thus blocks PLCγ2 signaling, has become an important and effective treatment for CLL. However, a subset of patients become refractory to this treatment over time and relapse, with PLCγ2 as a primary driver of the recurrent disease , 123. MuPLCG2 were noted to be correlated with CLL resistance to Ibrutinib  signaling, a common relapse patient PLCγ2 mutation, suggesting SYK and LYN as alternative targets in Ibrutinib resistance  of brutinib . It was sistance . SYK/PLCsistance , 134. Fu disease .in vitro suppressed their proliferation and induced apoptosis and cell cycle arrest  . Treatmee arrest . Howevere arrest . Downrege arrest . The useCγ2 mice  in a modlymphoma . These m T cells . Mutatio T cells . These m T cells .via the virally encoded integral latent membrane protein 2A in combination with BLNK , osteosarcoma, esophageal squamous cell carcinoma, esophageal adenocarcinoma, cervical adenocarcinoma, Birt-Hogg-Dube tumors, non–small-cell lung cancer, and breast cancer. PLCγ2 was implicated in the Wnt signaling pathway necessary for kidney development and development of Wilms' tumor . In addiof PLCG2 . PLCγ2 wof PLCG2 . This isof PLCG2 . PLCγ2 wisplatin . Howeverisplatin . Populatisplatin , in non–isplatin , and as isplatin . Indeed,isplatin , 153.2, also resulted in PLCγ2 upregulation in human osteosarcoma cell lines  has been associated with decreased risk of AD by genome-wide association study  and neurofibrillary tangles. A rare variant in the on study . The inion study , 162. Reon study  Fig. 4)PLCG2 genon study . In conton study . On a fuon study .Figure 4PLCG2 gene expression in the cortex of AppNL-G-F/NL-G-F mice Fig. 4). Taken together, these results provide further evidence that PLCγ2 may play an important role in AD pathophysiology. Future studies investigating how these variants in PLCγ2 modulate its function and microglia phenotypes in AD could lead to the identification of novel therapeutic strategies.In summary, PLCγ2 is a fascinating enzyme regulating diverse biological functions through critical immunological molecules used by a variety of extracellular receptor signaling pathways. Conclusive genetic evidence demonstrates how important normal PLCγ2 function is to immune health, the overaction of which can lead to immunodeficiency, autoimmunity, or autoinflammation. Some of these same mutations can also drive hematopoietic cell proliferation, for example, in Ibrutinib-refractory CLL, and so inhibition of PLCγ2 could be considered as a therapeutic modality in some malignancies and immune disorders. However, such intervention would need to be approached with caution, as the constitutive roles of PLCγ2 are important, as are other gain-of-function variants that provide protection against neurodegenerative disease. Therefore, improved understanding of PLCγ2 functions could inform cellular and temporal targeting of this pathway for therapeutic benefit in the future.S. L. M. is a scientific advisor for IFM Therapeutics. J. T. J., E. M., and S. L. N. declare no conflicts of interest.


Fibroblast growth factors (FGFs) comprise a large family of growth factors, regulating diverse biological processes including cell proliferation, migration, and differentiation. Each FGF binds to a set of FGF receptors to initiate certain intracellular signaling molecules. Accumulated evidence suggests that in early development and adult state of vertebrates, FGFs also play exclusive and context dependent roles. Although FGFs have been the focus of research for therapeutic approaches in cancer, cardiovascular disease, and metabolic syndrome, in this review, we mainly focused on their role in germ layer specification and axis patterning during early vertebrate embryogenesis. We discussed the functional roles of FGFs and their interacting partners as part of the gene regulatory network for germ layer specification, dorsal–ventral (DV), and anterior-posterior (AP) patterning. Finally, we briefly reviewed the regulatory molecules and pharmacological agents discovered that may allow modulation of FGF signaling in research. Early embryogenesis in vertebrate embryos involves the irreversible developmental process. As the ovum receives the male haploid genome from a sperm to become a diploid cell, the process of fertilization is started with the fertilized ovum then being referred to as a zygote. The zygote goes through several key developmental stages including mid-blastula transition (MBT), gastrulation (germ layer formation), and neurula, for establishing the overall body axis and generating the anterior CNS and posterior PNS . These aN-terminal hydrophobic peptides and they include FGF3, 4, 5, 6, 7, 8, 10, 17, 18, 19, 21, and 23 [N-terminal hydrophobic peptides and are endoplasmic reticulum-Golgi-independent for their secretion. These include FGF1, 2, 9, 16, and 20 [N-terminal signal peptide rather than being secreted  [FGFs interact with specific FGF receptors to initiate intracellular signaling and there are four cell membrane tyrosine kinase FGF receptors, FGFR1, 2, 3 and 4, which are members of the larger receptor tyrosine kinase (RTK) group ,18. Eachdomains) . There idomains) . Heparandomains) , and it domains) . Ligand domains) . For FGFdomains) . The spldomains) ,20.In this section, we briefly summarized the activation modes of FGFR associated cytosolic effectors and linked components that act as intermediates. Several partner receptor proteins may be associated with the cytosolic domain of an FGFR, such as cell adhesion molecules (CAMs) and G-protein coupled receptors (GPRCs) . Signal-Cellular diversification is an essential process in generating a complex, multicellular organism. This process begins with gastrulation as the three germ layers of endoderm, mesoderm, and ectoderm are specified. For an amphibian embryo, the first embryonic germ to specify is the endoderm from the vegetal pole. The formation of mesoderm, the second germ layer, is the result of active mesoderm inducing signals, originating from the vegetal region or the prospective endoderm, which triggers the mesoderm specification. During the mesoderm specification, generally two distinct signaling centers, namely the ventral and dorsal signaling centers, are established in the marginal region; these are based on presence and activation of maternal or zygotic factors at ventral and dorsal half of the embryo A. Once tvegt abolished endoderm formation and overall germ layer patterning [vegt dramatically reduced the expression of fgf3, fgf4, fgf8, Xnr1, Xnr2, and Xnr4; however, expression of bmp4 and bmp7 remained unaffected or were increased [endodermin (edd) and mixer expression [edd, mixer, and sox17β  in a activin/smad2-dependent manner [sox17 and endodermal defects [Sox17 [In amphibian embryo, the vegetal half  contains several important maternal factors  that might govern early primordial germ layer formation. To date, several transcription factors primarily located in the vegetal region have demonstrated consistent inductive activity for germ layer differentiation. One instance is Vegt , which is a maternal T-box transcription factor as depleted tterning . Similartterning . These rncreased . FGF sigpression . Recentlt manner . The one defects . Similar defects . There hs [Sox17 . These sThe role of FGF signaling, however, is not always endoderm inhibitory, at least in certain systems. From stem cell research, FGFs cooperate with activin signaling in promoting endodermal differentiation as the down-regulation of FGF signaling by SU5402 (an FGF inhibitor) abolished activin-A induced definitive endoderm formation in human embryonic stem cells (hESCs) . The actIn 1969, an inductive role for endoderm in inducing mesoderm formation was demonstrated. In this experiment, the section from the animal half  and the section from vegetal half were conjugated and cultured . Surprisxbra, chordin (chrd), noggin (nog), and goosecoid (gsc) expression [fgf3, fgf8, fgf20, fgfr1, and fgfr2 [Fgf3 expression is primarily in the ring around blastopore lip (mesoderm) of Xenopus gastrula, which then resolves into neural or brain tissues at a later stage of development [fgf4, fgf8, and fgf20 are largely expressed in early mesoderm or late mesodermal lineages [The active molecules with mesoderm inductive activity from the vegetal hemisphere include members of Xenopus nodal-related factors  and TGFβ family (activin βB), now known as endogenous mesoderm inducing factors in various animal models, including Xenopus, zebrafish, and mouse ,108,109 pression . Activinpression . In compnd fgfr2 . Supportelopment . Similarlineages ,115,116.ventx1.1 and xhox3) [gsc, chrd, and nog) genes in Xenopus embryos [xbra expression [xbra transcription in animal explants of Xenopus embryos, wherein Xbra makes an autocatalytic regulatory loop to maintain the Fgf4 expression [fgf4, but its activation is most likely thought to depend on Nodal signaling; this is similar to fgf8 activation largely depending on nodal/activin signaling [Brachyury (Bra/Xbra) is a T-box transcription factor and signature mesodermal  factor, and is actively involved in gene regulation process required for mesoderm induction and differentiation ,118. Simd xhox3) ,121,122.d xhox3) ,124. Eom embryos . Xbra eapression . In turnpression ,125. Xbrignaling . FGF8, hignaling .chrd, nog, gsc, and siamois (sia) are highly expressed in dorsal mesoderm [gsc expression is sustained under FGF inhibitory conditions [chrd and nog (from the organizer) have been reported to be both sensitive and insensitive to FGF signaling, thus complicating the interpretation of their underlying mechanism [FGF signaling has an indispensable role in mesoderm induction and specification, but the detailed molecular mechanisms by which different FGF ligands regulate a different subset of mesodermal markers is not fully understood. Based on their spatiotemporal expression patterns, these markers may be divided as dorsal, ventral, and pan-mesodermal markers. In dorsal mesoderm of Xenopus gastrula, mesoderm , in whicnditions . Howeverechanism ,128. As echanism ,109,110,xcad3 and hoxb9 (posteriorizing factors) expression and normal axis development in Xenopus [ventx1.1, a ventral mesodermal/ectodermal inducer and a BMP/Smad1 target transcription factor [With the early ventral mesoderm, which later leads to development of posterior tissues, several pieces of evidence indicate that FGF signaling plays an upstream role to the RAR (retinoic acid receptor) . In part Xenopus . Cdx  levels via Xbra and Xcad2. These findings imply a context-dependent  function for FGF on ectoderm formation. The regulation of each modality, however, remains to be further studied.BMP signaling is essential for ectoderm  specification during embryonic development as BMPs activate ectoderm specifier mal fate ,136. Prerylation ,137,138.pression . In zebrondition . Also inondition , again scription , and Xcapression . These schrd, nog, and Follistatin, abolishing the neural plate formation [Neuroectoderm formation starts from the superficial ectodermal layer near the organizer , in a reormation . Triple ormation ,150. Thiormation ,152.zic3 expression in animal explant of Xenopus embryos [zic3 and foxd5a; with BMP inhibition. Zic1 transcription was also increased [Requirement of FGF signaling in neuroectoderm formation could be considered in two different ways: first, being an instructive signaling, in addition to BMP inhibition, FGF signal itself induces neuroectoderm via activation of neural specific genes. Second, being inhibitory to BMP signaling, to guarantee the neuroectoderm formation, FGF and Map kinase participate in inhibition of Smad1 via linker region phosphorylation. Both ways may function together in neuroectoderm formation. However, the first one is essential and the second one is an additional one since the BMP inhibited condition still requires an FGF input. This implies that intact FGF signaling is necessary as an instructive signal in addition to BMP inhibition for neural fate. Although which FGF member(s) instructs naive ectoderm to neuroectoderm remains to be investigated further, ectopic expression of FGF2 induces the neural genes  embryos . Anotherncreased . BMP signcreased . This obfgf8a/b in animal explants without xbra and fgf4 expression [Xsox3 and N-tubulin via FGFR4a signaling [More recently, Foxd4l1.1, an immediate early neural marker, was shown to be induced following inhibition of BMP signaling . Foxd4l1pression . FGF8b cignaling . In miceignaling . Similarignaling  again imignaling , in retiignaling , and antignaling . Togethexbra [Xenopus embryos provide an easy and powerful tool to investigate the regulatory mechanisms in germ layer specification. In this system, the ventral region (A4 blastomere) of 16 or 32 cell-stage embryo fate mapping has this region differentiate into epidermal cells ,149. Howxbra . Knockdoxbra . The reqxbra ,158,160.For an amphibian embryo, the first embryonic axis is the dorsoventral (DV) axis that is established prior to germ layer formation. The anteroposterior (AP) axis is an extension of the DV axis, in which the dorsal expands to the anterior, and the ventral expands to give the posterior end B. The legsc, chrd, follistatin, and xnot, resulting in formation of the secondary axis [xbra and xcad2 known to promote ventral/ectodermal fate via activation of ventx1.1 expression [At late blastula and early gastrulation, Xenopus embryos exhibit a dorsoventral (DV) axis that is established prior to mid-blastula transition (MBT) and it involves shifting maternal morphogens or molecular gradients in a process known as cortical rotation . The venary axis . We havepression ,130. On pression ,38,115. pression ,164,165.pression ).xcad3, hoxB9, and hoxA7 [The dorsoventral (DV) axis typically expands to continue with anteroposterior (AP) axis development. The dorsal animal region reaches to the anterior and the ventral vegetal region extends to the posterior. The majority of evidence point to FGF signaling as being a posterior inducer. Dissociated cells from an ectodermal explant of Xenopus embryos were shown to adopt an anterior neural fate. However, additional FGF converted their fate to a posterior neural one ,168,169,nd hoxA7 ,171. In nd hoxA7 ,171.Within the FGF family, there are also variants of FGFs that may have varying functions in axis development, although they are categorized under the same FGF signaling pathway. For example, FGF8 can generate the highest number of splice variants (FGF8a-h) among FGF family in mice, and these variants are subject to relative expression differences during development (reviewed ). FGF8a efgf and wnt8 during the Xenopus embryonic development [hox genes during neural tube development [Both genes family of caudal homologs and Hox have been reported as the targets of FGF and Wnt signaling during AP elongation in vertebrates ,177,178.elopment ,184. Anoelopment . In miceelopment ,187. Botelopment ,187.nodal and pitx2 expression and activates cSnR (chick snail-related gene) expression [The dorsoventral (DV) axis is followed by gastrulation organizing the anteroposterior (AP) axis. The left–right (LR) axis is formed and defined when in vertebrate embryos the anatomy or positions of visceral organs with respect to the midline become apparent. In mice, initiation of embryonic LR patterning may be defined in two crucial steps (reviewed ). In thepression . Interespression .foxj1, rfx2, ier2, ift88, and fibp1) responsible for functional nodal cilia are regulated by FGF signaling [The most critical event in the symmetry-breaking event known to direct LR asymmetry in mammals is nodal flow. The nodal flow generally refers to leftward extracellular fluid movement powered by nodal cilia in the ventral nodes (review ). Nodal ignaling ,196, andSeveral agents have been discovered or experimentally developed that are relatively specific in modulating FGF signaling, and chemically, these include proteins and small molecules. Based on their activities, these modulators may be placed into three groups: The first includes the endogenous activator(s) as those agents having a positive or compensatory activity relative to FGF signaling. Second are the endogenous factors showing inhibitory or negative activity towards FGF signaling. Third includes the group of relatively small molecular-weight compounds, chemically synthesized to act as inhibitors and with use intended mostly for therapeutic or research applications. Prominently known endogenous activators/inhibitors of the FGF pathway were chosen to be briefly discussed.A positive feedback regulatory mechanism plays an important role in embryonic and post embryonic development in vertebrates. In this process, several modulators may act as co-activators, interacting with a specific domain of an FGF or an FGFR; this interaction can in turn significantly accelerate or amplify FGF signaling. Notably, heparan sulfate proteoglycans (HSPGs) and glypicans are essential extracellular key modulators, which interact with a variety of growth factors including various FGFs and morphogens and their receptors . These ifgf genes, which is capable of interacting with the cytosolic domain of FGFR and blocking the intracellular effectors interacting with FGFR. An ectopic gain of function of sef, for example, has been shown to reduce phosphorylation of Raf1 and MEK1/2 [A negative feedback mechanism is an essential regulatory strategy for nearly all signaling pathways and this is particularly important during embryonic development. FGF signaling has demonstrated as having an active functional role at multiple steps across embryonic development. To date, a group of endogenous proteins have been identified that show antagonist activity to FGF signals and are generally associated with the negative feedback loop in downregulating FGF signal transduction. One is Sprouty, an intracellular protein that interacts with Drk and Gap1, being components of Ras/MAPK pathway, and blocks their interaction ,198. Anod MEK1/2 . Other ad MEK1/2 , Pyst1/Md MEK1/2 , Dusp1 [d MEK1/2 , and FRSd MEK1/2 , all endlzme) gene where lzme encodes an enzyme required for glycosaminoglycan biosynthesis) causing defective mesoderm and endoderm migration [gpc4 (Glypican 4) shows pleiotropic developmental aberrant phenotypes including defected primary axis and forebrain patterning [FGF modulators are essential in early embryogenesis as several studies have reported knockdown of various HSPG genes causing defective development in vertebrates and invertebrates (reviewed ). The inigration . Gene exigration . Similartterning . A detaitterning ,206,207.Aberrant FGF/FGFR signaling triggers numerous congenital disorders and many cancer types. Evidence obtained from gain-of-function/loss-of-function experiments demonstrates that proper FGF/FGFR function is required for correct embryonic development in vertebrates. The findings we discussed here suggest that various FGF ligands can trigger distinct germ layer specifications exclusively or in coordination with other signaling pathways. Genetic manipulation using recombinant techniques allowed us to investigate the genetic and functional diversity of FGF/FGFR signaling in early embryogenesis and postnatal development of vertebrates. The current understanding of the gene regulatory network (GRN) in embryonic development has made great progress, and we summarized some of the data within our line of investigation particularly for FGF signaling and its crosstalk with other signaling pathways. A number of particular FGFs were characterized that participate in early embryogenesis, and additional investigations for a more complete picture of their role in active GRN are required. The impressive array of additional modern techniques that have now become mainstream will be utilized for this purpose; these include single-cell RNA/DNA sequencing, efficient knock-in/knock-out technology of CRISPR, and characterizing hESCs along with vertebrate embryos. Taken together, this review provides an overview of the findings of the current research on embryonic development and lineage specification concerning FGF signaling in vertebrates.


Over the past decade, widespread concern has emerged over how environmental governance can be transformed to avoid impending catastrophes such as climate change, biodiversity loss, and livelihood insecurity. A variety of approaches have emerged, focusing on either politics, technological breakthrough, social movements, or macro-economic processes as the main drivers of change. In contrast, this paper presents theoretical insights about how systemic change in environmental governance can be triggered by critical and intellectually grounded social actors in specific contexts of environment and development. Conceptualising such actors as critical action intellectuals (CAI), we analyze how CAI emerge in specific socio-environmental contexts and contribute to systemic change in governance. CAI trigger transformative change by shifting policy discourse, generating alternative evidence, and challenging dominant policy assumptions, whilst aiming to empower marginalized groups. While CAI do not work in a vacuum, nor are the sole force in transformation, we nevertheless show that the praxis of CAI within fields of environmental governance has the potential to trigger transformation. We illustrate this through three cases of natural resource governance in Nepal, Nicaragua and Guatemala, and Kenya, where the authors themselves have engaged as CAI. We contribute to theorising the ‘how’ of transformation by showing the ways CAI praxis reshape fields of governance and catalyze transformation, distinct from, and at times complementary to, other dominant drivers such as social movements, macroeconomic processes or technological breakthroughs. It is now well accepted that the climate crisis, biodiversity loss, social inequalities, and livelihood insecurity cannot be tackled without systemic change in how environment and natural resources are governed O’Brien . Three mStudies have linked the prospects for transformational change to drivers including: globalisation , people who contribute to systemic change though their intellectual work and political engagement (praxis) in relation to fields of environmental governance. Our previous work has shown that critical action research that challenges hegemonic knowledge, and experimental actions to demonstrate alternatives, can promote lasting change Ojha . This kiThe motivation for our analysis comes from the absence of conceptually articulated, practice-based theorizing on transformational change. This work evolved from our critical action research on community-based forest governance in Nepal, where some of us were actively engaged as CAI, exposing the hegemony of techno-bureaucratic practices and working to empower disadvantaged communities Ojha . We drawThe burgeoning research on transformation has exposed a host of dilemmas around the interface between deliberate human agency and structure Feola . Feola ‘Critical intellectual action that challenges the status quo has long been recognised as triggering social change: examples include decolonisation Said , criticaTo link agency with the politics of knowledge and social action, we use Bourdieu’s conceptualisation of the ‘field’ to capture interwoven social relations, political economies and cultural norms Bourdieu , 1998 thAnalyzing CAI praxis in relation to fields allows us to explore the ‘how’ question of transformation that has too often fallen in the cracks between theory and practice  government laws : “In my job my interest was, or my politics were, to put forestry as a development agenda at local level,” (interview 26 December 2017).The first wave of profound change took place in the 1970s, when a few government forest officials working at the district level experimented with allowing communities to participate in creating forest management rules and to access forest products as usufruct rights. Perhaps the first was TBS Mahat, a widely respected CAI who pioneered community forestry from within the government system at a time when the policy insisted upon centralised control  for technical knowledge  transfer … related to nursery techniques, plantation establishment, silvicultural practices, etc. I was instructed not to get involved in policy matters, as that was something for the Nepal government (Interview March 2021).Nepali officials like Mahat were joined by foreign researchers in donor projects responding to the global outcry over the so-called Himalayan land degradation crisis Eckhom . The NepBoth international forestry experts and Nepalese officials continued to reshape the field of forest governance. All were clear in interviews with us that it was both their intellectual capability and on-the-ground experience of governing forests that triggered their desire to take professional risks against the status quo. These pioneering efforts on decentralising forest rights were joined by Nepali CAI such as Narayan Kaji Shrestha who initially worked for donor funded projects and later became active as a civil society activist pushing community-based forestry.These pioneers worked in tandem to persuade the Forest Department to allow community groups to have rights of access to forest areas, and created functioning models of community forest management across a wide swath of the Middle Hills region. First-generation CAI sparked the formulation of a new Forest Act 1993 (and its by-laws in 1996). This law was among the world’s most progressive, recognising community rights over government forest areas. As a result, by the late 1990s, the number of community forestry groups multiplied exponentially across the country, establishing community forestry as one of the most important ‘development successes’ in Nepal.A second generation of CAI emerged after the 1993 Forest Act, inspired by wider democractic political reforms. The number of CAI multipled and their work supported change through critical research, empowerment of community groups, and faciltiating dialogues between community leaders and the government. Some worked with donor projects to demonstrate the impact of community participation on forest cover (Branney and Yadam 1998), while others working from within the government highlighted the economic potential of community forestry Kanel . NGO basHowever, underneath the layers of progressive change and community empowerment, this story has a bleak side too. Changes in the organisational culture of the forest department have been extremely slow. The bureaucracy has never fully accepted the rights of communities, or certainly not to the extent that the 1993 Forest Act provided (nearly complete autonomy once a forest area was handed over), something which persists as of this writing. In the early 2000s, a new generation of trained forestry and environment intellectuals entered the field. Perhaps, most prominent were those who created ForestAction Nepal (FAN) in 2000 (the lead author of this paper is a co-founder). FAN quickly became a key institutional home of CAI wherein they: created significant space for research, policy engagement and the dissemination of critical knowledge by launching a practitioner-oriented research journal and a policy briefs series; experimented with various forms of stakeholder dialogues; and engaged in action research. FAN worked closely with FECOFUN and reformist forest officials to help cement community forestry as the main instrument of forest governance in the hill districts . FAN undertook a quick and strategic assessment of this proposal and published a Policy Note , Eastern Nicaragua became one of the first regions in the Americas in 1987 to establish autonomous polities that guaranteed indigenous and Black communities’ participation in a multicultural government. From the 1990s onwards, this autonomous status has been contested by the Nicaraguan state, claiming the region consists of “national lands”  the Interamerican convention of Human Rights does not recognize collective property, it only recognizes private property (…) but we decided to educate that tribunal [the Interamerican Court of Human Rights]. (…) we decided that I would not represent [legally] the community and I would be in the audience as an expert witness. We thought about (…) showing how justice is administrated from the indigenous perspective. (…) if the court did not understand us, how would it understand that for the indigenous community there is not only a material relation with the land but also a spiritual relation? (…) we wanted to connect the right to life to the right to land” .Besides such higher scale engagement in the environmental governance field, CAI’ partnership with local indigenous communities in Nicaragua has also aimed at demonstrating ways of working with ethnic minorities that overturn conventional, hierarchical ways. In the Nicaraguan case of Awas Tingni, the above-mentioned CAI collaborated with the indigenous community in a novel way and used alternative methods of articulating knowledge systems for achieving transformation in practice. For example, the CAI could have legally represented the indigenous community at the court. Instead, she supported the indigenous community to represent itself through twenty-five of its members. What was at stake was not only winning the case, but also showcasing decolonised ways of working together. This further showed the world that indigenous groups have legitimate and established ways to ensure justice and organizational systems to deliver it in practice. The CAI explained:In addition to the mobilisation of a justice and recognition narrative such as above, CAI have also used technical tools to generate alternative data to empower the voices of marginalised groups. While this type of effort led by CAI may not change all the historically and culturally embedded colonial practices that systematically disadvantage indigenous people, they envision practical pathways for socio-environmental transformations that can serve as benchmarks.CAI’ capacities to mobilise technical knowledge typically used to serve the interests of the powerful have been instrumental in the defence of indigenous people’s right to land in Central America. CAI in Guatemala have assisted indigenous communities to defend their land rights in the face of increasing oil palm plantations by using GPS to map community rights, but in accordance with their indigenous vision, and in a participatory way. Such maps helped to materialise the traditional system of land rights and to establish community land management plans, thereby empowering the community to develop counter propositions for the Guatemalan state and its World Bank funded land legalisation programme that did not recognise indigenous community rights  and the Community Land Act 2016). In 2013, newly constituted County level authorities were given financial resources and some decision-making authority in the management of forests, water and land   International to influence Turkana policy. The CAI leveraged their research backgrounds  emerged and engaged in praxis triggering systemic change in environmental governance. At its core, CAI praxis involves creating alternative and action-oriented knowledge to challenge dominant policy assumptions and empower disadvantaged groups in the processes of governance. CAI praxis is distinguished from other kinds of activism by emphasising the need to change discourses and reframe policy narratives, in addition to working towards concrete changes in access to and control over resources. They thus focus on knowledge reframing At least three theoretical insights are noteworthy. First, since systems of governance are rooted in the self-reproducing logic of fields, any attempt to effect systemic change requires exposing the epistemic foundations of the dominant order, such as through utilising the power of critical evidence, social experimentation, and building of critical epistemic communities. CAI have emerged as strong social agents to take on such challenges, and central to their success are their abilities to question existing ways of thinking and promote new ways of acting as well as playing with the underlying and self-reproducing logic of the field with the intention of overturning it. This means that systemic change inevitably requires some form of critical intellectual work that is capable of connecting practice, discourse, and politics within and beyond the boundaries of the field.Second, the work of CAI is itself relationally co-produced with the dynamics of fields, and hence the transformational potential of CAI praxis is significantly linked to the structural regularities and recurrent moments of crisis in the field. Through CAI praxis, the field is challenged, and when successful, reshaped. That reshaping in turn demands new forms of engagement and reflection by CAI, as realities of the field shift, or previous attempts at change fail. In the long and arduous trajectory of system change, CAI as individuals may change their career or engagements, but the field remains to give birth to a new generation willing to take on the challenge. An important conclusion here is that systemic change emerges not just from the strategic acts of critical action intellectuals, but also from the interplay between CAI praxis and the dynamics of the field, especially when moments of opportunity emerge in the temporal trajectory of the field.Finally, as transformation involves changes in systems, behaviours and mindsets, no single event or action is likely to bring about such profound change. Systemic change requires continuously pushing the work of knowledge and community mobilisation, roles which CAI take on, with their action oriented epistemic research as well as political will to engage in action for change. However, they are also part of the uneven political field, and their success is not just linked to what they do but also to their identities and positions in the field. CAI praxis is also contingent on the possibility of CAI sustaining their work in challenging times in the field. This means understanding the prospect of CAI praxis has to be contextual and a dynamic process of learning and contextual theorising, locating the prospect of transformation in the interface between CAI and their praxis in the field.


As mechanical injury is a primary aetiological factor in OA, we investigated miR-140-dependent mechanosensitive gene regulation using a novel CRISPR-Cas9 methodology in primary human chondrocytes.MicroRNA 140  assay. miRNA levels were confirmed by qPCR in chondrocytes and in wild type murine femoral head cartilage after acute injury. Predicted close match off-targets were examined. Mechanosensitive miR-140 target validation was assessed in 42 injury-associated genes using TaqMan Microfluidic cards in targeted and donor-matched control chondrocytes. Identified targets were examined in RNAseq data from costal chondrocytes from miR-140−/− mice.Primary (passage 1/2) human OA chondrocytes were isolated from arthroplasty samples (six donors) and transfected with ribonuclear protein complexes or plasmids using single guide RNAs (sgRNAs) targeting miR-140 (90–98%) was obtained when two sgRNAs were combined with double RNP-mediated CRISPR-Cas9 transfection. miR-140 levels fell rapidly after femoral cartilage injury. Of the top eight miR-140 gene targets identified (P < 0.01), we validated three previously identified ones . Novel targets included Agrin, a newly recognised pro-regenerative cartilage agent, and proteins associated with retinoic acid signalling and the primary cilium.High efficiency gene editing of miR-140 targets of disease relevance.We describe a highly efficient CRISPR-Cas9-mediated strategy for gene editing in primary human chondrocytes and identify several novel mechanosensitive  Osteoarthritis (OA) is generally accepted as a biologically driven disease where mechanical stresses, combined with other factors, lead to imbalance between catabolic and repair activities within the jointmiRs) are important regulatory molecules in all animal and plant cells. They are small endogenous RNAs, typically 20–25 nucleotides in length, that suppress specific mRNAs, by binding and targeting the mRNA for degradation or by suppressing protein translationmiR is formed of a 5 prime (5p) and 3 prime (3p) strand that forms a hairpin loop. miRs undergo a process of maturation using two key enzymes, Drosha and Dicer, deletion of which have profound developmental phenotypes,miR is then loaded onto the Argonaute protein to form the active RNA-induced silencing complexMicroRNAs  which is specifically, and highly expressed in articular cartilage,miR-140 is hosted by the gene WW domain containing protein 2 (WWP2), an E3 ubiquitin ligase. Genetic deletion of either miR-140 or Wwp2 in mice leads to skeletal abnormalities and accelerated OAmiR-140 influences chondrocyte proliferation by reducing Sp1, a transcription factor which controls the cell cycle regulator p15 miR-140 have also been linked to skeletal abnormalities in humansmiR-140 plays an important homeostatic and chondroprotective role in the developing skeleton and adult joint. miRs can be targeted for therapeutic gain and can themselves be used as therapeutic agents, so their biology is of particular interestmiRs and small interfering RNAs (siRNAs) bind to specific mRNAs to target them for destructionin vivo tool. miRs usually have multiple endogenous gene targets through which they modulate cell behaviour. A recent breakthrough molecular approach utilises clustered regularly interspaced short palindromic repeats (CRISPR), an antiviral (anti-bacteriophage) defense system of prokaryotic cells, forming part of their innate immune responseBoth ,CRISPR-Cas9 mediated gene editing of primary human chondrocytes is challenging and previous reported transfection efficiencies have varied between 16% and 70%miR-140 in primary human articular chondrocytes with high efficiency and without affecting WWP2 expression. We examine the regulation of miR-140 upon ex vivo murine cartilage injury and explore the regulation of a number of previously described and novel targets that are relevant to chondrocyte mechanobiology and OA pathogenesis.In this study we optimize a method to use CRISPR-Cas 9 to drive deletion of Human tissue: Osteoarthritic human articular chondrocytes were isolated from tissue obtained from individuals undergoing unicompartmental (UKA), or total knee replacement (TKR). There were no exclusion criteria. Samples, Kellgren and Lawrence grades 3–4, were obtained from the Oxford Musculoskeletal Biobank and were collected with informed donor consent in full compliance with national and institutional ethical requirements, the UK Human Tissue Act, and the Declaration of Helsinki .sgRNA design: For gene editing, the ALT-R® CRISPR-Cas9 system from Integrated DNA Technologies  was applied. The Cas9 protein, trans-activating CRISPR RNA (tracrRNA) and CRISPR RNA (crRNA) were all acquired from the same company. To form a functional sgRNA duplex, 3 μl of tracrRNA (5 nmol) were mixed with 3 μl of target-specific crRNA (2 nmol) in 94 μl IDT nuclease free duplex buffer and RNA quantified by nanodrop. While the universal tracrRNA forms the backbone, the crRNA is custom designed and target-specific. The required amount was then incubated for 5 min at 95°C and slowly annealed at room temperature for 10 min. Off and on-targets were predicted using a combination of two software packages: IDT (https://eu.idtdna.com/pages) and SANGER - https://www.sanger.ac.uk/htgt/wge/find_off_targets_by_seq.Transfections: For RNP transfections, sgRNA  was complexed with Cas9 (1 μg) for 5 min in 50ul reduced serum medium . Cas9 Plus reagent (2 μl) was added before incubation. Cas9 Plus is part of Lipofectamine CRISPRMAX . In another tube, Lipofectamine CRISPRMAX (3.5 μl) was added to Opti-MEM (50 μl), and incubated for 5 min. The Opti-MEM media containing the sgRNA/Cas9 complex was carefully added to the Opti-MEM containing Lipofectamine CRISPRMAX and incubated for another 10 min before slowly pipetting the Cas9/sgRNA/Lipofectamine into the cell media. For double transfection, cells were left for 48 h, exchanged into 10%FBS/DMEM for 24 h, then a second round of transfection by addition of freshly prepared Opti-MEM media containing the sgRNA/Cas9 complex/Lipofectamine CRISPRMAX was performed. Cells were incubated in 10%FBS/DMEM for a further 48 h before testing.T7 Endonuclease 1 (T7E1) assay: DNA was first amplified by quantitative polymerase chain reaction (qPCR) using a high-fidelity DNA Polymerase  and gene-specific primers  or unpaired. One-way analysis of variance (ANOVA) was conducted when comparing more than two groups, followed by Tukey's test (when comparing means with every other mean) or Dunnett's test (when comparing means with control mean) for comparison between the groups. RT-qPCR expression was determined by applying log 2 formula (2−ΔΔCT) using housekeeping genes RNU24 (for microRNA) or RPLP0 (pre-printed on customised microfluidic cards). Where relevant statistical significance was defined by a P value of <0.05.We first assessed the impact of two different transfection methods on isolated human osteoarthritic chondrocytes (OA hACs). Passage 1 hACs were transfected either with pX330 or using a Ribonucleoprotein (RNP) complex. The pX330 plasmid is an ∼8.5 kb plasmid with the coding sequence for Cas9 and a targeting sgRNA subcloned into it. The RNP complex consisted of the Cas9 protein and targeting sgRNA. RNP transfected and untreated control OA hACs exhibited continuous growth and increased confluency at 24 h and 48 h post transfection. However, plasmid transfected OA hACs appeared to stop proliferating and exhibited high numbers of non-adherent, typan blue positive cells indicating toxicity . ImmunobmiR-140 in hACs, we first confirmed that both arms of miR-140 (140-5p and miR-140-3p), were expressed in cells taken from OA cartilage samples from several different donors (each donor represented by a different colour) (miR-140-5p levels were slightly higher than miR-140-3p (P = 0.0445).Before exploring the role of  colour) . miR-140https://eu.idtdna.com/), were designed to target the hairpin structure of miR-140, which sits within an intron of WWP2 [miR-140 targeting sgRNAs were complexed with Cas9 as RNPs and transfected using Lipofectamine CRISPRMax. A 439 base pairs (bp) long PCR product was amplified using miR-140 flanking primers , with predicted high on-target and low off-target scores, using IDT software (miR-140 was targeted using sgRNA L3 (which had the highest IDT scores) or using the combination of either sgRNAs L2 and L3, or sgRNAs L1, L2 and L3, ensuring that the final concentration of sgRNA remained constant across groups. Three different controls were included: a NT sgRNA control (NT), Lipofectamine (LF) and Cas9 treated cells (no sgRNA) (LF and Cas9), as well as completely untreated cells (untreated). For each donor, each experimental condition was performed either in duplicate or triplicate. DNA cleavage was assessed by the T7E1 assay, after amplifying the 439 bp product described in miR-140 gene expression (a surrogate read out of editing efficiency) was examined in the same samples by qPCR. 50–60% reduction in gene expression of miR-140-3p [mir-140-5p [miR-140 was greatest (>80%) when using the combination of sgRNAs L2+L3 , albeit reaching statistical significance only for miR-140-3p ; miR-140-5P = 0.0541  [miR could be directly contributing to the injury response. To test this, we examined a number of genes that were either previously identified miR-140 targets or shown to regulate or to be strongly regulated by cartilage injury (n = 5) after miR-140 deletion and compared with their respective paired NT control. Data are presented in SEPT2 (1.95 ± 0.51 fold), bone morphogenetic protein 2 (BMP2) (5.33 ± 2.19 fold) and RA receptor gamma (RARG) (2.42 ± 0.47 fold) were upregulated upon miR-140 deletion, displaying statistical significance after stringent correcting for multiple comparisons  data taken from costal cartilage of 7-day old constitutive miR-140 KO mice (available at NCBI GEO datasets with the accession number GSE144360). We considered the top eight genes from our qPCR analysis, whose unadjusted P values were <0.01 . BMP2, RARG and TTBK2 were not regulated in neonatal mouse costal chondrocytes by deletion of miR-140.To explore the biology of e injury . These iparisons . Interesed genes  indicatire <0.01 . Of thesmiR-140 in primary human chondrocytes by performing transfection of RNP complexes containing Cas9 and sgRNA targeting sequences. This method was superior to that using the pX330 plasmid which exhibited evidence of cell toxicity within 48 h of transfection. Editing efficiency using RNP appeared to be optimal when combining more than one sgRNA, and when double transfection was performed. Efficiency at this level is usually only obtained by clonal expansion of selected targeted clones, which risks losing the chondrocyte phenotypemiR-140 was suppressed by >80% for all donor samples.In this manuscript we describe a novel approach for efficient genomic editing of miR-140 in human chondrocytes we first assessed whether it was regulated upon cartilage injury, an important and clinically relevant stimulus for the tissue,,miR-140 was downregulated 4 h following murine cartilage injury, a time at which many other genes are upregulated. miR-140 might therefore influence the response of the tissue to OA-induced mechanical injury and might explain the enhanced OA phenotype seen in miR-140 knockout micemiR-140 deletion on chondrocyte genes that are known to be strongly regulated by injury, in addition to genes that had previously been identified as miR-140 targets by other groups  following a single round of transfection indicating that high level depletion is required to uncover biological function.To explore the biological significance of P value was <0.01, miR-140-dependent genes included those involved in the RA pathway , primary cilia biology , and anabolic factors . Several, but not all, of these were strongly miR-140-regulated in costal chondrocytes taken from neonatal miR-140 knockout mice, perhaps reflecting differences in species or relating to differences in chondrocyte site and maturity . SEPT2 was the most robustly miR-140-regulated gene in both human articular and costal chondrocytes and has previously been identified as miR-140-dependentSEPT2 in chondrocytes is unknown but it may be relevant that two other cilia-related genes (IFT88 and TTBK2) were also regulated by miR-140 in human articular chondrocytes. The primary cilium has previously been linked to OA through its established role in modulating hedgehog signallingin vitro possibly by controlling the distribution of the scavenger receptor LRP1in vivoConsidering those genes where the uncorrected miR-140 targets. FGF2 was strongly miR-140-regulated in human articular and murine costal chondrocytes, as described previouslySEPT2 and FGF2 possess a miR-140-5p seed sequence in their 3p-UTR, and so are predicted to be direct targets of miR-140. Two other anabolic molecules were identified as miR-140-dependent in human articular chondrocytes: BMP2 and AGRN. AGRN is a heparan sulfate proteoglycan, usually associated with the neuromuscular junctionin vivoAGRN, unlike BMP2, was also strongly miR-140-dependent in neonatal murine costal chondrocytes.Several genes with known pro-regenerative or anabolic roles were also identified as miR-140 targets in human articular chondrocytes (RARG and CYP26B1). CYP26B1 was also strongly miR-140-dependent in murine costal chondrocytes. We have recently observed that cartilage injury strongly regulates RA dependent genes, including the CYP26 enzymes, which are the key regulators of cellular RA levelsWe identified two novel RA regulated genes as ADAMTS5 was not regulated in miR-140 knockdown hACs in this study, even though 3′-UTR of ADAMTS5 contains a putative seed sequence for miR-140-3p. Its regulation might have been uncovered had we looked at chondrocytes stimulated with IL1 or equivalent catabolic stimulus. As miR-140 KO mice are characterised by short statureContrary to previous reportsWe recognise a number of limitations in this study. Firstly, the nature of both the T7E1 and MiSeq assays means that underestimation or overestimation of efficiency, respectively, may occurmiR-140 deletion by confirming previously identified targets and identifying new targets. We show, for the first time, that miR-140 is down-regulated upon cartilage injury, and that several injury induced genes are also miR-140 targets. This confirms the important role of miR-140 in cartilage homeostasis, and in the injured joint in the development of OA. As chondroprotective pathways are also regulated by miR-140, it would not seem prudent to regard miR-140 as a target in OA.To conclude, we have demonstrated that it is possible to get high levels of CRISPR-Cas9 mediated gene deletion in human primary articular chondrocytes. We have validated the functional effect of (1)The conception and design of the study , acquisition of data , analysis and interpretation of data .(2)Drafting the article or revising it critically for important intellectual content: all authors(3)Final approval of the version to be submitted: all authorsNo conflicts of interest relevant to this work are identified for any of the authors.Academic funding sources did not influence the project direction or decision to publish.


Fertility preservation is a novel clinical discipline aiming to protect the fertility potential of young adults and children at risk of infertility. The field is evolving quickly, enriched by advances in assisted reproductive technologies and cryopreservation methods, in addition to surgical developments. The best-characterized target group for fertility preservation is the patient population diagnosed with cancer at a young age since the bulk of the data indicates that the gonadotoxicity inherent to most cancer treatments induces iatrogenic infertility. Since improvements in cancer therapy have resulted in increasing numbers of long-term survivors, survivorship issues and the negative impact of infertility on the quality of life have come to the front line. These facts are reflected in an increasing number of scientific publications referring to clinical medicine and research in the field of fertility preservation. Cryopreservation of gametes, embryos, and gonadal tissue has achieved quality standards for clinical use, with the retrieval of gonadal tissue for cryopreservation being currently the only method feasible in prepubertal children. Additionally, the indications for fertility preservation beyond cancer are also increasing since a number of benign diseases and chronic conditions either require gonadotoxic treatments or are associated with premature follicle depletion. There are many remaining challenges, and current research encompasses clinical health care and caring sciences, ethics, societal, epidemiological, experimental studies, etc.  The preservation of fertility potential is an important issue for young people at risk of becoming infertile due to clinical conditions, diseases, or treatments. For patients with cancer, the current international guidelines recommend timely information on the risk of fertility loss associated with planned treatment, and referral to appropriate reproductive medicine centers to discuss further options for fertility preservation ,2,3. CanFertility preservation is also indicated when treatment of benign conditions may encompass a risk of infertility, either through medical treatments or surgery ,10. SomeCryopreservation of gonadal tissue aiming at its potential use in future reproductive treatments has been done successfully for many years in adult patients. In the case of pre-pubertal children, this method is their current only option to cryopreserve reproductive potential ,12. In aThe retrieval of gonadal tissue by invasive methods in prepubertal children is not a clinical standard practice for several reasons. An important issue is a risk for malignant cells in the tissue, often seen in children with leukemia, one of the most frequent pediatric malignancies . If re-tOn the other hand, girls after menarche could benefit from the same fertility preservation methods that are applicable to adults. However, difficulties arise in counseling young individuals that may not expect that fertility preservation may be of value for their future. The fact that counseling on fertility preservation after confirmation of a cancer diagnosis is usually managed as an emergency also makes communication difficult and lead to situations in which fertility preservation is finally not performed, even when it would have been possible and beneficial to the young patient at risk of infertility .There are several research areas that need to be developed to solve the technical problems with fertility preservation in prepubertal children. For teenagers, we need a better psychological understanding of how to help these young people make a decision that they will not regret in the future .One of the most characteristic symptoms of Turner syndrome, and the most distressing to the women with the syndrome, is infertility due to ovarian failure and the typical streak ovaries. There are, however, girls with Turner syndrome who present with signs of ovarian function in childhood and adolescence but have a risk of development of premature ovarian failure. Approximately 20% of girls with Turner syndrome develop some pubertal symptoms, usually breast development, but only 2–3% proceed to menarche . It is cThe recommended way to find the girls that have functioning ovaries and provide advice on fertility preservation at an early age is by taking repeated anti-Müllerian hormone (AMH) tests during childhood and adolescence ,24. AMH If AMH is detected it should be followed regularly, i.e., once a year or when other blood tests are needed, for example, those required under growth hormone treatment, hypothyroidism, or celiac disease, which are common in the management of girls with Turner syndrome.When the girl is mature enough to understand a discussion about her future fertility, she may be offered an ovarian biopsy via a minimally invasive laparoscopy . InformaIf the girl reaches spontaneous menarche, ovarian stimulation and collection of oocytes could be considered ,28. AfteBreast cancer is the most common malignancy affecting women of reproductive age . AdvanceFor adult women, established methods of fertility preservation include cryopreservation of oocytes, embryos, and ovarian tissue ,36,37,38Overall, the safety of fertility preservation in the setting of breast cancer, including the safety of COS, has been investigated in several recent studies ,48,49,50Long-term efficacy of fertility preservation in populations of young women with breast cancer can be measured as utilization rates of cryopreserved specimens after completed treatment or as subsequent pregnancy and live-birth rates. These types of data are yet scarce. Pregnancy rates after the use of cryopreserved embryos in survivors of breast cancer were reported to be comparable to those expected after in vitro fertilization in the general infertile population . In a coThe stage of maturity of the oocytes obtained is important since only mature oocytes at Metaphase II (MII) can be fertilized. If the oocytes will be cryopreserved, the method currently used is by vitrification, and immature oocytes are discarded ,36. HoweLong-term survival rates after gynecological cancer, especially in young patients, are increasing, and surgical techniques aimed at sparing reproductive organs and preserving fertility have been developed. The term most used for this type of surgery is fertility-sparing surgery (FSS), and it is often possible in early gynecological cancers . AlthougOvarian cancer is in general a disease appearing at an older age, and only about 8% of the women diagnosed with ovarian cancer are under 40 years of age . OvarianThe primary treatment of ovarian tumors is extensive surgery including hysterectomy and bilateral salpingo–oophorectomy, which results in infertility. Depending on the histologic subtype, surgery is combined with adjuvant chemotherapy. An FSS encompasses the practice of sparing the uterus and at least a part of one ovary, and it can be considered in women of childbearing age who wish to preserve their fertility, but this is also depending on the tumor type and the stage at diagnosis.In young women with early-stage non-epithelial ovarian cancer, FSS is now considered the standard surgical treatment ,58,59, aBorderline ovarian tumors (BOTs) are tumors with epithelial origin but without stromal invasion. Compared to malignant ovarian tumors, BOTs have better prognoses, and FSS is an accepted treatment for women of reproductive age ,62. EvenCervical cancer, which in most cases is caused by infection with high-risk oncogenic human papillomavirus (HPV), is commonly diagnosed in young women, and about one-third of all women who are diagnosed with cervical cancer in Sweden each year are under 40 years of age . In the In endometrial cancer, which is the most common gynecological cancer overall, only around 2% are diagnosed in women below 40 years of age . In the n = 262), hematological (n = 95), gynecological (n = 65) or other cancer (n = 75) were reported after a median follow up of 3.8 years [Since randomized clinical trials of fertility preservation in women at risk of infertility vs. no intervention may be unethical to perform, observational epidemiological studies following these two groups are key to evaluate the safety and efficacy of fertility preservation. The results summarized in the previous sections of this review indicate that ovarian stimulation for fertility preservation is safe for breast cancer patients and selected patients undergoing fertility-sparing surgery for the treatment of gynecological cancer. However, for other cancer types, the data available are limited. In a US study from 2018, no statistically significant difference in cancer recurrence or mortality after ovarian stimulation for fertility preservation in patients with breast  recently started collecting data on fertility preservation in an optional module of the ESHRE IVF-monitoring scheme . AccordiThere are numerous experimental studies currently ongoing aiming at developing possible pharmaceutical fertility preservative substances, which can be administrated prior to or during ovarian-toxic treatment to prevent its damage to ovaries . PublishAntioxidants or substances with antioxidative effects, bilberry, mirtazapine and hesperidin, mesna, sildenafil citrate, and hydrogen-rich saline have been shown to be able to reduce ovarian damage induced by cisplatin or cyclophosphamide in rats ,108,109.As regards human experimental studies, the fertility preservative effect of Gonadotropin-releasing hormone analogs (GnRHa) during chemotherapy has been investigated in several clinical trials. The data are still controversial. The guidelines for female fertility preservation of American Society of Clinical Oncology (ASCO) 2018, the ESHRE 2020, and the European Society for Medical Oncology (ESMO) 2020 do not recommend GnRHa as a proven fertility preservative method and stated that it should not thus be used in that place ,37. AlthAdditionally, there are several studies aiming to restore or rescue fertility competence after alkylating drug treatment using stem cells with mesenchymal origin. These stem cells could be isolated either from rodent femurs, tibiae, or adipose tissue or from human circulating blood, menstrual blood, amniotic fluid, placenta, or chorionic plate of the placenta. When different types of stem cells were injected into tail veins or ovaries of cyclophosphamide and/or busulfan or cisplatin sterilized/treated rodents, all of them showed improved fertility competence ,135,136.


Lack of vitamin A, carotenoids, and dietary fibers brings about serious health issues to this population. In this study, vegetables including bok choy, broccoli, cabbage, carrot, Chinese onion stalk (C_onion), mushroom, are added during the cooking of ugali, as nutritional supplements. The freeze-dried powder of each vegetable was used for its long storage, stable nutrients, and similar particle size. Sub-Saharan African assessors were trained and sensory evaluated the six different vegetable fortified ugali with the plain, unfortified as the control on five attributes. The plain ugali was indistinguishable with the C_onion stalk fortified in color, with the carrot and C_onion stalk fortified in odor, with all vegetables (except broccoli and mushroom) fortified ugali in taste, with carrot and C_onion stalk fortified in granularity, and with cabbage, carrot, C_onion stalk fortified in viscosity. Preference ranking analysis showed that the C_onion stalk fortified ugali is even more favorably preferred than the plain, unfortified ugali, probably due to the umami components in C_onion that serve as the taste enhancer. This study indicates that Chinese onion stalk is a potential vegetable supplement to population in the sub-Saharan Africa.Corn flour-based porridge like dough,  Ugali is a stiff, starchy porridge made from flour of various crops which is prepared by gradual mixing of the flour with boiling water. When the flour-boiling water mixture is allowed to simmer further, the starch-rich slurry transforms into a viscous porridge as a result of gelatinization of the starch [ugali is usually served with side dishes of stews containing various relish like fish, legume, cooked green leafy vegetables, beef, sour milk or other sources of protein [ugali itself is known to be extremely low in terms of nutrition content, it is the quality and quantity of these side dishes that can potentially improve their nutrition value [ugali is served with relish which is also low in nutrition value as is the case in most rural areas in sub-Saharan African countries, then the combined meal still remains of low caloric and nutritional value [In several sub-Saharan African countries, corn, cassava and sorghum are common crops whose yields merely satisfy local demands . These ce starch . Dependi protein ,4. Althougali, which is also named as bugali, gari, nshima, pap, etc. in different countries  cultivated in South Asia, sub-Saharan Africa and Latin America, nutrition enhancement in minerals and carotene could apparently help suppress the symptom of iron and vitamin A deficiency for local people. Meanwhile, their physical, mental and cognitive abilities were significantly improved [There are many names of this food according to the countries, regions, even tribes. It is most widely called ountries . Here weears ago . Normallproteins ,7, and cproteins . The low degrees . These wimproved . Promotiimproved ,12. Howeimproved ,14. Howeimproved .ugali is to add vegetables or fruits in ugali. Forsido et al. had tried adding Ethiopia banana to this Africa staple food, finding that the content of calcium and dietary fiber increased, so it was considered to have the potential to be applied in preparing this staple food [ugali for nutritional value increasing purposes, vitamin A or pro-vitamin A (β-carotene and carotenoids) should be considered with high priority as aforementioned. Bok choy, carrots, and cabbage have been supplied in meals to improve the total-body vitamin A pool size [Allium schoenoprasum), and mushroom (Lentinus edodes) are not only rich in vitamin A or carotenoids but also may add favorable flavors to ugali [ugali depending on the how the white corn is processed [Mageu was tried, however, fruit fortification does not supply dietary fiber as vegetables do [Another way to solve the nutrients deficiency in the staple food ple food . When onool size . Broccolto ugali ,19,20. Dto ugali ,22. Threrocessed . Whole mrocessed  Fortificables do . Thus, aables do . In thisugali [ugali to population with ugali as the staple food, as an evaluation of the feasibility of promoting vegetable fortified ugali in sub-Saharan Africa.Freeze drying of vegetables not only enhances storage stability, reduces transportation weight, maximally maintains micro-nutrients, but also enables grinding of vegetables to powders that allows fortification into ugali ,28. The Brassica rapa subsp. chinensis), broccoli, cabbage, carrot, C_Onion, mushroom  were purchased from Jiangsu QingGu Foods Co., Ltd. .White corn flour was purchased from the supermarket for international students at the west campus of China Agricultural University. The freeze-dried vegetable of bok choy  size before use. The white corn flour of 100 g was added gradually to 200 mL of boiling water and kept stirring all the time. Based on the nutritional value of each vegetable, 20 g could meet the daily requirement of nutritional intake of vitamin A, carotenoids, and dietary fiber. For vegetable fortified ugali was principally the same as previously performed on soymilk and chickpea [ugali, confidentiality of respondents and data, and the right to withdraw. Signed consent forms were collected from the assessors for record keeping purposes. After exclusion of assessors who were allergic to the abovementioned vegetables, a total of 24 assessors  were selected. The culinary traditions for ugali consumption in these countries are similar.Sensory analysis of chickpea ,30,31. Tugali samples in two sessions, 4 samples in each session and there was a 10 min break between sessions. In one session, assessors were required to clean their mouth with drinking water before and after assessing each sample, and take a break for 10 s between two samples. In each session, one plain ugali sample (with no vegetable addition) and three other vegetable fortified samples were offered with random sample codes  and the content of the sample information was kept unknown to assessors. The samples were presented in small, transparent plastic containers, marked with the corresponding code, served with a spoon and a cup of drinking water. For one ugali sample, assessors evaluated five attributes including: color, odor, taste, granularity and viscosity. Assessors used a nine-point hedonic scale . Finally, assessors were asked to rank the samples in order of preference . The questionnaire was collected and data was processed.A “questionnaire” was provided to each assessor and they were requested to fill their gender, age and nationality . Assessors were allowed to test 8 ugali were combined, and sample differences on each attribute were analyzed with least significant difference (LSD) test at a significance level of α = 0.05 with SPSS Statistic 26.0 . The violin plots were plotted with the GraphPad Prism .Sensory evaluation data of five attributes for two repeats of plain p < 0.05) [For median caregiver sensory rating, data of five attributes were compared by non-parametric Friedman’s two-way analysis of variance by ranks ( < 0.05) .F is calculated in Equation (1) where iR is the sum of ranks for sample i; n and K are the number of valid assessors and samples, respectively.For preference ranking analysis, the sum of the rank for each sample was calculated, and that of the two blank groups was averaged. The ranking data was analyzed by Friedman test at a significance level of α = 0.01 to evaluate significant differences among all samples . The tesF exhibits an asymptotic Chi-square distribution with K−1 degrees of freedom. At the α significance level, there are significant differences among all samples if F ≥ K−1 degrees of freedom. The significant differences between ranking data of two samples were analyzed with LSD test with SPSS Statistic 26.0 at a significance level of α = 0.05.The test statistic ugali samples before they try to taste them. And the initial observation and smelling of the sample profoundly impact personal willingness to taste. Thus, sensory evaluation data are divided into pre-tasting and tasting part for discussion.Generally, the sensory evaluation process can be divided into two steps. The assessors were allowed to observe and smell the ugali exhibited a white color with tint yellow , but not with ugali fortified with Chinese onion stalk. For ugali added with bok choy, broccoli and cabbage, there is no significant difference among them in presenting the green color. Interestingly, there is also no significant difference between ugali fortified with cabbage, carrot, and Chinese onion stalk. What’s more, ugali fortified with mushroom looks apparently less preferable by color from these fortified with cabbage, carrot, Chinese onion stalk, and the plain ugali (p < 0.05), but not with ugali fortified with bok choy or broccoli.Sensory evaluation of the color by assessors discriminated the color of plain ugali with these fortified with bok choy, broccoli, cabbage, and mushroom that showing less preferable hedonic scale (p < 0.05), but not these fortified with carrot and Chinese onion stalk. Meanwhile, there is no significant difference between carrot fortified ugali and these fortified with bok choy, broccoli, cabbage, Chinese onion stalk and mushroom. On the other hand, Chinese onion stalk fortified ugali distinguished itself from every other sample (p < 0.05) except carrot fortified- and the plain ugali.Sensory evaluation of the odor by the assessors discriminated the odor of plain ugali has no special flavor, different vegetables addition gives different flavor to ugali that may or may not satisfy its consumers. The taste was sensory evaluated to examine the catering to the potential consumers of each vegetable fortified ugali. Among all the vegetable added ugali, only the broccoli and mushroom added ugali exhibited significant less favorable responses in taste than the plain ugali (p < 0.05). Meanwhile, the taste is indistinguishable from broccoli added ugali and bok choy, cabbage, carrot, mushroom fortified ugali. And the taste of mushroom added ugali is indistinguishable from bok choy, broccoli, and cabbage added ugali.While the plain, corn flour based ugali. Among them, bok choy, broccoli, cabbage, and mushroom added ugali is significantly less preferred from the plain ugali as assessors evaluated (p < 0.05). And there is no distinguishable granularity difference between the plain, carrot and Chinese onion stalk added ugali.Although all vegetable powders were ground to the same size, they were all larger than the fine corn flour particles used in this study. The differences in the vegetable textures may introduce a variable in the mouth feeling that influences consumer experience. The granularity was sensory evaluated to exam the acceptance to the potential consumers of each vegetable fortified Ugali itself is a half-solid, half-liquid, viscous, porridge like food. Fortification with different vegetables may impact its viscosity that influences consumer mouth feeling. The viscosity was sensory evaluated to exam the acceptance to potential consumers of each vegetable added ugali. The cabbage, carrot, and Chinese onion stalk fortified ugali exhibited no differences in the mouth viscosity compared with the plain ugali. While there is a significant sensible viscosity difference between the plain ugali and the bok choy, broccoli, and mushroom added ugali (p < 0.05).ugali showed that all the sensory attributes, that may affect the acceptability of ugali, changed significantly with the addition of vegetables  as a food vehicle, caregivers were able to detect differences between fortified ugali for color, odor, taste, granularity, and viscosity (p < 0.05). For all the attributes, ratings were undesirable for every fortified ugali, except Chinese anion stalk that is almost as identically desirable as the plain, unfortified ugali.Caregiver evaluation of the five attributes of differently fortified getables A. Using ugali was: Chinese onion stalk-fortified, carrot fortified, cabbage fortified, blank (unfortified), broccoli fortified, bok choy fortified, and mushroom fortified , ranking among other fortified ugali was not significant. Similar survey with a much larger population might resolve the differences between the preferences of different fortified ugali in the future.The preference ranking sequence of the seven flavors of ortified A. Howeveugali, color, odor, taste, granularity, and viscosity, were evaluated by assessors. Since people usually sensed color and odor before they further taste it, which includes taste, granularity and viscosity, we decided to discuss color and odor before taste, granularity and viscosity. Plain ugali exhibited a white, yet tint yellow color that easily distinguish it from any fortifications except Chinese onion stalk due to the light and similar color of the later. Bok choy, broccoli and cabbage all endowed ugali green color due to the chlorophyll in them with non-distinguishable extent to consumers. It seems that color alteration with tint colors does not distinguish themselves, as evidenced by the indistinguishable hedonic scale of ugali fortified with cabbage, carrot and Chinese onion stalk. Deep brown color, endowed by addition of bok choy, broccoli, and mushroom does not differentiate each other for consumers. Although the odor of carrot (endowed by the presence of linden ether) added ugali is the same to consumers as the plain ugali, the color between them tells the difference [ugali was statistically unidentifiable from that of plain ugali [Five attributes of the plain and vegetable fortified fference . The odoin ugali .ugali and the Chinese onion stalk fortified one were unidentifiable from bok choy, cabbage, and carrot added ones. This is probably due to the rich flavor content in broccoli and mushroom that distinguished themselves [ugali often limits the nutrition intake, especially in children, leading to protein-energy malnutrition [ugali is the same to the potential consumers. This is probably due to the reason the highly rehydration process of freeze-dried carrot and onion stalk during ugali cooking that their texture differences between the plain ugali were eliminated [ugali is indistinguishable from that of cabbage, carrot, and Chinese onion stalk fortified ugali. This is probably due to the absence of starch or second thickener in the fortified ugali system [From the taste perspective, plain emselves ,36. Oralutrition . The oraiminated . The visi system .ugali exhibited no significant sensory differences in color, odor, taste, granularity and viscosity compared with the plain, unfortified ugali for assessors. Both the caregiver evaluation and preferencing ranking indicates that assessors even preferred the Chinese onion stalk fortified ugali over the plain one. Another Allium vegetable, Allium hookeri, significantly increased its sourness, sweetness and umami taste after hot air-dry [ugali is thus highly likely due to the presence of sweetness and umami components in this vegetable, not serving to change the taste, but as a taste/flavor enhancer for the plain ugali [In summary, the Chinese onion stalk fortified  air-dry . The ovein ugali ,42,43.ugali through the use of vegetable pastes proposed in this study and more. However, although these vegetable pastes have been found to be a potential source of nutrition when added to ugali, one anticipated limitation to their use would be their availability in the different African countries where ugali is the staple meal. If already readily available in most if not all of these countries, then follow up in-country studies should be conducted to determine the acceptability of such mixtures by the actual local people. Although the side dishes commonly served with ugali in various countries might have some nutritional value, the results of this study show some promising results that in the areas where such high-nutritional value side dishes are not guaranteed, consumption of ugali fortified with the vegetable pastes reported in this paper could be an ideal option.This study is one of its kind in this area and will hopefully result in more studies with larger sample sizes investigating the possibility of improving the nutritional value of ugali, specifically in vitamin A, carotenoids, and dietary fiber, freeze-dried powder of bok choy, broccoli, cabbage, carrot, Chinese onion stalk, and mushroom were added during ugali cooking. Fortification with these vegetables either changed the color, odor, or the taste, granularity, and viscosity that eventually lead to unfavorable assessor preferences. Except that the Chinese onion stalk fortified ugali cannot be distinguished from the plain, unfortified ugali while gained more favorable assessor preferences. This is probably due to the presence of umami components in the Chinese onion that serve as taste enhancer, thus renders this vegetable a promising candidate as vegetable supplement for the sub-Saharan Africa staple food. Meanwhile, on the basis of acceptance, bok choy, broccoli, cabbage and carrot variants had a comparable rating to that of plain ugali. Secondly, when the nutritional value was used as the basis, carrot, bok choy and onion stalk was found to considerably improve the micronutrient composition of ugali. Based on these findings, in addition to the Chinese onion stalk, bok choy and carrot options seem to be acceptable candidates for fortification which could be tested further in a follow up study involving a larger sample size.To increase the nutritional value of corn flour based