In this review, we summarize how zebrafish have successfully been employed to model human neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis, and Huntington’s disease. We discuss advantages and disadvantages of choosing zebrafish as a model for these neurodegenerative conditions.The zebrafish is increasingly recognized as a model organism for translational research into human neuropathology. The zebrafish brain exhibits fundamental resemblance with human neuroanatomical and neurochemical pathways, and hallmarks of human brain pathology such as protein aggregation, neuronal degeneration and activation of glial cells, for example, can be modeled and recapitulated in the fish central nervous system. Genetic manipulation, imaging, and drug screening are areas where zebrafish excel with the ease of introducing mutations and transgenes, the expression of fluorescent markers that can be detected  The zebrafish (Danio rerio) is a tropical freshwater teleost fish that is part of the Cyprinidae family that was introduced into biological research by George Streisinger in the 1970s. He selected zebrafish for three reasons: the generation of large numbers of embryos accessible from the one-cell-stage, the optical clarity of the developing fish, and a diploid genome allowing for genetic studies. Several large scale forward genetic screens by Nobel Prize laureate Christiane Nüsslein-Volhard and others firmly established the zebrafish as an ideal model system for developmental biology . Today, Zebrafish development is conveniently fast. Zebrafish are sexually mature by 12 weeks of age and can produce several hundred embryos each week  which deSNCA, PINK1, LRRK2, and Parkin), familial AD (PSEN1 and PSEN2), ALS , and HD (Huntingtin).Genetically, the zebrafish shares a high level of conservation with humans. A comprehensive sequencing study demonstrated that zebrafish share at least one ortholog with over 70% of all human genes  includinin vivo imaging of the entire CNS  and discuss on the background of recent advances in gene editing and manipulation of neuronal activity, how zebrafish can be a powerful model to investigate human neurodegenerative diseases.PINK1), Leucine Rich Repeat Kinase 2 (LRRK2) and the E3 Ubiquitin Protein Ligase Parkin. Environmental factors such as frequent exposures to harmful chemicals such as pesticides or previous brain contusions in individuals also play a substantial role in developing PD (Parkinson’s disease (PD) is one of the most common neurodegenerative diseases with an annual estimated global incidence rate of between 5 and 35 per 100,000 individuals. PD is characterized by the combined loss of dopaminergic neurons in the substantia nigra, and intracellular α-synuclein accumulation in neurons of various brain regions (Lewy pathology) . While Poping PD . CaffeinOf the plethora of different neurodegenerative diseases and movement disorders, PD is by far one of the most established in the zebrafish model and its promise as a PD animal model for the development of therapeutics has been highlighted in a recent review . High coSNCA gene encoding α-synuclein. α-synuclein mutations are linked to early onset familial PD, and α-synuclein aggregation in Lewy bodies is associated with sporadic cases of PD  gene that encodes for PINK1 protein plays a critical role in regulating oxidative stress and protecting neurons against mitochondrial dysfunctions  injection. Importantly, Acridine Orange assays and oxidative probes demonstrated increased neuronal cell death and accumulation of ROS, reflecting the increased susceptibility of neurons to oxidative insults and cell death following damage of cellular mitochondria (pink1–/–) mutant zebrafish were generated that showed persistent loss of dopaminergic neurons in the absence of severe morphological abnormalities  that encodes the LRRK2 protein account for 5–13% of familial PD and 1–5% of sporadic PD . Patients with loss-of-function of the human PARK7 gene that encodes DJ-1 present with early onset autosomal recessive PD  led to developmental abnormalities and deformations, significant loss of dopaminergic neurons, and behavioral as well as locomotor dysfunctions (Other genetic zebrafish models of PD include MO injections against the genes ssive PD . Zebrafissive PD . While ttoxicity . Knockdounctions .Vmat2) gene, in vivo study to demonstrate selective loss of dopaminergic neurons following MPTP exposure. Subsequent studies in the field have adopted the zebrafish MPTP model in studies aiming to develop PD therapeutics. Investigating the potential of a platinum nanoparticle using leaf extract of a plant (BmE-PtNP), In addition to the various genetic models of PD, several methods have been adopted to chemically induce neurotoxicity and the pathological hallmarks of PD in zebrafish . One of Furthermore, 6-OHDA administration in zebrafish resulted in selective loss of TH-positive dopaminergic neurons within days of application , as wellFinally, to investigate the link between pesticides and PD, Alzheimer’s disease (AD) is a chronic neurodegenerative disorder and the most frequent cause of dementia. AD is typically characterized by the presence of extracellular β-amyloid (Aβ) deposits generated from cleaved amyloid precursor protein (APP), as well as intracellular neurofibrillary tangles (NFTs) made up of aggregated hyperphosphorylated tau proteins. The disease leads to gradual hippocampal and parietal brain atrophy . GWAS g suggestiin vivo imaging that demonstrated rapid hyperphosphorylation and aggregation of tau as well as neuronal cell death -4 secretion by neurons and activated microglia  genes that have been implicated in the hereditary forms of AD  found in the human genome and these are ubiquitously expressed throughout development  was found to be lethal in mice, psen1–/– zebrafish were viable and did not exhibit gross morphological defects when compared to wild type zebrafish  application. OKA is a polyether C38 fatty acid toxin and a potent and selective inhibitor of the protein phosphatases 1 (PP1) and 2A (PP2A) . OKA-ind in vivo . Exposur in vivo . Further in vivo ,b led to in vivo . The eff in vivo . Supportdeficits .SOD1 (superoxide dismutase 1), TARDBP (encoding TAR DNA-binding protein-43 [TDP-43]), C9orf72 (open reading frame 72 on chromosome 9), and FUS (Fused in Sarcoma)  or Lou Gehrig’s disease, is a progressive neurodegenerative disease of the human motor system . ALS is Sarcoma) .The transparent bodies of zebrafish larvae allow for non-invasive visualization of motor neurons, from somas to their neuromuscular synapses, as well as their circuitry. This and the availability of genome editing, optogenetics and ease of drug screening facilitate functional analyses of ALS-associated proteins and consequential motor neuron degeneration with subcellular resolution .SOD1 , ubiquitinated and hyperphosphorylated TDP-43 C-terminal fragments accumulate in neurons and glia in brains of FLTD patients, but interestingly not in the spinal cord  found in ALS patients, proganulin, null mutations of which cause TDP-43 accumulation in FTLD, rescued motor neuron degeneration in the TDP43-A315T zebrafish line. Zebrafish express both independent orthologs of TARDBP (tardbp and tardbpl) throughout development, and single deletion of one ortholog is compensated for by increased expression of a splice variant of the other  were generated to establish the impact of TDP-43 loss-of-function  hexanucleotide repeats in the s of ALS . While hVarious mutant, deletion and transgenic C9orf72 zebrafish models have since been generated to investigate the functional role of C9orf72 in ALS pathogenesis . Multi-fin vivo C9orf72 loss-of-function model was developed via a MO-induced knockdown leading to signs of motor neuron degeneration with axonopathy phenotypes and abnormal motor behavior  is an autosomal-dominant inheritable neurodegenerative disorder that results from a CAG repeat extension in exon 1 of the huntingtin gene (HTT), which translates into a long polyQ repeat in the huntingtin protein. The length of these polyQ repeats correlate with the age of onset, penetrance and severity of the disease . HD has htt depletion on early development in the zebrafish  in the cytoplasm, thereby reducing its repressor function of REST-regulated gene transcription and in turn facilitating BDNF expression  is a major contributor in neural development including formation, differentiation, and survival of neurons . Wild typression . Post-mopression , 2007. Spression  the effepression . This capression . By combThe accumulation of mHTT in neurons indicates that protein quality control is compromised. The C-terminus of Hsc70-interacting protein (CHIP) plays a crucial role in ensuring the proper folding and conformation of proteins, linking molecular chaperones with the ubiquitin-protease system by acting as both co-chaperone and ubiquitin ligase . The co-in vitro chemical screen to identify novel enhancers of autophagy-inducing pathways independent of mTOR . Autophagy is a major clearance mechanism for intracellular protein aggregates which can be upregulated via the administration of the mTOR inhibitor rapamycin  or intact N17 and 97Q expansion alone (mHTT-exon1). The mHTT-ΔN17-exon1 embryos showed robust and rapidly progressive movement disorders, reminiscent of that seen in other animal models and human patients while the mHTT-exon1 embryos displayed a delayed onset and slower progression of movement deficits. Importantly, accumulation of mutant HTT was observed in neurons expressing the combined N17 deletion and 97Q expansion, but not in neurons expressing the 97Q expansion construct alone  amino acid fragment of HTT adjacent to the polyQ expansion domain regulates protein stability, toxicity, and sub-cellular localization . Investict alone . Recentlebrafish , and in ebrafish , these zThe zebrafish model is widely used to study various aspects of the CNS, from development and physiological function to degeneration and disease. We have highlighted many advantages, but we also want to address areas where zebrafish models show limitations.casper  and crystal  mutants, may be of use to future investigations that require in vivo imaging in adult zebrafish  or response time toward a tactile-evoked escape response. These tests may not be a true reflection of the more subtle muscular and movement disorders resulting from neurodegeneration. For example, it is difficult to establish balance or movement coordination in the zebrafish model, whereas it is straightforward in quadruped rodent models. Similarly, while anxiety and fatigue tests, as well as gait analyses have been routinely carried out in rodents, those assays have been more difficult to establish in the zebrafish model. Therefore, a need remains for the design of behavioral assays in zebrafish that more accurately mirror the repertoire of progressive motor disorders in neurodegenerative disorders.in vivo, such as the VAST (Vertebrate Automated Screening Technology) BioImager system developed for the zebrafish  have recapitulated the classic symptoms of the disease such as loss of dopaminergic neurons, neuroinflammation, locomotor and behavioral dysfunctions. Similarly, neurotoxin and pesticide exposure  resulted in loss of dopaminergic neurons as well as locomotor impairments. The zebrafish model thus presents a platform to investigate mechanisms of aggregate formation and consequences of gene mutation or pesticide exposure in the CNS. Several studies also included behavioral tests to measure depression, anxiety, social interaction aversion and aggression, features that are present in many PD patients. However, the results were quite variable and there is a need to establish better behavioral screens for the psychiatric symptoms of PD in the zebrafish model.Research into PD pathology has already integrated the zebrafish model well . TransgePSEN genes were viable and did not exhibit morphological or neurological defects. Tau phosphorylation and NFTs formation were successfully induced, but only appeared in the spinal cord and not in the brain of the larval zebrafish. Aβ aggregation could only be observed when human Aβ was injected into the fish brain, but not from endogenous protein. Additionally, Aβ overexpression induced phenotypes atypical of clinical AD, such as increased neuronal progenitor plasticity and proliferation, and enhanced neurogenesis. However, treatment of fish larvae with the known protein phosphatase inhibitor OKA successfully induced tau hyperphosphorylation, as well as deposition of Aβ, formation of senile plaques, and induced learning and memory deficits in zebrafish, mimicking the classical hallmarks of AD. With the number of AD cases expected to continually rise over the coming years, the need for drug discoveries is imperative. Thus, recent efforts have focused on the advantages of the zebrafish as a cost-effective and rapid in vivo pharmacological model for AD drug discovery. In this context, the use of CRISPR/Cas gene editing may help to establish better zebrafish models of AD.Many recent studies have employed zebrafish to model AD, and despite successful recapitulation of Aβ-sheet aggregation, increased neuronal toxicity and death, modeling AD in the zebrafish has thus far been limited . DeletioFUS, TDP-34, C9orf72, and SOD1 all displayed hallmarks of ALS such as muscle atrophy, motor axon abnormalities, motor neuron loss, as well as behavioral and motor deficits. Importantly, zebrafish studies have greatly advanced our understanding of the molecular mechanisms involved in ALS pathogenesis. For example, analysis of tardbp null fish revealed that the actin cross-linking protein Filamin C was upregulated as part of TDP-43 mediated pathogenesis, a fact later confirmed in the analysis of human samples. Investigation into FUS function in zebrafish revealed that FUS is essential for the interaction between U1 snRNP and RNA Polymerase II (RNAP II) during splicing, thus physically and functionally coupling transcription to splicing (250). BPA treatment of zebrafish not only induced motor axonopathy and loss of neuromuscular junction integrity, but was also associated with increased numbers of apoptotic motor neurons and the presence of activated microglia. Importantly, the zebrafish may prove to be particularly useful when disease phenotypes fail to manifest in classical mammalian models. For example, while knockdown of C9orf72 in zebrafish led to typical symptoms of ALS, previous studies in rodent models failed to show adverse effects of C9orf72 deficiency on neural health , and disturbed neural tube formation. Importantly, zebrafish are viable in the absence of Huntingtin in contrast to mice, which has helped to better understand the neurodevelopmental aspects of HD. Studies in zebrafish revealed that impaired HTT function in early development could have limiting effects on precursor cell numbers of neurons affected by the loss of normal HTT in later development. Other studies identified REST as a molecular target of HTT in regulating BDNF expression and strikingly, found that the soluble mutant forms of HTT mRNA and protein were the toxic components causing cell death. Two anti-prion compounds were identified that successfully inhibited poly-Q aggregation. Finally, zebrafish transgenic for a truncated form of mHTT (mHTT-ΔN17) displayed a rapidly progressive movement disorder, reminiscent of that seen in other animal models and human patients (In comparison to other disease models, not many studies have been carried out to explore HD pathology in the zebrafish. The overexpression of human mHTT, and different patients .To find new therapeutics for neurodegenerative diseases, the blood brain barrier constitutes a major hinderance for drugs to reach their targets in the brain. Nanotechnology is becoming a promising field of research for brain drug delivery using nanosized particles. Here the zebrafish is also proving an advantageous model for assessing blood brain barrier permeability with respect to novel neuro-specific technologies. Zebrafish provide an excellent model to test nanoparticle biocompatibility and toxicity , and recIn summary, current data encourage the use of zebrafish as a model for neurodegenerative diseases. While we have described some of the limitations, the zebrafish presents a rapid, cost effective, and highly practical platform compared to classical mammalian models. Recent analyses employing cutting edge technology such as optogenetics have provided new insights into processes such as aggregate formation and the downstream consequences on a molecular level. Moreover, new tools such as very early cell death reporters make zebrafish an asset in neurodegeneration research. The plethora of cellular fluorescent reporters and cutting-edge imaging techniques, combined with the accessibility for drug testing, underscores the potential of zebrafish for the development of therapies for human neurodegenerative diseases.KC, AK, DS, and JP contributed to the conceptualization and writing – review and editing. DS and JP contributed to the resources, supervision, and project administration. KC and AK contributed to the writing – original draft and visualization. JP contributed to the funding acquisition. All authors contributed to the article and approved the submitted version.


Ulcerative colitis (UC) is a chronic nonspecific inflammatory disease with a complex etiology. Intestinal mucosal injury is an important pathological change in individuals with UC. Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5+) intestinal stem cells (ISCs) exhibit self-renewal and high differentiation potential and play important roles in the repair of intestinal mucosal injury. Moreover, LGR5+ ISCs are intricately regulated by both the Wnt/β-catenin and Notch signaling pathways, which jointly maintain the function of LGR5+ ISCs. Combination therapy targeting multiple signaling pathways and transplantation of LGR5+ ISCs may lead to the development of new clinical therapies for UC. Ulcerative colitis (UC) is a chronic nonspecific inflammatory disease of the colorectal mucosa that manifests as chronic inflammation and repeated ulceration, seriously affecting the quality of life of patients. The incidence of colorectal cancer is increased in patients with UC over the long course of this disease . IntestiThe gut is one of the most complex organs and is critical for the absorption and digestion of nutrients . MultiplThe crypt–villus axis is the basic unit of the intestinal epithelium. A crypt is an area where ISCs and progeny cells proliferate, and a villus is an area where various functional cells differentiate . The cryin vitro, and Wnt and Notch signaling pathway activity was modulated by small molecules  and epidermal growth factor (EGF) signaling pathways, which play important roles in maintaining the self-regeneration, colonization and differentiation of colonic stem cells. Wnt signaling is the main driving force maintaining epithelial cells in the colon, and Wnt is expressed in a gradient along the crypt–villus axis . The actolecules . Withoutolecules . These rolecules . Moreoveolecules . Furtherolecules . In addiolecules . Howeverolecules . Taken tolecules .The links between basal factors and signal transduction and transcription factors must be explored to obtain a better understanding of the mechanisms that differentiate basal factors from signal transduction and transcription factors. Using DNA microarray technology, the heterogeneous expression of genes in mouse crypt–villus axis epithelial cells has been described, and other differentially expressed genes, including those associated with the differentiation and migration of cells, have been identified . Motifs As a specific nuclear transcription factor expressed in IECs, tail-type homeobox transcription factor 2 is widely expressed in distal compartments of villi and mainly regulates the proliferation, differentiation and migration of IECs. Moreover, the expression of the hydrogen/peptide coupling transporter (Pept1) gene in human IECs was regulated by CDX2. Because the Pept1 initiator motif does not contain a canonical T–A sequence region for binding CDX2, Pept1 expression is modulated by an interdomain region connected to the Sp1-binding region in the initiator motif. CDX2 bound to the promoter region of a short-chain lipoic acid transporter, and its mRNA expression was subsequently upregulated. CDX2 is also involved in regulating the expression of several other factors related to IEC proliferation and the intestinal epithelial structure. Studies have shown that CDX2 is activated through cis-acting mucin binding to mucin in the intestinal tract. However, MUC2 is expressed only in goblet cells; therefore, CDX2 plays an important role in goblet cell differentiation . As a meThe transcription factor GATA belongs to the zinc finger protein family. GATA-4/-5/-6 are expressed in the epidermis of the mature colon, and GATA-4 is present from high to low abundance from the proximal to distal end of the colon. GATA transcription factors inhibit or activate Lct, invertase, GATA-4 and GATA-6, which play important roles in maintaining the structure and differentiation of IECs. Conditional knockout of GATA-4 and GATA-6 in colonic epithelial cells of adult mice led to the proliferation of stem cells in the duodenum and jejunum, a significant decrease in the numbers of intestinal endocrine cells and Paneth cells, and a substantial increase in the number of goblet cells . GATA trChanges in the function of IECs may lead to changes in nutrient metabolism, and the metabolites produced by these cells may also regulate the colonization and development of IECs to a certain extent. The levels of EGF, TGF, amino acids and their derivatives are regulated and controlled by different mechanisms to promote the proliferation and regeneration of IECs after injury .via phosphorylation, initiating the activation of a series of downstream signaling pathways (such as PI3K/AKT and RAS/RAF/MEK/ERK) that ultimately activate intracellular transcription factors and regulate downstream gene transcription to modulate the proliferation and differentiation of cells. In crypt cells, the RAS/RAF/MEK/ERK is activated downstream of EGF  is a nonessential amino acid and the main functional substance of the intestinal tract. Gln deficiency induces the death of IECs and promotes the differentiation of ISCs to form new epithelial cells . SupplemThe Hippo-YAP pathway, also known as the Hippo pathway, is mainly composed of three parts: the upstream regulators MST1/MST2, core factors LATS1/LATS2, and downstream effectors YAP/TAZ . In contThe process of Notch signaling activation requires membrane contact between two cells, where signaling cells express Notch ligands  and signal receiving cells express Notch receptors  . After l1) negative regulation to prevent stem cell differentiation and thus maintain the stem cell pool; and 2) promoting differentiation in one direction and inhibiting differentiation in other directions to regulate the balance between secretory and resorptive cells. In terms of maintaining the stem cell pool, fast-cycling CBC stem cells are characterized by high Notch signaling, which promotes ISC proliferation , followed by phosphorylation and dimerization of receptor-activated SMAD proteins . They thIn the intestinal tract, BMP antagonizes the proliferation signal of the ISC niche and promotes cell differentiation. When BMP signaling is inhibited, ISCs proliferate excessively and exhibit polypoid changes . For exaThe BMP concentration gradient in the gut crypt-villus axis makes EECs subject to increased BMP signaling during their migration from the bottom of the crypt to the villus, which may lead to changes in its hormonal lineage and specific transcription of hormone genes . EEC subIECs include absorptive IECs, goblet cells, Paneth cells and intestinal endocrine cells . Most ceUnder normal circumstances, IECs and the mucus layer form a physical barrier protecting against the invasion of pathogenic microorganisms. Goblet cells play an important role in this process because they secrete highly glycosylated mucins, of which MUC2 is the most abundant, as the first line of defense in the intestinal epithelium . StudiesIn UC, the MUC2 glycosylation sequence is changed, which further aggravates the inflammatory response in individuals with UC . Recent ,  are produced mainly by Paneth cells in the intestinal tract, and among AMPs, α-defensin, which supports the mucus layer, is the most abundant component . In subjImmune abnormalities, including the production of autoantibodies and aberrant cellular immunity, cytokines, oxygen free radicals and nitric oxide, are the main pathological characteristics of UC. A study examining the proportion of Paneth cells in the colons of 10 patients with UC and 10 patients who underwent surgical resection to treat other diseases revealed that the colonic crypts of patients with UC containing more than one Paneth cell accounted for 19.6% of all Paneth cells in intestinal crypts, and in the most extreme case, the proportion reached 30%. No Paneth cells were observed in crypts near the areas of acute inflammation or abscess formation. Another researcher studied 44 patients with UC and found that 41 (93%) presented with Paneth cell metaplasia (PCM) in the colon or rectum . In addiLGR5, also known as GPR49, HG38, FEX, GPR67 and MGC117008, is a seven-transmembrane (TM) protein belonging to the G protein-coupled receptor (GPCR) family. The extracellular region of GPCR family proteins contains 17 leucine-rich repeats that interact with glycoprotein ligands, a 7-alpha helix transmembrane region that is highly conserved, and N-terminal and C-terminal tails enriched in cysteine residues . No phys,  and possesses extensive stem-like pluripotency and self-renewal properties . In 2007, . The imbThe classical Wnt signaling pathway is characterized by the accumulation and transcription of β-catenin in the nucleus; hence, it is also called the Wnt/β-catenin signaling pathway . The WntIn vitro, Rspos support the survival, expansion and budding of ISCs, and a lack of Rspo1 leads to crypt cell and ISC apoptosis. Enhanced Wnt/β-catenin signaling induced by Rspos is mediated mainly through the formation of a complex with ubiquitin ligase and LGR family factors  by enema. The intestinal mucosa injury in these mice was treatable, and the regeneration and repair of the intestinal epithelium was promoted . These rin vitro and then autologously transplanting them into injured sites of the colon may ameliorate UC. These results and assumptions may provide researchers with new ideas and research directions for the treatment of UC in the clinic  and glycogen synthase kinase 3 (GSK3) targets β-catenin, leading to phosphorylation and proteasome degradation and ultimately resulting in the inactivation of the Wnt signaling pathway . The WntStudies have shown that LGR5 is the downstream target gene in the most important β-catenin signaling pathway . In addiWnt signaling is closely related to the regulation of ISC proliferation. ISCs express LGR proteins and Fzd. When the ligand Wnt and Fzd bind Rspo and LGRs, the destruction complex is inhibited, followed by β-catenin accumulation. Groucho enters the nucleus to displace this complex and then binds to TCFs to regulate the expression of genes by driving transcription, thereby regulating the proliferation of stem cells. In the absence of Rspo and Wnt, Fzd is degraded after ubiquitination mediated by the RNF43 or ZNRF3 protein, and the destruction of the complex will induce β-catenin degradation, resulting in its inability to bind to TCF and ultimately leading to the loss of LGR5+ stem cells. Activation of Wnt signaling leads to ISC expansion .Wnt concentration gradients regulate stem cell proliferation. Paneth cells produce the ligand Wnt3, which decreases as stem cells begin to differentiate and leave the stem cell region, creating a concentration gradient of Wnt3 as they move along the crypt-villus axis and creating a dynamic negative feedback loop. When stem cells proliferate faster, a large number of cells move toward the villi to reduce the Wnt concentration in the stem cell region and then reduce the proliferation of stem cells and the output of cells in the crypt. When stem cells are lost, their proliferation is slow, the Wnt concentration gradient is shortened, and the Wnt level at the bottom of the crypt is increased, which is conducive to the symmetrical differentiation of stem cells or the dedifferentiation of progenitor cells into stem cells to achieve the purpose of stem cell proliferation.The regeneration of the intestinal epithelium depends on the rapid response of ISCs, which are amplified and replenished by adjacent crypt stem cells in cases of injury and driven by LGR5+ stem cells to quickly complete repair . StudiesThe Wnt/β-catenin signaling pathway is essential for ISC proliferation. Recently, an experiment with a human colonic crypt model showed that the classical Wnt pathway and the inhibitory TGFβ/BMP pathway promote the regeneration of the human colonic epithelium and repair injured intestinal mucosa . Howevervia a C-terminal protein domain, which promotes the nuclear translocation and aggregation of β-catenin and contributes to the activation of the Wnt pathway (Another important link between the Wnt pathway and intestinal mucosal homeostasis is the bidirectional regulation of MUC expression and Wnt pathway activation . MUC2 in pathway . β-Caten pathway .In summary, these studies suggest that the Wnt pathway is the main driving force for ISC proliferation and that it maintains intestinal homeostasis. In clinical applications, inhibitors and agonists of the Wnt pathway may be therapeutic targets to regulate the overactivation and inhibition of the Wnt pathway, respectively, under pathological conditions to restore normal Wnt pathway activity and maintain a normal ISC proliferation rate as a method to establish intestinal epithelial homeostasis  Figure .The Notch signaling pathway consists of receptors, ligands and DNA-binding proteins. Notch signaling is activated by the interaction of Notch receptors with ligands on two adjacent cells . The binNotch signaling regulates the differentiation of ISCs into absorptive cell types. The conditional deletion of RBP-J and gamma-secretase inhibitor treatment to block NICD release resulted in the differentiation of crypt ISCs into secretory cells . SimilarBased on these results, Math1 promotes the differentiation of ISCs into secretory cell types, while Hes1 promotes the differentiation of ISCs into absorptive cell types . Both ofAlthough the Wnt and Notch pathways have been confirmed to play important roles in intestinal homeostasis, the specific mechanisms by which the two pathways interact to regulate ISC activity and directed differentiation remain unclear . AccordiRecently, the Wnt and Notch signaling pathways and their interactions were reported to regulate the proliferation and differentiation of ISCs. However, researchers have not clearly determined whether other pathways also play a role in these processes, and further experiments are needed .In conclusion, intestinal mucosal injury is an important pathological change in individuals with UC . The sel


Over the past decade, momentous progress has been made in the characterization of late actinide compounds. Here the authors highlight how advances in spectroscopic and computational tools have developed our understanding of fundamental transplutonium bonding interactions, and discuss whether covalency and heterogeneity changes in 5f-orbital bonding could be harnessed in environmentally and industrially relevant systems. Over the past decade, momentous progress has been made in the characterization of late actinide compounds. Here the authors highlight how advances in spectroscopic and computational tools have developed our understanding of fundamental transplutonium bonding interactions, and discuss whether covalency and heterogeneity changes in 5f-orbital bonding could be harnessed in environmentally and industrially relevant systems. Many of the chemical and physical properties of the transplutonium elements have not been characterized, even for those available in sufficient quantities for classical macroscopic experiments: americium (Am), curium (Cm), berkelium (Bk), and californium (Cf). Despite characterization limitations, each of these heavier actinides have found applied uses including as radioisotope thermoelectric generators2, as targets for superheavy element discovery3, and as neutron activation sources4.Trends in structure and bonding that are well-defined elsewhere in the periodic table are underdeveloped in the actinide series, and in particular for the transplutonium elements, which are recognized for their scarcity and inherent radioactivity. Nevertheless, the last ten years have brought about a surge in interest in the chemistry of the later 55 who combined extended X-ray absorption fine structure (EXAFS) spectroscopy with Monte Carlo simulations to resolve the solvation of Cf3+ ions, and experimentally confirm the actinide contraction extends to Cf. Since then, EXAFS has been applied to explore the solution chemistry of several transplutonium complexes, and significant deviations from predicted behavior, based on lanthanide results, have been observed8. For instance, our group investigated late actinide binding with the well-known chelator diethylenetriaminepentaacetic acid (DTPA), and found a larger than expected decrease in M–O bond distances at Cf  and thiocyanate ligands feature M–O bonds that are primarily ionic, as probed via electronic structure calculations and Raman spectroscopy, respectively13.In parallel, X-ray diffraction (XRD) techniques have advanced to the point where obtaining results with transplutonium complexes is feasible, which has proven valuable in improving our understanding of the role of valence orbitals in late actinide bonding. Pioneering work in this area was done by Albrecht-Schmitt et al. who synthesized and characterized single crystals of dipicolinate (DPA) and borate complexes with Bk15. This work has provided insight into the biological coordination of transplutonium elements while also offering new, qualitative information on metal-ligand covalency as protein recognition changes the electrostatic character of bonds in the actinide small molecule complexes. Beyond Scn, several endogenous proteins, including the iron transporter transferrin, are capable of directly binding to late actinides. Leveraging the presence of amino acid chromophores in the protein scaffold, which can act as sensitizing antennae, one can study metal-ligand coordination via time-resolved luminescence spectroscopy methods .To better understand the extent and impact of covalency on transplutonium compounds, actinide chemists have expanded their structural and spectroscopic toolbox, thereby taking advantage of techniques that offer unique characterization capabilities. We have taken inspiration from biological systems, using macromolecular crystallography to study the binding of late actinide small molecule complexes by a mammalian protein siderocalin (Scn) Fig. 14,15. Thods Fig. 16. Of no17. Density functional theory calculations did predict a decrease in 5f-orbital energy as one moves across late actinides for dipicolinate complexes; however, this meant covalency was driven by energy degeneracy  and electron paramagnetic resonance (EPR) spectroscopy to the late actinides. Harnessing this improved knowledge of covalency and bonding heterogeneities in transplutonium coordination chemistry has the potential to not only solve pressing needs in energy generation and waste management or to continue the quest for new elements, but also for new applications that will address pivotal contemporary issues. A prime example would be to leverage the large magnetic anisotropies and covalent bonding character observed in late actinide compounds19 for the development of new transplutonium materials with long spin-lattice relaxation times and enhanced qubit coherence times20, leading to the next-generation of quantum computing devices.The development of transplutonium coordination chemistry is still nascent and determining the role of 5


Liver cirrhosis is the 11th most common cause of death, causing more than 1 million deaths globally each year, and together with liver cancer, it accounts for 3.5% of all deaths worldwide.Nature Genetics, titled “Loss of FOCAD, manipulated through the SKI messenger RNA surveillance pathway, leads to a pediatric syndrome with cirrhosis,” Traspas and colleagues uncovered the essentiality of the FOCAD gene in maintaining liver health and provided evidence that loss-of-function mutations in FOCAD may contribute to cirrhosis in children.Cirrhosis is traditionally considered a late-onset disease that appears in adults following environmental factors, such as viral infection, a high-fat diet, or chronic alcoholism.The authors reported 14 children from 10 unrelated families in seven countries presenting with a multisystem syndrome characterized by severe neonatal cirrhosis. By combing genome/exome sequencing, a novel animal model of the human disease, and in vitro biological systems, the team identify that the FOCAD gene is indispensable for maintaining human liver health. Mutations in this gene cause a form of early-onset cirrhosis that has not been documented before. Using CRISPR-Cas9 technology, they established in vitro and in vivo FOCAD knockout models to further study the cellular and molecular mechanisms of pediatric cirrhosis. Phenotypic replication of human disease in FOCAD-deficient zebrafish reveals features of altered messenger RNA degradation processes in the liver. FOCAD deficiency in patient primary cells and human liver cell lines impairs the SKI mRNA surveillance pathway by reducing levels of the RNA helicase SKIC2 and its cofactor SKIC3. Compared with other cell types, hepatocytes rely heavily on this mechanism. Hepatocytes exhibited a decrease in albumin expression and overproduction of the cytokine CCL2 following FOCAD knockout, which may play a key role in the progression of cirrhosis. These findings reveal the importance of FOCAD in maintaining liver homeostasis and the potential for therapeutic intervention by inhibiting the CCL2/CCR2 signaling axis.There are few reports on the biological function of FOCAD, mainly involving focal adhesions, microtubule dynamics, cell cycle regulation, and cancer susceptibility.Monocyte chemoattractant protein 1  is the major ligand for the chemokine receptor C-C chemokine receptor 2 (CCR2) and is involved in a variety of diseases, including autoimmune diseases, viral infections, and cancer. During liver injury, CCL2 and CCR2 promote liver fibrosis by activating inflammatory signaling and immune cell infiltration. CCL2 is a potential therapeutic intervention point for patients with cirrhosis.Overall, this study confirms the clinical impact of recessive loss-of-function variants in the FOCAD gene and highlights the importance of the SKI mRNA surveillance pathway for liver homeostasis, and discloses a possible therapeutic intervention point via inhibition of the CCL2/CCR2 signaling axis. Although this study aimed to identify the underlying genetic causes of hepatic syndrome in children, it also has profound implications for the exploration of the pathogenesis of cirrhosis in adults. The relationship between abnormal FOCAD function and the susceptibility and severity of cirrhosis in adults remains to be further explored. In addition, further analysis of cirrhosis cases in different environmental backgrounds is promising to elucidate the genetic component of cirrhosis and its dependence on the environment. Furthermore, the knowledge and tools generated from this study have the potential to help discover and validate innovative targets for other common liver diseases such as fatty liver and hepatocellular carcinoma.


Basement membranes (BMs) are highly specialised extracellular matrix (ECM) structures that within the heart underlie endothelial cells (ECs) and surround cardiomyocytes and vascular smooth muscle cells. They generate a dynamic and structurally supportive environment throughout cardiac development and maturation by providing physical anchorage to the underlying interstitium, structural support to the tissue, and by influencing cell behaviour and signalling. While this provides a strong link between BM dysfunction and cardiac disease, the role of the BM in cardiac biology remains under-researched and our understanding regarding the mechanistic interplay between BM defects and their morphological and functional consequences remain important knowledge-gaps. In this review, we bring together emerging understanding of BM defects within the heart including in common cardiovascular pathologies such as contractile dysfunction and highlight some key questions that are now ready to be addressed. Basement membranes (BMs) are thin, sheet-like structures of the extracellular matrix (ECM) which serve as a barrier and anchorage point between the cell and interstitial ECM, and can influence cell differentiation, signalling, and migration in the vast majority of tissues . Within BMs have a wide array of biological functions, occur in almost all tissues, and underlie multiple cell types including epithelial and ECs, adipocytes, and myocytes. This physical demarcation compartmentalises tissues and cell types from one another A. BMs arDrosophila development showed diffusive properties of some BM components and that components can be secreted and deposited by cells that do not reside on the BM .,80.Hspg2idogen 2 ,82, comp dilated . Reducti dilated . However dilated .in utero via foetal shunts such as the foramen ovale, ductus arteriosus, and ductus venosus , w, w105], NID1 and LAMC1 have been associated with a variety of severe congenital malformations, which are collectively referred to as Dandy–Walker syndrome . U. U134].  and α10 . Integri and α10 . In the  and α10  , indicating systolic dysfunction and impaired contractility [Given the contribution of the BM to the structural stability of the heart, we can begin to appreciate how impairments in the relationship between cardiomyocytes and the BM can be a feature in congenital and adult cardiac diseases. Heart failure is characterised by left ventricular insufficiency either due to a failure of the myocardium to contract (systolic dysfunction) or relax (diastolic dysfunction). Despite similar clinical presentations, systolic and diastolic dysfunction arise through different remodelling processes of the left ventricle . BM compactility .−/−Col15 mice identified the first impaired heart contractility due to deficiency of a BM component [−/−Col15a1 mice have a reduction in EF and FS indicative of systolic dysfunction [−/−Col15a1 mice uncovered a marked reduction in left ventricular pressure in response to isoproterenol which pharmacologically increases heart rate. This indicates a decreased cardiac response following β-adrenergic stimulation, a feature which is associated with congestive heart failure and ageing [−/−Col15a1 mice established that BM components are required for heart contractility and the maintenance of contractile function throughout life.Functional analysis of omponent ,174. Intd ageing ,175,176.COL6A1 mutations. Mutations in collagen VI result in two muscle-related disorders; Bethlem myopathy and Ullrich congenital muscular dystrophy (UCMD) [−/−Col6a1 mouse. Interestingly, following cardiac injury, adverse remodelling was reduced in knock-out animals suggesting collagen type VI contributes to the initial injury response [This is similarly reflected in patients with y (UCMD) . Bethlemy (UCMD) . UCMD hay (UCMD) . It is cy (UCMD) . Howevery (UCMD) , and perresponse .Diastolic heart failure, or heart failure with preserved EF, differs from systolic dysfunction as the myocardium cannot relax efficiently leading to an increase in end diastolic pressure. Unlike systolic heart failure, diastolic heart failure often occurs when the ventricular wall is thickened as the cardiomyocytes undergo hypertrophy. However, diastolic dysfunction can also arise from other aetiologies including restrictive cardiomyopathy, constrictive pericarditis, and tension pneumothorax. In addition, the interstitial ECM undergoes remodelling as the production of fibrillar collagens (chiefly collagens I and III) increases, which is traditionally what is referred to when describing adverse ECM remodelling. Interestingly, and less frequently discussed, is that the expression of BM components such as collagen IV, laminin and fibronectin are also increased which contributes to the development of myocardial fibrosis and contractile dysfunction . Models COL4A1, COL6A1, and LAMA2 , inhibits apoptosis of cardiomyocytes induced by β1 adrenoceptor activation possibly by reducing excessive intracellular Ca2+ release by L-type Ca2+ channel [COL4A2 levels also altered cardiac electrical activity and action potential [2+ signalling, the mechanisms linking the L-type Ca2+ channel and canstatin remain unclear [The disorganisation of the cardiac dyad, in particular due to structural changes to the t-tubule, impairs regulation of Ca failure . Defects release . Impaire release ,199. Int channel . Reducedotential . While t unclear . SimilarWithin the adult heart, the BM appears to play several important roles from providing mechanical support to cardiomyocytes to assisting in excitation–contraction coupling. This has mainly been inferred from defects in BM structure in cardiac pathologies in which contractility and electrical conduction become impaired. However, whether this reflects a functional role of the BM in these aspects, or the pathological remodelling of the BM could be consequential and not a causal factor in these cardiac defects remains unknown. One main factor which has prevented the mechanistic investigation of this link has been the lack of appropriate models of BM disruption, mostly due to the embryonic lethality associated with BM component knock-out mouse models. Conditional mouse models as well as animal models that recapitulate mutations seen in patients  will be in vivo models that appropriately recapitulate the human disease to shed light on the causal mechanisms. The continued and more intensive investigation of the influence of the BM and ECM on cardiac development and function will be critical to gain a sufficient in depth understanding of the fundamental biology to underpin the identification, design, and development of novel therapeutic targets for several cardiovascular diseases.There is now a large body of evidence highlighting how the BM and mutations in its individual components can cause a spectrum of congenital heart defects in patients and animal models. Despite this and the growing evidence of BM disruption and remodelling in various common cardiovascular pathologies, there are major unanswered questions regarding the influence of BM defects on heart development, adult cardiac function and, importantly, the underlying pathomolecular mechanisms of cardiac defects. Several studies discussed in this review have highlighted correlations between the expression and deposition of BM components, and cardiac development and cardiomyocyte differentiation. However, the difficulties associated with studying systemic knock-out models has made investigating the causal relationship problematic. Refinement of animal models such as conditional mouse models and zebrafish will stand to unlock the door towards understanding the role of the BM in cardiac biology and disease. For example, employing cardiac specific mouse knockout models of BM components would greatly accelerate the investigation of the functional role of the BM, while overcoming the difficulties associated with BM disruption and embryo viability. Another factor which has slowed progress in answering these questions is the lack of clinical association of cardiac defects with disorders relating to BM components. Patients with mutations affecting BM components often receive limited evaluation of cardiac function and/or the outcomes have not been reported. In patients where a cardiac defect is identified, almost no mechanistic analysis is performed due to the obvious impracticality of obtaining cardiac tissue to carry out this analysis. This again emphasises the need for well-designed


Colletotrichum that affect a wide range of crop plants. Strategies to improve crop resistance are needed to reduce the yield losses; and one strategy is to manipulate protein kinases that catalyze reversible phosphorylation of proteins regulating both plant immune responses and fungal pathogenesis. Hence, in this review, we present a summary of the current knowledge of protein kinase signaling pathways in plant-Colletotrichum interaction as well as the relation to a more general understanding of protein kinases that contribute to plant immunity and pathogen virulence. We highlight the potential of combining genomic resources and phosphoproteomics research to unravel the key molecular components of plant-Colletotrichum interactions. Understanding the molecular interactions between plants and Colletotrichum would not only facilitate molecular breeding of resistant cultivars but also help the development of novel strategies for controlling the anthracnose disease.Anthracnose is a fungal disease caused by members of  Arabidopsis, more than 1,170 phosphopeptides from 472 phosphoproteins were identified after treatments with flg22 or xylanase, which are molecules that could trigger immune responses, suggesting a large amount of phosphorylation events occur during plant immune responses -dependent protein kinases and MAPKs  production, cellular Caathogens , 2.Arabidopsis include FLAGELLIN SENSING 2 (FLS2), EF-TU RECEPTOR (EFR), and PEP RECEPTORs (PEPRs), which recognize bacterial flagellin, bacterial elongation factor Tu and plant endogenous peptides, respectively  upon ligand perception, which is required for immune activation  is crucial for plants to defend against pathogens. In plants, PM-localized pattern recognition receptors (PRRs) are responsible for sensing conserved microbe-associated molecular patterns (MAMPs) or damage-associated molecular patterns (DAMPs) to initiate a series of immune responses  , which confers total resistance to the extremely virulent C. lindemuthianum strain 100 , 26 non-arginine-aspartate (non-RD) RLKs were identified by an in silico approach. By analyzing their expression patterns in both resistant and susceptible cultivars, CaRLK1, CaRLK15, CaRLK16 were found as candidate genes for resistance against C. truncatum  cascades are highly conserved signaling modules that play important roles in plant growth and defense  were identified to be involved in disease resistance against C. gloeosporioides  have been more thoroughly studied. In Nicotiana benthamiana, wound-induced protein kinase (NtWIPK) and salicylic acid-induced protein kinase (NtSIPK) have been reported to participate in PTI against the fungal pathogen C. orbiculare  or simultaneously silencing of both NtWIPK and NtSIPK could allow the full infection of the CoSSD1 knockout mutant pathogen   has been reported to mediate a pathway leading to the susceptibility to C. fructicola , which contributes to the degradation of SA  have indicated positive roles of MAPKs in regulating resistance. In silico and localization analysis revealed that GhMPK7 and GhMPK16 are localized in the nucleus, and the expression of both MAPKs are induced in response to pathogen infection  and late stages  were analyzed. The results showed that MAPK and leucine-rich repeat (LRR) RLKs were expressed in all infected samples, whereas CDPKs were expressed in both unharvested and harvested samples during early stages but only in the harvested samples during late stages, suggesting that different protein kinases may function at different stages of pathogen infection  and serine/threonine protein kinase aurora-3 (AUR3) were significantly induced in the roots during local infection   and cv. Gala (susceptible) in response to C. musae, C. gloeosporioides and C. lentis. Banana anthracnose is caused by C. musae, and exogenous melatonin treatment could significantly reduce the incidence of anthracnose. Transcriptomic analysis of banana peels showed that the MAPK signaling pathway was significantly enhanced after melatonin treatment, which might be involved in the enhanced fruit resistance  pathway, morphogenesis-related NDR kinase network (MOR) and two-component system have been reported to play important and conserved roles in the infection of Colletotrichum pathogens  and pathogenic growth in planta, suggesting conserved roles of Fus3/Kss1 MAPKs in regulating the fungal development and pathogenesis   Levin, . The corC. gloeosporioides showed that the deletion of CgHog1 resulted in enhanced sensitivity to osmotic stress and increased resistance to fludioxonil. Further transcriptomic profiles of wild type and Cghog1 mutant in response to sorbitol and fludioxonil indicate that CgHog1 may regulate the adaption to osmotic stress by controlling the synthesis and accumulation of osmolytes; and the growth defect caused by fludioxonil in Cghog1 mutants may be associated with disruption of endocytosis , ChRgf or ChCdc25, exhibit reduced intracellular cAMP levels and defects in vegetative growth, conidial germination and virulence, which could be restored by exogenous cAMP treatment  protein kinases are core members of the morphogenesis-related NDR kinase pathway (MOR), and control the cell polarity, which is crucial for plant cell invasion, the production of specialized penetration structures, and the spread of the fungi through the host tissues  has been reported to be required for osmotic stress resistance and function upstream of the Hog1 MAPK pathway. In Colletotrichum species, the HK family orthologous to Sln1 has only been examined in C. lindemuthianum. ClSln1 contains a N-terminal transmembrane domain, five typical domains of HKs, and a C-terminal RR domain. Loss of ClSln1 causes a deficiency in the production and melanization of appressoria, as well as complete loss of pathogenicity on bean leaves  function at the head of two-component phosphorelay systems (TCSs), a conserved signaling module in both prokaryotes and eukaryotes , Young Elite Scientists Sponsorship Program by CAST (Project No. 2020QNRC001), and Young Talents' Science and Technology Innovation Project of Hainan Association for Science and Technology (QCXM202001).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.


Fragile X Syndrome is a neuro-developmental disorder caused by the silencing of the FMR1 gene, resulting in the loss of its protein product, FMRP. FMRP binds mRNA and represses general translation in the brain. Transcriptome analysis of the Fmr1-deficient mouse hippocampus reveals widespread dysregulation of alternative splicing of pre-mRNAs. Many of these aberrant splicing changes coincide with those found in post-mortem brain tissue from individuals with autism spectrum disorders (ASDs) as well as in mouse models of intellectual disability such as PTEN hamartoma syndrome (PHTS) and Rett Syndrome (RTT). These splicing changes could result from chromatin modifications  and/or splicing factor alterations . Based on the identities of the RNAs that are mis-spliced in these disorders, it may be that they are at least partly responsible for some shared pathophysiological conditions. The convergence of splicing aberrations among these autism spectrum disorders might be crucial to understanding their underlying cognitive impairments. Neurodevelopmental disorders (NDDs) including those that lie on the autism spectrum are diagnosed primarily based on behavioral presentations in early childhood. NDDs vary in severity of clinical presentations and most commonly occur as intellectual disability (ID) . ChildreAlternative pre-mRNA splicing generates multiple transcript isoforms and protein variants for a single gene by co-transcriptionally altering the exon composition of the mature mRNA . Alternade-novo genetic mutations resulting in cryptic splice sites or mutations in RNA binding proteins   . RTT maiut (KO)] . SynaptiAlternative splicing regulation is a highly variable process that depends on complex interactions between cis-acting splicing signals, splicing factor recruitment, RNA motifs, combinatorial action of RNA binding proteins, epigenetic marks on the chromatin, speed of polymerase movement, and other molecular features. The mechanisms contributing to alternative splicing in the mammalian nervous system, which have been reviewed in Ref. , may expPTEN gene might disrupt interactions of Pten with the spliceosomal protein U2AF2, thereby altering the local splicing program.Similar to ASD, a decrease in expression of splicing factor SRRM4 has also been identified in a PTEN mouse model along with decreased expression of other proteins such as NOVA2 and RBFOX1. Another hypothesis explaining the local splicing defects in PTEN, is that mutations in the SETD2, resulting in increase of SETD2 (a lysine methyltransferase) protein levels. Increased SETD2 alters the H3k36me3 marks on the chromatin, which correlated with alternative splicing defects in mRNAs critical for proper synaptic function , and DNA modifications such as 5-hydroxymethylcytosine (5hmC). In Rett syndrome, MeCP2-dependent spliced exons show differential epigenetic signatures based on their tendency of inclusion or exclusion. Excluded exons display the DNA modification 5hmC, and the presence of the histone modification H3K4me3 (histone H3 lysine 4 trimethylation) H3K4me3. Exons differentially included in the mature mRNA demonstrate enrichment of H3K36me3 (histone H3 lysine 36 trimethylation) marks in Mecp2-knockdown neurons . SimilarFmr1 KO mouse tissues were between 10 and 30%, most of the mRNAs were involved in synaptic vesicle recycling function in the hippocampus. Thus, the synaptic plasticity deficit in this tissue may in part be due to small splicing changes in many mRNAs in the same pathway. Furthermore, splicing analysis results may be subject to the choice of bioinformatic programs used to assess splicing changes, depth of sequencing, ΔPSI threshold, read length and type of sequencing, reference genome version, and sample characteristics such as age, tissue, and cell type studied. Consolidating alternative splicing patterns from different studies requires careful assessment of all factors that may contribute to a bias in data generation. Identification of similar alternative splicing changes in genes belonging to specific biological pathways such as synaptic function or brain development in different monogenic and complex neurological disorders, suggests that similar upstream regulatory pathways may be perturbed.The functional significance of alternative splicing deficits in neurodevelopmental disorders is becoming increasingly clear owing to the ease of transcriptomic analysis and the use of mouse models mimicking exon mis-splicing events. An in-depth analysis of consolidated transcriptomic data from a large cohort of patients diagnosed with ASD, schizophrenia and bipolar disorder have provided significant insights enriching our understanding of the pathophysiology of psychiatric disorders . A key fDysregulated pre-mRNA splicing is a rapidly developing research focus in understanding pathophysiology of neurodevelopmental disorders. Several questions arise based on current research and answering them is vital to our understanding of how neurodevelopmental disorders manifest and show such phenotypic diversity in patients. For example, is aberrant splicing a cause or consequence of changes in the epigenetic landscape, splicing factor levels, or RNA binding proteins? Can restoring alternative splicing defects result in reversing the course of the disorder? If not, can the alternative splicing changes be used as biomarkers to inform therapeutic strategies and disease progression? Can the alternative splicing perturbations explain commonalities and differences between IDs and therefore predict the severity of the disorder? The genome-wide perturbations of alternative pre-mRNA splicing is a recent finding, and it is of particular importance to specifically study isoform level gene regulation to fully understand the molecular mechanisms of neurodevelopmental disorders.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The manuscript is written by SS. JR reviewed and provided substantial inputs to the manuscript.This research was supported by NIH Grants GM46779 and GM135087. SS was supported by a FRAXA Foundation postdoctoral fellowship.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.


Varicella-zoster virus (VZV) is a human herpes virus which causes varicella (chicken pox) as a primary infection, and, following a variable period of latency in neurons in the peripheral ganglia, may reactivate to cause herpes zoster (shingles) as well as a variety of neurological syndromes. In this overview we consider some recent issues in alphaherpesvirus latency with special focus on VZV ganglionic latency. A key question is the nature and extent of viral gene transcription during viral latency. While it is known that this is highly restricted, it is only recently that the very high degree of that restriction has been clarified, with both VZV gene 63-encoded transcripts and discovery of a novel VZV transcript (VLT) that maps antisense to the viral transactivator gene 61. It has also emerged in recent years that there is significant epigenetic regulation of VZV gene transcription, and the mechanisms underlying this are complex and being unraveled. The last few years has also seen an increased interest in the immunological aspects of VZV latency and reactivation, in particular from the perspective of inborn errors of host immunity that predispose to different VZV reactivation syndromes. Varicella-zoster virus (VZV) is a pathogenic human alphaherpesvirus which is a significant cause of morbidity. VZV causes a primary infection, usually in children, called varicella (chicken pox) following which it establishes ganglionic latency in neurons ,2. Latenzoster sine herpete, which is the presence of herpes zoster in the absence of a rash, Ramsay Hunt syndrome and meningoencephalitis, [Post-herpetic neuralgia (PHN), a condition highly refractory to treatment, is the most well-known complication of herpes zoster, causing severe and constant pain in the affected dermatome lasting longer than three months after the zoster rash . It has halitis, ,5. A poshalitis, . The structure of VZV is now well established. It is, in common with the other herpesviruses, a double-stranded DNA virus and it has a genome comprising approximately 125,000 base pairs. Though it has long been thought that its genome contains 68 open reading frames (ORFs), with three of them repeated, it is possible that there may be, in reality, more than this number of ORFs, with a recent genome-wide transcriptomal study suggesting that VZV may encode more than just 68 ORFS . FurtherIn this overview, the intention is to highlight some recent developments in our thinking about alphaherpesvirus latency, and VZV in particular; thus, this review will be selective rather than comprehensive. Different, but related, aspects of VZV latency will be considered, namely viral gene expression in latently infected ganglia, epigenetic control of viral transcription and immunological aspects of VZV latency and reactivation, the latter mainly based on studies of patients with severe VZV infection and underlying inborn errors of immunity.As indicated, after primary infection, VZV readily establishes latency in neurons ,10,11 ofThere are two main reasons why knowledge of VZV gene expression during ganglionic latency is potentially important. First, since the functional properties of several VZV genes are known, it is possible that identification of a specific viral transcript that is expressed during latency may give clues as to its functional importance in the process. Second, if it is known that a particular VZV transcript (or transcripts) is or are present during latency, then that may represent a potential therapeutic target in individuals with recurrent VZV reactivations producing herpes zoster. However, it should be appreciated that if a lytic transcript is detected, then it may function completely differently in latency. Moreover, knowledge of a specific transcript being present would only be an initial stage in potential drug development, which would certainly not be guaranteed, and further knowledge including the identification of a corresponding protein would be important.It is established that latent VZV, such as herpes simplex virus-1 (HSV-1), is present episomally in a circular form in ganglionic neurons . While iThe result of lytic infection and the following virus reactivation is the same: the production of progeny virions. However, this result is obtained through pathways that are different at critical stages. The following discussion is centered on VZV but includes results obtained with another alphaherpesvirus . VZV and HSV-1 both establish latent infection in neurons of the peripheral nervous system , even wiSince VZV is strictly a human pathogen  and, in During lytic infection, HSV-1 capsids contact the host nuclear pore via the virus portal protein and the encased virus DNA under 30 atmospheres of internal pressure is explosively injected into the nucleoplasm ,41. At tBased on these considerations, latent infection can also be seen as host defenses overcoming the virus lytic program resulting in silencing of the virus and halting the disease process. Alternatively, latency can be seen as the virus remaining silent until an immunologically naïve population arises, and further virus spread is possible. This teleological approach is based on the premise that the virus genome must remain transcriptionally silent (to avoid immune clearance) except for a region of the virus genome that can sense the status of the host neuron. However, it should be appreciated that there are many critical differences between initial lytic infection and reactivation, which could be related to latency 3 nucleotide primary transcript from which a 1.9 × 103 nucleotide intron is removed and stabilized against rapid degradation through the use of non-conical splice sites [During latency, both VZV and HSV-1 contain a region that remains transcriptionally active. The varicella latency transcript (VLT) for VZV and the latency associated transcript (LAT) for HSV-1 are transcribed during latency when the rest of the virus DNA contains histones with PTM indicative of heterochromatin and, thus, is transcriptionally silent. Both VLT and LAT are spliced transcripts mapping antisense to the immediate-early virus gene involved in disarming the host response to foreign DNA; open reading frame (ORF) 61 encoding immediate-early protein 61 (IE61) for VZV and RL2 encoding infected cell protein 0 (ICP0) for HSV-1. VZV VLT is a set of transcripts that originate from the same transcriptional start site (TSS) but differ in intron and transcription termination site usage . Notablyce sites . Multiplce sites ,54. 3 nucleotides downstream of the HSV-1 origin of DNA replication. A further interesting feature shared by VLT and LAT is their mapping location. Transcription of both VLT and LAT terminates in the repeat region of their respective unique short DNA segments. Both VLT and LAT transcription start sites are located adjacent to the unique short repeats. LAT transcription begins in the internal repeat of the unique long segment, but since this 9212 base pair region in HSV-1 is only 88 bp in VZV, VLT transcription begins within the unique long segment of the virus DNA. Nonetheless, both virus latent transcripts span the long/short junction of the virus genome. Additionally, both latent transcripts map close to the virus origin of DNA replication. VZV spans one of the two origins of VZV DNA replication, while LAT maps approximately 5 × 10As indicated above, the VLT primary transcript is antisense to VZV ORFs 61 and 62, while LAT maps antisense to HSV-1 RL2, the HSV-1 homology to VZV ORF61. VLT transcripts downregulate the transcription of VZV ORF61 in transfection assays . While iEpigenetics is defined as the process by which a stably heritable phenotypic change results from changes in the chromosome without alterations in its DNA sequence . HoweverNucleosomes compact ~150 bp of DNA about two copies each of histone proteins H2A, H2B, H3 and H4. Histones are evolutionally conserved and encoded by intronless genes that use primordial 3’ stem/loop structures instead of 3’ polyadenylation to stabilize the mRNA ,62,63. HThe complex interplay between histone PTM is seen during early-stage virus reactivation when multiple HSV-1 ,70,71,72CpG islands are defined segments of DNA where this dinucleotide occurs at a higher than random frequency . MethylaSupplementing and extending studies that describe the nucleosome content of latent virus DNA are the analyses into how these structures are maintained in the context of genomic topology. As a result, it has emerged that control of transcription increasingly focuses on the 3-dimensional structure of gene sets. CCCTC-binding factor (CTCF) is a site-specific DNA binding protein that has been called the “master weaver”, in that it has emerged as the key element maintaining linear domains (enhancer blocker and boundary elements) and intrachromosomal as well as interchromosomal loops ,89,90. HThe precise immunological determinants of protective immunity against VZV have remained incompletely understood. This is partly because VZV is a strict human pathogen and only limited in vivo data from (humanized) mouse models are available. In this setting, the study of human inborn errors of immunity conferring increased susceptibility to VZV have proven particularly valuable ,102. It Some information has been gained from studying the ganglionic histopathology from 15 individuals with latent HSV-1 or VZV post-mortem and comparing trigeminal ganglia (TG) with dorsal root ganglia (DRG) . When evSeminal studies on the genetics of inborn errors of immunity (IEIs) or primary immunodeficiencies (PIDs) in humans conducted over the past twenty-five years have revealed monogenic defects in the Toll-like receptor (TLR)3 pathway associated with susceptibility to herpes simplex encephalitis in children and adults . FollowiPOLR3A and POLR3C subunits, and it was demonstrated that these missense mutations caused an inability to convert the AT-rich DNA as present in the VZV genome into 5’triphosphorylated RNA; this led to defective type I and III IFN responses to DNA and VZV infection as well as increased viral ORF expression in patients’ PBMCs. Moreover, reconstitution of patient cells with WT POLR3A and/or POLR3C allowed DNA sensing, type I IFN production and viral control comparable to healthy control cells, thus functionally validating the disease-causing potential of the gene variants [POLR3F, in monozygotic adult twins with recurrent VZV meningoencephalitis [In 2017, the identification of an autosomal dominant loss-of-function pol III defect in four otherwise healthy children with severe VZV encephalitis or pneumonitis as part of primary varicella was reported . RNA polvariants . This wophalitis . In younphalitis ,120. TheAmong several unresolved questions related to VZV pathogenesis and immunity to this virus is the major issue as to whether pol III plays a direct role in controlling VZV latency through the production of type I and III IFNs, or whether pol III may be involved in the maturation of adaptive immunity during primary infection in more indirect/subtle ways. In the former case, it is not clear whether the role of pol III is exerted in the peripheral sensory ganglia, i.e., the PNS, or rather after reactivation into the CNS. Although several pertinent questions remain, these and other studies provide evidence that type I (and III) IFNs play important roles in VZV infection and reactivation. Intriguingly, in a clinical observation of a patient with disseminated VZV eruption/varicella who had circulating autoantibodies against IFNα, it was first reported in 1981  that typATG4A and LC3B2 in two adult patients and was the first study to associate defective autophagy with viral infection in humans [The majority of VZV disease manifestations associated with PIDs involve increased susceptibility to both primary disseminated infection and reactivation, in the latter case to cause herpes zoster or CNS involvement in the form of meningitis, encephalitis and vasculitis. It may, therefore, be informative to consider a medical condition involving exclusively alphaherpesviruses reactivation. Mollaret’s meningitis, the molecular and immunological pathogenesis of which is largely unexplored, is such a condition, and consists of recurrent lymphocytic meningitis caused by HSV-2 reactivation, in rare cases by VZV or HSV-1 ,122,123.n humans ,125. HSVBased on these findings, it is tempting to speculate that autophagy may also play a role in maintaining VZV latency, and that autophagy defects in humans may predispose to severe VZV infection and reactivation from latency. Indeed, in vitro studies have demonstrated both pro-and antiviral roles of autophagy in VZV immunity and pathogenesis. Briefly, several studies have provided evidence of interactions between VZV replication and autophagy pathways during lytic infection and shown that these virus–host interactions take place in a highly cell-type dependent manner. Whereas autophagy seems to exert antiviral roles in some contexts, the virus may also directly utilize autophagy molecules for its own benefit to enhance replication and viral egress (related to the endoplasmic reticulum and other cell organelles) and, thus, evade autophagy and subvert this process ,128,129.During the last decade there has been a significant increase in our detailed knowledge of the complex mechanism underlying VZV latency, as well as an emerging appreciation of the diversity of the clinical syndromes caused by VZV reactivation. The advances in the understanding of VZV latency have been extensive but largely incremental, and a true paradigm shift has yet to be defined. This increased understanding has largely been driven both by new molecular virological technologies and an increased interest in viral latency among the scientific community. The emergence of epigenetic mechanisms influencing VZV latency is likely to lead to an explosion of interest in this aspect. There has also been much recent interest in immunological aspects of VZV latency, mainly in terms of determining the host immune response to the virus and the role of immune protective mechanisms in preventing viral reactivation. This knowledge has been gained, in particular, by studying the clinical manifestations together with the genetics and cellular immune phenotype of individuals with inborn errors of immunity presenting with severe disseminated VZV infection. Since alpha human herpes virus latency is established so early in life, it is unlikely that viral latency can be completely prevented, but a significant consequence of current research is more likely to lead to therapies that may treat or prevent viral reactivation in both immunocompetent and immunocompromised individuals.


Key differences in established model systems have also led to controversy surrounding the role of viral gene products in latency establishment, maintenance, and reactivation. This review will discuss the details and challenges of various models including hematopoietic progenitor cells, monocytes, cell lines, and humanized mice. We highlight the utility and functional differences between these models and the necessary experimental design required to define latency and reactivation, which will help to generate a more complete picture of HCMV infection of myeloid-lineage cells.Human cytomegalovirus (HCMV) is a highly prevalent beta-herpesvirus and a significant cause of morbidity and mortality following hematopoietic and solid organ transplant, as well as the leading viral cause of congenital abnormalities. A key feature of the pathogenesis of HCMV is the ability of the virus to establish a latent infection in hematopoietic progenitor and myeloid lineage cells. The study of HCMV latency has been hampered by difficulties in obtaining and culturing primary cells, as well as an inability to quantitatively measure reactivating virus, but recent advances in both  Human cytomegalovirus (HCMV) is a prototypical beta-herpesvirus that infects a majority of the world’s population. Although most HCMV infections are asymptomatic in healthy individuals, the virus is the leading cause of congenital abnormalities following fetal infection and is a significant cause of morbidity and mortality following hematopoietic stem cell transplant and solid organ transplant –6. HCMV –+ hematopoietic progenitor cells (HPCs) are a critical reservoir of latent HCMV  how culture conditions mimic the bone marrow environment and support the pluripotency of HPCs, (2) how the heterogeneity of the CD34+ HPC population can affect experimental outcomes and why the source and purity of the cells is important, and (3) the controls required to demonstrate establishment of latency and reactivation.There are several key aspects of this model that are necessary to the successful study of HCMV latency in HPCs. Below, we highlight some of the important considerations when using the CD34in vitro culture model of HPCs is to mimic the in vivo conditions as closely as possible in order to maintain their progenitor cell phenotype, typically through use of cell types that act as surrogates for the bone marrow microenvironment. The gold standard for maintaining the functionality of stem and progenitor cells is to culture HPCs on top of stromal cell lines that provide cytokine support to mimic the bone marrow niche  supports a higher frequency of persistent infection and does not establish latency (+CD90+CD38+ and CD34+CD90−CD38+) can establish latency and reactivate, albeit with different reactivation frequencies  a subpopulation of lytically infected cells, (2) that culture conditions have resulted in changes in the cell population(s) that now support persistent infection, or (3) that the specific virus being tested is unable to establish latency. To test for the presence of infectious virus immediately prior to reactivation , half the cells are lysed and plated onto permissive fibroblasts. The frequency of infectious virus production is then measured by extreme limiting dilution assay (ELDA). ELDA is a mathematical improvement to limiting dilution assays that provides one-sided confidence intervals for populations that produce 0% or 100% responses. ELDA was developed for the study of stem cell populations in which cell numbers follow non-Poisson distributions and utilizes statistical methods that accommodate small numbers of replicates   . These red cells , 51. TheESCs  are plur+ HPCs (+ HPCs (as described above) to assess the ability of hESC-derived CD34+ HPCs to support HCMV latency and reactivation  establishment of latency, (2) maintenance of the viral genome, (3) reactivation of infectious virus following stimuli, (4) recapitulation of the function of viral gene products using recombinant viral mutants, and (5) induction of viral-induced myelosuppression via upregulation of TGF-β ; however, direct infection of monocytes with HCMV has been shown to prolong the life of the cells by induction of specific antiapoptotic and proautophagic signaling  promoter, resulting in increased IE gene expression and ultimately production of new virus  . iPSCs cin vitro culture, homogenous genetic background, and ease of manipulation. Because of these advantages, model systems using the myeloid cell line THP-1 , also known as phorbol 12-myristate 13-acetate (PMA), which promotes differentiation to mature monocytic lineages. An increase in HCMV gene expression can be measured following PMA treatment of THP-1 cells . THP-1 cells most closely resemble primary monocytes in both morphology and differentiation properties, while Kasumi-3 cells express CD34 and are more representative of a progenitor cell . Both ce-1 cells , 86, alt-1 cells , 81, sug-1 cells . Moreove+ HPCs found that treatment with HDAC inhibitors had differing effects on IE1 transcription  or CRISPR/Cas9, both cell lines have limitations as models for HCMV latency , 87. Impcription , 89. Morcription ; therefoin vivo. While murine CMV and rat CMV have served as surrogates for in vivo studies, they differ substantially from HCMV in their genome organization  mice. At 8 weeks postengraftment, approximately 5% of peripheral blood mononuclear cells (PBMCs) were human monocytes (huCD45+/huCD33+/huCD14+), which is approximately half the proportion of monocytes found in the PBMC of healthy humans. Following IP injection of HCMV-infected fibroblasts, HCMV DNA was detectable in samples from organ tissues repopulated with human hematopoietic lineage cells. Based on clinical studies  in addition to matched human fetal liver and thymus tissue (huBLT)  . The huBin vivo. First, Hakki et al. adapted the huNSG model to use G-CSF-stimulated peripheral blood stem cells from HCMV-seropositive donors as the human cell engraftment source  will open new avenues for investigation to better understand the intricate and balanced relationship between HCMV and its host.Overall, the HCMV humanized mouse models have been instrumental in validating viral factors important to latency and reactivation + HPCs is necessary to investigate the role of viral and cellular gene products in latency establishment and maintenance (+ monocytes can be used to investigate gene products that drive differentiation from monocytes to macrophages or dendritic cells (The study of HCMV latency and reactivation in myeloid-lineage cells is fraught with potential pitfalls as cellular differentiation state, virus genetic background, and culture conditions can all have significant impacts on experimental outcomes. These challenges make comparison of HCMV latency studies difficult without a precise understanding of the cell type, culture conditions, and experimental controls used. When designing an HCMV latency experiment, it is important to choose the model system that best addresses the state of latency to be measured . Infectintenance  and F, antenance  and F, wic cells  and F, tic cells  and F. Sic cells . Finallyin vitro and in vivo model systems, coupled with careful experimental design, can provide new and exciting avenues to understand the mechanisms of action of viral gene products and their interplay with host factors important for regulation of HCMV latency and reactivation.The viral and cellular gene products required for all aspects of latency and their mechanisms of action remain an understudied area of HCMV biology. Recent advances in


Objectives: Sonic hedgehog (SHH) signaling is important in bladder development. Mice with defective hedgehog signaling develop bladder anomalies. Clinically, urinary tract malformations are reported in human fetuses and infants with mutations of SHH and related signaling pathway genes. Information on the expression of SHH and associated signaling genes in normal human bladder development is fragmentary. This study determined the temporal and spatial expression patterns of SHH signaling pathway components in human fetal bladders by immunohistochemistry (IHC).Material and Methods: Twenty-four bladder specimens from 16 male and 8 female human fetuses aged 12- to 36-week (wk) were obtained from the First Affiliated Hospital of Xi'an Jiaotong University. The tissue slides were processed for IHC staining with SHH, Patched1 (PTC-1), Patched2 (PTC-2), Smoothened (SMO), GLI1 and proliferating cell nuclear antigen (PCNA). The expression levels of each gene were analyzed by semi-quantitative histological scoring system.Results: High intensity of SHH and SMO expression was detected in developing bladder urothelial cells, with no staining in lamina propria (LP), but with minimal expression of SMO in differentiating smooth muscle (SM) layers. The spatial distribution pattern of PTC1 and GLI1 was more complex with minimal expression in the LP layer, moderate expression in the SM layer, and high expression in the urothelium. PTC2 expression was mainly localized in the urothelium and LP, but no expression in the SM layer. All of the SHH signaling components were detected in fetal bladder tissues throughout the development, with expression peaks at 12- and 23-wk, coinciding with high cell proliferation as indicated by PCNA staining in the cell nuclei of urothelium and SM.Conclusions: The autocrine SHH signaling in the developing urothelium, and paracrine SHH signaling in the developing smooth muscle layer, mediated by SMO, PTC-1 and GLI1 were demonstrated during human bladder development. Expression of SHH signaling components peaked at 12-and 23-wk. The first expression peak at 12-wk may relate to urothelium growth, SM induction, and dilation of the bladder cavity. The second expression peaked at 23-wk may relate to urothelium and SM layer differentiation. The human urinary system originates from the intermediate mesoderm of the developing embryo, and bladder is one of the most important organs in human body for collecting and storing urine. Human bladder and ureterovesical junction develop from urogenital sinus (UGS) and derived from the hindgut during the fourth to seventh weeks of gestation , 2. The Sonic hedgehog (SHH) is widely studied among three types of hedgehog homologs in human body, and it is related to many biological phenomena. Previous studies have demonstrated that SHH, transforming growth factor-β (TGF-β), bone morphogenetic protein 4 (BMP4), and fibroblast growth factor receptor 2 (FGFR2) are the key signaling factors in bladder development, and they are interconnected in a complicated regulatory network. Some animal experiments have shown that a certain concentration of SHH is particularly indispensable for the development of bladder musculature and its function , 6. SHH Previous studies based on animals have shown that SHH and its downstream signaling molecules are involved in bladder development, relevant study  has idenin vitro studies cannot accurately represent human beings. The expression of SHH signalings in different stages of normal human bladder development is largely unknown. In this study, we determined the temporal and spatial expression patterns of SHH signaling components in the developing human fetal bladder tissue samples by immunohistochemistry, and we explored the possible role of SHH signaling in the formation and development of human urinary bladder.However, it is not easy to acquire normal human developing bladder specimens, and results from mice or Twenty-four fetal bladder specimens were obtained with the approval of the Institutional Review Board of the First Affiliated Hospital of Xi'an Jiaotong University. Informed consent was obtained by the consulting obstetricians, and all specimens used in this study were considered discarded tissues with devoid of any identifiers. Gestational age was determined from the date of the last menstrual period and was confirmed with crown-rump length and body length measurements. The gestational age distribution and the total number of tissue specimens (in parenthesis) were: 12 weeks (2 cases), 16 weeks (2 cases), 18 weeks (2 cases), 21 weeks (2 cases), 23 weeks (2 cases), 24 weeks (2 cases), 26 weeks (2 cases), 28 weeks (2 cases), 31 weeks (2 cases), 32 weeks (2 cases), 33 weeks (2 case), 36 weeks (2 cases). All those fresh fetal bladder specimens were taken and fixed in 4% paraformaldehyde for 24 h at least, embedded in paraffin, and sliced at 4 μm thickness for immunohistochemical study.Mouse anti-human SHH (BD-610182) primary antibody was purchased from American BD company, mouse anti-human SMO (SC-8424), PCNA (SC-6260), GLI1 (SC-8598), PTC-1 (SC-3039), PTC-2 (SC-4853) primary antibody was purchased from Santa Cruz, USA, and immunohistochemistry kit was purchased from Dako, USA.The EnVision two-step method was used for immunohistochemistry staining, and the experimental procedure was carried out according to the kit instructions. The tissue slides were dewaxed by xylene, hydrated by gradient ethanol (100-30%), rinsed with distilled water for 3 min, and then placed in 0.01 mol/L sodium citrate buffer (pH 6.0) and microwaved for 5 min for antigen retrieval, and naturally cooled at room temperature. The slides were washed with 0.01 mol/L PBS (pH 7.4) for three times, and subjected to Dual Endogenous Enzyme Block for 10 min. After washing in 0.01 M PBS (pH 7.4) for three times, the slides were incubated overnight with the diluted primary antibody or the PBS solution as the negative control. After washing with 0.01 mol/L PBS (pH 7.4) for three times, the slides were subjected to 30 min DakoCytomation EnVision+ HRP reagent incubation for anti-mouse and anti-rabbit secondary antibodies, or 15 min Biotinylated Link coupled with 15 min Streptavidin-HRP incubation for goat antibodies. Signals were detected by adding substrate hydrogen peroxide using diaminobenzidine as a chromogen and followed by 2 min hematoxylin counterstaining. In the end, all the slides were routinely dehydrated, transparence, sealed and observed under Nikon Eclipse 600 Microscope .All of the immunostained slides were analyzed by two histopathologists, and eight fields in each slide were randomly selected and examined under a 40× objective lens of the microscope. All fields were assessed using a semiquantitative histological scoring system (H-score) described previously . In eachFigure 3).SHH were mainly expressed in the fetal bladder urothelial cells but not in the lamina propria and differentiating smooth muscle layers. The expression of SMO could be largely detected in the developing bladder urothelium with minimal staining in the mesenchymal cells of the fetal bladder smooth muscle layers. However, the expression of PCNA could be detected in the nucleus of the developing urothelium and mesenchymal cells of the fetal bladder with stronger intensity than that of SHH and SMO . With thThe spatial distribution patterns of PTC1 and GLI1 were characterized by minimal expression in the lamina propria, moderate expression in the smooth muscle layer, and high expression in the urothelium of the developing bladder. The expression of PTC2 was mainly localized in the urothelium and lamina propria, but not in the smooth muscle layer . With thNormal human bladder development requires two indispensable aspects, including the precise temporospatial distribution of key morphogenetic and regulatory signals to generate radial patterning for the subsequent compartmentalization of distinct cellular and tissular phenotypes; on the other hand, it is highly depending on the cross-talk between urothelium and its surrounding mesenchyme, which is called epithelium-mesenchyma interaction. Tissue recombination experiments have proved that bladder urothelial epithelium is required to form mesenchyme and differentiate into smooth muscle through diffused signaling molecules in cellular microenvironment .The expression of SHH in the early development of organs has their temporal and spatial specificity, and it gives rise to regulating the morphogenesis of organs through autocrine and paracrine effects on both epithelial tissues and surrounding mesenchymal cells –16; howe−/− and Gli2−/− bladders were hypoplastic; and furthermore, SHH signaling could regulate bladder morphogenesis in a dose-dependent manner. In this study, we examined the SHH signaling components in normal human bladders, and no anatomical or histological abnormalities were observed. The above animal studies as well as transgenic mice have some similarities to our findings in human fetal bladders by using IHC examination. All of the studies indicated that SHH/GLI axis might regulate mesenchyme proliferation and differentiation.Previous studies , 7, 19 hAdditionally, previous studies predicted that high levels of SHH and GLI2 expressing cells in the mesenchyme could support urothelial cell proliferation but suppress smooth muscle differentiation. Autocrine SHH through PTC could drive alterations of cell morphology but not relevant to GLI-mediated transcription . AlternaTasian et al.  illuminain vitro studies to summarize that paracrine SHH from the urothelium regulates smooth muscle differentiation. Hence, we speculated that autocrine SHH in the developing urothelium, and paracrine SHH signaling in the developing smooth muscle layer, mediated by SMO, PTC1 and GLI1, were demonstrated during human bladder development.The autocrine action and paracrine action are two ways for cytokines playing biological effects, and both classical and non-classical hedgehog signal transduction pathways involve these two approaches. Many studies , 26, 29 Clinically, urinary tract malformations, such as ureteral duplication and ectopy, vesicoureteral reflux, bladder exstrophy are reported in human infants with mutations of SHH or its related signaling pathway genes. Birth defects are structural or functional abnormalities that existed before birth. Genetic counseling, prenatal screening and diagnosis are very important tools for the prevention and treatment for birth defects. With the development of medical technology, prenatal diagnosis can be made before birth of the fetus with a variety of advanced detection methods to explore the fetal development situations in utero, observing whether the fetus has malformation, making diagnosis of congenital diseases, and creating conditions for fetal intrauterine treatment or selective abortion. Between 18 and 24 weeks of gestation, amniocentesis, percutaneous umbilical cord blood puncture and fetal tissue biopsy can be safely performed, and then the molecular genetic examination can be carried out for the evaluation of possible developmental defects. Under the help of the identification of abnormal expression or mutations of some development related essential genes, such as SHH/GLI, the obstetricians might timely terminate the pregnancy with those problem genes to avoid of the possible birth defects, such as bladder exstrophy, or they can make full consideration for post-natal treatment or active intervention.We would like to underline the limitations in this study. Firstly, we should have applied statistical tests to evaluate expression level of SHH signaling pathway components in fetal human bladder; however, it was inappropriate for studies with very small sample size, just like ours. Frankly speaking, fresh embryonic tissues with different gestational ages by induced labor were extremely difficult to obtain and to be used for experimental research. Hopefully, we are still going to stick with this study in the future, so as to look forward to more in-depth results when the sample size could be increased. Secondly, we did not examine the abundance of these signalings; however, once we collect more tissue samples in the future, we would like to use more accurate methods, such as western blot and quantitative RT-PCR, to analyze SHH signaling components at different stages of human bladder development and conduct the statistical analysis.In summary, autocrine SHH in the developing urothelium, and paracrine SHH signaling in the developing smooth muscle layer, mediated by SMO, PTC1 and GLI1, were demonstrated during human bladder development. The first expression peak of SHH signaling at 12 wks might relate to urothelium growth, smooth muscle cell induction, and the second expression peak of SHH signaling at 23 wks might relate to smooth muscle layer differentiation.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by the Institutional Review Board of the First Affiliated Hospital of Xi'an Jiaotong University. The patients/participants provided their written informed consent to participate in this study.GZ and DH conceived and designed this study. HZ and SX conducted specimen collections and all the immunohistochemical staining. XW implemented quality control. HZ and GZ wrote and revised the manuscript. All authors read and approved the final manuscript.This study was supported in part by the grants from the Fundamental Research Funds for the Central Universities of China (No. xjj2018zyts34) and the Research Funds on Social Development from the Department of Science and Technology of Shaanxi Province (No. 2020SF-119) to GZ.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.