Astrocytes are the most prevalent glial cells in the brain. Historically considered as “merely supporting” neurons, recent research has shown that astrocytes actively participate in a large variety of central nervous system (CNS) functions including synaptogenesis, neuronal transmission and synaptic plasticity. During disease and injury, astrocytes efficiently protect neurons by various means, notably by sealing them off from neurotoxic factors and repairing the blood-brain barrier. Their ramified morphology allows them to perform diverse tasks by interacting with synapses, blood vessels and other glial cells. In this review article, we provide an overview of how astrocytes acquire their complex morphology during development. We then move from the developing to the mature brain, and review current research on perisynaptic astrocytic processes, with a particular focus on how astrocytes engage synapses and modulate their formation and activity. Comprehensive changes have been reported in astrocyte cell shape in many CNS pathologies. Factors influencing these morphological changes are summarized in the context of brain pathologies, such as traumatic injury and degenerative conditions. We provide insight into the molecular, cellular and cytoskeletal machinery behind these shape changes which drive the dynamic remodeling in astrocyte morphology during injury and the development of pathologies. Astrocytes have classically been depicted as star-like cells , S100β, Aldh1L1, Sox9, AldoC, Glt1 and glutamine synthase  and brain tumors, but follow a more complex response pattern during the progression of these diseases. During the later phases of AD, reactive astrocytes form prominent glial scar-like barriers around amyloid plaques and disrupt the anthropoid-specific architecture of non-reactive astrocytes in the neocortex  and, in particular, during astrogliosis. Available data indicate distinct differences in the actin organization of polarizing astrocytes after injury, compared to other extending or migrating cells Figure . In cellng Table . The parAlthough the relevance of actin dynamics in palisading astrocytes is not clear, studies in astrocytes in culture and in tissues indicate a role of the actin cytoskeleton in controlling hypertrophy and alterations in astrocyte complexity. Acute inhibition of the Arp2/3 complex in brain slices increases the astrocyte cell body size and abundance of large processes analogous to reactive astrocytes in diffuse trauma. Moreover, inactivating the Arp2/3 complex accelerates the hypertrophy of stellate astrocytes in the oxygen/glucose-deprivation model of stroke, whereas overactivating Arp2/3 by depleting its endogenous inhibitor PICK1 or overexpressing the activator N-WASP suppresses cell body expansion Figure . This isin vivo. The substitution of murine astrocytes through the implantation of glial progenitors derived from human-iPSC makes it possible to investigate the specific role and contribution of abnormal astrocytes in diverse pathologies. A better understanding of the plasticity of astrocyte morphology and function will help us to gain insight into the fundamental properties of this profound shapeshifter in both normal cells and diverse human pathologies.The morphological features of astrocytes are integral to the normal shape and functioning of the CNS from late embryonic development throughout all stages of an organism’s life. Despite their critical relevance in both normal and disease states, the molecular mechanisms behind the plastic morphology of astrocytes are poorly understood. Nonetheless, improvements in cell culture methods and the development of new tools and elaboration of sophisticated microscopy techniques will allow observation and manipulation of more authentic astrocyte settings. In this context, nanoscopic PAP-synapse interactions are becoming increasingly accessible with super-resolution light microscopy, and as such, provide an appealing experimental alternative to sophisticated electron microscopy (Heller et al., JS, BJE and KM wrote the manuscript. JS and KM created the figures.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.


PRKN, PINK1, DJ-1 and HTRA2 and are associated with mitochondrial impairment. This phenotype can be caused by the dysfunction of mitochondrial quality control machinery at different levels: molecular, organellar or cellular. Mitochondrial unfolded protein response represents the molecular level and implicates various chaperones and proteases. If the molecular level of quality control is not sufficient, the organellar level is required and involves mitophagy and mitochondrial-derived vesicles to sequester whole dysfunctional organelle or parts of it. Only when the impairment is too severe, does it lead to cell death via apoptosis, which defines the cellular level of quality control. Here, we review how currently known PD-linked genetic variants interfere with different levels of mitochondrial quality control. We discuss the graded risk concept of the most recently identified PARK loci (PARK 17–23) and some susceptibility variants in GBA, LRRK2 and SNCA. Finally, the emerging concept of rare genetic variants in candidates genes for PD, such as HSPA9, TRAP1 and RHOT1, complete the picture of the complex genetic architecture of PD that will direct future precision medicine approaches.Mitochondrial impairment is a well-established pathological pathway implicated in Parkinson’s disease (PD). Defects of the complex I of the mitochondrial respiratory chain have been found in post-mortem brains from sporadic PD patients. Furthermore, several disease-related genes are linked to mitochondrial pathways, such as  Patients with Parkinson’s disease (PD) experience motor impairments such as resting tremor, bradykinesia, rigidity and postural instability but also non-motor symptoms such as sleep perturbations, constipation, cognitive impairment or depression  or other disease-associated genes  arise as risk factors and relate hereditary to the more common sporadic form of PD  including common risk factors such as SNCA polymorphisms.Based on linkage studies and next-generation sequencing technologies like whole-exome or whole-genome sequencing, more recently an increasing number of genes has been linked to monogenic PD, expanding the current total of  23 Lill . MoreoveMitochondria are highly dynamic organelles that are essential to maintain cellular function. These organelles maintain neuronal function and integrity via sustained energy supply for important cellular functions including synaptic activity or calcium buffering after depolarisation  entry in the mitochondria via the mitochondrial Ca2+ uniporter. The balance between Ca2+ accumulation in mitochondria and release via mitochondrial Na+/Ca2+ and H+/Ca2+ exchangers allows mitochondria to have a high capacity of buffering cytosolic Ca2+ , which encode the proteins PINK1, a mitochondrial localised kinase and Parkin, a ubiquitin-E3 ligase  and Parkin  have been found in juvenile PD patients . Loss of function mutations in the DJ-1 gene have been associated with autosomal recessive early onset PD and are far rarer than PINK1 or Parkin mutations   have been found in PD patients from a German cohort  has been proposed as a PD-linked protein by Chartier-Harlin and collaborators  borators . Even thborators , EIF4G1 borators .mtUPR promote an increase of mitochondrial chaperones  and proteases transcription to support the recovery of the mitochondria   were the first identified genetic cause for PD , are the most common risk factor for sporadic PD with 3–7% of all PD patients harbouring a mutation in this gene  and are known to lead to autophagolysosomal dysfunction, accumulation of glucosylceramide and lipid metabolism impairment  are the most frequent cause of autosomal dominant PD  and may disturb interaction with binding partners or diminish dimerization of LRRK2  domain of LRRK2. The amino acid exchange may lead to conformational changes and could affect dynamic interaction among LRRK2 domains  degeneration of dopaminergic neurons in the substantia nigra. Therefore, novel approaches in defining access to causative neuroprotective therapies have to account for this heterogeneity and need to define criteria for stratification of sporadic PD into different subgroups. Among the different signalling pathways described in PD, mechanisms implicating impaired mitochondrial homeostasis get more and more into focus. Also, an increasing number of monogenic forms of PD have been identified that have helped to dissect the different instances of mitochondrial quality control involved in the neurodegenerative process. Here, the PINK1/Parkin-mediated pathway for organellar quality control is representing an important building block in the understanding of the molecular underpinnings of PD. Based on genetically defined models of PD, new approaches can now be taken to develop neuroprotective treatment strategies. Using patient-derived fibroblasts from Parkin mutation carriers, the first successful compound screening campaign was recently published that allowed to define bile acid derivatives as potential candidates for reverting PD-associated mitochondrial phenotypes like energy deficiency and altered mitochondrial morphology  that are directly involved in the shuttling of vesicles and maturation into lysosomes for degradation of not only mitochondria but also other organelles and protein aggregates (Edvardson et al. However, even all monogenic forms of PD taken together only contribute to a minority of all patients and therefore strategies to define the mitochondrial subtypes within the most common sporadic form of PD need to be developed. Besides rare genetic variants that interfere with proper mitochondrial function and confer substantial risk to develop typical PD, also more common variants, e.g., in the


Adenylate cyclase 1 (ADCY1 or AC1) is a member of ADCY superfamily and was primarily found to be expressed in the brain. ADCY1 is responsible for catalyzing ATP to cyclic AMP (cAMP). As a secondary messenger, cAMP can regulate plenty of cellular activities. cAMP can perform its regulation in cellular transport through the binding to cAMP dependent protein kinases (PKAs), cAMP-activated guanine exchange factors (EPACs) and cyclic nucleotide-gated channels functioning in transduction of sensory signals (CNGs). Lung cancer is one of the leading factors of cancer-related death worldwide. Platinum-based chemotherapy is the first-line treatment for advanced lung cancer patients. In addition, surgical treatment, radiation treatment, and molecular targeted therapy are also therapeutic options for lung cancer patients in clinical settings. However, drug resistance and toxicity are the major obstacles that affect chemotherapy outcome and prognosis of lung cancer patients. And the therapeutic efficiency and adverse effects are varying with each individual. In recent years, investigations based on genetic sequencing have revealed the emerging role of ADCY1 mutations in affecting drug efficiency in various cancers such as lung cancer, esophageal cancer and colorectal cancer. The potential function of ADCY1 in chemotherapy resistance is of great importance to be noticed and investigated. Lung cancer is one of the leading causes of cancer-related death worldwide in recent years Adenylate cyclase 1 (ADCY1 or AC1) is a member of ADCY superfamily and is primarily found to be expressed in the brain There are several report about the relationship between ADCY1 and drug resistance. ADCY1 was investigated to be significantly associated with the overall survival of patients with melanoma, and plays an important role in the metastases of melanoma In this review, we summarized the recent advances of ADCY1 in lung cancer and its underlying mechanisms. First, we overviewed the relationship between ADCY1 and cAMP, as ADCY1 is a vital regulator which can catalyse ATP to cyclic adenosine 3', 5'-monophosphate (cAMP). cAMP is an important second messenger involved in diverse cellular responses including cell growth, cell differentiation, cell proliferation, apoptosis and metabolism. Then, we focused our eyes on the downstream effectors of cAMP signaling pathway adjusted by ADCY1, and their relationship with drug resistance in lung cancer. We also explored the three classical signaling pathways and other signalling pathways modified by cAMP in the regulation of drug resistance in lung cancer. Moreover, we summarized the probable lncRNAs modulated by cAMP which may contribute to drug resistance in lung cancer patients. Finally, we highlight the feasibility of ADCY1 as a potential biomarker to predict drug resistance, which may provide an important clue for future lung cancer treatments.ADCYs are activated by the stimulatory GTP-binding proteins (G proteins) which can response to various external signaling molecules through the regulation of G protein-coupled receptors, such as neurotransmitters and hormones PKAs and EPACs are the major targets of cAMP signaling pathway, activated PKAs-phosphorylated-enzymes can regulate relevant metabolism and transcription factors such as cAMP response element binding protein (CREB) 2+. The interaction of ADCY1 with specific signaling proteins and regulatory factors can generate privileged domains in cAMP signaling pathways ADCY1 can influence cAMP biosynthesis and degradation through the coupling by CaThe cAMP signaling pathway plays an essential role in diverse cellular responses including cell growth, cell differentiation, cell proliferation, apoptosis and metabolism. cAMP signaling can also modulates cancer cell death induced by anticancer drugs and γ-rays cAMP has been identified to participate in response to various DNA damaging statuses. It has been widely acknowledged that cAMP can regulate cell apoptosis by modulating the expression of Bcl-2 family proteins, the inhibitor of apoptosis proteins (IAPs) and the repair protein (XRCC1) of γ-ray-induced DNA damage in human lung cancer cells cAMP signaling can decrease the phosphorylation of Bcl-2 through the inhibition of JNK activation, which will result in the down-regulation of cell autophagy and phosphorylation, and then reduce the ubiquitination and increase the expression of histone deacetylase 8 (HDAC8) As a novel inhibitor of apoptosis proteins that inhibit apoptosis triggered by a variety of stimuli, IAPs can play important roles in suppressing cell death and regulating DNA damage in the cell cycle. The translation initiation regulation of the internal ribosome entry sites is one of the mechanisms that can regulate the expression of IAP The X-ray repair cross-complementing protein 1 (XRCC1) can affect cellular sensitivity to ionizing radiation. The expression and polymorphisms of XRCC1 play an important role in DNA repair and it may be a prognosis biomarker for lung cancer patients treated with radiation or chemotherapy In conclusion, cAMP can modulate DNA damage and repair to regulate cellular survival and the prognosis of lung cancer patients. It may regulate the sensitivity to radiation and chemotherapy in lung cancer patients through the modulation of these three popular effectors: Bcl-2, IAPs and XRCC1 Investigations have also revealed that cAMP can regulate radiation-induced DNA damage by other effectors. The ataxia-telangiectasia mutated (ATM) protein kinase plays a vital role in harmonizing the cellular response to radiation-induced DNA damage 2+ binding protein, sorcin can induce the expression of MDR1/P-gp markedly through the activation of cAMP response element (CRE) located in the MDR1/p-gp promoter, and then regulate the CREB pathway which is associated with the MDR phenotype Multidrug-resistance (MDR) phenotypes are a major obstacle to the efficiency of cancer. The ATP-binding cassette transporter MDR1/P-glycoprotein (MDR1/P-gp), as an ABC transporter, has been identified to be associated with MDR in various cancers, such as lung cancer, ovarian cancer, laryngeal cancer MEK is activated in multiple cancer types such as non-small-cell lung cancer (NSCLC) and colorectal cancer (CRC). Selumetinib is a selective MEK1/2 inhibitor and is currently widely used in the treatment of NSCLC and SCLC Parathyroid hormone-related protein (PTHrP)-(1-34) and PTHrP-(140-173) can protect lung cancer cells from apoptosis after the expose to radiation or chemotherapy drugs. A slight increase of cAMP was sufficient to protect lung cancer cells from apoptosis In conclusion, cAMP or its analogue (8-Cl-cAMP) can regulate multidrug-resistance in lung cancer patients. There are plenty of downstream effectors participating in the cAMP signaling pathway such as the classical PKA and CREB. These complex but well-ordered signaling pathways can regulate the majority of cellular activities such as cell apoptosis, cell growth, cell cycle, cell proliferation, which may have an effect on multidrug-resistance in the treatment of lung cancer patients Long noncoding RNAs (lncRNAs) play a diverse range of biological roles, including regulation of gene expression. In recent years, lncRNAs have been recognized as emerging regulators in tumorigenesis As a second messenger in multi-cellular activities, cAMP can regulate various lncRNAs effectors. COX-2 is an anticancer target; however, the drug resistance of COX-2 inhibitors is a major obstacle limiting their therapy efficiency in clinical treatment There are many LncRNAs highly up-regulated in liver cancer (HULC) known as Lnc HULC. They play important roles in tumorigenesis and metastasis. And they can be act as biomarkers to predict drug efficacy and prognosis of various cancers In summary, cAMP can regulate multidrug resistance in lung cancer and other malignant tumors through the regulation of particular lncRNAs involving in different signaling pathways, which is of great value in clinical treatment for lung cancer therapy Figure .Here we reviewed for the first time of ADCY1 and its association with drug resistance in lung cancer. As a member of adenylate cyclase (ADCY), ADCY1 was first found to be expressed abundantly in brain and subsequent studies was mainly focused on its role in brain diseases. Mutations of ADCY1 were revealed to be associated with hearing impairment or other disorders in the nervous system. In this review, we focus on the function of ADCY1 in the regulation of multidrug resistance in lung cancer mediated by cAMP, which is a second messenger participating in plenty of cellular activities. cAMP has also been reported to be involved in the regulation of drug resistance in lung cancer through the downstream effectors in different signaling pathways such as the effectors belonging to DNA damage signaling pathway and specific lncRNAs. Taken together, ADCY1 is of great significance to be a novel biomarker to predict drug resistance in lung cancer patients. However, the specific mechanism of ADCY1 regulating drug resistance need to be further investigated.


RLIM/Rnf12 is an E3 ubiquitin ligase that has originally been identified as a transcriptional cofactor associated with LIM domain transcription factors. Indeed, this protein modulates transcriptional activities and multiprotein complexes recruited by several classes of transcription factors thereby enhancing or repressing transcription. Around 10 years ago, RLIM/Rnf12 has been identified as a major regulator for the process of X chromosome inactivation (XCI), the transcriptional silencing of one of the two X chromosomes in female mice and ESCs. However, the precise roles of RLIM during XCI have been controversial. Here, we discuss the cellular and developmental functions of RLIM as an E3 ubiquitin ligase and its roles during XCI in conjunction with its target protein Rex1. Xist plays crucial roles during both forms of XCI and paints the X from which it is expressed , which silences exclusively the paternally inherited X (Xp), directly follows zygotic genome activation (ZGA) at the end of the 2-cell stage. While this pattern of XCI is maintained in extraembryonic tissues including trophoblast and primitive endoderm, epiblast cells which give rise to the embryo proper reactivate the Xp (XCR) and undergo a random form of XCI (rXCI) around implantation . The lonxpressed . InitiatCI state . Prior t pairing . Xist tring Rex1  and in c to Xist .Rlim gene  encodes a RING finger ubiquitin ligase (E3)  . During  tissues . In cell tissues  but in m. RLIM cain vivo functions of Rlim have been discovered in female mice. In mammary glands of pregnant and lactating females, RLIM serves as a survival factor specifically for milk-producing alveolar cells  developed Xist clouds and initiated XCI upon differentiation in vitro and in vivo, and in tetraploid complementation experiments these ESCs were able to undergo rXCI and form post-gastrulation embryos  is expressed from the “KO” Rlim locus (RlimKO or WT) leads to nuclear accumulation of Rex1, indicating gain-of-function activity. Moreover, while Rnf12KO ESCs (Rlim333f) express Rex1 in the nucleus, cell derivatives with a CRISPR/Cas9-mediated deletion of RLIM333  are XCI competent at least to some degree and displayed mostly cytoplasmic Rex1 (Rlim in ESCs does not necessarily lead to nuclear accumulation of Rex1 (Rlim ORF (referred to as Rnf12CR–/CR–) and is unable to undergo XCI, XCI activity was restored upon additional deletion of Rex1  die  die  and not  failure .Rlim in the maintenance of iXCI at blastocyst stages appears clear. This role was genetically established by inducing the Rlim cKO in embryos after ZGA via a paternally transmitted Rosa26-Cre transgene as well as by data obtained from RNA-seq experiments following expression profiles of X-linked genes and Xist transcription in single embryos across pre-implantation stages comparing RlimKO with WT embryos  controlling Rex1, thereby explaining different baseline levels and localization of Rex1 protein in cells. Thus, while RLIM appears the only factor controlling nuclear exclusion of Rex1 at early pre-implantation stages, in ESCs Rlim represents only one of multiple pathways. These data suggest that the dynamics of both Rlim and Rex1 regulation during mouse development is not always paralleled in ESCs in culture, and more work is needed to elucidate the cellular regulation of Rex1 protein.Concerning XCI in various female ESC models, the ability of female ESCs to undergo XCI correlates with low nuclear levels of Rex1. However, nuclear exclusion and overall cellular levels of Rex1 is controlled by RLIM in some ESC lines, but not in others, indicating the existence of alternative pathway(s) that may be sensitive to culture and/or differentiation conditions and possibly also involving multiple mechanisms on RLIM regulation such as E3 ligase activity and differences in the epigenetic make-up selected via clonal line selection. Moreover, various ESC lines might represent different developmental states with possibly leaky suppression of Rnf12KO-derived systems is based on a cloning artifact. RLIM333 causes nuclear accumulation of Rex1, an effect overlapping with that of the RlimKO and thus, this gain-of-function activity will be eliminated by an additional Rex1KO. However, because RLIM333 lacks specific protein domains, its activity is no longer associated with pathways regulating the full-length protein in terms of expression, stability and E3 ligase activity. In Rnf12KO-derived model systems including mice, there is likely enhanced effects on nuclear Rex1 due to the lack of RLIM and the presence of RLIM333, potentially influencing various aspects of the iXCI process in a temporary, qualitative and quantitative manner, and the effects of RLIM333 on XCI cannot be distinguished from those induced by the lack of Rlim. Outside the Rex1-Rlim axis, RLIM has other E3 ligase targets most of which are also targeted by other E3s. While the KO has little/no effect on such targets, the presence of RLIM333 might, and thus other phenotypes are likely. This is why the reported lethality in male Rnf12KO (RlimKO models including germline RlimKO models (Cre-recombinase negative) no lethality has been observed in males lacking RLIM over more than 10 years. Therefore, data obtained by the Rnf12KO model need to be interpreted not only from the KO perspective, but also from a gain-of-function perspective. Because the terms “KO” or “−/−” are incorrect and cause confusion, derivatives of the Rnf12KO ESCs should be renamed to account for the presence of RLIM333.Concerning RLIM333, the presence of this truncated protein in  Rnf12KO  is conceRlim-Rex1 axis in controlling XCI both in vivo and in vitro. Going forward, it will be important to elucidate the functional evolutionary conservation of this module in other species, the dynamics between both factors specifically in epiblast cells during implantation including effects on Xist/Tsix, and to identify factors that mediate rXCI in vivo.In summary, recent results have assigned a crucial importance to the All datasets generated for this study are included in the article/The animal study was reviewed and approved by UMMS Institute of Animal Care and Usage Committee.Both authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The World Health Organization/International Union of Immunological Societies (WHO/IUIS) Allergen Nomenclature Sub-Committee was established in 1986 by leading allergists to standardize names given to proteins that cause IgE-mediated reactions in humans. The Sub-Committee's objective is to assign unique names to allergens based on a critical analysis of confidentially submitted biochemical and clinical data from researchers, often prior to publication to preserve consistency. The Sub-Committee maintains and revises the database as the understanding of allergens evolves. This report summarizes recent developments that led to updates in classification of cockroach group 1 and 5 allergens to animal as well as environmental and occupational allergens. Interestingly, routes, doses, and frequency of exposure often affects allergenicity as does the biochemical properties of the proteins and similarity to self and other proteins. Information required by the Sub-Committee now is more extensive than previously as technology has improved. Identification of new allergens requires identification of the amino acid sequence and physical characteristics of the protein as well as demonstration of IgE binding from subjects verified by described clinical histories, proof of the presence of the protein in relevant exposure substances, and demonstration of biological activity . Names are assigned based on taxonomy with the abbreviation of genus and species and assignment of a number, which reflects the priority of discovery, but more often now, the relationships with homologous proteins in related species. Advances in molecular biology, recombinant protein technology, methods of genomic, and proteomic analysis, structural biology, and high throughput screening have led to a greater understanding of allergenic proteins over the past 30 years. Identification of new allergens and improved characterization dictate the need for revision of some allergens and updated requirements for allergen nomenclature.In the 1980's key clinical and experimental allergists led by D Marsh at Johns Hopkins University, USA, L Goodfriend , TP King , H Lowenstein (University of Copenhagen (Denmark) and TAE Platts-Mills  framed basic rules for naming allergenic proteins in common allergenic sources .Allergen names are assigned with the first 3 or sometimes 4 letters of the genus, one or sometimes two letters for the species followed by an Arabic numeral, based on order of discovery . HoweverExpression of recombinant proteins for IgE tests and characterization has led to more accurate identification. Changes to the nomenclature have attempted to incorporate an increased understanding of allergens. Higher resolution studies and isolation techniques of previously defined allergens now shows some to be composed of multiple subunits from different genes; Fel d 1, or multimers; collagen, vicilins, and glycinins. This manuscript focuses on recent challenges and underlying reasons for name assignment by the WHO/IUIS Nomenclature Sub-Committee. We refer those working on allergen identification to Blatella germanica genome  of Tenebrio molitor  are common enzymes present in multiple organisms from different taxonomic groups. One of the main functions of GSTs is detoxification by catalyzing the addition of a glutathione (GSH) molecules to other commonly toxic compounds that are ultimately removed from the cell , including those from cockroaches (Bla g 5 and Per a 5), mites , helminths , fungi (Alt a 13), and plants (Bet v 8)  .Blattella germanica) was the first cockroach GST to be identified (O18598), and it is a sigma class GSTs (Periplaneta americana) were identified as binding IgE, and they belong to the delta class of GSTs [MG255130  represent a large protein family found in mammals and marsupials. Common characteristics are secretion in many body fluids, small size, alpha-helical, and dimeric structure creating a hydrophobic binding pocket , 15. MajOther examples are recently added allergens, barramundi collagen Lat c 6 and salmon allergen Sal s 6 proteins. Collagen is a triple-stranded rope-like coiled structure, composed of 3 protein chains. Each chain has been given an isoallergen name, despite the fact that they do not exist as monomers. Currently it is only possible to evaluate separate IgE binding if using recombinant monomeric proteins and thus it is not possible to assess the allergenicity of each of the entire native proteins. The committee may reevaluate this policy in the future.Cross-reactive carbohydrate determinants (CCDs) are carbohydrate epitopes carrying an α-1,3-fucose and, or β 1,2 xylose on complex carbohydrates of some glycoproteins are the main targets of IgE recognition. They are present on many plant and insect proteins and responsible for extensive IgE cross-reactivity between different allergen sources . HoweverSalmon salar) based on public genomic sequences  . It woulMost outdoor aeroallergens are derived from grass, weed and tree pollen or fungal spore sources. Although Blackley discovered that pollen caused hay fever in the nineteenth century , it was Ligustrum vulgare (Common privet), Syr v 1 and Syr v 3 of Syringa vulgaris (Lilac) and Hum j 1 of Humulus japonicus (Japanese hop).The IUIS Allergen Nomenclature Subcommittee recognize 43, 55, and 45 allergens for pollen of grasses, trees and weeds, respectively. Another four pollen allergens of herbaceous plants included; Lig v 1 of Urochloa mutica (Para grass). Group 2 and 3 grass pollen allergens share approximately 30% identity with the carboxyl domain of the beta-expansin protein family. Consequently, grass pollen group 2/3 pollen allergens are smaller in size (10–12 kDa), but their function is unknown. Sor h 2 and Ory s 2 are described in subtropical species, but their clinical importance is less well-demonstrated. Several isoforms and multiple variants exist for Group 5 allergens  and these share between 53 and 78% identity between six species for which these allergens have been characterized , mugwort (Artemisia vulgaris), pellitory (Parietaria judaica), amaranth, thistle , and sunflower (Helianthus annuus) and belong to 10 allergen families. Clinically important allergens of the Asteraceae family include pectate lyases (Amb a 1 and Art v 6), defensin-like proteins (Amb a 4 and Art v 1), non-specific lipid transfer proteins (Art v 3 and Par j 2), whereas allergens of the Amaranthaceae family include Ole e 1-like proteins and Che a 1, and pectin methylesterase .Weed pollen allergens arise from 16 species including ragweed (Betula verrucosa), Lamiales; Ash (Fraxinus excelsior) and Olive (Olea europaea), Proteales; Plane (Plantanus acerifolia) and Cupressales; Japanese Cedar (Cryptomeria japonica), encompassing 21 species contribute allergens belonging to 18 protein families. Clinically important allergens include Pathogenesis Related protein-10 ; Fra e 1, polygalacturonase; Cry j 2 and Pla a 2, and pectate lyase; Cry j 1. Profilins are ubiquitous and highly conserved in sequence and structure  and polcalcins  are pan-allergens common to grass, weed and tree pollen, though these allergens are generally less potent than PR-10 proteins.Trees from multiple orders; Fagales; for example, Birch (Aspergillus fumigatus including the ribotoxin (Asp f 1), peroxisomal protein (Asp f 3), metalloprotease (Asp f 5), cyclophilin (Asp f 11), vacuolar serine protease (Asp f 18), and enolase (Asp f 22), which are all major allergens. Alternaria alternata spores, one of commonly recognized fungal spores contains 12 listed allergens including its major allergens (Alt a 1), and, a glutathione S transferase (Alt a 13).A variety of fungi produce the 112 listed WHO/IUIS allergens from as many as 36 protein families. While fungal hyphae or spores may be used as food or drug sources, many are common sources of indoor and outdoor allergy and a small number colonize human tissues including skin and lungs. Spores of 15 Ascomycota species belonging to diverse fungal families; Aspergillaceae, Cladosporiaceae, Didymellaceae, Nectriaceae, and Pleosporaceae produce 72 airborne outdoor fungal spore allergens. There are 23 allergens listed for Occupational sensitization has been observed for centuries in historical and medical writings. The first description of baker's asthma was probably by Ramazini around 1700; the first report of allergic responses to fish was in 1937 by De Besche relating to a fisherman who developed allergic symptoms when handling codfish . SensitiThe most common workplace allergens are relatively high-molecular-weight proteins derived from cereal flour, natural rubber latex, wood dust, livestock and laboratory animals, seafood, industrial enzymes and mold . Animal Over 400 occupational allergenic sources are identified and documented as sensitizers, derived from plants, animals and microbes. However, few have been characterized on the molecular level and only a limited number of purified native or recombinant allergens are available for testing . The curTriticum aestivum), where 28 allergens are registered with the WHO/IUIS, however only 13 are confirmed food allergens. The route of sensitization is categorized as “unknown” for 15, with 14 being relevant in baker's asthma and several allergens are relevant for grass-pollen cross-reactivity, including profilin (Tri a 12) and a subunit of the tetrameric heterologous a-amylase inhibitor chloroform/methanol-soluble CM17 protein (Tri a 40).A good example of the complex nature of identifying “occupational allergens” among plants is wheat  often leads to speculation about route of exposure in sensitization. Of the 35 registered tropomyosins, 22 are known food allergens, while 13 are not known to cause food allergies. Of these 10 are derived from insects or mites and the route of sensitization is most likely via inhalation. Among the food allergens 19 tropomyosin are derived from different seafood species and are relevant occupational allergens, however not identified as such (Chironomus thummii bind IgE from sera of those who raise the insects for fish food (Tenebrio molitor) as a source of protein for processed human food (Among animal derived allergens, tropomyosin is an abundant and clear food allergen from crustacean shellfish and molluscs that is also implicated in IgE binding and possibly in triggering airway allergy from mites and some insects.  as such . Some anish food . A few rman food . Some inin vivo proof that new allergens illicit allergic responses to date, only in vitro evidence and a good clinical history of symptoms.Many other foods have been reported in single case reports as the cause of occupational allergies and asthma, and sensitization by inhaled allergen exposure is therefore quite likely . ImportaThough molecular and scientific advances have led to improved characterization of allergens and with-it increased clarity in naming and categorizing them, the heterogeneous nature of human data remains a challenge. This Sub-Committee has in the past asked for minimal data characterizing protein sequences, clinical symptoms of serum donors or test subjects, IgE binding and allergic functionality. We see great diversity in data submitted with candidate proteins. Our recent publications are intended to help researchers focus on relevant questions to improve the evidence that individual proteins are allergens, and not simply cross-reactive in IgE binding with only mild clinical consequences. Sequences of the allergens and demonstration of IgE binding from relevant symptomatic individuals are essential for defining allergens. Some researchers would like to simply submit to the Sub-Committee information about recombinant proteins based only on genomic sequences of potential allergen sources. While that activity may help predict possible allergens, our goal is to call for direct proof of allergenicity using subjects with clear clinical reactivity to source materials, and with clear IgE binding to proteins presented at representative concentrations.The purpose of the allergen nomenclature system is to help researchers, clinicians, pharmaceutical companies, regulators, and the public clearly understand the identity of clinically important allergens for diagnosis and to help ensure compliance for improved safety.However, our system is not perfect. Recent research shows that some specific glycans are important allergens including α-gal that is bound to tick and mammalian proteins and to glycol-lipids. We do not define these structures as allergens but hope to help publicize risks and educate consumers and allergists about risks.Each author contributed a significant portion of writing for the manuscript and reviewed the final document. This collection of authors is a representative segment of the WHO/IUIS Allergen Nomenclature Sub-Committee. AK is not a Sub-Committee member, but has contributed to identification and characterization of allergens in this manuscript.AP is employed by the company Indoor Biotechnologies, Inc. The remaining 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.


Motor control and body representations in the central nervous system are built, i.e., patterned, during development by sensorimotor experience and somatosensory feedback/reafference. Yet, early emergence of locomotor disorders remains a matter of debate, especially in the absence of brain damage. For instance, children with developmental coordination disorders (DCD) display deficits in planning, executing and controlling movements, concomitant with deficits in executive functions. Thus, are early sensorimotor atypicalities at the origin of long-lasting abnormal development of brain anatomy and functions? We hypothesize that degraded locomotor outcomes in adulthood originate as a consequence of early atypical sensorimotor experiences that induce developmental disorganization of sensorimotor circuitry. We showed recently that postnatal sensorimotor restriction (SMR), through hind limb immobilization from birth to one month, led to enduring digitigrade locomotion with ankle-knee overextension, degraded musculoskeletal tissues , and clear signs of spinal hyperreflexia in adult rats, suggestive of spasticity; each individual disorder likely interplaying in self-perpetuating cycles. In the present study, we investigated the impact of postnatal SMR on the anatomical and functional organization of hind limb representations in the sensorimotor cortex and processes representative of maladaptive neuroplasticity. We found that 28 days of daily SMR degraded the topographical organization of somatosensory hind limb maps, reduced both somatosensory and motor map areas devoted to the hind limb representation and altered neuronal response properties in the sensorimotor cortex several weeks after the cessation of SMR. We found no neuroanatomical histopathology in hind limb sensorimotor cortex, yet increased glutamatergic neurotransmission that matched clear signs of spasticity and hyperexcitability in the adult lumbar spinal network. Thus, even in the absence of a brain insult, movement disorders and brain dysfunction can emerge as a consequence of reduced and atypical patterns of motor outputs and somatosensory feedback that induce maladaptive neuroplasticity. Our results may contribute to understanding the inception and mechanisms underlying neurodevelopmental disorders, such as DCD. From this, it seems likely that early atypical sensorimotor experiences in children should lead to the emergence of atypical movements, motor control problems and disorganization of sensorimotor circuitry that persist into adulthood. The impact of disuse or immobilization  during child development has mainly been studied in the presence of brain lesions6. Only a very few studies have focused on the impact of early disuse occurring in the absence of brain damage. For example, swaddling, the ancient practice of wrapping infants in cloth so that limb movements are restricted, appears to delay the onset of several motor skills8. Swaddling has become popular again, such as in neonatal intensive care units using elastic cloth, as a means to reduce sudden infant death syndrome or crying, promote sleep, or improve muscle tone9. Disturbances in the planning, execution and control of body movements in the absence of brain damage are now termed developmental coordination disorders (DCD) and usually coexist with various deficits in executive functions in 5–6% of school-aged children10. Patients with DCD show reduced abilities to produce consistent movements11, poor motor coordination and kinaesthetic acuity13, broad impairments in sensorimotor representations and perception14, each reflected by disrupted central networks15 Children with autism spectrum disorder (ASD) also exhibit gross or fine motor abnormalities, motor learning deficiencies and difficulties executing sequences of actions16. Most children with DCD or ASD show similar sensorimotor impairments, reduced physical activity and interactions with their environment, and atypical motor development, the latter detected as atypical spontaneous or general movements (GMs)18.It is now understood that development of movement repertoires, motor control and body representations in sensorimotor circuitry are achieved through early spontaneous movements, sensorimotor experiences and reafference in children3. Arising from spontaneous, self-generated and evoked movements during maturation, early somatosensory feedback drives electrical activity patterning from the spinal cord to the cortex. This feedback guides the development and refinement of the anatomical and functional organization of sensorimotor circuitry in rodents20. Atypical, disturbed GMs reflect impaired connectivity and functional disorganization in the brain22. Accordingly, we hypothesized that limited and abnormal patterns of somatosensory inputs during development may lead to abnormal anatomical and functional organization of the sensorimotor circuitry in adulthood as a result of maladaptive cortical plasticity. Such sensorimotor disorganization may in turn alter somatosensory and bodily perceptions, motor outputs, and musculoskeletal structure and physiology.During typical development, the repertoire of GMs in limbs increases over time in variation, fluency, amplitude and complexity into a continuous stream of small and elegant movements. Atypical GMs correspond to rigid, cramped synchronized and stereotyped movements that exhibit limited fluency, variation and complexity with increasing age23. SMR rats displayed not only increased variations in the kinematic parameters of treadmill locomotion, mentioned above, but also reduced variations of hind limb joint angles; these variations persisted over time23. These results appear to recapitulate the “toe walking” and increased spatiotemporal variations of movements observed in children with ASD25. SMR also led to musculoskeletal histopathology and increased stretch reflexes, suggestive of muscle hyperreflexia and spasticity. We postulated that reduced and atypical patterns of both motor outputs and somatosensory reafference during development likely contributed to the emergence of movement disorders and musculoskeletal pathologies that persisted into adulthood23. To further explore this concept that adult movement disorders may have developmental origins even in the absence of brain damage, we investigated here the impact of postnatal SMR on the neuroanatomical and functional organization of hind limb somatosensory and motor representations in adult rats using microelectrode cortical mapping techniques, and brain histology and immunochemistry. We also assessed the balance between excitation and inhibition in the sensorimotor cortex using in vivo microdialysis and western blotting methods. To further understand the processes and interactions underlying any maladaptive neural plasticity, we performed principal components analyses (PCA) to summarize the many variables recorded within the same animals and linear correlations between these variables.We recently showed that transient postnatal sensorimotor restriction (SMR), experimentally produced using 28 days of transient (16 hours/day) hind limb immobilization of developing rats, lead to degraded locomotion on a treadmill that persisted for more than 30 days after cessation of the immobilization. This long-lasting degradation was characterized by reduced length, amplitude and velocity, overextended knees and ankles, and digitigrade locomotion that resembled true pes equinus or “toe walking”23), as well as electrophysiological procedures , in vivo microdialysis  and Western-blotting , and stereological brain histology and immunohistochemistry studies  from P90 to P120, i.e., about 60 to 90 days after cessation of the SMR. Since we did not provide anatomical or histological features of the region of cortex in which we recorded or stimulated, we will refer to somatosensory and motor maps originating from the cortex, as opposed to primary somatosensory and motor cortices26.We used 17 rats that were exposed to sensorimotor restriction , whereas the normalized hairy representation of the foot did not differ between the two groups . In addition to the increased number of multiple RFs after SMR, their size also increased . Specifically, RFs located on glabrous foot skin surfaces were about twice as large in SMR rats as in controls , whereas the size of hairy foot RFs did not differ significantly between the two groups   was 10 times greater in SMR rats  than in control rats . In contrast, the percent of double RFs did not differ between SMR  and control  rats. Concomitantly, the proportion of single RFs located on a single hind paw location was decreased in SMR rats , compared to controls , more multiple RFs located on three or more foot subdivisions, and numerous RFs located on both sides of the hind paw. In fact, the delineations between toe and plantar pad representations were difficult in SMR rats, so that map sites with three or more RFs had to be pooled together in specific map sectors  and the normalized area of such cortical sectors relative to the total map area (expressed as percentages) was about 16 times larger after SMR . Thus, SMR induced a drastic degradation of both the topographical organization of the somatosensory hind limb maps and cortical neuronal properties. Interestingly, there was an emergence of RFs covering both sides of the leg in SMR rats  yet no such double-sided RFs in controls . However, the area of the somatosensory map devoted to the leg representation did not differ significantly between SMR rats (0.49 ± 0.28 mm²) and controls . The proportion of excellent responses were greater in SMR rats  than in controls , the percent of clear responses decreased in SMR rats  compared to controls , while weak responses did not differ between SMR rats  and controls . SMR seemed to increase the neural responsiveness to tactile stimulation, indicative of changes in neuronal properties and increased excitability in the somatosensory hind limb representation.Next, neural responsiveness originating from the cortex to light tactile stimulation was qualitatively classified on the basis of the magnitude of the signal-to-noise ratio, as in a previous studyThus, SMR favored the emergence of numerous multiple, disjunctive and enlarged RFs, and a decrease in the hind limb map area, resulting in degradation of the somatotopy and topographical organization of the somatosensory cortex, as well as altered neuronal properties. SMR induced-map changes appeared to be specific to glabrous foot skin areas, and the appearance of RFs covering both sides of the hind limb or foot is indicative of degraded neuronal properties after SMR.29. In control rats, the motor hip representation occupied on average 70.9% of the total area of the hind limb motor map  of neuronal motor responses was drastically reduced after SMR ; whereas, the extracellular concentration of GABA did not differ between the two groups  and vesicular GABA transporter (vGAT), reliable markers of excitatory and inhibitory transmission, respectively. The amount of vGLUT1 increased approximately 90% in SMR rats relative to controls  or hind paw motor representation . In the same line, the numerical density of GAD positive cells did differ not between the two groups of rats in either the hind limb somatosensory representation  or the hind limb motor representation . Thus, SMR appeared to have no significant impact on brain neuroanatomy, compared to controls.The numerical density of NeuN positive cells did not differ significantly between the two groups of rats in either the hind paw somatosensory representation  were used to describe 7 sets of variables: somatosensory (n = 23 variables) and motor (n = 17) hind limb map features, neurotransmission (n = 4) and neurohistopathology (n = 6), as well as locomotion at P30 (n = 24) and P65 (n = 24) and musculoskeletal histopathology (n = 7) as previously reportedp < 0.0001), indicating a clear separation of the two groups along PC1  and P65 , to musculoskeletal histopathology  and tended to correlate with neurotransmission findings  and somatosensory map features, but not with the other sets of variables. The motor hind limb map features along the PC2 axis significantly correlated to the highest contributing variable, which was locomotion at P30 , and to musculoskeletal histopathology , but not to the other sets of variables (Table p < 0.0001), as well as at P65, although to a lesser level . In contrast, locomotion features at both ages were highly interrelated , suggestive of a self-perpetuating interplay between these variables, as described previously23. Neurohistopathological variables did not correlate significantly to any other variables, confirming the lack of impact of SMR on brain neurohistology. Unfortunately, we could not assess the relationships between somatosensory and motor map features as we could not map both cortices in the same animals due to the use of different anesthetics.Second, to evaluate the relationships between the 7 sets of variables, individual animal scores for each set of variables . SMR-related somatosensory map reorganization was highly related to locomotion impairments, excitation/inhibition imbalance and musculoskeletal histopathology. Surprisingly, motor map features and neurotransmission contributed the least to group differentiation. Reorganization of the motor hind limb representation was also related to locomotion impairments, although only at P30, and to histopathology, although to a lesser extent than the degradation of the somatosensory hind limb representation. Peripheral histopathology and locomotion impairments were highly correlated, confirming the self-perpetuating interplay between these variables.23.We also found that sex had no specific impact on the features explored here and previouslyTo investigate a hypothesis of a developmental origin of adult disorders, we showed in the present study that reduced and abnormal patterns of somatosensory inputs after hind limb movement restriction during postnatal development had no significant impact on adult brain neuroanatomy yet did effect the functional organization of the sensorimotor cortex. Compared to controls, SMR led to: 1) a degradation of both somatosensory hind limb map organization and neuronal properties; 2) a decrease in the total motor map area devoted to hind limb movements concomitant with an overall increased excitability; 3) increased glutamate release, yet no changes in GABA levels in the sensorimotor cortex; yet 4) no neuroanatomical changes in the sensorimotor hind limb cortex.23); whereas, 3) motor map alteration was weakly influenced by these variables and not by neurotransmission.To evaluate interactions underlying the observed neural maladaptive plasticity, we used PCA and correlational statistics. We found that SMR primarily effected locomotion at P30 and P65, as well as somatosensory map features and musculoskeletal features in decreasing order; yet had lesser effect on motor map features, neurotransmission or neurohistology. This meta-analysis of all data from the same rats also outlines that: 1) deterioration of somatosensory maps is tightly related to locomotion impairments, neurotransmission imbalance and musculoskeletal histopathology; 2) locomotor disorders and musculoskeletal pathology likely interplay tightly in a self-perpetuating cycle  process of plasticity34. In the same line, activity-dependent changes in excitation-inhibition balance leads to masking or unmasking of intracortical motor connections that acts upon motor map boundaries and thresholds, i.e., neuronal tuning36. Decreased hind limb sensorimotor maps and increased size of RFs did not match with the overall hyperexcitability found in the hind limb sensorimotor area, so that the hind limb map reorganization may be explained by alternative mechanisms of plasticity. The absence of neuroanatomical pathology after SMR allows us to rule out its impact on the neurotransmission changes and map reorganization. In addition, SMR induced a long-lasting increase in the stretch reflex evaluated with post-activation depression23, that is supposed to reflect hyperexcitability within the lumbar spinal cord and spasticity38. The latter finding fits with the present observation of enduring cortical hyperexcitability.We investigated the excitation-inhibition neurotransmission in the sensorimotor cortex because it appears to govern cortical map reorganization. Use-dependent regulation of glutamatergic excitation underlies expansion or retraction of somatosensory maps, while use-dependent GABAergic inhibition promotes cutaneous RF changes, corresponding to the modulation of neuronal tuning on the basis of synaptic potentiation/depression and unmasking/masking of thalamocortical divergent and convergent connections, respectively39, may explain the increased cortical excitation observed in adult SMR rats. An alternative explanation for such an increase could reside in the persistence of dense dendritic arborisation that are usually pruned during the first few postnatal weeks40. For instance, postnatal whisker removal reduces the pruning of supernumerary dendrites that are normally present in S1 layer V pyramidal cells41, thus preserving abnormally important levels of excitatory synapses involved in increased cortical excitability42. Thus, a co-existence of both Hebbian and homeostatic processes of plasticity may explain the observed differences in remodelling of somatosensory and motor maps in relation to hyperexcitability in the sensorimotor cortex.In this long-term study, a slow homeostatic (non-Hebbian) increase in global synaptic strength  and/or intrinsic excitability, which in turn enhances responses to remaining inputs23, which resembles “toe walking” or true pes equinus, observed in children with ASD24. According to the literature44, typical plantigrade locomotion in control rats45 produces asynchronous and distributed stimulation through the foot skin surfaces from proximal to distal parts during the stance up to the beginning of the swing. During typical motor development, spatiotemporally distributed cutaneous inputs of the foot in controls likely contribute to the differentiated representation of the different foot skin surfaces, such as individual toes and plantar pads. In contrast, we postulate that digitigrade locomotion during postnatal SMR provides synchronous and colocalized tactile inputs from several toe and/or pad surfaces, leading to enlarged RFs that cover abnormally large skin surfaces. This enlargement results in the dedifferentiation of the somatosensory representation of contiguous foot territories and finally to the disruption of the somatotopic and topographic organization of the somatosensory foot maps, also observed in animal models of focal hand dystonia46. In addition, unrepresented skin surfaces in control rats, such as the proximal phalanges of toes and skin surrounding pads, became represented in the somatosensory maps after SMR. The very large RFs covering the entire leg of SMR rats may result from synchronous activations of cutaneous mechanoreceptors during chronic co-contractions of antagonist leg muscles (see27). Taken together, abnormal, specific tactile stimulation during digitigrade locomotion induced a maladaptive reorganization in the somatosensory cortex, in which only the glabrous representation was altered by SMR, not the hairy representation (see27). Neural maladaptive plasticity of the somatosensory maps also appeared highly related to musculoskeletal histopathology and neurotransmission.Based on PCA and correlations within the same animals, our meta-analysis indicates that the SMR-related deterioration of the somatosensory map organization and neuronal properties was tightly related to locomotor disorders evaluated at P30 and P65. SMR led to severely degraded kinematics of gait on a treadmill in relation to knee and ankle overextension, and smaller footprints that revealed a digitigrade locomotion23. Surprisingly, only locomotion disorders at P30 and musculoskeletal histopathology correlated weakly with the motor map reorganization. In addition, neurotransmission appeared correlated primarily with somatosensory map features and secondarily with musculoskeletal pathologies, but not with motor map features or locomotion. Taken together, our results emphasize the key role of the functional degradation of the somatosensory hind limb representation, which appears to mainly depend on abnormal patterns  of somatosensory inputs resulting from both postnatal SMR and enduring degraded reafference resulting from disturbed locomotion rather than decreased levels (quantity) of inputs, which likely seems involved in the excitation/inhibition imbalance48. These results proceed from dynamic selection, competition and collaboration of primary neuronal groups on the basis of afferent inputs resulting from behaviors and experience that induce changes in the strength of synaptic connections, thus governing the topographic organization of cortical maps and the corresponding tuning of neurons, and leading to diverse repertoires of movements51. Thus, the development and continuous refinement of neural connectivity is influenced interactively by feedback from neural targets52, supporting the interplay between neural connections, maps and functions intermingled in regulatory loops and self-perpetuating cycles sustained by experience.Those correlations do not indicate a directionality of the relation, so that it means that locomotor disorders and musculoskeletal histopathology may be attributed to the somatosensory map degradation. Yet, the high correlation between locomotion degradation and musculoskeletal histopathology after SMR confirms their interplay in a self-perpetuating cycle Fig. , in whic23). Our meta-analysis suggests the existence of a self-perpetuating cycle, in which musculoskeletal pathologies likely contribute to muscle overactivity and locomotor disorders. Spasticity and related muscle disorders may contribute in turn to enhanced musculoskeletal and locomotion disturbances . As a consequence, degraded movements due to spastic muscles and digitigrade locomotion likely produce atypical somatosensory feedback to the immature sensorimotor circuitry, including the lumbar spinal circuitry involved in automatic gait, and primary somatosensory and motor cortices associated with other subcortical structures in the sensorimotor loop19. In the absence of brain damage, this atypical somatosensory reafference probably triggers and maintains the functional disorganization of the sensorimotor maps and neuronal properties, and likely that of the lumbar spinal cord , involved in both motor commands during automatic locomotion and the emergence of muscle overactivity and paresis . The disorganized sensorimotor circuitry may in turn produce atypical or aberrant motor commands, such as abnormal muscle synergies involved in gait disorders observed in rats after hind limb unloading55 or in patients with stroke57. Thus, we propose the existence of three interplaying, self-perpetuating cycles: the first cycle is related to peripheral changes, including gait disorders, increased stretch reflex and musculoskeletal pathologies , increased stretch reflex  and musculoskeletal tissue degradation, compared to typical sensorimotor development in controls Fig. 23. Our mces Fig. 23. As a sis Fig. 53,54. Th52. Limited and abnormal patterns of somatosensory inputs arising from SMR in early development, in the absence of neuroanatomical histopathology, induced long-lasting locomotor disorders, hind limb musculoskeletal histopathology, hyperreflexia and functional disorganization of the adult sensorimotor cortex. Taken together, this recapitulates several symptoms and neurological features found in patients with ASD, and DCD in general22. Adults with DCD that have no evident brain damage, show reduced abilities to produce consistent movements11 and impairments in kinesthesic acuity13. Children with ASD also exhibit gross and fine motor abnormalities, motor learning deficiencies, inabilities in executing sequences of actions16 and deficits in tactile localization that may reflect broad impairments in multisensory body representations15. Thus, these children share early aberrant sensorimotor experience resulting from atypical spontaneous or general movements (GMs) observed in children with DCD58, along with its related brain disorganization, especially inabilities in processing and integrating sensory information16. GMs are predictive of neural, motor and cognitive outcomes in children with DCD58. Our animal studies on postnatal SMR supports the idea that developmental maladaptive plasticity may lead to long-lasting motor disturbances, as observed in DCD and point out the pertinence of early rehabilitative programs. Further studies are needed for a better understanding of the possible impact of early SMR on behavioral and cognitive abilities and on the postnatal development of the lumbar spinal circuitry.The present study based on postnatal SMR confirms and emphasizes the preponderant role of individual, early experience in shaping the body and brain, and modulating their interplay from maturation to adulthood, depending on physical and environmental constraintsAll experiments and animal use were carried out in accordance with the guidelines laid down by NIH (NIH Publication #80-23) and EC Council Directive (2007/526/EEC). This research involving animals was approved by the Direction Départementale des Services Vétérinaires – Préfecture des Bouches du Rhône, France (permit # C13-055-18).23 for details).Sprague-Dawley rat pups of either sex from different litters were pseudo-randomly assigned to two groups: 1) a group subjected to transient hind limb immobilization from P1 to P28 for 16 hours per day, thus producing sensorimotor restriction (SMR); and 2) a control group (Cont). Feet of SMR pups were gently bound together with medical tape. Their hind limbs were then immobilized in an extended position and taped to a cast made of epoxy putty sticks  and motor (M1) cortices were examined in rats from 90 to 120 days-old, using either microelectrode electrical recordings or stimulation, respectively. Hind limb maps were derived from the left hemisphere, as these maps correspond to the right contralateral hind limb whose locomotion kinematics were previously recorded and reported46 . Briefly, each animal was anaesthetized with sodium pentobarbital ; supplemental doses  were provided as needed to maintain a deep and stable level of anaesthesia throughout the mapping session. A craniotomy was made over the S1 hind limb representation located between −1 and + 3 mm from bregma in the rostrocaudal axis and between 1 and 5 mm in the mediolateral direction. Multiunit recordings were made using tungsten microelectrodes  in the upper layer IV (650–700 µm) of the S1 cortex. The low-threshold receptive field (RF) corresponded to the cutaneous area whose barely visible indentation or gentle hair movements elicited clear bursts of S1 neuronal activity. High-threshold responses elicited by taps on the skin were classified as cutaneous responses; stroking of hairs and manipulations of muscles and joints were classified as non-cutaneous responses. Unresponsive cortical sites exhibited spontaneous discharges only. To elaborate maps of the hind limb representation we drew boundaries encompassing cortical sites whose RFs were restricted to a common hind paw subdivision . When RFs were located on distinct and separate skin subdivisions of the foot, borders were drawn midway between adjacent recording sites. When a single or multiple RF included different but adjoining skin subdivisions of the hind limb, a boundary line was drawn that crossed the cortical sites. Map borders were placed midway between responsive and unresponsive sites . Animals were anaesthetized with ketamine hydrochloride  and xylazine . Supplemental doses of ketamine  and acepromazine  were delivered as needed to maintain a deep and constant level of anaesthesia. A craniotomy was made over the M1 hind limb representation located between −1 and +3 mm from bregma in the rostrocaudal axis and between 1 and 5 mm in the mediolateral direction, indicative of large overlap of somatosensory and motor hind limb maps  for each rat and normalized relative to either the ventral or dorsal foot skin surfaces for somatosensory maps only. Relative areas of joint movements are normalized relative to the total motor hind limb map area for each rat. Average values were computed for both groups of rats. The absolute sizes of glabrous and hairy receptive fields (RFs), measured in mm², were normalized relative to the ventral and dorsal hind paw skin surfaces, respectively, and expressed as percentages. The relative RF areas measured in each rat were averaged, and mean RF sizes in each group were computed. Mean RF size per rat was computed after discarding the large RFs located on the dorsal surface of the hind pawin vivo microdialysis within the sensorimotor cortex located contralateral to the mapped side, at 2 hours before mapping. In other rats, Western blot analysis was used to quantify intracellular amounts of transporters for both glutamate (vGLUT1) and GABA (vGAT) in sensorimotor cortical tissues collected contralateral to the mapped side, at one hour after mapping.To gain insight into the long-term impact of SMR on extra- and intracellular levels of glutamate and GABA, in subsets of rats, we performed Carnegie Medecin microdialysis probes  were implanted within the hindpaw representation of the right S1-M1 area using stereotaxic coordinates . An implantation site that avoided large blood vessels; traces of blood were not found after probe withdrawal. The terminal ends of the probes were covered with a polycarbonate membrane. The membrane had a diameter of 0.24 mm, a length of 2 mm and a 6 KDa molecular mass cut-off. The membrane acted like a blood vessel, with extracellular molecules passing through it by diffusion gradient. A new probe was used for each rat. This procedure is detailed in Supplementary Methods.−1 pH 7.4, NaCl 150 mmol.L−1) containing protease inhibitors  using a Potter homogenizer and a ratio of 100 µg of brain pieces per mL of buffer. Homogenates were centrifuged at 500 g for 3 min at 4 °C. Supernatants were aliquoted and stored at −80 °C until use. Proteins present in each supernatant were resolved by SDS-PAGE and transferred onto nitrocellulose membranes. Proteins of interest were revealed with specific antibodies and then enhanced using chemiluminescence . For illustration purposes, image editing was performed using ImageJ software (http://rsb.info.nih.gov/ij/) and was limited to linear brightness/contrast adjustment , before undergoing transcardial perfusion with 4% paraformaldehyde in phosphate buffer . The brains were then harvested, postfixed in the same fixative solution for 3 h, cryoprotected in sucrose 30%, and frozen for cryosectioning. Sections to be immunostained for glial fibrillary acidic protein (GFAP), amyloid-ß precursor protein (APP), DAPI , NeuN  and GAD (GABAergic interneurons expressing GAD67) consisted of 15 µm coronal sections .After mapping one side of the brain, and microdialysis or collection of tissues for western blot analysis of the contralateral side, the animals received terminal doses of anaesthesia  equipped with a motorized stage and Stereo Investigator software-controlled computer system . Cell counting was performed at x40 magnification using an optical fractionator method in semi serial sections (see Supplementary Methods). In both groups, the number of samples from the left and right hemispheres was roughly equal.28.The heights of the genu of the corpus callosum, and S1, M1 and entorhinal cortices were measured in haematoxylin and eosin (H&E) stained sections using a 20x objective and the Auto-Width tool of Bioquant Image Analysis program in which the inner and outer boundaries of structures were traced, and then mean layer thicknesses of a length of genu or cortex were automatically generated. Alternate sections were stained with Fluoro-Jade using previously described methodsData normality and homogeneity of variance were determined with Shapiro test, Bartlett test and var.test before to apply parametric  or nonparametric  tests with normal approximation with continuity correction or correlations  using only R . The investigators were blinded to rearing conditions throughout the different experimental sessions until statistical comparisons were performed.59. Then, in order to identify variables that most optimally separate the two groups, we performed a PCA with all dataset variables (n = 105). We usually kept the first two PCs with Kaiser’s criterion over 1 that in combination explained at least 35 to 70% of the variance59. In the PC analysis of this dataset, we kept the factor loadings over |0.35| of variables with significant contributions (p < 0.05) to reduce dataset dimensionality and to optimally separate animals of each group  made it difficult to easily extract parameters that accounted for SMR-related changes. To identify variables that contributed the most to the overall variability of our data, we used principal components analysis (PCA) to reduce high dimensionality . PCA is defined as an orthogonal linear transformation of a dataset into a reduced subspace with new coordinates, so that the variance is maximized on each new coordinate axis or principal component (PC), thus minimizing the loss of information. A normalization method, in which the data mean was adjusted to 0 and the standard deviation (SD) was adjusted to 1, was applied to allow comparisons of data with disparate values and variancesSupplementary data


The broad range of information obtained from MEMRI has led to the use of Mn2+ in many animal models of neurodegeneration which has supplied important insight into brain degeneration in preclinical studies. Here we provide a brief review of MEMRI use in neurodegenerative diseases and in diseases with neurodegenerative components in animal studies and discuss the potential translation of MEMRI to clinical use in the future.MRI has been extensively used in neurodegenerative disorders, such as Alzheimer’s disease (AD), frontal-temporal dementia (FTD), mild cognitive impairment (MCI), Parkinson’s disease (PD), Huntington’s disease (HD) and amyotrophic lateral sclerosis (ALS). MRI is important for monitoring the neurodegenerative components in other diseases such as epilepsy, stroke and multiple sclerosis (MS). Manganese enhanced MRI (MEMRI) has been used in many preclinical studies to image anatomy and cytoarchitecture, to obtain functional information in areas of the brain and to study neuronal connections. This is due to Mn For example, diffusion MRI is sensitive to early tissue damage due to ischemia which can influence treatment decisions, whereas long-term injury to tissue due to stroke can be measured with T1 or T2 based MRI contrast , other forms of dementia such as frontal-temporal dementia (FTD) and mild cognitive impairment (MCI), Parkinson’s disease (PD), Huntington’s disease (HD), and amyotrophic lateral sclerosis (ALS). In addition, MRI is used for the neurodegenerative components of diseases such as epilepsy, stroke and multiple sclerosis (MS) that are not primarily caused by neurodegenerative processes. MRI applications to neurological diseases can be broadly characterized as those where the brain changes have large effects on MRI and can be used for diagnosis, medical decisions and trials with small numbers of participants. These studies rely on well-established MRI contrast mechanisms including enhancement in T3 (0.5 mm linear resolution). Smaller volumes of hippocampal subfields such as the subiculum, CA1, CA3 and dentate gyrus (DG), have been measured in Alzheimer’s patients compared to MCI and healthy patients  plaque formation and the presence of neurofibrillary tangles (NFTs) occurs at late stages of the disease . Treatment of MPTP rats with ceftriaxone, was shown to prevent these changes , prior to MPTP intoxication did not result in increased manganese enhancement in the SNc  induces death of dopaminergic neurons in the SNc. Systemic administration of manganese in a PD rat model, 2 weeks after MPTP lesioning in the SNc was induced, resulted in decreased Mn2+ uptake was evident in the developmental stage (P17), prior to the onset of photoreceptor damage, throughout the retina, but increased at later ages. This was attributed to ionic dysregulation during pathological retinal thinning  rats, a model of photoreceptor degeneration, underwent MEMRI before degeneration onset [developmental stage Postnatal day (P) 17] and during the course of degeneration, . The retinal thickness was significantly reduced only in the pathological stage, P57 rats, compared to age matched control rats. Decreased Mn−/−) mouse model  mouse model, Mn2+ injection into CA3 region of the hippocampus, showed reduced Mn2+ transport from the hippocampus to the amygdala and basal forebrain and to the contralateral hippocampus , a microtubule interacting compound that protects against tau pathology, resulted in similar axonal transport rates as for control mice in this mouse model of ALS , and the thalamic anteroventral compared to control rats  rat model of PD, where lesions are induced in the SNc. MnGenetic mutations are associated with axonal transport impairment that is commonly found in neurodegenerative diseases, such as APP and tau in AD, SOD1 in ALS, huntingtin in HD and Parkin in PD  and is characterized with lesions that can appear throughout the brain and in later stages with demyelinated axons and progressive neurodegeneration. In stroke, following the acute stage where an ischemic core is formed, the later stages are characterized with ongoing vascular impairment and neurodegeneration. Epilepsy is characterized by spontaneous recurrent seizures with the development of hippocampal sclerosis, neuronal cell loss, inflammation and neurodegeneration.2+ transport in the first few days after stroke was induced  and photothrombotic cortical injury (PCI). Systemic administration of manganese in rats and mice that undergo unilateral MCAO and PCI showed increased manganese accumulation in the perilesional tissue and reduced MnLocal injection of manganese into the brain was used to study connectivity in animal models of stroke. Manganese injection into sensorimotor cortex, 2 weeks after unilateral stroke induced by MCAO in rats, exhibited reduced and delayed manganese enhancement in the ipsilateral thalamus and SN which indicates loss of connectivity of areas in the sensorimotor cortex , which uses active immunization with CNS homogenates, myelin, or myelin-derived antigens, that induce autoimmune mediated responses. As optic neuritis is one of the first symptoms of MS, MEMRI was used to assess axonal transport in the optic nerve (ON) in EAE animal models. In an EAE rat model, systemic administration of Mn2+, showed decreased signal intensity in the hippocampus that was attributed to decreased neuronal activity in a KA rat model  and pilocarpine induced status epilepticus (SE) models that represent the acute phase of epilepsy. In the latent phase, animals are seizure free until the onset of spontaneous recurrent seizure in the chronic phase. These phases are associated with different neurobiological changes such as, hippocampal sclerosis, mossy fiber sprouting, inflammation and neurodegeneration. MEMRI studies after systemic administration of Mn2+ injection into the lateral ventricle also resulted in increased manganese enhancement in the hippocampus, CA1 and DG, but was inversely correlated with seizure frequency , showed very high whole blood manganese concentrations and in some cases developed parkinsonian-like symptoms. No signal change was detected in the brain of control patients and low manganese exposure patients  studies rather than rely on MRI. Recent advances in preparation of high energy radiotracers allow for the production of two manganese radiotracers for PET imaging; manganese-51 (51Mn) and manganese-52 (52Mn) with half-lifes of 46 min and 5.6 days, respectively. As PET has higher sensitivity than MRI, a lower concentration of Mn2+ can be used for PET, that may enable its use in clinical studies to obtain information that has been shown to be useful in preclinical MEMRI studies. Indeed, manganese-54, a gamma-emitter  has been used to study manganese accumulation of tissues in the body of mice and monkeys (Dastur et al., 54Mn is not likely to be clinically useful.Another approach would be to use radioactive Mn52Mn allows its use in longitudinal biodistribution studies in the body and brain (Topping et al., 52Mn a suitable radiotracer for imaging of brain anatomy and function. 52Mn2+ infusion in rats showed manganese accumulation of the radiotracer in the head and brain with a similar contrast to that seen in MEMRI (Brunnquell et al., The half-life of 5.6 days, of the positron emitter 52Mn should also allow neuronal tracing studies. Intranasal administration of 52Mn2+ traces the olfactory pathway over the course of several days in monkeys (Figure 52Mn2+ radioactivity was localized in the nasal turbinates with no manganese accumulation in the brain. By 4 days later 52Mn2+ could be traced to the amygdala and prefrontal areas of the cortex (Figure 52Mn2+ into the ventral tegmental area and the dorsal striatum, respectively. The ability of 52Mn2+ to follow neuronal pathways after direct injection of the 52Mn2+ PET tracer to a specific area in the brain similar to MEMRI, can be used to study all the neuronal pathways already studied with MEMRI such as the visual and BG pathways (Pautler et al., 2+ to any area of the human brain, as has already been shown in rodents (Howles et al., 52Mn2+ for PET imaging acts so similar to that of Mn2+ in MEMRI, even though the PET dose is ~2,000-fold lower (Saar et al., 52Mn2+ may be useful in human studies for imaging neurodegeneration by tracking changes in neuronal connections and anatomical changes at clinically safe doses.The ~6 days half-life of s Figure  similar s Figure . Immediax Figure  showed i2+, care must be taken in assigning the cellular mechanism for any changes in MEMRI after systemic Mn2+ administration. However, tracing studies that use Mn2+ to measure changes in axonal transport have been more straightforward. MEMRI has been used to assess pharmacological treatments in animal models of neurodegeneration and can serve as a screening tool to identify potential therapeutics. Development of MRI agents that can be used at safe doses or use of PET manganese radiotracers might enable the translation of these exciting results to humans.In conclusion, there is a large growth of preclinical literature that has used MEMRI to study different aspects of neurodegeneration. Due to the wide variety of mechanisms that the brain uses for accumulation of MnGS wrote the manuscript. AK proposed the topic and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


SHH), in tissues that constitute the eye directly and also in those tissues that exert indirect influence on eye formation. After a brief overview of human eye development, the many roles of the SHH signaling pathway during both early and later morphogenetic processes in the brain and then eye and periocular primordia will be evoked. Some of the unique molecular biology of this pathway in vertebrates, particularly ciliary signal transduction, will also be broached within this developmental cellular context.Mutations in effectors of the hedgehog signaling pathway are responsible for a wide variety of ocular developmental anomalies. These range from massive malformations of the brain and ocular primordia, not always compatible with postnatal life, to subtle but damaging functional effects on specific eye components. This review will concentrate on the effects and effectors of the major vertebrate hedgehog ligand for eye and brain formation, Sonic hedgehog ( These are regularly found with mutations of Aphakia results from the interruption of any signals involved in optic cup invagination, lens induction, differentiation or survival—if early enough, the absence of lens will have repercussions for the remaining optic vesicle. Congenital cataract results from affecting later lens differentiation and secondary fiber formation and matrix composition. NCC are indispensable for anterior chamber development, particularly the cornea, iris, trabecular network and ciliary process . This malformation has been classically defined as the failure of the forebrain to divide into two cerebral hemispheres. Clinically, this covers a phenotypic spectrum in the brain that always features ocular and related craniofacial malformations, which can range from complete anophthalmia to cyclopia to synophthalmia to microphthalmia, hypotelorism and coloboma  and dorsoventral axis in all vertebrates studied. As the optic vesicles grow, they keep this complementary expression, so that the most proximal region keeps expressing Pax2, while the distal-most region expresses Pax6  in the optic stalk. mid1 encodes a E3 ligase involved in the ubiquitination and proteasomal degradation of Pax6  axis of the evaginating optic vesicle is determined by midline Shh signaling, the primordium also gets patterned along the nasal/temporal (NT) and dorsal/ventral (DV) axes. Zebrafish optic vesicle evagination occurs in a very reduced window of time as compared to this process in mammals. The eye field becomes split in two domains by the same morphogenetic movements driving the lateral evagination of the optic vesicles  syndrome  and mesenchymal  eye progenitors is essential for the correct induction and patterning of each, as has been examined in numerous animal models over the years. Such studies have been a source for candidate genes and pathways in human ODA pathogenesis.PTCH1 and SOX2, account together for at most one in five micro/anophthalmia cases   and Indian hedgehog . Although Dhh has been reported to be the major ligand present and involved with corneal homeostasis in the adult mouse, it has not to date been implicated in human ODA or later ocular phenotypes , in contrast to most other molecules reviewed here that are present in target tissues, encodes a protein needed for the secretion of the mature SHH peptide  are the direct transcriptional effectors of Shh signaling. The three homologues in humans, GLI1, GLI2 and GLI3, are on chromosomes 12q13.3, 2q14.2, and 7p14.1, respectively. These TFs act in both repressor and activator forms, post-transcriptionally dependent on Hh signaling activity. This dual role was first established in Drosophila  and hypertelorism. Further abrogation of GLI3 may be embryonic lethal even earlier in humans, such that its potential ocular phenotype would not have been reported.However, the activator form of GLI3 is also necessary for human health, though not implicated specifically in eye formation. Pallister–Hall syndrome (PHS) is a rare, often lethal condition comprising massive benign hamartomic tumors of the hypothalamus, effects on the pituitary gland and subsequent adrenal function, and effects on digit numbers (polydactyly or syndactyly). Such effects on digits had been anticipated in experimental embryology by directly or indirectly manipulating Shh levels in the outgrowing limb  dates to the mid-twentieth century (James Smoothened (Smo) is a G protein-coupled receptor that partners intracellularly with scaffolding proteins such as Suppressor of Fused  to relieve the constitutive processing of Gli TFs into their N-terminal truncated repressor forms, and allow their transcriptional activator forms to predominate. Cyclopamine is a historic and well-known Hh signaling antagonist acting on SMO, which is initially bound and repressed by PTCH1 at the cell membrane , seven have no ocular symptoms aside from strabismus, while patient 258,033 with a nearly 7-Mb deletion comprising 74 genes has an epibulbar dermoid tumor, proptosis, and an eyelid coloboma, and patient 261,321, with a larger deletion yet, is reported simply with abnormal scleral morphology. Thus, SMO haploinsufficiency does not clearly affect the human eye or brain, reflecting possible rescue by the remaining allele.A few patients with presumably heterozygous deletions comprising the Smo null (homozygous) mutants also do not demonstrate HPE, but they do have ocular defects that correlate with mispatterning of the DV axis, reminiscent of loss of the Gli2 or Otx2 effectors of Shh signaling. In these mutants, Bmp4, usually expressed in the dorsal portion of the developing eye, expands ventrally, accompanied by reduction in transcription of Vax2 and Pax2 transcription factors and subsequent impairment of ventral eye formation , through its fibronectin domains. Missense substitutions in BOC has only recently been identified as a true HPE modifier gene in concert with SHH mutations  or glycogen synthase kinase 3 (GSK3). Mutations in In the absence of SHH, SUFU constitutively sequesters GLI2/3 proteins at the base of ciliary microtubules. Translocation of this complex to the tips of cilia then promotes Gli TF cleavage into repressor forms, and Sufu has recently been shown to accompany these to the nucleus as far as the chromatin itself. In the presence of SHH, SUFU instead traffics GLI1/2/3 activator forms into the nucleus and the GLI2/3 repressor forms out (Zhang 2017).The kinesins are a superfamily of molecular motors that hydrolyse ATP to translocate cargoes along the microtubules of primary cilia, towards the tip. Mutations affecting intraflagellar transport and kinesin proteins can cause ocular ciliopathies like retinitis pigmentosa (RP) by preventing the transport of proteins necessary for photoreceptor development, function and survival. They also can cause misregulation of Shh signal transduction by playing havoc with the trafficking of Gli TFs  while Ift122 is required for retrograde transport (toward the base). Mouse mutants in Ift172−/− optic cups show expansion of Pax2 and Sox1 expression at the expense of Pax6, which is reduced to a small patch at the distal-most region of the primordium, indicating relative reduction in optic cup- and expansion of optic stalk-fated territories. Likewise, expression of the TFs Mitf and Otx2, markers of the RPE, abnormally extends into the inner layer of the optic cup, with a concomitant reduction in the NR-specific TF Chx10, indicating that loss of Ift-mediated Shh signaling leads to expansion of proximal RPE fate at the expense of the more distal NR fate. Curiously, Ift122−/− optic cups show a more severe phenotype, with no invagination of the optic vesicle at all. Pax2 and Sox1 are dramatically expanded throughout the mutant optic vesicle, while Pax6 expression is virtually absent, and neither RPE nor NR is specified at all. Thus, Ift122-negative optic primordia adopt a full optic stalk fate. The phenotypes observed in Ift177 and Ift122 mutant embryos suggest overactivation of the HH pathway under conditions in which cilia formation is compromised, correlated in phenotypes with the extent of stimulation. Astonishingly, the concomitant knockout of Ift122 and Gli2 can partially rescue optic cup patterning and morphogenesis  and thereby, protein kinase A (PKA) early in development to further the proteolytic processing of Gli repressor forms may also counter these effects -null mouse mutants show abnormal, shorter cilia, and display a number of corresponding developmental defects, amongst which microphthalmia due to fully penetrant coloboma and frequent loss of the lens primordium (Lupu et al. Ccrk knockouts, the Gli1 expression domain of the distal optic vesicle is expanded and Mitf is ectopically expressed within the optic cup, promoting a RPE fate. Furthermore, Ccrk mutation rescues the effect of Smo mutants, in which Pax6 is expressed throughout the entire optic vesicle following loss of Shh signaling, demonstrating that Ccrk acts downstream of Smo. The analysis of both Ccrk−/−;Gli2−/− double mutants showed compensation for the excess distal Shh signaling activity mediated by the loss of Ccrk in terms of ocular vesicle morphology, although at the expense of ventral and midline tissues with normally highest exposure to Shh. Likewise, Ccrk−/−;Gli3−/− double mutants show exacerbation of the distal RPE phenotype at the expense of neural retina fate, and total loss of lens specification (Lupu et al. Recently published work demonstrates that The vertebrate eye is a highly complex structure whose assembly is coordinated over time from four distinct sources of embryological cells. Hedgehog signaling plays a role from the very first stages of specification of the neural epithelium, to determining the eye fields, to the maintenance of healthy functional ocular tissues in the adult organism. Over the last 25 years, the number of molecular participants in this pathway has grown exponentially, as have demonstrations that changes in their activity or availability cause human ocular disease. The primary cilium has been shown to be the principal organelle necessary for hedgehog signal reception and integration. The presence of the cilium is cell cycle-dependent, implying that Hh activity may also be cell cycle-dependent, adding one more level of complexity to the modulation of cellular functions by this signaling pathway. A “rheostat” model, first used to describe the gradation in signaling levels of the TF Mitf and in opposition to a binary on–off switch model (Carreira et al.