Sarcomas are a heterogeneous group of mesenchymal malignancies. In recent years, studies have demonstrated that inhibition of angiogenic pathways or disruption of established vasculature can attenuate the growth of sarcomas. However, when used as monotherapy in the clinical setting, these targeted antiangiogenic agents have only provided modest survival benefits in some sarcoma subtypes, and have not been efficacious in others. Preclinical and early clinical data suggest that the addition of conventional chemotherapy to antiangiogenic agents may lead to more effective therapies for patients with these tumors. In the current review, the authors summarize the available evidence and possible mechanisms supporting this approach. Sarcomas are a heterogeneous group of malignancies, including soft tissue sarcomas (STS) and tumors of bone and cartilage. Conventional chemotherapy regimens for advanced or metastatic sarcomas have low survival rates, substantial toxicity, and frequent emergence of resistance, making alternative novel treatment approaches a priority.Sarcomas express proangiogenic factors that may represent therapeutic targets, with vascular endothelial growth factor (VEGF) being the best characterized. In animal models of human sarcomas, inhibitors of angiogenesis have shown promising antitumor activity –3. AntiaThis review summarizes evidence supporting the use of targeted AA in combination with cytotoxic chemotherapy in sarcomas. We performed an extensive review of the available medical literature using the US National Library of Medicine’s PubMed search function to find relevant primary articles based on key search terms including “angiogenesis”, “antiangiogenic”, “antiangiogenesis”, and “antivascular”. These search terms were searched with “chemotherapy” and “sarcoma”, “bone tumor”, or “soft tissue cancer”. The “Related Articles” function of PubMed and reference lists from relevant articles were used to identify additional articles. Additionally, in order to identify recent trials not yet published, we also performed a search of abstracts presented at the American Society of Clinical Oncology (ASCO) annual meetings from 2013 to 2015.In the current review, we provide the results of this search beginning with the preclinical data supporting AA in combination with chemotherapy in this diverse group of diseases. The review concludes with an assessment of the completed and ongoing clinical studies that have treated patients with sarcoma using this therapeutic strategy.Angiogenesis is tightly regulated at the molecular level. Dysregulation of angiogenesis occurs in various pathologies and is one of the hallmarks of cancer. Concentrated efforts in this area of research are leading to the discovery of a growing number of pro- and anti-angiogenic molecules, many of which are already in clinical trials. The complex interactions among these molecules and how they affect vascular structure and function in different environments are now beginning to be elucidated , 8–10. TWith the discovery of VEGF as a major driver of tumor angiogenesis, efforts have focused on novel therapeutics aimed at inhibiting VEGF activity. Unfortunately, clinical trials of anti-VEGF monotherapy in patients with solid tumors have resulted in only modest responses. Intriguingly, the combination of anti-VEGF therapy with conventional chemotherapy has improved survival in cancer patients compared with chemotherapy alone .The proposed mechanisms of benefit from combined AA and chemotherapy include: 1) normalization of the tumor vasculature by altering vascular permeability and increasing drug accessibility O]H2O positron emission tomography (PET) imaging in a mouse model of lung cancer showed that treatment with the VEGFR/platelet-derived growth factor receptor (PDGFR) inhibitor PTK787 created a 7-day window of improved tumor blood flow when tumor vessels are transiently normalized [rmalized . An imprrmalized . Initiatrmalized . TogetheIn contrast, a study by Van der Veldt et al. in non-small cell lung cancer (NSCLC) showed that pretreatment with bevacizumab reduced both perfusion and net influx rate of radiolabeled docetaxel as measured by PET with effects persisting after 4 days . This stOther preclinical studies reporting the impact of AA upon delivery of cytotoxic therapies include sunitinib, an inhibitor of VEGFR and PDGFR, combined with temozolomide in orthotopic glioma models , 21. SunInterestingly, when used as monotherapy, several preclinical studies have shown that the normalization of blood vessels by AA may result in paradoxical increased invasion of local vessels by the tumor and resulting metastases. A recent study of the effects of combination therapy in breast cancer model suggest that the addition of chemotherapy to AA can help prevent local invasion of vessels promoted by the AA and result in lower metastatic rate. Antiangiogenic therapy with DC101 (VEGFR2 inhibitor), while blunting tumor volume growth, was found to increase local invasion in multiple primary tumor models, including a patient-derived xenograft . This efSynergistic effects of combination therapy of AA with chemotherapy have been seen in several preclinical models of solid cancers Fig. b. For exSorafenib increased apoptosis in melanoma-derived cell lines treated with melphalan or temozolomide . The molThe timing and sequence of AA with chemotherapy can also be critical in determination of synergy or antagonism. Troiani et al. demonstrated the sequence-dependent interactions of ZD6474  with oxaliplatin in colon cancer cell lines in vitro using three combination schedules . TreatmeAlternatively, Naumova and colleagues demonstrated that paclitaxel and SU6668, a VEGFR2/PDGFR inhibitor, synergistically inhibited the proliferation and increased apoptosis of endothelial cells . These fAnother mechanism of combination therapy involves overcoming chemoresistance Fig. c. AcquirFor example, a series of in vitro and in vivo studies using preclinical models of human pancreatic cancer characterized the synergistic effects of combination therapy with gemcitabine with masitinib, a selective inhibitor of PDGFR . The masTargeted AA and cytotoxic chemotherapy have been combined in several laboratory models of sarcoma, mainly STS, as summarized in Table 165 in STS growth, metastasis, and chemoresistance, Zhang and colleagues generated stably VEGF165-transfected STS cell lines to study the effect of VEGF overexpression in vitro and in vivo. VEGF165-transfected xenografts formed highly vascular tumors with shorter latency, accelerated growth, enhanced chemoresistance, and increased incidence of pulmonary metastases [165-overexpressing xenografts, inhibited angiogenesis, increased the vessel maturation index, and suppressed tumor cell proliferation compared to monotherapy-treated mice. The addition of DC101 induced endothelial cell sensitivity to doxorubicin and suppressed the activity of matrix metalloproteinases secreted by endothelial cells [To elucidate the role of recombinant human VEGFtastases . Combineal cells . These ral cells .Likewise, the enhanced antitumor effects of combination therapy using low-dose topotecan and pazopanib in mouse models of osteosarcoma and rhabdomyosarcoma are thought to be related to augmented antiangiogenesis . The metConcomitant use of a dual VEGFR2/EGFR inhibitor (vandetanib) with doxorubicin resulted in additional cytotoxicity and endothelial cell growth inhibition with lowered doxorubicin doses compared to vandetanib monotherapy in leiomyosarcoma, fibrosarcoma, and uterine sarcoma models . In addiOutside the field of sarcoma, AA have been combined with chemotherapy with varying outcomes. A retrospective study of patients with advanced solid malignancies treated on phase 1 protocols between 2004 and 2013 showed that chemotherapy concomitant with VEGF(R) inhibitors was associated with significantly higher odds ratio for clinical benefit compared with chemotherapy without VEGF(R) inhibitors .For example, in lung, breast, and colorectal carcinoma, AA have shown increased activity when combined with standard chemotherapy, as highlighted below. In advanced non-small cell lung cancer, a randomized phase 2 trial showed a trend towards increased response rate and time to progression when bevacizumab was combined with paclitaxel and carboplatin . SeveralPerhaps the disease in which bevacizumab has had the greatest impact in combination with chemotherapy is metastatic colorectal cancer. After a randomized phase 2 study showed encouraging results when bevacizumab was combined with fluorouracil and leucovorin , a randoTargeted AA and chemotherapy have been combined in numerous early phase clinical trials in children and adults with advanced solid tumors. Phase 1 studies that included patients with sarcoma are summarized in Table There have been four reported phase 2 studies evaluating the combination of AA with chemotherapy in sarcoma. The combination of bevacizumab with doxorubicin was evaluated in 17 patients with metastatic STS . While tThe Children’s Oncology Group (COG) evaluated bevacizumab or temsirolimus in combination with vinorelbine (V) and cyclophosphamide (C) in a randomized phase 2 study in patients with advanced rhabdomyosarcoma. Both treatment regimens were well tolerated and without unexpected toxicities. In a preliminary report, patients randomized to VC plus temsirolimus had a superior event-free survival compared to VC plus bevacizumab  . As a VCRay-Coquard and colleagues examined the addition of bevacizumab added to paclitaxel in a randomized phase 2 study of patients with angiosarcoma. While the combination antiangiogenic regimen was shown to be active in patients with angiosarcoma, the PFS and overall survival was similar in both arms . NeverthRecently, the Spanish Group for Research on Sarcomas presented their findings of a phase 2 study of sorafenib and ifosfamide in 35 patients with advanced STS . This coAdditional clinical trials evaluating combination therapy with targeted AA and cytotoxic chemotherapy in patients with sarcoma are ongoing Table . With eaIn adults, phase 2 studies are evaluating pazopanib and topotecan in patients with high-risk sarcomas (NCT02357810); pazopanib plus gemcitabine in advanced STS ; pazopanib and paclitaxel in advanced angiosarcoma (NCT02212015); sorafenib, epirubicin, ifosfamide, and radiotherapy followed by surgery in high-risk STS (NCT02050919). Lastly, there is one open randomized phase 3 trial evaluating bevacizumab versus placebo combined with docetaxel and gemicitabine in the treatment of advanced uterine leiomyosarcoma (NCT01012297).Outside of the context of formal clinical trials, several retrospective case studies/series have also highlighted the potential efficacy of these combination regimens. A child with transformed malignant angiosarcoma was successfully treated with bevacizumab, gemcitabine, and docetaxel, which resulted in temporary tumor regression with progression free survival of 12 months . DramatiExtensively reviewed elsewhere , 54, metAdvances in the biology of sarcomas have established the critical role of tumor angiogenesis and multiple signaling pathways involved in tumor development, growth, and therapy resistance. Numerous preclinical studies have demonstrated that targeting proangiogenic mechanisms in combination with cytotoxic chemotherapy may provide a valid approach to overcoming chemoresistance and inhibiting growth of these tumors. Early clinical data are still inconclusive, but some reports suggest that the use of these AA in combination with chemotherapy may be beneficial in the treatment of patients with advanced sarcoma.Similar to various targeted therapeutic approaches that looked straightforward initially, antiangiogenesis has turned out to be more complex and nuanced than originally thought. Although VEGF seems to have a critical role in angiogenesis, our knowledge of the other molecular determinants of angiogenesis is still in its infancy. In fact, many of these pro- and antiangiogenic molecules are context- and dose-dependent. Additional studies are needed to understand these mechanisms and expand these findings to determine how to optimize these strategies for use in the management of patients with sarcoma. Ultimately, randomized studies are needed to demonstrate the benefit of angiogenesis inhibitors combined with chemotherapy.


Neurogenesis, the process of generating new neurons in the brain, fascinates researchers for its promise to affect multiple cognitive and functional processes in both health and disease. Many cellular pathways are involved in the regulation of neurogenesis, a complexity exemplified by the extensive regulation of this process during brain development. Toll-like receptors (TLRs), hallmarks of innate immunity, are increasingly implemented in various central nervous system plasticity-related processes including neurogenesis. As TLRs are involved in neurodegenerative disorders, understanding the involvement of TLRs in neurogenesis may hold keys for future therapeutic interventions. Herein, we describe the current knowledge on the involvement of TLRs in neurogenesis and neuronal plasticity and point to current knowledge gaps in the field. Neuronal development is regulated by a myriad of proteins with functions intimately linked with tissue formation such as cell migration  adaptor proteins, namely, MyD88, Mal, Trif, TRAM and one inhibitory TIR adaptor protein, SARM  and murine SARM1 suppress Wallerian degeneration cell-autonomously for weeks after axotomy. This indicates that the functionality of Sarm1 as a pro-degenerative agent is conserved in mammals , a TLR-related adaptor protein, which was recently studied for its roles in neuronal plasticity. Sarm1 is a multidomain adaptor molecule containing two sterile α motifs and one Toll/interleukin-1 receptor homology domain. Sarm1 was originally identified in humans as a negative regulator of the TRIF-dependent TLR3 and TLR4 pathways in innate immunity  and their adaptor proteins  exhibit distinct effects on the different aspects of neurogenesis throughout the development of the brain. This may not come as a surprise, given the critical role of the TLR2 typically forms heterodimers with TLR1 or TLR6. While a deficiency of TLR2 has no impact on NPC proliferation, it is not known whether deficiency of TLR1 or TLR6 affects NPC proliferation.The impact of TLRs 5, 7, 8 or 9 has not yet been examined for an effect of NPC proliferation.So far, the only TIR adaptor protein examined for their expression pattern during brain development were Trif, MyD88 and Sarm1. It is interesting to examine the expression pattern of Mal and TRAM during brain development. Further, it will be interesting to assess whether these adaptor proteins have an active role in brain development.Developmental deficiency for Sarm1 exhibits pleiotropic effects on cognitive behavior. It is not clear, however, whether these effects are solely developmental or whether Sarm1 affects adult cognitive behavior independently of its effects on neuronal plasticity.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.


Recent identification of synthetic lethal interactions involving several proteins of the SWI/SNF complex discussed in this Research Highlight has opened the possibility of new cancer therapeutic approaches. Research over the two last decades has shown that epigenetic deregulation is a common feature in carcinogenesis. With the latest advances in cancer genome sequencing, the occurrence of inactivating mutations in SWI/SNF chromatin-remodeling genes in several tumor types has attracted a great deal of interest. The SWI/SNF complex alters the interactions between DNA and histones using the energy of ATP hydrolysis, modifying the availability of DNA’s information to the cellular machinery. In this way, the SWI/SNF complex can modify gene expression, such as by controlling the accessibility of DNA to transcription factors, thereby controlling transcription as a whole.Various studies have determined that genes encoding subunits of the SWI/SNF chromatin-remodeling complex are mutated in cancer about 20% of the time. Initial insights into the role of the SWI/SNF complex in tumorigenesis arose from the identification of the core subunit SMARCB1/SNF5 in malignant rhabdoid tumors, following demonstration of its tumor suppressor function in cell lines and animal models. Subsequently, mutations and/or loss of expression of the catalytic subunit SMARCA4 have been reported predominantly in non-small cell lung cancer (NSCLC), as well as other cancers. Furthermore, mutations in the accessory core subunit ARID1A have been reported in ovarian clear cell and endometrial carcinomas, among other cancers, and PBRM mutations have been reported in clear renal cell carcinomas.Recent understanding of the role and importance of the SWI/SNF complex in tumor development has opened the door to new potential therapeutic strategies based on the concept of synthetic lethality .Synthetic lethality is defined as a type of genetic interaction where the co-occurrence of two genetic events results in organismal or cellular death. The term also can be applied to cancer biology. During their transformation, cancer cells acquire genetic and epigenetic variations that distinguish them from their wild-type counterparts. Consequently, cancer cells have been reprogrammed, exposing new genetic and epigenetic vulnerabilities. Synthetic lethal interaction partners of cancer-associated molecular changes should therefore offer therapeutic opportunities. In recent years, synthetic lethality has attracted attention in the field of oncology, as it provides a new angle for therapy and may explain the sensitivity of cancer cells to certain drugs.In this Research Highlight, we will discuss some of the potential therapeutic strategies that can be developed by targeting the SWI/SNF complex using the concept of synthetic lethality -5 Figur.Figure 1SMARCA4-mutant lung tumors [The SWI/SNF complex contains one of the two mutually exclusive DNA-dependent ATPases, SMARCA4 , and SMARCA2 . Although SMARCA4 and SMARCA2 display high homology and presumably have overlapping functions, other observations suggest that they have different roles in cancer. For instance, tumor sequencing has unveiled some of these functional differences: SMARCA2 inactivation by mutation is infrequent in tumor development; however, SMARCA4 has been found mutated in primary tumors and cancer cell lines. The first somatic mutation of SMARCA4 was described in an NSCLC tumor , and expg tumors .et al. screened siRNA against SMARCA2 in a panel of SMARCA4 mutant NSCLC cell lines and found that the inhibition of SMARCA2 significantly decreased cell viability compared with control siRNA, whereas SMARCA4 wild-type cells were unaffected. They extended these results to a xenograft mouse model of NSCLC [SMARCA4 inactivation leads to greater incorporation of the nonessential SMARCA2 subunit into the SWI/SNF complex [TP53, in SMARCA4 mutant cancer cell lines [In a pioneer study, Oike of NSCLC . Indepenet al. also identified SMARCA2 as being essential for the growth of tumor cells that harbor loss of function mutations in SMARCA4 [Hoffman SMARCA4 and that could be exploited as a putative therapy in small cell lung cancer (SCLC) [MAX) gene in SCLC, they found that about 6% of the tumors have homozygous and tumor-specific mutations in MAX. Interestingly, the authors observed that the alterations in MAX and amplification of the MYC genes were mutually exclusive, and none of the MAX-mutant cells carried concomitant mutations of SMARCA4. The researchers observed that SMARCA4 was able to regulate MAX expression by binding specifically to its promoter, and that depletion of SMARCA4 strongly hinders cell growth, specifically in MAX-deficient cells, indicating a synthetic lethal interaction that could be therapeutically exploited [Recently, Romero and colleagues found another putative synthetic lethality that also involves r (SCLC) . Studyinxploited .ARID1A, which encodes AT-rich interactive domain 1A, is frequently mutated across a variety of human cancers  and also has bona fide tumor suppressor properties. Using a broad screening approach, Helming et al. identified ARID1B, an ARID1A homolog whose gene product is mutually exclusive with ARID1A in SWI/SNF complexes [ARID1A-mutant ovarian cancer cell lines. The authors showed that loss of ARID1B in ARID1A-deficient backgrounds destabilizes SWI/SNF and impairs proliferation in both cancer cells and primary cells, thus acting as a synthetic lethal and opening new therapeutic opportunities.Recent studies have revealed that another member of the SWI/SNF complex, omplexes , as the Epigenetic proteins have been intently pursued as targets for cancer therapy. However, it was thought that SWI/SNF complex proteins were not good targets for cancer therapy due to the tumor suppressor activities of the complex. Nevertheless, the research highlighted here describing synthetic lethality interactions between members of the SWI/SNF complex with putative therapeutic applications may have changed this point of view.SMARCA4 mutant tumors. In the same way, the inhibition of SMARCA4 could have therapeutic benefits in MAX mutant tumors.A new therapeutic approach includes the use of inhibitors that specifically target bromodomains. The proof of concept of these small cell-permeable inhibitors came from a report where the inhibitor I-BET151 was able to bind specifically to the bromodomains of BRD3/4 and be therapeutically active in MLL-fusion leukemia . The broSMARCA4 is known to have a tumor suppressive function, leukemia cells instead rely on SMARCA4 to support their oncogenic transcriptional program [Additionally, these drugs may be exploited in other genetic contexts where the SWI/SNF activities are necessary to maintain the tumor phenotype. For example, recent reports have observed that while in most of the tumor types studied,  program ,11.The recent identification of synthetic lethal interactions involving several subunits of the SWI/SNF complex has opened the possibility of new therapeutic approaches that can be exploited using a new generation of epigenetic inhibitors. A recent report suggested that the incipient microRNA-based technology could be also in the arsenal to target SWI/SNF proteins . As the


The exact mechanisms that generate levodopa-induced dyskinesias (LID) during chronic levodopa therapy for Parkinson’s disease (PD) are not yet fully established. The most widely accepted theories incriminate the non-physiological synthesis, release and reuptake of dopamine generated by exogenously administered levodopa in the striatum, and the aberrant plasticity in the cortico-striatal loops. However, normal motor performance requires the correct recruitment of motor maps. This depends on a high level of synergy within the primary motor cortex (M1) as well as between M1 and other cortical and subcortical areas, for which dopamine is necessary. The plastic mechanisms within M1, which are crucial for the maintenance of this synergy, are disrupted both during “OFF” and dyskinetic states in PD. When tested without levodopa, dyskinetic patients show loss of treatment benefits on long-term potentiation and long-term depression-like plasticity of the intracortical circuits. When tested with the regular pulsatile levodopa doses, they show further impairment of the M1 plasticity, such as inability to depotentiate an already facilitated synapse and paradoxical facilitation in response to afferent input aimed at synaptic inhibition. Dyskinetic patients have also severe impairment of the associative, sensorimotor plasticity of M1 attributed to deficient cerebellar modulation of sensory afferents to M1. Here, we review the anatomical and functional studies, including the recently described bidirectional connections between the cerebellum and the basal ganglia that support a key role of the cerebellum in the generation of LID. This model stipulates that aberrant neuronal synchrony in PD with LID may propagate from the subthalamic nucleus to the cerebellum and “lock” the cerebellar cortex in a hyperactive state. This could affect critical cerebellar functions such as the dynamic and discrete modulation of M1 plasticity and the matching of motor commands with sensory information from the environment during motor performance. We propose that in dyskinesias, M1 neurons have lost the ability to depotentiate an activated synapse when exposed to acute pulsatile, non-physiological, dopaminergic surges and become abnormally receptive to unfiltered, aberrant, and non-salient afferent inputs from the environment. The motor program selection in response to such non-salient and behaviorally irrelevant afferent inputs would be abnormal and involuntary. The motor responses are worsened by the lack of normal subcortico–cortical inputs from cerebellum and basal ganglia, because of the aberrant plasticity at their own synapses. Artificial cerebellar stimulation might help re-establish the cerebellar and basal ganglia control over the non-salient inputs to the motor areas during synaptic dopaminergic surges. Levodopa-induced dyskinesias (LID) seldom occur with the first dose of levodopa, but chronic exposure to the drug results in LID in 20–30% of PD patients in ∼2 years and in 80% within 5 years (In spite of being the most efficacious drug for the relief of motor symptoms of Parkinson’s disease (PD),  5 years . The ris 5 years , higher  5 years , dopamin 5 years –9, and b 5 years .Based on the timing of their appearance in a levodopa cycle, LID are termed as “peak-dose dyskinesia” or “biphasic dyskinesia.” Peak-dose dyskinesias are associated with high plasma concentrations of levodopa  and the The neural mechanisms of LID, those that determine the clinical type of LID  and those allowing the co-occurrence of Parkinsonism and biphasic dyskinesias, are still not fully understood –16.831 and Ser845 in the membranes of medium spiny neurons (MSNs) after levodopa treatment in dyskinetic rats  neurotransmitter systems. The degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc) is associated with a series of changes in the nigro-striatal synapses such as a loss of tonic release of dopamine, diminished dopamine storage, and reuptake capacity [for review see Ref. ]. Positrtic rats . Thus, aHere, we review the theories of LID related to striatal and cortical maladaptive plasticity in PD in the light of (1) the recent studies on motor cortex plasticity in different stages of evolution of PD –31, (2) Based on current understanding, the cortico–basal ganglia–cortical circuit functions as a complex, integrated network with multiple feed-back and feed-forward loops . The motA major recent advance has been the identification of topographically organized, reciprocal links between basal ganglia and the cerebellum in non-human primates. Retrograde viral transporter studies in primates showed direct projection from the dentate nucleus, a major output of cerebellum, to the CM/PF complex intralaminar nuclei in the thalamus and subsequently to the dorsolateral putamen , 50. ThiDopaminergic fibers from the SNc innervate the striatum and all structures in the basal ganglia, including the STN and GPi, as well as the prefrontal, motor, and sensory cortices . StriataNeuroanatomical studies have confirmed the presence of direct dopaminergic innervations from the midbrain to M1 in rats , 66, monIn human beings, studies in which plastic changes in M1 were artificially induced with non-invasive stimulation techniques  revealed that dopamine has a non-linear dose-dependent effect on M1 plasticity –81, whicA receptor subtype Patients with stable clinical response to levodopa with no fluctuations in their clinical response to levodopa and without LID could express LTP and LTD even when tested without levodopa. This suggests a beneficial and persistent treatment effect of levodopa therapy on M1 plasticity when compared to the severe loss of plasticity in the untreated state.(2)Patients with wearing-off motor fluctuation showed a persistent treatment effect of chronic levodopa treatment on LTP, but not on LTD. In these patients, exposure to an acute boost of dopamine, by administering their regular dose of levodopa, revealed a detrimental effect of dopamine on both LTP and LTD.(3)Patients with both motor fluctuations and dyskinesias have no persistent treatment effect on either LTP or LTD. Acute boost of levodopa did not restore LTP or LTD. Interestingly, acute dopamine boost led to a paradoxical LTP in the motor cortex of these patients in response to an intervention that normally induces LTD in healthy subjects. The paradoxical facilitation was more in those with more severe disease and with higher clinical response to levodopa, suggesting that both are consequences of the acute dopamine replacement. Moreover, PD patients with LID are unable to depotentiate an already established LTP in M1after an acute dosing with even a small dose of levodopa . This fiIn human PD, changes in motor cortex plasticity have been studied using transcranial magnetic stimulation (TMS), delivered alone as theta-burst (TBS) or as a paired associative stimulation (PAS), in which magnetic pulses delivered over M1 are classically associated with sensory pulses delivered on a peripheral nerve Figure . The plailateral . There wlevodopa :(1)Patide novo PD patients  is highly increased with respect to healthy controls and correlates only with LID scores and not with severity scores of Parkinsonian signs . Sigma rde novo human PD as in de novo animal model of PD and if this cerebellar defect contributes to the genesis of specific manifestations like tremor or dyskinesias. If it were the case, then this metaplastic state of the cerebellar cortex could be reversed or delayed by early artificial inhibitory stimulation of the cerebellar cortex.Further evidence of cerebellar involvement in LID comes from TMS studies. In patients with PD, with mild to moderate LID, repeated sessions of bilateral cerebellar inhibitory stimulation after regular doses of levodopa induce a sustained reduction of dyskinesia lasting at least 2 weeks , 129. ReEarlier neuroimaging studies have found increased activity of motor and premotor areas in dyskinetic PD patients when compared to non-dyskinetic patients , 134. Th(1)Does DBS of the STN influence both STN–thalamo-cortical and STN–cerebellar transmission? Though the exact mechanisms by which DBS of the STN improves PD are not fully established, the fact that effective DBS of the STN improves cerebellar hyperactivity , 139 sug(2)The dose of administered levodopa has to be reduced after immediately after DBS of the STN to reduce dyskinesia. Does this indicate that DBS of the STN by itself cannot normalize the aberrant plasticity within M1 caused by dopamine surges, which would be necessary to alleviate dyskinesia? Preliminary data showing that sensorimotor cortex plasticity improves only after long-term synergistic combination of DBS with reduced doses of levodopa, but not after DBS alone . That lo(3)Does selective DBS of the ventral GPi but not of the dorsal GPi or STN simultaneously inhibit the propagation of pro-dyskinetic signals through both cortico-striatal synapses and cerebello-striatal synapses  on D1-bearing MSNs? It is already known that GPi has functional somatotopy  and thatThis model is based on the view that a physiological level of dopaminergic stimulation is critical for maintaining normal plasticity at the glutamatergic terminals in the interconnected large motor network involving the basal ganglia, motor cortices, thalamus, and sensorimotor areas of the cerebellum. This model considers M1 only as the final target of this network and proposes LID as the product of a cascade of changes triggered by the altered dopaminergic signaling in this network. M1 neurons deprived of the ability to depotentiate activated synapses due to abnormally high synaptic dopamine levels during peak-dose, are rendered indiscriminately receptive to non-salient or even aberrant afferent inputs from the environment conveyed by other neural structures . This defect may be additionally amplified by the aberrant signals reaching M1 from the thalamic relays erratically modulated by the basal ganglia and cerebellum (Figure 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.


This review describes genetic factors that may contribute to PROP sensitivity including: (1) the variants of the TAS2R38 bitter receptor with their different affinities for the stimulus; (2) the gene that controls the gustin protein that acts as a salivary trophic factor for fungiform taste papillae; and (3) other specific salivary proteins that could be involved in facilitating the binding of the PROP molecule with its receptor. In addition, we speculate on the influence of taste sensitivity on energy metabolism, possibly via modulation of the endocannabinoid system, and its possible role in regulating body composition homeostasis.Taste sensitivity to the bitter compound 6- Taste is the sensory modality that enables organisms to distinguish nutrient-rich food from noxious substances, and acts as a final checkpoint for food acceptance or rejection behavior ,2. In huTaste sensitivity varies greatly among individuals and may be one of the most important determinants influencing food choice and therefore the nutritional status and health of the individual . It is wWhether taste sensitivity plays a role in controlling the metabolism of ingested nutrients is still debated. It is also unclear under which mechanisms taste sensitivity may influence macronutrient intake. Importantly, foods are consumed as macromolecules  not as isolated components. However, the gustatory system is configured to respond to single units and breakdown products such as amino acids, free fatty acids, and mono- and di-saccharides. With the exception of the saccharides, these components are not abundant in human diets. Digestive enzymes such as amylase and lipases are present in saliva that degrade macronutrients into these single units . These mTaste in humans begins with the activation of the epithelial-derived taste cells where taste reception and signal transduction mechanisms are located. Groups of taste cells (50 to 100) are organized into taste buds situated on the surface of papillae. There are three different functional taste papillae types that are topographically arranged mostly on the superior surface of the tongue. Fungiform papillae are found in the anterior two-thirds of the tongue, foliate papillae on the lateral sides, and circumvallate papillae on the posterior one-thirds.ca. 25 T2Rs encoded by clusters of genes located on chromosomes 5p, 7q, and 12p [+ and K+ channels as well as hemichannels (Panx1) involved in the generation of action potentials and in the taste-induced ATP secretion to excite specific ATP receptors in nervous fiber and taste cells. These “receptors cells” do not form specialized synapses with taste sensory nerve fibers, which exist in close proximity to their basal pole. Type III cells are labeled “presynaptic cells” as they form synaptic junctions with gustatory nerve terminals [2) solutions by way of ion channels [Taste buds show an elegant functional organization in which different cell types with opposing effects of positive and negative feedback are integrated to shape the neural output transmitted to the hindbrain . Bud cel and 12p . T2Rs re and 12p ,30,31,32erminals ,34,35,36channels ,38,39,40Stimulants evoke a series of chemical signals that are integrated in the taste bud before taste information is transmitted to gustatory nerve fibers. The activation of Type II cells by sweet, bitter or umami stimuli induces secretion of ATP through Panx1 hemichannels. The extracellular ATP exerts three different functions mediated by ATP receptors : activation of gustatory afferent nerve fibers; activation of adjacent presynaptic cells which release 5-HT and/or NE; and autocrine signaling via a positive feedback mechanism onto receptor cells that increases their own secretion.Taste signals integrated in taste buds are transmitted by fibers of the three cranial nerves  to the rostral portion of the solitary tract nucleus (NST) of the medulla. This information is transferred to the thalamus , and hence to the gustatory areas of the cortex in the insula where it gives rise to the taste sensation. Local projections from the NST within the brainstem mediate non-cortical behavioral responses, such as those related to food ingestion or rejection. Gustatory signals from the NST also project to feeding centers in the amygdala and the hypothalamus where they can modulate hunger and fullness.How the taste signals integrated within taste buds are translated into a neural code for the perception of different taste qualities remains an open question. Three theories have been widely discussed: the labeled line (LL) theory which states that dedicated fibers transmit each sensory quality; the across fiber pattern (AFP) theory which posits that qualities are encoded by patterns of activity across several fibers; and finally a theory of temporal coding. According to the temporal coding theory, taste qualities are deciphered by different frequencies and/or timing patterns of action potential discharges ,42. AlthThe minimal gustatory circuitry and basic taste models described above are not sufficient to explain complex behavioral taste-induced processes. Moreover, the taste network in the brain is too extensive (over 20 brain regions are implicated in taste processing) and heavily interconnected (over 40 connections) via reciprocal pathways, to be adequately described by traditional feed-forward models of taste coding . The intThe genetic basis of taste variability was accidentally discovered by Arthur L Fox in 1931, while he was working in his laboratory to synthesize non-nutritive sweeteners. Fox found that people varied in their response to the bitter synthetic compound phenylthiocarbamide (PTC). Subsequent tests showed that about 30% of individuals could not taste PTC (non-tasters), whereas the majority could taste it as moderately or intensely bitter (tasters) . These sSome studies have consistently reported that individuals who differ in their taste response to PROP/PTC are also anatomically different. In particular, there is considerable evidence that super-tasters have a greater density of fungiform taste papillae on the anterior surface of the tongue, when compared to the other PROP taster groups ,52,53,54 et al. showed that PROP bitterness intensity was strongly associated with mRNA expression of the PAV-TAS2R38 allele, an (indirect) index of receptor protein production [The ability to taste PROP is a heritable trait . The genoduction . Finallyoduction ,61,62.Individual differences in PROP bitterness have also been attributed to many other factors including the chemical composition of saliva, its physical properties and the number, size and morphology of taste papillae. Taste stimuli, in the mouth, must diffuse through the salivary fluid layer to penetrate the taste pore. Once they gain access to the taste pore cavity, taste molecules interact with receptor sites on the plasma membrane of microvilli, which extend from the apical portion of taste cells . This prAs early as the first half of the twentieth century, Fox  suggeste et al. [l-Arg and l-Lys) that selectively interact with the PROP molecule by facilitating its binding with the TAS2R38 taste receptor.The functional meaning of these proteins in saliva and the physiological mechanisms by which they facilitate the perception of PROP bitterness have been further investigated by Melis et al. . These a in vitro experiments, that isolated cells thrive better when exposed to saliva from AA subjects or the corresponding active iso-form (Ser90) of the protein, thus reinforcing the association between the gustin gene and the formation and function of papillae. Although gustin and TAS2R38 have been shown to have independent effects on PROP tasting, together they account for up to 60% of the variance in PROP bitterness intensity. In contrast, only 40% of the variance in PROP taste threshold is due to the combined effects of gustin and TAS2R38 [The multiplicity of genetic and environmental factors that appear to influence PROP sensitivity, as well as the inherent genetic diversity in this trait across populations, make it difficult to identify individual, relevant factors that contribute to PROP tasting. In this regard, the study of genetically homogeneous populations are valuable since they tend to minimize background noise associated with the characterization complex traits. We studied a homogenous genetic cohort on the island of Sardinia and showed that a key factor strongly associated with PROP taste sensitivity is the polymorphism, rs2274333 (A/G), located in the gustin (CA6) gene that controls the zinc-dependent salivary protein of the same name ,71. Gust TAS2R38 . In addi TAS2R38  failed tControversial data exist on the involvement of gender in individual differences in PROP perception. Some studies showed that women are more frequently tasters compared with men ,76,77. WBrassica vegetables, and also avoid strong-tasting versions of foods that do not contain the thiourea groups including sweets, spicy foods and alcoholic beverages [Several studies in human nutrition have suggested that the PROP phenotype may serve as a general marker for oral sensations and food preferences, thus influencing dietary behavior and nutritional status . It has everages ,94,95,96everages ,99,100 beverages ,102, repeverages  showed teverages ,104 and everages ,105. FinThese findings support the hypothesis of an inverse correlation between PROP tasting and calorie consumption and/or BMI, which has been reported in several studies ,107,108.Several studies have focused on identifying the factors that may lead to divergent conclusions about the involvement of the PROP phenotype in food preferences, dietary choice and BMI . One of The role of hormonal fluctuations due to the menstrual cycle on taste sensitivity is conflicting . Variati et al. [It is known that the cognitive control of eating behavior plays an important role in determining the relationship between PROP phenotype and BMI, especially in women ,106. Sin et al.  showed tN-arachidonoylethanolamide  and 2-arachidonoylglycerol (2-AG), and their high affinity cannabinoid receptors (CB) 1 and CB 2 [Factors that influence energy metabolism may also affect the relationship between PROP status and BMI. One example is the endocannabinoid system that may work to fine-tune body metabolism in response to dietary exposure to taste stimuli. The endocannabinoid system regulates “on demand” production and degradation, by specific pathways, of arachidonic acid derivatives, and CB 2 . These rand CB 2 . Dietaryand CB 2 , by affeand CB 2 . Moreoveand CB 2 ; endocanand CB 2 . These dand CB 2 . InteresFinally, many bioactive compounds that are regularly present in very low amounts in our diet, or added to foods as spices, may influence energy balance. For example, terpenes and polyphenols, with strong taste and flavor components, possess sympathomimetic properties that increase thermogenesis and boost energy metabolism . CapsaicRecent discoveries elucidating the role of PROP and other taste phenotypes, such as gustin, in body weight provide critical insights for understanding the influence of taste sensitivity on eating behavior, energy metabolism, BMI and health. Gustin may be of particular importance because of its role as a trophic factor for taste bud density and maintenance. Future studies should be aimed at evaluating genetic, metabolic and anthropometric variables that interfere with the relationship between taste sensitivity and BMI in diverse physiological or pathological conditions that may induce substantial changes in BMI.While genetic factors are not modifiable by dynamic environmental changes, other factors, such as the endocannabinoid system is responsive to diet changes, and may function to fine-tune body metabolism according to nutritional content. A more comprehensive approach to weight management should consider the influence of dietary fats on the endocannabinoid system as well as the role of bioactive flavor compounds in energy metabolism. Thus, better characterization of the factors that modify these systems would help us to target populations at-risk and to design diets with tailor-made supplements and/or functional foods, to optimize health.


Smoking causes infertility in men, and children born to male smokers are at increased risk of childhood cancers and birth defects, such as cardiac defects, cleft palate, renal agenesis, and spina bifida. These effects are due to the accumulation of toxic constituents of cigarette smoke (CS) in the systemic circulation and seminal plasma.2 CS contains more than 7000 chemicals, including at least 539 polycyclic aromatic hydrocarbons (PAHs), of which 69 are proven carcinogens that are also products of incomplete combustion of fossil fuels and environmental pollution.3Tobacco causes nearly six million deaths per year worldwide; in the US, smoking and second-hand smoke exposure causes one in every five deaths and incur almost $300 billion annually in total economic costs.4 Whereas the endogenous activation of AHR by growth factors and dietary metabolites like kynurenic acid maintains tissue homeostasis and regulation of normal immune function, its exogenous activation by PAHs leads to spermatogenic arrest, suppression of meiosis, cell cycle arrest, spermatocyte cell death, and male infertility.6 However, the precise role of AHR in spermatocyte susceptibility to CSC is still emerging. In an earlier study,7 we demonstrated that the CSC induced several molecular and phenotypic changes in the spermatocyte cell line, (GC-2spd(ts)).8 By using a competitive AHR-specific pharmacological inhibitor, CH223191,9 we observed that many of the gene expression changes were dependent on AHR signaling. In the subsequent work, we showed that inhibition of AHR abrogated cell cycle arrest in spermatocytes.10 However, given that AHR is required for normal sperm development in mice,11 our recent report published in Cell Death Discovery12 compared the effects of pharmacological inhibition of AHR to knockdown of AHR expression, yielding intriguing findings  or tar, exert their cytotoxicity by activating the ligand-inducible transcription factor, aryl hydrocarbon receptor (AHR).findings .13 Pretreatment of the cells with CH223191 significantly reduced several of these effects, including DNA damage, caspase activation, and membrane alteration.We first characterized the effects of CSC on GC-2spd(ts) spermatocytes and found that CSC caused oxidative stress both in the cytoplasm and the mitochondria, though it did not affect mitochondrial membrane potential. Additionally, CSC treatment of spermatocytes promoted apoptosis, as indicated by increased expression of both anti- and pro-apoptotic genes, DNA damage, activation of caspases 3 and 7, and plasma membrane damage. Others have reported similar results in different cell types.Ahr in spermatocytes by 70%. Strikingly, we observed that Ahr knockdown did not abrogate many of the CSC-mediated effects that were suppressed by AHR inhibition. For example, Ahr knockdown did not reduce CSC-mediated caspase activation or plasma membrane damage. Moreover, spermatocytes in which Ahr was knocked down had higher levels of DNA damage than control spermatocytes. This finding suggests a regulatory role for AHR in the early stages of DNA damage or repair7 and supports an earlier finding that loss of AHR triggers DNA damage.14 To confirm these findings, we compared control and Ahr-null mouse embryonic fibroblasts (MEFs) and found that complete loss of Ahr was unable to prevent the CSC-mediated increase in caspase activation and membrane damage.To compare the effects of AHR inhibition to reduction of AHR expression, we used siRNA to knockdown expression of 10 that the cross talk between AHR–NRF2 pathway and MAPK signaling is required for CSC-induced spermatocyte cell death. However, here we found that MAPK inhibitors could not prevent CSC-induced membrane damage in spermatocytes. We interpret this finding to mean that the membrane damage is directly caused by the CSC-induced increase in free oxygen radicals and the involvement of other signaling mediators in addition to the MAPK pathway as reported by others in rat lung.15 Our results reveal that pharmacological inhibition and genetic manipulation of AHR have different effects; this fact should be kept in mind in future studies. More importantly, our data indicate that inhibition of AHR by using drugs such as CH223191 may be a viable prophylactic strategy to prevent germ cell death mediated by exposure to CS and other environmental pollutants containing PAHs.We demonstrated earlier


Fibrosis is the aberrant deposition of extracellular matrix (ECM) components during tissue healing leading to loss of its architecture and function. Fibrotic diseases are often associated with chronic pathologies and occur in a large variety of vital organs and tissues, including skeletal muscle. In human muscle, fibrosis is most readily associated with the severe muscle wasting disorder Duchenne muscular dystrophy (DMD), caused by loss of dystrophin gene function. In DMD, skeletal muscle degenerates and is infiltrated by inflammatory cells and the functions of the muscle stem cells (satellite cells) become impeded and fibrogenic cells hyperproliferate and are overactivated, leading to the substitution of skeletal muscle with nonfunctional fibrotic tissue. Here, we review new developments in our understanding of the mechanisms leading to fibrosis in DMD and several recent advances towards reverting it, as potential treatments to attenuate disease progression. Duchenne muscular dystrophy (DMD) is a fatal, X-linked recessive disorder characterized by a progressive loss of muscle mass and function . DMD hasRecently, however, attention has also begun to focus on understanding and modifying the pathological background of the disease, it is now well established that many of the pathological features of DMD are not only caused by the lack of dystrophin and/or the failure of muscle stem cells  but are also due to the complex interactions of these cells with the surrounding environment. Changes in this environment can delay muscle repair and regeneration and enhance inflammation, leading to disease exacerbation and fibrosis development. Important contributors to DMD pathogenesis, and potential obstacles or targets for achieving better therapeutic outcomes, include the inflammatory components of the damaged and regenerating muscle and the auxiliary cell mediators such as fibroblasts that support satellite and inflammatory cells, as well as the milieu of soluble factors produced by them. In particular, recent studies have highlighted the importance of all these cells and factors in the development of not only fibrosis arising during aberrant regeneration and DMD progression, but in other diseases such as diabetes and in aging (reviewed in ).Fibrosis is defined as the excessive or unregulated deposition of extracellular matrix (ECM) components and is a particular hallmark of DMD and abnormal repair processes in several other tissues upon injury including liver, lung, kidney, and pancreas. Controlled deposition of ECM components during growth and repair is critical for providing a scaffold to build and structure new tissue, but alterations in the timing, the intensity, and/or the components of this process can lead to excessive ECM deposition (fibrosis) and loss of tissue function. Fibrosis has a double negative consequence for the potential treatment of DMD in that it not only alters muscle function, but also reduces the amount of target muscle available for therapy and repair. Therefore, a better understanding of the components and processes leading to the development of fibrosis is important for our ability to improve muscle repair, treat DMD, and potentially restore muscle function. Although this review focuses on DMD, there is evidence from other myopathies that a dysregulated and/or disordered ECM may also contribute to disease progression , 10.β signaling pathway, perhaps the most critical effector of fibrosis, and in particular its interaction with connective tissue growth factor (CTGF) and the renin angiotensin system (RAS). We also review recent efforts to lineage trace cellular sources of ECM production and assess the novel fibrogenic role assigned to other nonsatellite cell types that have been identified in muscle. Finally, we will also describe some of the recent progress in the development and characterization of animal models for the study of fibrosis in vivo and some potential therapeutic approaches to combat and diminish fibrosis in DMD.A review of all of the contributing factors that lead to fibrosis is beyond the scope of this report, although several recent reviews are available , 12. Ins in vivo is transforming growth factor-beta (TGFβ) as reviewed in [β is initially generated as a latent precursor of one of three isoforms TGFβ1, TGFβ2, and TGFβ3 [β is stored in the ECM where it is activated upon tissue damage or specific growth signals , and one TGFβ type II receptor. Importantly, TGFβ is expressed in regenerating muscle after injury, as well as in the dystrophic muscle of DMD patients and mdx mice [β and other profibrogenic polypeptides can be produced by infiltrating immune, inflammatory, mesenchymal, and tissue-specific cells (reviewed in [One of the most potent profibrogenic factors describediewed in . TGFβ isnd TGFβ3 . Latent iewed in ). Activamdx mice , 16, 17,iewed in ).β is liberated it can signal via the canonical TGFβ pathway (see below) or through several alternative pathways  signaling in IGF-1R(+/−) heterozygous mice deleted for the IGF-1 receptor in skeletal muscle using a muscle specific MyoD-Cre driver resulting in impaired regeneration, depressed expression of MyoD and myogenin, and increased expression of TGFβ1, α-SMA, and collagen I and fibrosis [β1-stimulated Smad3 phosphorylation and increase phosphorylated-AKT- (P-AKT-) Smad3 interactions, thus impeding nuclear translocation of Smad3 and thereby reducing the expression of fibrotic genes. Conversely, a reduced amount of IGF-1R diminished the levels of P-AKT, allowing dissociation and nuclear translocation of Smad3 and enhancement of the TGFβ1 signaling pathway and fibrosis [Recently, several indirect interactions between TGFfibrosis . Furtherfibrosis .β can also decrease the production of enzymes that degrade the ECM, while simultaneously increasing production of proteins like tissue inhibitors of metalloproteinases (TIMPs) and plasminogen activator inhibitor type-1  that inhibit ECM-degrading enzymes. Consistent with this, injection of recombinant TGFβ into skeletal muscle in vivo has two effects. Firstly, it stimulates TGFβ expression in myogenic cells (among other cells) in an autocrine fashion and secondly, it induces connective tissue production in the area of the injection , which regulates the release and bioavailability of TGFβ from the ECM, was recently shown to modulate fibrosis in mdx mice although its role in human DMD, if any, has not yet been established [TGFiewed in ). The saantation . This pration [β . Similarfibrosis . Finallyablished .β on fibrosis. For example, it can induce collagen type 1, α5 integrin, and fibronectin much more potently than TGFβ in fibroblasts [β, decorin may also negatively regulate CTGF activity. Even if the exact mechanism is not known, it has been shown that the inhibitory action of decorin on CTGF activity depends on its capacity to directly bind to CTGF [β and CTGF in muscular dystrophies by directly inducing fibrotic processes and inhibiting myogenesis [β treatment of C2C12 myoblasts was also shown recently to significantly increase AT2 expression [β signaling and CTGF expression. Interestingly, while Ang 2 seems to be profibrotic, recent data indicates that angiotensin 1–7 (Ang 1–7), an endogenous bioactive peptide derived from Ang 2, has opposite effect to Ang 2. Indeed, it has been shown that Ang 1–7 inhibits TGFβ-Smad signaling in vivo, which in turn leads to the reduction of the profibrotic microRNA- (miR-) 21, decreasing greatly fibrosis development in dystrophic muscles of mdx mice [Connective tissue growth factor (CTGF) is a nonstructural regulatory protein present in the ECM that has an important role in fibrosis. Skeletal muscle from DMD patients, dystrophic dogs, and mdx mice all show elevated levels of CTGF . Fibrosiroblasts . Moreoveroblasts . Like fo to CTGF . Interesogenesis . A possiogenesis . ACE is ogenesis . Interesogenesis  and thatogenesis . TGFβ trpression , therefomdx mice .β and CTGF that promote ECM deposition, normal muscle repair also requires factors that regulate the proteolytic degradation of the ECM, for example, to facilitate satellite cell and myoblast migration, to remove the temporary scaffold laid down during regeneration, and to allow fiber growth. Clearly, if these factors become dysregulated, then ECM deposition, fibrosis, and loss of muscle function could ensue. We will only consider here recent developments to this group of molecules, which include serine proteases of the plasminogen activation (PA) system, the broad family of matrix metalloproteinases (MMPs), and their inhibitors, PAI-1 and the TIMPs, respectively . Seiewed in . The MMPiewed in . MMPs faiewed in  or plasmiewed in .β in muscle fibroblasts. Secondly, it also activated miR-21 expression, which in turn inhibited PTEN and enhanced signaling via the AKT pathway, which controls cell proliferation and survival, thus endowing TGFβ with a remarkable ability to promote cell proliferation. However, age-associated fibrogenesis and muscle deterioration in mdx mice, as well as advanced muscle fibrosis in young mdx mice lacking PAI-1, could be prevented by direct interference with miR-21 or the PAI-1 substrate uPA. Consistent with this, forced miR-21 overexpression in mdx muscle accelerated fibrosis and enhanced disease severity, whereas treatment of aged mdx mice with an inhibitor of miR-21 improved muscle homeostasis and reduced fibrosis. This is an important observation since fibrosis is normally considered irreversible at advanced ages. Further studies are needed to refine our understanding of the role of the PAI-1/miR-21 fibrogenic axis in skeletal muscle fibrosis and the disease course in DMD patients, but as the full role of the axis emerges, so too will new therapeutic targets. From all these findings, it seems that the pronounced fibrosis in human dystrophic muscles is at least partially related to an altered proteolytic activity in the dystrophic muscles due to imbalances in expression and activity of PA/MMP system components [Regulation of MMPs is thus complicated and involves not only expression, but also activation. In addition, in some cases the activity of MMPs can amplify or synergize with serine proteases of the plasminogen activation system to mediate ECM remodeling during tissue repair. The main function of the PA system is to degrade fibrin and at its core is the zymogen plasminogen which is converted into the active enzyme, plasmin, by two plasminogen activators (PAs): tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). There are several inhibitors of the PA system including plasminogen activator inhibitor 1 (PAI-1) and alpha2-antiplasmin that operate at the level of the PAs or plasmin, respectively . The PA mponents .One of the hallmarks of DMD is the chronic cycles of myofiber necrosis and repair, which histologically manifest as a sustained infiltration of mononuclear cells in muscle tissue. Consequently, considerable efforts have gone into characterizing these inflammatory cells to define their functions and their differences between the various acute and chronic (transgenic/genetic) models of skeletal muscle injury. One of the first events after tissue damage is the invasion of inflammatory cells to the site of injury. In response to acute injury, the first cells infiltrating the muscle are mainly leukocytes belonging to the myeloid lineage, principally neutrophils and monocytes/macrophages , which is expressed on myeloid cells, and induces expression of proinflammatory cytokines and chemokines ([αMβ2-binding motif [Although M1 macrophages are important for satellite cell proliferation (reviewed in ), they cmokines  and expressing high levels of the ECM protein osteopontin  and cytotoxic T cells (CTL) in fibrosis development has been raised recently. One of the most interesting animal models, to at least partially reveal the role of these cells in regeneration and fibrosis, is the scid/mdx mouse model. These mice are deficient in functional lymphocytes (both B and T cells), allowing the study of the function of these cells in the progression of the disease. Interestingly, scid/mdx mice develop less fibrosis in the diaphragm at one year of age, which correlates with a decrease in TGFc muscle . In addic muscle , and shoc muscle . When anopontin , nonendothelial (CD31−), and nonsatellite cells (α7 integrin- or SM/C2.6- depending on the study), but they did express markers of progenitor cells such as CD34 or Sca-1 [α [α+ progenitors in the dystrophic muscle of mdx mice [The existence of progenitor cells sharing characteristics of mesenchymal stem cells (MSCs) in skeletal muscle has been recently discovered , 66. The in vivo , and weror Sca-1  or the fSca-1 [α , 68 and Sca-1 [α , but it mdx mice .α+, Sca1+, and gp38+, which also transiently expresses Adam12 upon acute injury [ in vitro and in vivo. These results suggest that the Adam12+ fraction might be a subpopulation of the FAPS described by Joe et al., which would be more committed to become bona fide fibroblasts. Indeed, depletion of the Adam12+ fraction of MSCs reduced the ECM accumulation induced by cardiotoxin injection. Although this study was performed in an acute model of injury, these findings are consistent with the fact that overexpression of Adam12 in mdx mice aggravates fibrosis [Interestingly, a recent study characterized a subpopulation of cells expressing PDGFRe injury . Using afibrosis  and suggUnlike organs such as kidney or heart, where distinct cell types have been shown to contribute to fibrosis , 74, in As it becomes increasingly accepted that fibrosis is a crucial component in the pathogenesis of DMD, the need for appropriate mouse models that reflect the human disease has become more and more urgent, since disease progression is less severe in mdx mice than in human patients. In the mdx mouse, fibrosis develops extensively and exclusively in the diaphragm muscle during adulthood , while i+/− mice (mdx mice with haploinsufficiency of utrophin) were generated and were found to have increased inflammation of the hindlimb muscles at 3 and 6 months and in the diaphragm at 3 months only [α-sarcoglycan-deficient mice [β in muscle fibroblasts; loss of PAI-1 also activated miR-21 expression (through a nongenomic TGFβ-induced Smad activity), which in turn inhibited PTEN (phosphatase and tensin homologue) and enhanced AKT signaling, thereby endowing TGFβ with a remarkable cell proliferation-promoting potential on fibroblasts.In order to exacerbate or hasten fibrosis, different genetic mouse models have been generated. Utrophin is a large sarcolemmal protein with many structural and functional features similar to dystrophin that is upregulated in muscle of DMD patients and may be able to partially compensate functionally for the loss of dystrophin. Transgenic mdx/utrnths only . Howeverths only . In addiths only . Anotherent mice , 83. Indent mice  showed tConsistent with the notion that arginine metabolism by arginase-2 in M2 macrophages can drive fibrosis, and that the numbers of arginase-2-expressing M2 macrophages are elevated in the muscle and hearts of dystrophic mice, ablation of arginase-2 in mdx mice resulted in significantly less fibrosis in limb and diaphragm muscles . ConversA study by Fukada et al. showed that mice in the DBA/2 background exhibit a poor regeneration process after repeated injury. Furthermore, mdx mice in the DBA/2 background (D2-mdx) show severe loss of skeletal muscle weight and higher muscle weakness, while fibrosis and fat accumulation are greatly increased in comparison with mdx mice in the C57Bl10 background .Nevertheless, neither of these mdxmouse models mimics precisely the pathophysiology of human dystrophic disease, and this is one of the reasons why it is difficult to study therapies to stop the progression of fibrosis in DMD.In addition to genetic models, the drive to overcome our poor understanding of the molecular mechanisms underlying fibrogenesis in DMD has prompted the development of new experimental procedures that can be used to efficiently boost or advance fibrosis in young mdx mice in both hindlimbs without having to wait for its natural physiological onset.Mechanical muscle injury by daily repeated micromultipunctures for 14 days has been demonstrated to trigger fibrotic lesions in mdx hindlimb muscles that consequently display a similar pattern to diaphragm muscles . In thisFibrosis development is a consequence of the chronic degeneration and impaired regeneration of dystrophic muscle, which is itself caused by loss of the dystrophin gene. While the core aim of gene or cell therapy remains to replace the missing gene and thereby cure the disease at the roots by targeting the cause of muscle degeneration, preventing fibrosis progression should be considered an adjunct therapy for several reasons. Firstly, fibrosis development may negatively interfere with cell and gene therapies by reducing the amount of target tissue available for repair. Secondly, preventing fibrosis can also potentially improve quality of life and lifespan of dystrophic patients on its own. Several pharmacological treatments targeting fibrotic cells or molecules are currently being tested and some are showing promising effects in human and animal models .α2-deficient congenital muscular dystrophy [One of the signaling pathways involved in fibrosis development is the RAS system (as discussed above). Several components of this system have been used as targets to decrease dystrophic muscle fibrosis in animal models. Administration of the angiotensin 2 type 1 receptor antagonist losartan, which is commonly used to treat high blood pressure, has been shown to improve muscle strength and ameliorate fibrosis in dy(2J)/dy(2J) mice with laminin-ystrophy . Other systrophy , decreasystrophy , and decystrophy .β signaling and concomitantly decreasing the number of fibroblasts [Other molecules target the RAS by inhibiting ACE. For example, lisinopril is an ACE-inhibitor that has been shown to preserve cardiac and skeletal muscle integrity in mdx mice . Indeed,roblasts . Taken tβ neutralizing antibody improves the phenotype of mdx mice by delaying fibrosis development [β and CTGF are pleiotropic, molecules, targeting them often induces undesired side effects such as increasing the amount of proinflammatory CD4+ T cells infiltrating the muscle [Targeting profibrotic growth factors or cytokines to slowdown fibrosis development has also showed promising results. Administration of the antibody FG-3019, which neutralizes CTGF, or the administration of an anti-TGFelopment . Howevere muscle .α, which was used originally for human cancer therapy. It has been shown that its administration is beneficial for muscular dystrophy in mdx mice [ in vitro [NCT01847573, http://clinicaltrials.gov/show/NCT01847573) has assessed the safety and tolerability of halofuginone in DMD patients, but no data on efficacy will be available for some time. However, despite several drugs showing promise in animal models, few have currently progressed to clinical trials in DMD patients.Imatinib is an inhibitor of tyrosine kinase receptors, including PDGFRmdx mice . Interesin vitro . Other min vitro . Halofugin vitro . Interesin vitro , suggestβ with antibodies, the more recent implication of the RAS system in fibrosis may also prove important mechanisms as there are already many approved drugs on the market that are able to modulate this system and potentially produce beneficial effects. However, despite all the promises shown in treating mice, there have been few clinical trials to date. While this is frustrating to patients and families, there is also a need for caution in advancing too quickly since there are still many unknowns and many clear differences in muscle pathology and fibrosis development between human DMD patients and mdx mice that may respond unexpectedly to treatment. Nonetheless, these differences are growing smaller as new animal models and new molecular tools are developed.Fibrosis is an excessive deposition of ECM components that sometimes occurs as a result of dysregulated or chronic damage and repair processes. Human DMD patients have characteristic signs of muscle necrosis and repair with persistent fibrosis in muscles at young age, which play a significant role in the progressive nature of the disease and the reduced life expectancy. However, the mdx mouse model of DMD does not normally develop fibrosis extensively in the limbs, particularly at young age, and has several other differences from the human disease. Here we have reviewed some of the recent literature that has tried to bridge this species difference in order to develop better models to study the human condition, to improve existing treatments, and to open the door for new ones. Recent animal models have been shown to be better tools for unraveling the roles of new cell mediators in repair and fibrosis, including inflammatory and mesenchymal cell subpopulations, but the identity of a real ECM-producing cell in skeletal muscle remains elusive. Moreover, cell sorting techniques in mice are often complicated to reproduce in human patients due to differences in cell surface markers and the unavailability of sufficient sample material to extract significant numbers of cells. Advances have been made in the identification of new growth factors and cytokines and their downstream signaling components, which are also important targets for ameliorating fibrosis. As well as new advances in targeting TGF


Although all Quaternary mammal food webs were highly nested and robust to secondary extinctions, general food web properties changed in the Holocene. These results highlight the ability of communities to re-organize with the arrival of phylogenetically similar species without major structural changes, and the impact of climate change and super-generalist species (humans) on Iberian Holocene mammal communities.Most evidence of climate change impacts on food webs comes from modern studies and little is known about how ancient food webs have responded to climate changes in the past. Here, we integrate fossil evidence from 71 fossil sites, body-size relationships and actualism to reconstruct food webs for six large mammal communities that inhabited the Iberian Peninsula at different times during the Quaternary. We quantify the long-term dynamics of these food webs and study how their structure changed across the Quaternary, a period for which fossil data and climate changes are well known. Extinction, immigration and turnover rates were correlated with climate changes in the last 850 kyr. Yet, we find differences in the dynamics and structural properties of Pleistocene  Climate change is one of the major drivers affecting the diversity, composition, structure and functioning of ecological communities. Species respond in different ways to climate change which directly affects their persistence within the food web and, consequently, the composition and structure of the community Because the Quaternary fossil record is extensive and the climate changes during this period are relatively well understood, the examination of the Quaternary fossil record could be key to understanding long-term dynamics and structure of ancient biological communities Bos primigenius, Crocuta crocuta), others remained for more than one million years , and certain species appeared for short periods . Quaternary climate changes co-occurred with extinctions, migrations and the arrival of new species in the Iberian Peninsula that have likely impacted the structure of mammal communities living in this region.In the Iberian Peninsula, fossil records show that the composition of species within communities changed during the Quaternary following climate changes e.g. . WhereasIn spite of the observed long-term species changes during the Quaternary, few studies have analysed long series of species turnovers but see , even feSecond, the information from one single fossil site is limited for studies at the community scale where several species are considered. Because of a number of biases that affect the fossil record In this work, we assemble six large mammal communities in the Iberian Peninsula spanning 850,000 years (Pleistocene and Holocene time periods). To obtain a complete species pool for large Iberian mammals, communities were constructed using published data from 71 fossil sites. Thus, the results presented here describe how large mammal communities at a regional scale changed during the Quaternary climate changes. The aim of this study is to explore the long-term and community structure of a large mammal food web across 850,000 years of climate changes. We specifically address the following questions: (a) how have Quaternary mammal communities changed in terms of dynamics  and food web structure?, and (b) are the changes in the structure and dynamics of these communities associated with Quaternary climate changes?We assembled six different large mammal food webs across the Quaternary: one for the Early Pleistocene , one for the Middle Pleistocene , plus two food webs during the Late Pleistocene, one for the last interglacial maximum, LIM; 120 kyr BP, and one for the last glacial maximum, LGM; 21 kyr BP), and two food webs for the Holocene . For each time, we identified the large mammals (>20 kg), including hominids, that were present using fossil records from several Iberian fossil sites , a phenomenon that seems to be widespread across evolutionary time 2) To examine food web structure we use standard metrics: species richness, number of links and connectance  or Megaloceros giganteus (Irish elk) went extinct, followed by large carnivores like Panthera spelaea (lion). This is consistent with the observation that modern mammalian communities are remnants of larger Pleistocene mammal communities After more than 800,000 years of relative stability, food web properties changed in two steps in the Holocene: species richness and generality first decreased and then increased. The first step was caused by the extinction of large mammals after the Last Glacial Maximum (21–10 kyr BP), when prey such as Bos taurus (cattle) or Equus caballus (horse) appeared as new species in the food webs. These introductions slightly increased the number of species and the mean number of prey species per predator, the latter being also a consequence of the existence of generalist predator species, especially the humans. In contrast to Pleistocene communities, the introduced species were not phylogenetically related to the extinct species, and this has likely impacted ecosystem functioning, as certain ecosystem functions may not have been completely replaced by phylogenetically related species.In the second step, during the Holocene only human-introduced herbivores, such as Our analysis of robustness reveals across-time similarities in Pleistocene and Holocene communities. Despite the significant extinction and turnover rates across the Quaternary, and the reported changes in community size and food web connectivity, the robustness of mammal communities remained fairly constant for nearly one million years. On the other hand, all our mammal food webs present highly nested structures. Simulations have shown recently that nestedness is a destabilizing force in predator-prey food webs Homo neanderthalensis were present earlier, but our results suggest that they had a smaller impact on food webs compared to Holocene populations of Homo sapiens.The high nestedness reported in Quaternary mammal communities is explained by the presence of highly generalist predators. Both theoretical and empirical evidence show that super-generalist species become central nodes (most connected species) in the core of the nested community and may increase the overall nestedness The high impact of omnivore humans on food webs has also been found in contemporary communities. A recent study explored a spatial  gradient of human impacts in Serengeti food webs, average body mass and species richness also decreased with increasing human impact. Food webs tended to be more generalist following the human-induced extinction of the least-connected, most specialized species Paleoecological community studies provide insight into the relationships between biological communities and perturbations such as climate change over long time scales. This work presents a first attempt to track temporal changes in food webs over long time periods. Although the spatial and temporal scales are coarse, the patterns revealed are significant. We found that large mammal communities in the Pleistocene were able to re-organize despite the high extinction rates reported in the last 850,000 years. However, our results indicate that Holocene mammal communities experienced changes that were related to the arrival of new species introduced in the communities by humans (cattle and game species). Future work should aim at investigating the actual changes in temperature at local scales and the duration of those cycles; this information could be used to make cross-comparisons of the species’ extinction rates within and among continents, and their effects on ancient food webs, and to understand the mechanisms driving the likely spatial differences in the extinction rates.Collectively, our results suggest differences between the Pleistocene and Holocene time periods in the structure and dynamics of large mammal food webs. These conclusions have consequences for understanding the current-day sixth extinction event, and to what extent modern communities will be able to re-organize during current climate change. Not only are current rates of change in climate high, but also they interact with other drivers of extinction such as habitat loss and overexploitation, and the feedbacks generated by these processes and biotic interactions may result in higher extinction rates than those expected only from climate change File S1Table S1, Reference sources used for determining the presence of species in each of the 71 sites. Table S2, The species present (indicated by 1) at each period. Weight categories: 1 = <45 kg, 2 = 45–90 kg, 3 = 90–360 kg, 4 = 360–1000 kg, 5 = >1000 kg. Phylogenetic replacement categories: 0 = Not replaced by a species in the same genus in the next time period, 1 = Replaced by a species in the next time period, 2 = Species still present. Table S3, Prey weight classes. This information, together with spatio-temporal co-occurrence and actualism (see main text) was used to establish the links between species in the food web. Figure S1, Geographic location on the Iberian Peninsula of the Quaternary fossil sites used for constructing the ancient food webs. Figure S2, Large mammal food webs of the Iberian Peninsula during the Quaternary. (a) Early Pleistocene ; (b) Middle Pleistocene (450 ky BP); (c) Last Interglacial Maximum (120 ky BP); (d) Last Glacial Maximum (21 ky BP); (e) Early Holocene (10 ky BP); and (f) Present. Each node  is a species, and each link indicates a trophic interaction. Figure S3, Number of prey per predator in each time period, with species sorted in descending order. The time periods are the same as in Figure S4, A random extinction experiment demonstrate that the distribution of the number of links, connectance and link density observed Holocene food web is not expected by chance. 1000 food webs have been created extracting randomly 11 species from last glacial maximum food web (the observed number of extinct species in the Holocene).(DOCX)Click here for additional data file.


Xenopus and zebrafish are segmented in a similar fashion as the mouse metanephric nephrons. Here we review functional and molecular aspects of nephron segmentation with a particular interest on the signaling molecules and transcription factors recently implicated in kidney development in these three different vertebrate model organisms. A complete understanding of the mechanisms underlying nephrogenesis in different model organisms will provide novel insights on the etiology of several human renal diseases.The nephron is the basic structural and functional unit of the vertebrate kidney. To ensure kidney functions, the nephrons possess a highly segmental organization where each segment is specialized for the secretion and reabsorption of particular solutes. During embryogenesis, nephron progenitors undergo a mesenchymal-to-epithelial transition (MET) and acquire different segment-specific cell fates along the proximo-distal axis of the nephron. Even if the morphological changes occurring during nephrogenesis are characterized, the regulatory networks driving nephron segmentation are still poorly understood. Interestingly, several studies have shown that the pronephric nephrons in  All vertebrates possess an excretory organ, the kidney that regulates fluid balance, osmolarity and pH but also performs blood filtration in order to excrete metabolism end products and drugs. Most of these diverse tasks are accomplished by the nephrons, the functional units of the kidney.During vertebrate development, the kidney emerges from intermediate mesenchyme (IM) and progresses either via two or three stages: pronephros, mesonephros, and metanephros. Although each of these kidney forms differs in their overall organization and complexity, they all have the nephron as their basic structural and functional unit.Xenopus, zebrafish, and mouse.Nephrons in amphibians, fish, and mammals are organized into discrete segments that are composed of distinct renal epithelial cell types carrying highly specific functions for glucose, solute transport and acid/base balance. Therefore, correct segmentation of the nephron is crucial for kidney function. The similarity in segmental organization of nephrons existing between the mammalian metanephric kidney and the frog and fish pronephros raises new possibilities to explore and dissect regulatory pathways controlling nephron patterning. This article reviews recent advances on nephron patterning in In lower vertebrates, such as amphibians and fish, pronephric kidney is functional during embryonic and larval life and consists of two bilateral nephrons. Mesonephros will develop later on and additional nephrons will appear to ensure appropriate level of waste excretion in adult organisms. In amniotes, such as birds, reptiles, or mammals pronephros and mesonephros are only transient structures that will induce the formation of the functional metanephric kidney. Interestingly, pro-, meso-, and metanephros share similar organization: a filtration unit, a nephron tubule for solutes reabsorption and secretion, and a collecting duct to transport the filtrate product to the excretion site.Xenopus is a simple nonintegrated nephron composed of three major compartments: the glomus, the tubule, and the duct. In contrast to zebrafish and mammals, the glomus does not connect with the most proximal part of the tubule but projects into the coelomic cavity, where the filtered blood is released. The tubules are formed opposed to the glomus. The pronephric anlagen are first induced from the intermediate mesoderm at about stage 12.5 (early neurula) by surrounding tissues to form a proximal condensate. This structure elongates into the dorso-ventral direction establishing the future pronephric tubule. By stage 24 (tailbud), the cells undergo a mesenchymal-to-epithelial transition (MET) to form an epithelial tubule, the pronephric duct. This duct elongates, becomes highly folded in the proximal region, while it grows caudally and fuses to the rectal primordium to subsequently open to the exterior via the cloaca. In the most proximal region of the pronephros, three ciliated funnels, the nephrostomes, open into the coelom and force coelomic waste into the proximal tubules. The glomus morphogenesis starts at stages 29–30 (late tailbud): a heap of capillaries arises from the dorsal aorta and associates with splanchnopleuric cells budding into the coelom. The blood starts to be filtered at stages 35–36 (tadpole) [The pronephros in Xenopus, the zebrafish pronephros comprises a pair of nephron tubules attached at their anterior ends to a single glomerulus. The pronephros arises from bilateral stripes of a renal progenitor field, emerging from the intermediate mesoderm precursors 12 hours post-fertilization (hpf). These cells are localized adjacent to a cell-field that generates a mixture of angioblasts and primitive blood cells. The pronephric tubule differentiates between 16 and 24 hpf through an antero-posterior MET transition process. The most proximal part of the nephron arises from the IM under somites 3 to 8, whereas the distal part comes from a small portion of tubules forming along the IM , together with appearance of well-developed podocytes  Figure B.The zebrafish mesonephros will form during the juvenile stages around the length of the pronephros, which acts as a scaffold for mesonephros formation . It contIn mice, the first morphological renal development hint occurs at E8 when paired epithelial nephric ducts (ND) (or Wolffian ducts) arise dorsally from the IM under the fifth somite. These bilateral ducts then reach the cloaca. As the ducts grow, they sequentially induce the formation of pronephric and mesonephric tubules from the adjacent IM. At the most anterior part, few mesenchymal cells adjacent to the nephric duct form rudimentary tubules, referred to as pronephros, which is a transient structure not functional in mammals. As pronephros degenerates, mesenchymal cells of the mid part of the ND  condensate and give rise to mesonephric tubules. These tubules, consisting of well-developed glomeruli and proximal tubule like structures, are transient filtration units that start to degenerate as the metanephric or definitive kidney develops .Even if subsequently the mesonephric duct regresses, it has additional inductive functions for Mullerian duct development, while parts of its ductal system participate in the male reproductive organs. The Mullerian duct forms at embryonic day 11.5 (E11.5) close to the cranial part of the ND. Caudal elongation and maintenance of the Mullerian duct depends on ND integrity. In males, the Mullerian duct degenerates and the Wolffian duct (or ND) gives rise to the vas deferens, epididymis, and the seminal vesicles. In females, oviducts, uterine horns and the upper part of the vagina differentiate from the Mullerian duct while the Wollfian duct degenerates .The definitive kidney, or metanephros, arises from the caudal part of the Wolffian duct under 25th somite by reciprocal interactions between the epithelium and the adjacent condensed mesenchyme. At E11, the ureteric bud emerges from the ND and invades metanephric mesenchyme (MM). Signals coming from both the ureteric bud and MM are necessary for the bud to extend and branch and subsequently give rise to the entire collecting duct system. Each time a new branch is formed, signals from the UB tip epithelium induce a subset of surrounding mesenchymal cells (cap mesenchyme) to undergo an epithelial transition in order to establish a polarized renal vesicle (RV) . In miceSpecification of IM cells and renal primordium formation.Epithelialization and differentiation of renal primordium.Patterning of nephrons to make functional and specialized segment tubules.Formation of glomerular capillaries for blood filtration.Therefore, even if the non-amniotes pronephros and the amniotes metanephros have different shapes and adaptive functions, their development is similar and occurs through four stages:In the case of the pronephric kidney, these steps will occur only once to form one pair of bilateral nephrons. In mammals, these processes will repeat numerous times to give rise to a highly branched system connected to thousands of nephrons.Slc) gene family appears to be expressed in similar fashion among vertebrates [In mammals, proximal, intermediate, and distal tubules are three major nephron segments but they can be further subdivided in regard to their morphology and molecular signature. The glomerulus is connected to the proximal convoluted tubule by the neck. The proximal tubule can be subdivided into three segments: the convoluted tubule is formed by segments S1 (PS1) and S2 (PS2) and is followed by the straight tubule (or S3). The loop of Henle is further divided into the descending thin limb (DTL) and the ascending thin limb (ATL). The distal tubule comprises the thick ascending limb (TAL) and the distal convoluted tubule (DCT). The connecting tubule makes the connection between the nephron and the collecting duct. Terminal differentiated metanephric nephrons are therefore highly specialized structures characterized by a specific combination of solute transporters and tight junction elements. Several studies have pointed to the regional expression of solute transporters along the proximo-distal axis of metanephric kidney. In particular, the solute carrier  is divided into three segments , whereas the intermediate tubule (IT) and the distal tubule (DT) are both composed of two segments IT1 and IT2, and DT1 and DT2, respectively. In contrast, the connecting tubule does not appear to be further subdivided. As illustrates the schematic representation of nephron segments in slc5a2 and slc5a1 sequentially expressed along the proximo-distal axis of the proximal tubule [slc5a2 localizes to the S1 and S2 segments of the mouse proximal convoluted tubule and to the Xenopus PT1 and PT2 segments, whereas slc5a1 is expressed in the mouse S3 and the Xenopus PT2 and PT3 segments. No water transporter was found in the Xenopus intermediate segment. Thus, urine is not concentrated within the intermediate tubule and stays hypo-osmotic with plasma, suggesting the absence of an intermediate tubule. However, the intermediate segment expresses cldn8, also present in the mouse descending thin limb. Moreover, functional studies have further shown that the Irx transcription factors are present in both organisms in the future intermediate segment (see below). The Xenopus intermediate pronephros displays in addition distal tubule characteristics, since it expresses markers such as slc12a1, slc12a6, or kcnj1 [Xenopus DT1 and the mouse TAL segments express similar markers, such as the Na-K-Cl transporter slc12a1, while slc12a3 (NCC) is restricted to the Xenopus DT2 and the mammalian DCT. Finally, as in the mouse, the Xenopus connecting tubule showed expression of the transporter slc16a7 and the tight junction markers calb1, clcnk, and kcnj1. This further emphasizes the similarities between the connecting tubules of the pronephros and metanephros  displays an apical brush border and expresses transporters necessary for acid-base homeostasis as well as for glucose and amino acids reabsorption, such as in mammals’ PCT. The PCT and the proximal straight tubule (PST) express slc9a3, which is a proximal tubule marker in mammals [In zebrafish, interesting similarities have also been found in several differentiated cell types between pronephros and the mammalian metanephros. Within the glomeruli, endothelial cells, are fenestrated and podocytes interdigitate with foot processes . At the  mammals .tjp2a and tjp3, are expressed in the distal pronephros [slc12a1, a marker of the mouse TAL, and the DL expresses markers that are also specifically found in mammalian DCT and collecting duct such as clck, slc12a3, and cldn8 [Xenopus  determine whether cells are fated towards the proximal tubule or the distal tubule [wt1, foxc2, lmx1b, mafb, tcf21, and hey1. Simultaneously, knockdown of wt1 and foxc2 leads to an absence of podocytes. The regulatory network appears to be highly orchestrated since only double or triple knockdowns of these transcription factors affect podocyte development [irx1 and 3 are regulated by retinoic acid (RA) during nephron segmentation [Xenopus pronephros patterning remains unclear, since downregulation of RA leads to a failure of pronephros formation [In nt fates . The Iro and IT2 . Loss oformation ,30. Notcl tubule ,32,33,34entation . Howeverormation .hnf1b is expressed in the pronephric field and is maintained throughout the entire pronephros with highest levels in the proximal part. We have recently shown that hnf1b is required for both proximal and intermediate tubule fates in Xenopus. Our own experiments further suggest that it may act on these patterning events through the Notch pathway and the Iroquois genes within a complex regulatory circuit [hnf1b during nephron segmentation have not yet been determined. However, studies of the mouse Hnf1b promoter and enhancer have shown that Hnf1b is a direct target of RA [hnf1b acts downstream of RA in the developing Xenopus neural tube, further suggesting a similar regulatory circuit during pronephros segmentation [The transcription factor  circuit . The epiet of RA ,38. Moreentation  , the Notch ligands Dll1 and Jag1, as well as Bmp2, whereas the proximal domain is characterized by the high expression of Wt1 [Wt1 is required for glomerulus podocyte layer specification [Pax2 exhibits segmented expression during nephron morphogenesis and is strongly reduced in the podocyte progenitor territory, where Wt1 expression is high. It has been shown that Wt1 promotes podocyte development through direct repression of proximal Pax2 activity [Lhx1-deficient RVs fail to regionalize along the proximo-distal axis, lack the expression of the transcriptional targets Brn1 and Dll1, and do not progress to the CSB stage [Brn1, in turn, is involved in loop of Henle elongation and distal convoluted tubule formation [Brn1 and Lhx1 are required for distal and medial nephron segment formation. It is still unclear what are the first signals driving this polarization. However, polarized expression of Wnt4 and Lef1 within renal vesicle suggests that a Wnt-signaling gradient may contribute to this process [The first indication of polarity and proximo-distal axis appears quite early. Indeed, just after MET, several genes begin to be expressed in a polarized manner in the RV. The distal domain is defined by the restricted expression of many genes, including the transcription factors n of Wt1 ,58,59. Cfication . The traactivity . Lhx1-deormation . These o process  . Recent studies have further shown that the ligand Jag1 plays a dominant role relative to Dll1 for both podocyte and proximal tubule development [Several studies have implicated the Notch pathway in podocyte and proximal tubule fate acquisition. Accordingly, Notch pathway components display polarized expression patterns within the SSB: the Notch ligands n region ,63  leads to similar defects in nephron segmentation [Hnf1b is required for the acquisition of a proximo-intermediate segment fate in the vertebrates, thus uncovering a previously unappreciated function of a novel SSB subcompartment in global nephron segmentation and further differentiation.Interestingly, members of the Iroquois gene family ebrafish . We haveand Irx2 C. Moreovse genes . Interesentation . These rLgr5 is expressed in the distal segment of the SSB and in the distal convoluted tubule and thick ascending limb of the nephron. Lineage tracing studies have shown that Lgr5 marks progenitor cells of these segments. Lgr5 deletion did not lead to any kidney phenotype, but Lgr4/5 double knockout causes the loss of distal progenitors and reduced proliferation in the nephron tubules [in situ hybridization analysis has shown that Evi1 is expressed in the developing SSB [Evi1-null embryos impairs further analysis of its role in nephron patterning [The surface receptor  tubules ,71. A laping SSB . Unfortutterning .Wnt-signaling patterns the proximo-distal axis of the nephron, with proximal regions differentiated in regions with lowest Wnt signaling [Wnt-β-catenin gradient and which are the direct targets involved in nephron patterning.More recently, a β-catenin signaling gradient has been involved into nephron patterning. Pharmacological inhibition of β-catenin activity favored proximal cell differentiation. Reciprocally, proximal fate was repressed when β-catenin activity was increased  Figure C. Consisignaling . HoweverXenopus and zebrafish nephron patterning, RA has not been linked to proximo-distal patterning of the mouse metanephric nephron. Further studies are needed to elucidate the possible involvement of RA signaling in nephron segmentation, since Raldh1 and 2 are expressed in a restricted manner in the rat nephric duct and early nephron structures [In contrast to ructures .Xenopus and zebrafish studies are therefore crucial for our understanding of this critical field in developmental biology. These studies show that the molecular circuits involved in the proximo-distal nephron patterning in vertebrates are highly similar. Nevertheless they are not completely conserved, in particular the timing of appearance and/or requirement of some regulators. Several invertebrate models have also been studied, such as the fruit fly D. melanogaster, and obtained results that are further highlighting the amazing degree of conservation among the excretory cell types across the phylogenetic tree [Nephron patterning is a complex process that ensures the formation of highly specialized segments and their following physiological functions. The fundamental conservation of molecular anatomy between pronephric and metanephric nephrons has a great potential for discovering genes and regulatory pathways during nephron segments establishment. tic tree .Although the key transcriptional players of nephron segmentation have been identified, many of them remain to be discovered as well as the epistatic relationships and interactions that may exist among them. An additional and important issue will be the identification of the upstream signaling pathways acting on key regulators to establish distinct nephron segment fates. Systematic approaches such as RNA-seq and ChIP-seq on specific nephron segment progenitors combined with the use of simpler vertebrate models will certainly provide new insights onto the regulatory programs underlying theprocess of nephron segmentation.