Radiotherapy (RT) plays a fundamental role in the treatment of glioblastoma (GBM). GBM are notoriously invasive and harbor a subpopulation of cells with stem-like features which exhibit upregulation of the DNA damage response (DDR) and are radioresistant. High radiation doses are therefore delivered to large brain volumes and are known to extend survival but also cause delayed toxicity with 50%–90% of patients developing neurocognitive dysfunction. Emerging evidence identifies neuroinflammation as a critical mediator of the adverse effects of RT on cognitive function. In addition to its well-established role in promoting repair of radiation-induced DNA damage, activation of poly(ADP-ribose) polymerase (PARP) can exacerbate neuroinflammation by promoting secretion of inflammatory mediators. Therefore, PARP represents an intriguing mechanistic link between radiation-induced activation of the DDR and subsequent neuroinflammation. PARP inhibitors (PARPi) have emerged as promising new agents for GBM when given in combination with RT, with multiple preclinical studies demonstrating radiosensitizing effects and at least 3 compounds being evaluated in clinical trials. We propose that concomitant use of PARPi could reduce radiation-induced neuroinflammation and reduce the severity of radiation-induced cognitive dysfunction while at the same time improving tumor control by enhancing radiosensitivity. They can be divided into acute, subacute, and late effects, of which the late effects have the greatest impact. These arise months or even years after treatment and in many cases culminate in irreversible cognitive decline. While adverse neurocognitive effects of RT are common, the specific impact of RT on quality of life in long-term survivors of GBM has not been well characterized. Predicting the clinical impact of RT is complicated since both acute and late effects of RT on quality of life are context dependent, being influenced by numerous radiobiological , physiological (preexisting brain function), and patient-related factors . Although RT remains the most effective nonsurgical treatment modality for GBM, its efficacy is confounded by the innate and adaptive radioresistance of these tumors which is reflected in their inevitable recurrence.Radiotherapy (RT) plays a fundamental role in the treatment of patients with brain metastases and primary central nervous system (CNS) tumors, extending survival for many of these patients. Of the primary malignant brain tumors, glioblastoma (GBM) is the most common in adults and carries a very poor prognosis . GBM are notoriously invasive, infiltrating the brain parenchyma extensively, so high doses of RT  are often delivered to large volumes of brain in order to delay recurrence and extend survival.2 A critical subpopulation of GBM cells that retains stem cell characteristics exhibits marked upregulation of the DDR, which renders them resistant to DNA damaging treatments including RT.3The fundamental rationale for using RT in cancer treatment is based on the rapidly proliferating phenotype of cancer cells which, in addition to defects in the DNA damage response (DDR), makes them sensitive to fractionated courses of radiation. RT damages cellular DNA by causing single strand breaks (SSBs) and double strand breaks (DSBs). This initiates the DDR, an intricate signaling network of proteins that interrupts cell cycle progression and facilitates DNA repair. Failure to repair DNA breaks can result in cell death either through apoptosis or via mitotic catastrophe, the former being more prevalent in nonmalignant cells and the latter predominating in cancer cells.4 As such, it is hypothesized that the neurogenic niches in the brain are the most susceptible to radiation-induced DNA damage and play key roles in the pathogenesis of neurotoxicity and associated cognitive deficits.Different cell populations within the normal brain also exhibit markedly different levels of radiation sensitivity. Neural stem cells and undifferentiated neural progenitor cells (NPCs) are highly radiosensitive, undergoing apoptosis, proliferative arrest, and premature differentiation into other neural cell types in response to relatively low radiation doses.5 Neuroinflammation is the inflammatory response within the CNS and is critical in the resolution of cellular damage and tissue injury upon exposure to a variety of noxious stimuli. The magnitude and duration of the neuroinflammatory response, and hence its functional impact, are strongly dependent upon the nature of the stimulus with potential for both positive  and negative effects  on brain physiology and behavior. In the context of brain RT, the general consensus is that radiation induces neuroinflammation through activation of microglia, astrocytes, and endothelial cells in response to neural cell damage.5 The microglia-mediated neuroinflammation that ensues is similar in nature to that observed in a variety of neurological diseases including Alzheimer’s disease (AD), Parkinson’s disease (PD), cerebral ischemia, multiple sclerosis (MS), and amyotrophic lateral sclerosis.6Beyond this well-documented phenomenon, the precise mechanisms of RT-induced neurotoxicity are still being investigated and appear to be multiple and complex. Recently, emerging evidence cites radiation-induced neuroinflammation as a critical predictor of the long-term effects of RT on cognitive decline.7 To a lesser extent, PARP proteins also interact with kinases such as ataxia–telangiectasia mutated (ATM), nibrin (NBS1), and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to mediate DSB repair through the homologous recombination (HR) and nonhomologous end joining (NHEJ) repair pathways.7One of the hallmarks of both the DDR and neuroinflammation is activation of the poly(ADP-ribose) polymerase (PARP) family of proteins. PARP proteins are well characterized for their integral role in the DDR, primarily initiating base excision repair (BER) in response to SSBs. Binding of PARP-1 to DNA breaks activates its catalytic function, which is to generate long, branching chains of poly(ADP-ribose) (PAR) on itself and a variety of target proteins. These modifications stimulate recruitment of BER-associated proteins and dissociation of PARP-1 from the damaged sites, promoting DNA repair and cell survival.8–10 However, this role for PARP has not been studied in the context of radiation-induced neuroinflammation. PARP hyperactivation is implicated in several diseases sharing inflammatory/immune-mediated pathways including those associated with cancer progression and neurodegeneration.11 Therefore, PARP activity represents an intriguing mechanistic link between radiation-induced activation of the DDR and subsequent neuroinflammation.The mechanisms linking PARP activity to neuroinflammation are complex and less well characterized than its DDR functions. Accumulating evidence implicates PARP-mediated secretion of inflammatory mediators from damaged immune cells as the primary mechanism in multiple CNS diseases, including stoke, brain trauma, and neurodegenerative processes.12–15 Likewise, multiple studies in various preclinical tumor models have demonstrated that PARP inhibitors (PARPi) have modest but consistent radiosensitizing effects; indeed at least 3 PARPi are currently undergoing early phase clinical evaluation in a variety of cancer types including GBM.16 While PARPi have been found to exacerbate acute radiation toxicity in rapidly proliferating normal tissues such as the esophageal or oropharyngeal mucosa,17 early data indicate that they can be combined with brain RT without adverse effects.19 Utilizing PARPi in combination with brain RT offers the exciting prospect of sparing normal tissue from radiation-induced neuroinflammation whilst enhancing tumor responses to radiation. However, our current understanding of the suppressive effects of PARP inhibition on neuroinflammation is constrained by the overreliance on the use of animal models harboring PARP genetic defects (KO) and the relative lack of pharmacological PARPi capable of penetrating the brain parenchyma. PARPi utilized in clinical and preclinical studies block the enzymatic function of PARP by competing with its nicotinamide adenine dinucleotide (NAD+) substrate at the catalytic domain. This interferes with the DDR by suppressing BER function but also by preventing automodification of PARP thus delaying its release. This phenomenon of “PARP trapping” is increasingly recognized as playing a key mechanistic role in the therapeutic effects of PARPi, and accounts for many of the differences observed between pharmacological inhibition and genetic depletion of PARP.20Despite the relative lack of mechanistic understanding, there is extensive data showing that pharmacological inhibition or genetic depletion of PARP exerts beneficial effects by alleviating neuroinflammation in several animal models of inflammatory CNS conditions including stroke, traumatic brain injury, meningitis, and experimental autoimmune encephalomyelitis (EAE).The aim of this review is to provide an overview of the physiological and clinical impact of radiation-induced neuroinflammation and the specific role of PARP enzymes in this response. Of the many PARP family proteins, PARP-1 is by far the most abundant, accounting for up to 90% of poly(ADP-ribosylation) in most cells studied to date. PARP-2 and PARP-3 are much less abundant but have been shown to make measurable contributions to the DDR. Most studies in the neuroinflammation field have focused on PARP-1. Of note, the vast majority of PARPi currently available are active against both PARP-1 and PARP-2. This review will focus primarily on the role of PARP-1 in the pathophysiology of radiation-induced neuroinflammation. We intend to emphasize the importance of PARP hyperactivation in neuroinflammation and discuss the clinical significance of PARPi both as radiosensitizing agents in GBM cells and as potential mitigators of radiation-induced and inflammatory-mediated neurotoxicity.5 Neuroinflammation is a complex process, the nature and extent of which depend on the context, duration, and anatomical location of the primary insult. Although the neuroinflammatory response has evolved to mitigate triggering factors in order to restore and maintain homeostasis, it can also be damaging and may result in adverse physiological, biochemical, and behavioral consequences. This inappropriate response is not well understood but seems to be a consequence of the complex mechanisms controlling microglial activation in different brain regions.The cellular and molecular mechanisms involved in radiation-induced brain injury and secondary cognitive dysfunction are beginning to be elucidated. Although vascular abnormalities, demyelination, NPC death, decreased neurogenesis, and direct glial activation play important roles, recent studies have revealed that irradiation (IR) can trigger immune responses within the CNS, leading to chronic neuroinflammation.5 For many years, neuroinflammation was considered a self-contained process, predominantly regulated by astrocytes and resident immune cells such as microglia; this contributed to the dogma that the CNS was an “immune-privileged” organ. However, research in the last few years has clearly demonstrated that this is not the case. Neuroinflammation involves interplay between several resident CNS cell types and peripheral immune cells that migrate into the brain parenchyma through the blood–brain barrier (BBB), the permeability of which is often compromised upon endothelial cell insult and subsequent activation.5 These discoveries have stimulated a comprehensive reevaluation of neuroinflammatory responses in neurodegenerative diseases, traumatic brain injury and, to a lesser extent, the pathophysiology of radiation-induced brain injury.The pathophysiology of the neuroinflammatory response to ionizing radiation involves primary structural damage and secondary effects on cell dysfunction that lead to progressive changes and associated cognitive decline long after the initial injury.21 Microglia can become activated by alarmins secreted by neurons and endothelial cells that have suffered radiation damage. One well-described example is high mobility group box 1 (HMGB1), expression of which is upregulated following IR exposure and thought to contribute to microglial activation by binding to the toll-like receptor 4.22 Another possible mechanism for microglial activation is by direct IR-induced DNA damage causing modification of transcription factors such as nuclear factor κB (NF-κB) and activating protein 1 (AP-1), which control expression of several genes involved in initiating the inflammatory cascade.23 This is followed by a surge in inflammatory mediators consisting of cytokines, chemokines, and reactive oxygen species (ROS), which are produced by microglia and astrocytes.5 These secretions promote microglial phagocytosis of damaged neural cells and particulate matter, resulting in increased neuronal and progenitor cell death. Inflammatory mediators secreted by microglia and astrocytes, such as interleukin-6 (IL-6), stimulate the endothelium to increase expression of adhesion molecules on the luminal surface, such as intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule (VCAM-1), compromising BBB integrity.24 Brain microvascular endothelial cells can also be directly activated by IR, increasing expression of adhesion molecules. Leukocytes then attach to endothelial cells and, together with microglia, secrete a panel of matrix metalloproteinases (MMPs) that destabilize the parenchymal basement membrane,24 enabling leukocyte migration across the BBB and perivascular spaces into the brain parenchyma to further aggravate neuroinflammation.25 Alarmins, such as HMGB1, also contribute to radiation-induced endothelial barrier injury by activation of the mitogen-activated protein kinase signaling pathway.22 Among chemokines, CCL2 allows infiltration of peripheral macrophages, altering the brain microenvironment and playing a prominent role in the development of radiation-induced cognitive alterations  exhibited an initial dynamic activation microglia that was followed by significant recovery after 1 week.29 This observation is in keeping with extensive clinical data showing that elderly patients are at increased risk of chronic radiation-induced neurotoxicity.30Microglial activation can persist for months after IR, creating a chronic and self-sustaining neuroinflammatory process characterized by a vicious cycle of microglial activation, secretion of proinflammatory agents, neuronal damage, and cell death.31 These and other studies have identified that selective inhibition of microglia-mediated neuroinflammation was able to ameliorate radiation-induced cognitive impairment, making it an interesting therapeutic target.27More specifically, neuroinflammation in the hippocampus has been shown to influence the functional integration of hippocampal neurons. Radiation-induced neuroinflammation disrupts microvascular niches in mouse models, depleting neurogenesis and causing long-term cognitive deficits such as memory loss.7 PARP also contributes to a wide range of cellular and biochemical pathways (reviewed in Weaver and Yang32) with increasing evidence indicating a critical role in modulating the inflammatory response both within and without the CNS.PARP-1 is best known for its participation in DNA repair processes, where it is involved chiefly in BER. It also plays a role in alternative NHEJ, which is thought to act as a backup pathway in cells deficient in canonical NHEJ, and interacts with the HR pathway both directly and indirectly via its role in maintenance of DNA replication fork stability.33 NF-κB is one of the best characterized transcription factors, regulating the expression of multiple genes involved in immunity and inflammation. Under basal conditions, NF-κB is localized in the cytoplasm, but once activated it undergoes nuclear translocation allowing DNA binding and increased transcription of inflammatory cytokines, chemokines, adhesion molecules, and inflammatory mediators including inducible nitric oxide synthase (iNOS), ROS, and tumor necrosis factor α (TNFα).34 Several studies have reported that nuclear translocation of NF-κB requires PARP-1 function.36 Following radiation-induced DNA damage, PARP binds to SSBs and recruits BER proteins to induce PARylation and initiate DNA repair. During this process, PARP undergoes automodification which promotes its disassociation from DNA and enables it to form a stable nucleoplasmic protein complex comprised of SUMO1, P1ASγ, NEMO (IKKγ), and ATM. ATM-mediated phosphorylation of NEMO triggers sumoylation of the inactive NF-κB complex in the cytoplasm and subsequent ubiquitination of NEMO from the activated complex. In the nucleus, PARP physically binds to the p65/p50 subunits through p300-CBP histone acetyltransferase, allowing NF-κB-driven transcription of proinflammatory molecules  and alarmins  is regulated by PARP-1.43 PARP-1 can be activated by DNA damage, usually secondary to reactive oxygen and nitrogen species (ROS/RNS), but also in response to ERK1/2-mediated phosphorylation as a consequence of raised intracellular Ca2+.44In vitro and in vivo studies have shown that the response of microglia and astrocytes to inflammation is mediated by PARP-1 and its activation stimulates protein synthesis and proliferation.46 Although the exact mechanisms are unclear, it has been proposed that PARP upregulates matrix metalloproteinase-9 (MMP-9) which digests tight junctions and basement membrane proteins, contributing to BBB disruption.47 PARP-mediated inflammation also increases the expression of adhesion molecules controlling leukocyte migration across the BBB.45 Interestingly, PARP activation in monocytes promotes their adhesion to the brain microvascular endothelium and subsequent migration across the BBB by promoting cytoskeletal rearrangements.25 PARP-1 also regulates microglia-mediated control of endothelial tight junction integrity influencing BBB permeability.48BBB dysfunction is also widely observed in neuroinflammatory-associated diseases. PARP-1 activity is heavily implicated in this phenomenon, with several studies citing associations between PARP activation, edema formation, and increased infiltration of peripheral immune cells into the brain parenchyma.+ followed by adenosine triphosphate (ATP) depletion that results in cell death and necrosis, promoting further microglia activation.9 At the same time, excessive PARP-1 activation also leads to PAR accumulation in the cytoplasm, translocation of apoptosis-inducing factor from the mitochondria to the nucleus and cell death by parthanatos, which is a specific form of cell death that occurs as a result of overactivation of PARP-1.49Finally, PARP-1 is a key mediator of neuronal cell death associated with excitotoxicity, ischemia, and oxidative stress. Hyperactivation of PARP-1 can lead to energy failure due to consumption of NADAs previously mentioned, hyperactivation of PARP can be detrimental, causing neuronal death as well as chronic microglial activation and neuroinflammation. These observations led to the evaluation of PARPi as potential mitigators of neurotoxicity in animal models of CNS pathologies in which neuroinflammation plays a key role.50–57 Ischemia followed by reperfusion appears to be a potent stimulus of PARP overactivation which results in neuronal death, as described above.57 In addition to reducing necrosis and apoptosis in stroke models, inhibition of PARP has been reported to reduce inflammation, astrogliosis, and microglia activation.59 PARP inhibition decreases proinflammatory cytokines such as interferon gamma (IFNγ), TNFα, IL-6, and IL-17 and increases anti-inflammatory cytokines such as IL-4, IL-10, and transforming growth factor beta 1 (TGFβ1).61 It also reduces the levels of transcription factors such as NF-κB and inflammatory prostaglandins such as cyclooxygenase 2.61 Regulatory T cells (Tregs) play a modulatory role in immune responses and can improve outcomes after ischemic strokes. One recent study showed that PARP inhibition upregulates circulating Treg cells, which could help alleviate neuroinflammation by altering cytokine levels.60 PARP inhibition has also been shown to protect the vasculature and decrease BBB damage by downregulating VCAM-1, ICAM-1, E-selectin, and MMP-9 expression and reducing extravasation of immunoglobulins.61–63Many studies have empirically demonstrated protective effects of various PARPi in rodent and primate models of stroke.64–70 ROS/RNS produced after the initial trauma cause upregulation of PARP activity leading to secondary additional neuronal injury which can be reversed by PARP inhibition.71 Apart from the attenuation of early neuronal injury, PARP inhibition in brain trauma models also reduces microglial activation and reactive astrogliosis in rodent and pig (Sus scrofa) models.72 As in stroke models, it has been shown that PARP inhibition decreases levels of NF-κB in the cortex, reducing inflammation and downregulating MMP-9 thus preserving BBB function.64The neuroprotective properties of PARPi have also been extensively studied in animal models of traumatic brain injury.73–77 PARP inhibition attenuated oligodendrocyte depletion and decreased demyelination in a rodent model that uses cuprizone.74 Of direct clinical relevance, high PARP-1 expression has been identified in blood leukocytes from patients with MS and PARP inhibition was associated with decreased expression of factors such as TGFBR1/TGFBR2/BCL6 in B cells.73 As in stroke models, the immunomodulatory effects of PARPi appear to involve both increased numbers of Tregs and enhanced function.76 Consistent with these findings, expression of ICAM-1, NF-κB, and the inflammatory mediators interferon-γ, TNFα, and iNOS were decreased in CNS tissues from rodent models treated with PARPi.76The role of PARP in neuroinflammatory disease has also been studied in MS and its preclinical model experimental autoimmune encephalitis (EAE), as well as bacterial meningitis.78–82 Reduced microglial activation in response to PARP inhibition has been reported in all these diseases.82Evidence supporting important pathogenic roles for PARP-1 now extends to neurodegenerative diseases, in which it has been observed to promote neuronal cell death, mitochondrial function, and neuroinflammation. Preclinical studies have shown therapeutic benefits of a variety of PARPi in rodent models of PD, AD, and Huntington’s disease.Much less evidence is available in the context of radiation-induced neuroinflammation. However, the intracellular mechanisms implicated in this phenomenon share many of the same signaling pathways as the CNS diseases described above, and there is a prominent inflammatory component. As described earlier, radiation has been shown to regulate NF-κB transcription factors in the brain, causing microglial activation and increasing transcription of inflammatory cytokines, chemokines, adhesion molecules, and inflammatory mediators including iNOS, ROS, and TNFα. All these factors are implicated in the many neurological conditions in which PARP inhibition has shown promising results. In this context, it seems entirely reasonable to propose that PARP inhibition could play a neuroprotective role in suppressing radiation-induced neuroinflammation and thus reducing the associated cognitive decline.84 In keeping with these roles, PARP inhibition causes accumulation of DNA breaks in replicating cells and PARPi have potent single agent activity against cells harboring defects in HR repair.85 Of greater relevance to their potential roles in neuro-oncology, PARPi also exhibit potentiating effects when combined with DNA damaging agents such as TMZ and RT, which together form “standard of care” for GBM patients. Multiple studies in preclinical models have demonstrated that PARPi potently augment the cytotoxic effects of TMZ and consistently enhance radiosensitivity across a broad range of cancer types including GBM. Importantly, these radiosensitizing effects are only observed in proliferating cells, in which unresolved SSBs are converted into DSBs during replication, thus enhancing the cytotoxic effects of radiation.86 In the context of GBM, therefore, PARP inhibition is predicted to enhance the capacity of RT to ablate rapidly proliferating tumor cells while having minimal effect on cells in the surrounding normal brain, which are almost exclusively nonreplicating.As previously mentioned, PARP-1 plays an important role in DNA damage repair, predominantly by stimulating and facilitating function of the BER pathway, and with additional roles in alternative NHEJ, HR, and replication fork restart.88 However, a recent phase I clinical trial reported that olaparib  penetrates both tumor core and margin regions in patients with recurrent GBM, despite failing to penetrate the BBB of mice and rats. These findings support the concept that the BBB is sufficiently disrupted in and adjacent to GBM that even “nonbrain penetrant” PARPi may have clinical activity.89Until recently, one of the main limitations to evaluating PARPi in the treatment of brain tumors concerns their ability to penetrate the BBB. Some newly developed PARPi such as pamiparib (BeiGene) and niraparib  have shown improved BBB penetration in mice, making them attractive options.90–94 The radiosensitizing effects of PARPi have also been reported in other CNS tumors such as medulloblastoma and ependymoma.90Several studies using different PARPi provide strong evidence of radiosensitizing activity both in vitro and in vivo in a variety of preclinical models of adult and pediatric glioma.16These preclinical data underpin multiple phase I/II clinical trials that have been completed or are actively recruiting, evaluating 3 different PARPi  in combination with RT and/or TMZ in patients with gliomas, predominantly but not exclusively GBM. A comprehensive list of the current clinical trials can be found in a recent review.Taking all this clinical and preclinical together, we propose that the strategy of combining RT and PARP inhibition for the treatment of GBM holds exceptional promise for this cancer of extreme unmet need. Tumor cell death will be enhanced by inhibition of DNA repair pathways, whilst normal brain tissue may be protected both from radiation-induced neuroinflammation, via suppression of glial activation and inflammatory mediators, and from radiation-driven apoptosis of NPCs and neurons, via avoidance of ATP depletion .95 While one might speculate that PARP inhibition would further suppress microglial activity, thus protecting tumor cells, a recent abstract described the opposite effect.96 This illustrates the importance of assessing the neuroinflammatory effects of PARP inhibition in microenvironments that recapitulate the clinical scenario. More specifically, the presence of a tumor might alter the immunomodulatory effects of both radiation and PARP inhibition. Adding further complexity, combining DNA damaging agents such as radiation with PARPi has potential to enhance the immunogenicity of the tumor cells by promoting neoantigen release, increasing tumor mutational burden, and enhancing immunogenic signaling via the cGAS-STING pathway.97Having made this proposal, we would also like to acknowledge the crucial role played by the tumor microenvironment in influencing treatment responses of GBM and other brain tumors, and the additional layers of complexity that this creates. Tumor-associated macrophages and microglia have been shown to play an important role in creating an immunosuppressive tumor environment that protects tumor cells from recognition and destruction by the immune system.Considering these complex interactions, we would like to emphasize the urgent need to develop new, orthotopic, immunocompetent models of GBM that would allow detailed study of the multifaceted effects of PARP inhibition and their consequences on cell survival, neuroinflammation, and cognition in the context of a tumor bearing brain with an intact immune system.99 ATM inhibitors are potent tumor radiosensitizers that are currently being evaluated in clinical trials in GBM patients; they may have neuroprotective effects by modulating oxidative stress and preventing neuronal apoptosis.100 ATR and CHK1 exert modest radiosensitizing effects similar to PARPi.98 Neither ATM, ATR, nor CHK1 inhibitors have been shown to regulate neuroinflammation.While this review focuses on PARP, it is important to mention that other proteins of the DDR pathway, including ATM, ataxia–telangiectasia, and Rad3-related protein (ATR) and checkpoint kinase 1 (CHK1) have also shown radiosensitizing effects in preclinical models of GBM.Although, our understanding of the pathophysiology of radiation-induced cognitive dysfunction is currently limited, there is strong evidence supporting a major role of neuroinflammation. Recent data have identified neuroinflammation as a common feature in many CNS diseases including brain trauma, stroke, and multiple neurodegenerative processes, and preclinical studies have provided robust evidence to support the use of PARPi to alleviate neuroinflammation in these conditions. At the same time the use of PARPi as radiosensitizers for the treatment of brain tumors has generated promising preclinical data and multiple early phase clinical trials are ongoing. This, in combination with the availability of brain penetrant PARPi, offers the intriguing opportunity to radiosensitize brain tumor cells, while simultaneously reducing radiation-induced neuroinflammation. The translation of these exciting preclinical findings to the clinic offers the promise of improving survival and quality of life for brain tumor patients receiving RT. It will be important to include neuroinflammatory markers, cognitive function scores, and quality of life measures when designing future clinical trials, to help elucidate the potential neuroprotective role of PARPi when combined with radiation.


In zebrafish, the spatiotemporal development of the vascular system is well described due to its stereotypical nature. However, the cellular and molecular mechanisms orchestrating post-embryonic vascular development, the maintenance of vascular homeostasis, or how coronary vessels integrate into the growing heart are less well studied. In the context of cardiac regeneration, the central cellular mechanism by which the heart regenerates a fully functional myocardium relies on the proliferation of pre-existing cardiomyocytes; the epicardium and the endocardium are also known to play key roles in the regenerative process. Remarkably, revascularisation of the injured tissue occurs within a few hours after cardiac damage, thus generating a vascular network acting as a scaffold for the regenerating myocardium. The activation of the endocardium leads to the secretion of cytokines, further supporting the proliferation of the cardiomyocytes. Although epicardium, endocardium, and myocardium interact with each other to orchestrate heart development and regeneration, in this review, we focus on recent advances in the understanding of the development of the endocardium and the coronary vasculature in zebrafish as well as their pivotal roles in the heart regeneration process. The zebrafish is a popular small vertebrate model organism used to study embryonic development and, more recently, regeneration, as it is capable of functionally restoring appendages and organs after injury. In this review, we first describe the key stages of endocardium and coronary vessel development and then focus on how these different endothelial cell populations contribute to heart regeneration.ex utero development. Within 48 h post fertilisation (hpf), the major organs and body plan are completely formed, and the first circulatory loop is established by 24 hpf [In the past 25 years, the zebrafish model has facilitated a wealth of discoveries about angiogenesis and cardiac morphogenesis. It presents several advantages, such as the generation of large sample sets, with a single breeding pair producing ≥200 embryos per mating, their ease of genetic manipulation, and rapid early y 24 hpf . Zebrafiy 24 hpf ,4. https://zfish.nichd.nih.gov/Intro%20Page/intro1.html (accessed on 13 January 2021)) [Because zebrafish embryos are small in size and readily fit within conventional microscopy set ups, they are much easier to investigate than mammalian models. A seminal study by Isogai and colleagues, who used confocal microangiography to observe lumenised vessels, provided a comprehensive description of the first seven days of vascular development, publicly available online: (y 2021)) . Apart fThe generation of new transgenic fluorescent reporter lines, advances in microscopy techniques, and the development of morpholino technology, which transiently suppresses mRNA translation, allowed the initial characterisation of molecules central to the development of the vascular system. The use of live in vivo imaging of cardiovascular processes at single-cell resolution enabled the identification of the molecular mechanisms of vessel development in unprecedented detail. Studies describing how signalling pathways involving molecules such as Vascular endothelial growth factor (Vegf), Notch, and C-X-C motif chemokine ligand 12 (Cxcl12)—C-X-C chemokine receptor type 4 (Cxcr4) act together to establish the embryonic vasculature, as well as the discoveries of key angiogenic processes, such as pruning or lumenisation, and the role of perivascular cells have been extensively reviewed elsewhere ,10,11,12The use of morpholinos to simulate the “loss of function” of genes of interest involved in angiogenic processes has been challenged since the observation of Kok and colleagues that a number of genuine loss of function genetic mutant animals failed to phenocopy vascular deformations observed in the corresponding morpholino-injected animals . This caIn vertebrates, the cardiovascular system consists of the heart and all blood and lymphatic vessels in the body. It is the first organ system to become functional in the zebrafish embryo and is crucial to survival past 7 days post fertilisation (dpf). Heart tissue consists of three layers: the myocardium, epicardium, and endocardium. The myocardium is a functional syncytium made up of cardiomyocytes (CM) that contract in response to action potentials induced by pacemaker cells. The endocardium is the inner lining of the heart. It arises from vascular endothelial cells  and contcirca 24 hpf. Looping occurs from 36 hpf onwards and, by 48 hpf, the distinct curvatures of the atrium and ventricle can be identified. At 72 hpf, a superior valve leaflet has been generated, preventing retrograde blood flow, and by 5 dpf, the heart valve is fully functional, the epicardium fully covers the myocardium, and there is extensive trabeculation  and blood flow-responsive Heart development protein with EGF-like domains 1  are expressed at sites of high fluid shear stress such as the atrioventricular endocardium. They act there to suppress Kruppel-like factor 2a (klf2a) and notch1b expression during cushion formation [klf2a and notch1b activation [klf2 activity in the endothelial cells forming the valve and positively regulates yap1 in some endothelial cells and the smooth muscle cells surrounding the endothelium [notch1b [yap1 mutant embryos displayed valve defects, suggesting that smooth muscle cells contribute to valvulogenesis and require Yap1 to do so [Valve formation, cardiac conduction, ventricular trabeculation, and remodelling all contribute to the maturation of the heart into a fully functional organ and their molecular regulation is the subject of active research. Recently, it was shown that Krit1 ormation . Additiotivation . Two stutivation ,29. Piezothelium , indepento do so .Outflow tract development was described to be reliant on the flow responsive Transforming growth factor β (TGFβ) receptor, Activin A receptor-like type 1 (Acvrl1). Acvrl1 is essential for the accumulation of endocardial cells from aortic arches, the second source of endocardial cells forming the outflow tract . Other gThe endocardium is the layer of cardiac endothelial cells that lines the inner surface of the heart tube. Endocardial cells act as blood flow mechanosensors and are a source of angiocrine factors important in the regenerative response after cardiac injury. Endocardial cells also contribute to the overall development of the heart: they form the endocardial cushions, septa, and heart valves; they promote trabeculation, support cardiac conduction, participate in the remodelling of the outflow tract, and are the primary source of coronary vessels (recently reviewed in ). The exact origin of endocardial cells remains a source of debate and lineage studies suggest that it may differ between model organisms. In zebrafish, all endocardial and myocardial cells originate from the blastula . After gAfter this has occurred, endocardial cells remain on the inner lining but also contribute to different heart tissues, such as the heart valves , and developmental processes such as the trabeculation of the myocardium and the generation of the coronary vessels see . To contEndocardial cells also support trabeculation. In mice, this was recently reported to involve endocardial cells sprouting towards the myocardium, thus segmenting the ventricular chamber and encapsulating myocardial cells . In contgata5 gene [smarce1 mutants, endocardial cells fail to aggregate in the midline and the atrioventricular boundary is not maintained. Additionally, a study by El-Rass and colleagues found that disrupting platelet-derived receptor alpha (Pdgfra) caused very similar defects in cardiac fusion and endocardial migration to the midline, also affecting atrioventricular chamber development [Kmt2d-deficient zebrafish are a model of Kabuki Syndrome and mimic some of the congenital disorders observed in patients. Serrano and colleagues showed that Kmt2d, through the suppression of Notch signalling, enables endocardial cells to interact with myocardial cells and thus shape the ventricular lumen. Work from Bornhorst and colleagues further explored the biomechanics regulating overall heart growth during development [nkx2.5-deficient and wnt8a heat-shock overexpressing fish, Bornhorst et al. showed that myocardial size expansion causes mechanical tension in endocardial cell junctions, which is transduced by Cadherin-5, and thus causes the nuclear translocation of Yap1 to induce endocardial cell proliferation , and Wnt signalling ,53,54. Bta5 gene . In smarelopment . Anotherelopment . Kmt2d-delopment . The endelopment . Using neration  or Prospero Homeobox 1 (Prox1a) [It is important to note that, unlike mammals or birds, zebrafish do not form coronary vessels during embryogenesis. This is likely why this developmental process is little studied and in need of more attention by experts in the zebrafish cardiovascular development field. Similarities and differences across different model organisms were recently reviewed by Kapuria et al. . A semin(Prox1a) . More re(Prox1a) . Interesvegfaa-deficient zebrafish embryos, rescued by injection of vegfaa mRNA at one-cell-stage, were able to form coronary vessels normally. It is possible that vegfab and related genes compensate for this lack of angiogenic cue [What induces the initial outgrowth of the coronary vessels is unknown. One study demonstrated that coronary angiogenesis appears to be independent of Vegfaa, as enic cue . Similarenic cue . If and Despite the relative simplicity of the zebrafish heart compared to mammals—i.e., it only has one atrium and one ventricle—many processes involved in heart morphogenesis and homeostasis are conserved across species, making the zebrafish a useful tool for modelling congenital cardiovascular defects. Electrophysiological parameters of the zebrafish heart are also closely aligned with humans—e.g., the electrocardiogram of zebrafish is similar to humans with clearly distinguishable P, QRS, and T waves; ventricular action potentials are comparable; and the heart rate of adult zebrafish (145 bpm) is closer to the human heart rate (60–100 bpm) than that of mice (580–680 bpm) ,73. FurtCoronary blood vessel obstruction caused by atherosclerotic plaque rupture or embolism can leave the heart with insufficient blood supply and result in myocardial infarction (MI), more commonly referred to as a heart attack. It is estimated that 1 billion cardiomyocytes are lost during a heart attack in humans . FibrotiIn both the resection and cryoinjury models, a series of cellular events will take place to complete the regeneration of the damaged tissue , see Fig. inflammatory phase, during which local tissue necrosis and apoptosis result in the recruitment of leukocytes [First, immediately after the injury and during the next couple of days, an inflammatory response is accompanied by the activation of the endocardium and epicardium. This first step in the regenerative process can be described as the ukocytes ;reparative phase takes place. Activated endocardial and epicardial cells proliferate and “encase” the wound internally and externally, respectively. Epicardial cells undergo epithelial to mesenchymal transition and give rise to epicardial-derived cells (EPDCs) [Next, a  (EPDCs) , which,  (EPDCs) . As a reregenerative phase per se, pre-existing cardiomyocytes at the border of the injury dedifferentiate, re-express cardiac progenitor markers, and start to proliferate in response to paracrine molecules secreted by neighbouring cells [During the ng cells ,82;maturation phase occurs. The regenerated cardiomyocytes re-establish themselves as part of a functional cardiac syncytium and the scar is completely removed [Finally, a less well-studied  removed .The length of time necessary to complete all phases depends on the model used, with the regeneration following cryoinjury taking longer than the resection or the genetic ablation models because of the need to clear the necrotic tissue and fibrotic scar.To achieve complete regeneration and restore full cardiac function following MI, the reestablishment of the coronary vessel network is critical, as the delivery of oxygen and nutrients is essential to cater to the high metabolic demands of the heart ,85. In aIn the following part of this review, we discuss the cellular mechanisms and signalling pathways that have been reported to play essential roles in the restoration of the coronary vascular network and endocardial response in the regenerating zebrafish heart  . Neverthppargc1a) was reduced in actively sprouting and proliferating coronary ECs sorted from 96 hpci hearts [ppargc1a displayed increased coronary vessels density within the injured area, indicating its limiting role on EC proliferation [apelin, which is expressed in activated endothelial cells after injury [apelin and other glycolysis-promoting genes are regulated by hif expression in ECs in the ischemic heart [Endothelial cells in the cryoinjury border zone display an acute glycolytic switch. Expression analysis of the oxidative metabolism regulator gene, peroxisome proliferator-activated receptor gamma coactivator 1a  promoter [vegfaa−/− mutant fish displayed a reduced vessel density at the infarct zone, which was accompanied by a decrease in cardiomyocyte proliferation, permanent scar formation, and a failure to fully regenerate. Interestingly, vegfaa gene expression increases at 24 hpci, hours after the initial angiogenic sprouting; thus, Vegfaa bioavailability may originate from proteolytic release of local extracellular matrix-bound Vegfaa [vegfaa expression was reduced in 1-day post-amputation (dpa) zebrafish hearts that were treated with an anti-inflammatory glucocorticoid used to dampen the early inflammatory response, thus impairing revascularisation, and resulting in an incomplete regeneration [flt1−/− mutant zebrafish with enhanced Vegfaa signalling, the length and density of intra-coronary vessels in the injured area increased, and more cardiomyocytes were found to proliferate and associate with vessels [vegfaa drives CM hyperplasia and activates regenerative programmes in the endocardium (and epicardium) following cardiac injury [Vascular endothelial growth factor (VEGF) is a well-characterised and potent pro-angiogenic peptide essential for the development of the coronary vasculature ,101. It d Vegfaa  or couldd Vegfaa . Indeed,neration . Another vessels . Additioc injury . Therefore, the key role for Vegf in the angiogenesis/revascularisation processes previously described in other models was also validated in the zebrafish model of heart regeneration. As the number of specific zebrafish mutant lines increases, we are likely to find out more about the tight spatial and temporal control of Vegf and its role in heart repair.nrg1 activity was found to be sufficient to induce the heart regeneration programme and angiogenesis in uninjured hearts [reg cells recruited to the cardiac injury site also secrete Nrg1 and this was accompanied by a strong upregulation of its receptors  by CM [Neuregulin1 (Nrg1) is a transmembrane protein which, after proteolytic cleavage by metalloproteinases or A disintegrin and metalloproteinases (ADAM), becomes a signal peptide that binds to ErbB2 and ErbB4 receptor tyrosine kinases. Neuregulin signalling is essential for zebrafish cardiogenesis ,105 and d hearts . Gemberld hearts . Treg ceb) by CM . The exanotum1b and wif1 as Notch target genes that dampen Wnt signalling and ultimately boost CM proliferation [vegfc, id1, efnb2a, egr1), endothelial integrity , and endothelial cell differentiation . Interestingly, they also identified plasminogen activator inhibitor 1 (serpine1), a fibrinolysis inhibitor, as being upregulated upon Notch inhibition [Notch signalling is a well-characterised regulator of endothelial tip cell differentiation during mammalian and zebrafish developmental angiogenesis ,110,111.feration . Additiohibition . These studies demonstrate a central role for Notch in modulating CM proliferation and scar resorption via endocardial maturation. Secretion of endothelial paracrine factors, such as Wnt antagonists, to the myocardium is also key to successful cardiac repair ,113.Cxcr4a mutant zebrafish do not develop coronary vasculature and those few that survive to adulthood exhibit severely impaired cardiac regenerative capacity [hif1−/− zebrafish hearts displayed reduced epicardial cxcl12b expression after cryoinjury, along with a decrease in superficial coronary cell proliferation. Conversely, increasing Hif1a activity with Dimethyloxallyl Glycine (DMOG) induced endothelial proliferation, which led to the conclusion that hypoxia triggers epicardial cxcl12b expression via Hif1a [cxcr4a mutant hearts were largely devoid of new superficial coronaries at 7 dpci whilst control hearts were fully covered by this time point. This led them to postulate that the activated epicardium, secreting Cxcl12b, guides superficial coronary ECs expressing Cxcr4a to modulate revascularization [The EC chemokine receptor 4a (Cxcr4a) is essential for coronary vasculature guidance in zebrafish juvenile development. capacity . Marín-Jia Hif1a . They alrization . As descThe epicardium is a quiescent single mesothelial cell layer encasing the heart. It is activated upon cardiac injury. Epicardial cells start proliferating and a subpopulation displays morphological changes, migrates into the damaged tissue via a process called epicardial-to-mesenchymal transition (EpiMT), and thus participates in the regenerative process . The acttbx18 failed to migrate into the regenerating myocardium, coronary neovascularisation failed to occur, and a permanent scar was deposited [The supporting role of the activated epicardium in the revascularisation of the regenerating myocardium was initially demonstrated by Lepilina et al. . Fibrobleposited . Recent eposited . Erk phoeposited ; therefoeposited .fgfr1). Therefore, suppressing the early inflammatory response severely hinders the zebrafish heart regenerative capacity, at least in part via inhibiting Fgf signalling and its function in mediating revascularisation [Other cell types also mediate Fgf signalling in the zebrafish regenerating heart. Zebrafish treated with an anti-inflammatory glucocorticoid, thereby suppressing the early immune cells infiltration to the wound, had a significant reduction in cardiac regeneration capability. Huang and colleagues observed a decreased revascularisation of the injured tissue in parallel with a reduction of mRNA expression of fibroblast growth factor receptor 1  signalling pathway plays a critical pro-angiogenic role in zebrafish heart regeneration. An upregulation of esection . In theiesection ,115. Phaesection . Thus, Pnrps are strongly upregulated in the activated epicardium [nrp1a mutant fish, the impaired epicardial activation and a marked reduction in the number of new coronary vessels both likely contribute to the hindered scar resolution and delayed overall cardiac regeneration observed [The transmembrane glycoprotein neuropilins (NRP1 and NRP2) are co-receptors required for the development of the nervous and vascular system . Nrps moicardium , matchinicardium ,114. Nrpicardium . Becauseicardium ,132, it observed . Because Nrps are able to bind several co-receptors to mediate multiple ligands’ signalling pathways in different cell populations, but also because each Nrp isoform has two ohnologues in the zebrafish, compensatory mechanisms are likely to complicate the interpretation of the specific Nrp1 and Nrp2 roles in the zebrafish model. Further studies are needed to fully assess which parts of this complex signalling network contribute to cardiac regeneration.Runx1 loss of function affects multiple cell types within the regenerating heart but, in wild-type controls, was strikingly overexpressed by the epicardium and the endocardium following cryoinjury [runx1 mutants differs from wild-type control fish and its resorption is significantly faster. Interestingly, sorted Runx1-positive endothelial/endocardial cell populations were very heterogenous, with subsets of cells expressing collagens, EndoMT genes, and/or smooth muscle genes after injury. The runx1 mutation led to the loss of an endocardial cluster expressing smooth muscle cell myh11a and taglnI, potentially explaining the difference in scar composition [runx1 mutant fish also showed deregulated genes involved in fibrinolysis, including an increase in plasminogen receptor annexin A2 (anxa2) and a decrease in serpine1 expression in comparison to wild-type hearts. This led the authors to further suggest that, in addition to the differential scar composition, increased fibrinolysis in the runx1 mutant underlies the reduced amount of fibrin observed in the wound [Runx1 is a master transcription factor that plays a key role in modulating the proliferation of several cell types, both during development and adulthood. yoinjury . The scaposition . Additiohe wound .2O2). ROS, in turn, directly interact with key downstream molecules to trigger signalling in a broad range of cellular processes [2O2 catalysed by the NADPH-oxidase (Nox) enzymes, Duox and Nox2, promotes heart regeneration by a derepressing mechanism, whereby H2O2 weakens the activity of the redox-sensitive phosphatase Dusp6, thus increasing Erk1/2 phosphorylation in response to growth factor signalling [dusp6 expression in endothelial cells and the epicardium of regenerating zebrafish hearts [dusp6 mutant zebrafish hearts and this aligned with enhanced Erk phosphorylation and accelerated cardiac regeneration [2O2.Following zebrafish cardiac injury, the avascular damaged tissue becomes hypoxic and initiates an oxidative stress response via the production of reactive oxygen species (ROS) such as hydrogen peroxide -synthesising enzyme Aldh1a2 is rapidly expressed by the entire endocardium following resection of the apex of the heart before becoming localised to the injury zone the day after. The expression of eability . Aldh1a2ury site ,135. In Mammalian studies demonstrated that lymphatic vessels respond to myocardial infarction by increasing in length and density; the extent of this response is associated with an improved cardiac outcome ,138,139.An extensive cardiac lymphatic vascular network develops in mammalian embryonic hearts . In contvegfc expression until 42 dpci [de novo lymphatic vessels then sprout and form connections, providing the primary neolymphatic vessel cell source during the first 14 days after cardiac injury. Lymphatic vessels derived from the outflow tract take 1–3 weeks to reach the injury area after cryoinjury and contribute to a smaller proportion of lymphatic vasculature in the injured region [flt4 and sprout in response to Vegfc during lymphatic vessel development in the zebrafish; conversely, flt4−/−, vegfc+/− zebrafish hearts were unable to form neolymphatic vasculature in the cryoinjured area, resulting in increased scar tissue deposition and abolition of the regenerative capacity in cryoinjured zebrafish hearts [vegfc−/−, vegfd−/− double mutant zebrafish hearts failed to regenerate after cryoinjury and displayed a persistent scar [Apical resection generates a mild lymphangiogenic response, whereas neolymphangiogenesis is robustly stimulated globally in cryoinjured zebrafish ventricles, implicating differential requirements for lymphatic vessels when cardiac tissue is damaged in cryoinjury versus the removal of tissue in the resection model . Cryoinj 42 dpci ,146. Prod region . Leadingh hearts . A subseent scar . Vegfc−/− and vegfd−/− null zebrafish that fail to develop lymphatic vessels in the heart presented an accumulation of TUNEL staining in the injured tissue, indicating that dead cells remain in the injury site. These findings agree with a study by Harrison and colleagues that detected immune cells in the lymphatic vasculature of cryoinjured hearts but not in uninjured heart lymphatic vessels [Extensive inflammation, necrosis, and apoptosis of cardiac cells is observed following cardiac insult. The inflammation and cellular debris are resolved and subsequent scar resorbed in regenerating zebrafish hearts; thus, biological mechanisms orchestrating these clearance mechanisms must also influence heart regeneration . A study vessels . Further vessels . Re-establishment of the lymphatic vessels in the injured area of zebrafish hearts likely governs the clearance process during cardiac regeneration. Given that new blood vessels infiltrate the injured area within hours of initial cardiac insult and that LECs migrate alongside coronary vessels as a guidance ,145, it The zebrafish is a popular model organism that has allowed the observation of developmental processes in vivo in real time, greatly expanding our knowledge of the cardiovascular system. Increasingly sophisticated tools enable us to gain a better molecular and cellular understanding of how blood flow, contractility, and myocardial, epicardial, and endocardial cells all act together to generate a functional cardiovascular system. The importance of the revascularisation of the injured heart has only been elucidated recently. Indirect or direct impairment of the signalling molecules or cell types involved in revascularisation results in incomplete regeneration. Neovessel growth takes place very rapidly after cardiac damage and is a prerequisite for the subsequent regenerative processes. Multiple tissues and signalling pathways are key to achieving a mature vascular network, highlighting the importance revascularisation has on subsequent regeneration. Although several studies have identified angiogenic candidates that govern heart regeneration, it is still an undercharacterised field that warrants further investigation. The body of evidence gathered from studies thus far comes from different models of myocardial infarction. It is plausible that differential mechanisms of revascularisation between zebrafish models and regenerating mammalian models take place. To achieve translational benefits, it is important to tease out which pathways are conserved and able to stimulate revascularisation in adult mammalian hearts. Revascularisation is key to regeneration regardless of the organ, but it is only one of several processes that we need to fully understand and re-initiate if we are to successfully mend the broken heart.


Ig locus of B-cells in adaptive immunity. Many of the modifications alter base-pairing properties and may thus cause replicative and transcriptional mutagenesis. The best known and most studied epigenetic mark in DNA is 5-methylcytosine (5mC), generated by a methyltransferase that uses SAM as methyl donor, usually in CpG contexts. Oxidation products of 5mC are now thought to be intermediates in active demethylation as well as epigenetic marks in their own rights. The aim of this review is to describe the endogenous processes that surround the generation and removal of the most common types of DNA nucleobase modifications, namely, uracil and certain epigenetic modifications, together with their role in the development of hematological malignances. We also discuss what dictates whether the presence of an altered nucleobase is defined as damage or a natural modification.DNA of all living cells undergoes continuous structural and chemical alterations resulting from fundamental cellular metabolic processes and reactivity of normal cellular metabolites and constituents. Examples include enzymatically oxidized bases, aberrantly methylated bases, and deaminated bases, the latter largely uracil from deaminated cytosine. In addition, the non-canonical DNA base uracil may result from misincorporated dUMP. Furthermore, uracil generated by deamination of cytosine in DNA is not always damage as it is also an intermediate in normal somatic hypermutation (SHM) and class shift recombination (CSR) at the  The DNA of all living cells undergoes continuous structural and chemical alterations resulting from fundamental cellular metabolic processes and reactivity of normal cellular metabolites and constituents. Examples include base alterations caused by reactive oxygen species (ROS) from mitochondrial respiration, incorporation of non-canonical dNTPs in DNA replication, nonenzymatic DNA methylation by the cellular methyl donor S-adenosylmethionine (SAM), as well as hydrolytic decay of DNA in the aqueous environment of the cell. The resulting subtle modifications of DNA include oxidized bases, aberrantly methylated bases, and deaminated bases, the latter largely uracil from deaminated cytosine. In addition, the non-canonical DNA base uracil may result from misincorporated dUMP, since dUTP is a normal intermediate in the biosynthesis of dTTP ,2. Many Ig locus of B-cells in adaptive immunity. The seminal discovery of activation-induced cytosine deaminase  some two decades ago was the clue to understanding the mechanisms of SHM and CSR (The best known and most studied epigenetic mark in DNA is 5-methylcytosine (5mC), generated by methyltransferases that use SAM as a methyl donor, usually in CpG contexts. The oxidation products of 5mC are now thought to be intermediates in active demethylation, as well as epigenetic marks in their own rights. Furthermore, uracil generated by the deamination of cytosine in DNA is not always damage as it is also an intermediate in normal somatic hypermutation (SHM) and class shift recombination (CSR) at the  and CSR , althougIg genes at a rate almost a million times higher than spontaneous mutations rates in somatic cells. This process also requires one of the uracil DNA N-glycosylase (UNG)-proteins encoded by the UNG gene , which is the second most common hematologic malignancy. Moreover, MM is a progressive disease often preceded by an asymptomatic stage, monoclonal gammopathy of undetermined significance (MGUS), and by an intermediate stage, smoldering multiple myeloma (SMM). This provides a unique opportunity to study the multistep driver events of malignant evolution .Many modified nucleobases may have profound functional consequences for the cell. Their processing may be very different depending on whether the alteration is read as a DNA “damage” that must be repaired or a DNA “modification” that specifies a function. Damaged bases in DNA are most frequently processed by the BER pathway , which ic-Myc transcription [NEIL3−/− mice displayed impaired learning ability and memory retention, as well as reduced anxiety . Furthecription . Similarcription  and UNG cription . It is uiewed in ). Althouiewed in , and posiewed in .Mammalian DNA polymerases utilize dUTP and dTTP for DNA synthesis with equal efficiency . The inc4 abasic sites [U:A base-pairs are not directly mutagenic. However, their presence may lead to mutations when uracil is excised by uracil DNA glycosylase, leaving a cytotoxic and mutagenic abasic site. The mutagenicity of abasic sites resulting from incorporation of dUMP and removal of genomic uracil by UNG was demonstrated using yeast mutants defective in dUTPase and suppression of mutagenicity by simultaneous inactivation of Ung ,28. The ic sites ,31. AlthUNG−/− mice apparently replicate their DNA and grow normally and display no overt phenotype until late in life. Furthermore, isolated nuclei from UNG knockout cells [The rate of incorporation of dUMP in DNA replication, or in DNA repair processes, most likely depends on the dUTP to dTTP ratio, which displays surprisingly high variation between different cell types, both normal and malignant. In addition, drugs or knockdown of enzymes required for biosynthesis of dTTP increase dUTP/dTTP ratios several-fold. In unperturbed proliferating cancer cells in culture, the dUTP/dTTP ratio was found to be in the range of 0.01–0.05. However, in slowly proliferating or nonproliferating cells this ratio may be in the range of 0.5–1.9 and in human primary macrophages as high as 60 (reviewed in ). The exut cells , as wellut cells , do evenDNA methylation occurs mainly at C5 of cytosine and is involved in a range of diverse biological processes, including gene expression, which, in turn, has a profound impact on cellular identity and organismal fate. Methylation is catalyzed by DNA methyltransferases (DNMTs), which can be classified as de novo methyltransferases   and mainFinally, it has been suggested that canonical long patch BER as well as non-canonical mismatch repair may erase epigenetic methylation indirectly during the fill-in phase and that this may be initiated by AID-mediated deamination of unmodified cytosines ,46,47. A5hmC is thus a key player in active demethylation. Some data further suggest that it is not only a demethylation intermediate but also an epigenetic mark on its own . It has UNG gene so far precludes an evaluation of the possible development of lymphomas caused by UNG mutations in humans [Whereas mouse embryonal fibroblasts (MEFs) from UNG knockout mice and human lymphoblastoid cells display a 5–10-fold increase in genomic uracil , mouse on humans . Howevern humans .Furthermore, it has become apparent that UNG may also contribute to the development of B-cell malignancy, together with AID. Chromosomal translocations involving immunoglobulin switch regions is a hallmark of B-cell malignancies. In a mouse model, the generation of such translocations required both AID and UNG . FurtherReplication across U:G mispairs generated by AID directly generates C→T transition mutations observed in SHM. A lack of UNG increases the fraction of C→T mutations, as expected . GeneralIg genes also undergo high rates of mutation. Subsequent studies have demonstrated that AID deaminates cytosine moieties in 25% of the expressed genes of mouse germinal center B cells [Ig loci, but still much higher than the spontaneous mutation rate [One intriguing question was whether the unprecedented level of mutation is restricted to regions of the antibody genes or if expressed genes in activated B cells other than  B cells . Howeverion rate . It was ion rate . DysreguIg genes and/or in non-lymphoid cells, may contribute to genetic changes resulting in malignancy. Constitutive AID expression in transgenic mice causes the development of lymphomas [MYC) and the tumor suppressor gene TP53 [MYC gene induces MM in a mouse model of this malignancy [Aberrant AID expression, which can lead to genome-wide mutations in genes other than ymphomas . These aymphomas ,67,68. Fene TP53 . Moreovelignancy .TP53, strongly suggesting that it may be an early tumor-initiating event [Besides AID, there is another enzymatic source for the presence of uracil in DNA, namely, apolipoprotein B editing complex (APOBEC) enzymes. Of note, it was demonstrated that APOBEC upregulation was responsible for elevated uracil levels in cellular DNA and increased mutation rates in breast cancer cell lines . APOBEC3ng event  (revieweng event ). Of notng event .Recent studies concerning driver events in MM strongly suggest that aberrant AID activity, associated with a specific mutational landscape, is mostly involved in the tumor initiation, while later phases of MM development are linked to an APOBEC mutational process ,75. MoreGenerally, methylation of CpG islands limits binding of transcription factors at gene promoters. Global CpG hypomethylation is known to occur in human cancers and precancerous conditions, although hypermethylation is also observed at distinct loci   ,90.TET2 is among the most commonly mutated genes in adult myeloid malignancies, occurring in ~25% and ~50% of cases of myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML), respectively [TET2 is also mutated in MM [TET2 knockout mice developed lethal myeloid malignancies within their first year of life [TET mutations. The functional consequences of presumed aberrant epigenetic changes can be disease initiation and progression, the latter via interactions with other somatic mutations including those with a direct epigenetic impact or others that may have indirect effects. Recently published work has aimed to determine if there is a direct link between various mutations linked to TET activity and 5hmC, 5fC, 5caC, and 5hmU content with the goal to determine if such modifications can serve as predictive biomarkers of myeloid neoplastic disease process [DNA methylation is an important regulator of hematopoiesis, and several studies suggest that disturbed DNA methylation contributes to immune disorders and hematologic malignancies . TETectively ,94,95. Red in MM . However of life , a causa of life ). Nevert process .Accurate measurements of 5fC, 5caC, and 5hmU are challenging. The reasons for this include not only very low levels of these modified bases in mammalian genomes, but also levels of 5hmC 3–4 orders of magnitude higher, which impairs the detection and quantitation of less common metabolites. We recently developed a rapid, highly sensitive, and specific isotope-dilution automated two-dimensional ultra-performance liquid chromatography tandem mass spectrometry (2D-UPLC-MS/MS) method that is specifically tailored for simultaneous analysis of global levels of 5mC, 5hmC, 5fC, 5caC, and 5hmU (together with uracil and 8-oxodG) .TET2 mutations in patients with myeloid malignances (MDS) was 5fC [TET2 mutations were associated with decreased 5fC and 5caC but had only a moderate influence on 5hmC [TET2 mutations, likely resulting in a truncated protein, yielded similar epigenetic profiles. These results suggest that TET2 mutations alter protein structure and function in ways that weaken its activity specifically with respect to higher order oxidation products. This work is the first in vivo evidence for a direct link between endogenously generated TET2 mutations and the enzyme activity reflected in its product levels [Using this methodology, we found that the best predictor/marker of  was 5fC . It is e was 5fC  and thus on 5hmC . By usin on 5hmC  showed tt levels .TET2 mutations. More recently, 5fC was suggested to be a key epigenetic mark controlling gene expression [TET mutations focused on the 5hmC content as a first product of the dioxygenase reaction, emerging evidence suggests that levels of 5fC and 5hmU are also altered and may have pathogenetic significance. There is little consensus concerning links between TET mutations and clinical outcome. Therefore, the measurement of these alternately modified bases could potentially be helpful to improve accurate identification of the disease mechanisms and may prove valuable as prognostic and diagnostic tools in myeloid malignancies. To underline a meaning of the above described results in the context of future work, it should be mentioned that there is a relationship between MDS and MM evolution; it was found that MM patients had an 11-fold increased risk of myelodysplastic syndromes (MDS) and MGUS patients had an 8-fold increased risk of MDS [CCND1, CCND2, and MMSET, as well as at enhancers of CCND2 and MYC. The potential value of 5hmC as a prognostic marker was underscored by a recent study of circulating cell-free DNA from 184 newly diagnosed MM patients, in which 5hmC was mapped across an eight-gene panel and correlated with overall survival in the MM patients [5fC is enriched at active enhancers involved in tissue development/differentiation . This supression . In sum,k of MDS . A recenk of MDS . Here, gpatients .Ig gene diversification. Oxidatively generated derivatives of 5mC play an important role in the active process of DNA demethylation and may have specific regulatory functions. Our review demonstrates that for DNA the terms “damage” and “modification” have different implications for the cell, but what dictates whether the presence of an altered nucleobase is defined as damage, a lesion that may lead to malignant transformation, or a modification? Genomic uracil from misincorporated dUMP is not even a modified nucleobase, unlike deaminated C. As it is a normal intermediate in dTTP biosynthesis, it is present in significant amounts in cells and is probably incorporated in substantial amounts but rapidly removed, as outlined. It cannot be excluded that such incorporation has a physiological function yet to be understood.We propose that genomic uracil and aberrant profiles of demethylation intermediates might be driver events in the development of certain hematologic malignances. Intriguingly, genomic uracil and endogenously generated epigenetic DNA modifications seem to play important cellular regulatory roles (for a review see ); for exIt is also puzzling why a potentially mutagenic DNA damage may lead to malignancy in some tissues but not in others. What processes determine the outcome? Perhaps the epigenetic DNA nucleobase modifications, e.g., 5hmC and the higher order products of TETs as well as genomic uracil are more important than previously thought since they display considerable inter-tissue differences. Moreover, transcription factors or histone modifiers that sense these modified nucleobases likely vary among tissues and developmental stages and contribute to dictate outcome. Most likely, it is not only simply the level of damage, but also its location and context that is the key factor, as discussed in some detail in a previous review . The preThe differentiation of hematopoietic stem cells toward plasmocytes (PCs) is tightly regulated by epigenetic mechanisms, and flaws in this epigenetic control can result in various B cell related disorders including MM (reviewed in ). There


Approximately 70% of the cost of beef production is impacted by dietary intake. Maximizing production efficiency of beef cattle requires not only genetic selection to maximize feed efficiency ), but also adequate nutrition throughout all stages of growth and development to maximize efficiency of growth and reproductive capacity, even during gestation. RFI as a measure of feed efficiency in cattle has been recently accepted and used in the beef industry, but the effect of selection for RFI upon the dynamics of gestation has not been extensively studied, especially in the context of fluctuating energy supply to the dam and fetus. Nutrient restriction during gestation has been shown to negatively affect postnatal growth and development as well as fertility of beef cattle offspring. This, when combined with the genetic potential for RFI, may significantly affect energy partitioning in the offspring and subsequently important performance traits. In this review, we discuss: 1) the importance of RFI as a measure of feed efficiency and how it can affect other economic traits in beef cattle; 2) the influence of prenatal nutrition on physiological phenotypes in calves; 3) the benefits of investigating the interaction of genetic selection for RFI and prenatal nutrition; 4) how metabolomics, transcriptomics, and epigenomics have been employed to investigate the underlying biology associated with prenatal nutrition, RFI, or their interactions in beef cattle; and 5) how the integration of omics information is adding a level of deeper understanding of the genetic architecture of phenotypic traits in cattle. Feed is, by far, the largest financial burden for the beef industry . The cosCattle producers need to find the balance between meeting a herd’s feed or nutritional needs while doing so in a cost-effective manner. Analyzing the chemical composition of feed and forage to understand their nutrient content is another strategy for more optimal cow/calf feed management . This apIn addition to adopting optimized feed management strategies to reduce feed costs and increase profitability, genetic selection  has also been used to improve farm profitability. In particular, the selection for animals which eat less but produce the same amount of meat has attracted considerable attention from beef producers. One such measure of feed conversion efficiency is called residual feed intake (RFI). The use of RFI in livestock breeding programs is growing rapidly, especially in the beef industry, since those cattle which are genetically classified as Low-RFI  eat less and produce less methane per unit weight gain . HoweverAs noted earlier, the highest feed cost period typically occurs during winter when animals cannot eat fresh forage. In colder regions of the world such as Canada, northern regions of the United States and some European countries, winter usually coincides with the second half of a cow’s gestation period when the most critical nutrient needs of dams should be provided. Almost 75% of ruminant fetal growth occurs in late gestation, and therefore nutrient insults at this stage will have the greatest impact on fetal size  . In contAn improved understanding of the physiological basis of RFI, prenatal nutrition, and their effect upon progeny traits can be achieved through the help of multiple omics sciences including metabolomics, transcriptomics and epigenomics. This review focuses on: 1) the importance of RFI as a measure of feed efficiency and how it can affect other economic traits in beef cattle; 2) the influence of prenatal nutrition on physiological phenotypes in calves; 3) the benefits of investigating the interaction of genetic selection for RFI and prenatal nutrition; 4) how metabolomics, transcriptomics, and epigenomics have been employed to investigate the underlying biology associated with prenatal nutrition, RFI, or their interactions in beef cattle; and 5) how the integration of omics information is adding a level of deeper understanding of the genetic architecture of phenotypic traits in cattle.RFI is a feed efficiency measure calculated using the difference between an animal’s actual feed intake and its expected feed requirements for maintenance and growth over a specific time period . RFI is 2 = 0.29–0.46) in cattle, which makes it a good candidate for genetic improvement  is an important greenhouse gas that contributes to global warming. The global warming potential of methane is about 25-fold higher than that of CO2 , and emitted less daily methane  and CO2  compared to HRFI animals. Lower feed intake results in a host-mediated response in microbial communities  in LRFI animals favoring reduced methane production . ACTH is secreted from the anterior pituitary gland following a stressful event, which then stimulates the secretion of cortisol from the adrenal cortex , there are two stages of myogenesis in cattle development. During the first wave of myogenesis, which occurs within the first 2 months of gestation, the primary muscle fibres are formed . Since oSeveral studies have shown that poor prenatal nutrition is not only associated with reduced growth and body weight of cattle, but also negatively impacts carcass quality . GreenwoSeveral studies have been performed to understand the impacts of prenatal under- or over-nutrition on the health of cattle offspring. via transfer of maternal antibodies to the fetus through placenta and to offspring through high quality colostrum, is an important factor in determining the health of offspring  as well as events, exposures or phenomena occurring outside the cell or organism, which are dictated by the environment . As a reMetabolomics uses advanced analytical chemistry techniques to comprehensively detect and measure hundreds of small molecule metabolites in cells, biofluids, and tissues. Metabolomics uses a variety of analytical chemistry techniques including nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) as well as coupled techniques such as liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), and inductively coupled plasma-mass spectrometry (ICP-MS). Each technique enables the detection of specific classes of metabolites with differing levels of sensitivity and accuracy. Each technique has also been used to identify and quantify different metabolites within various bovine biofluids and tissues and all of these metabolites are available in public databases .via creatinine phosphate and typically is increased due to muscle breakdown, and therefore is an indicator biological pathways related to protein metabolism. In sheep, blood creatinine concentrations have been shown to have a negative correlation with fat depth and a positive correlation with muscle mass , carnitine , and beta-hydroxybutyrate (product of tissue fatty acid catabolism) have been found to be higher in the plasma of HRFI vs. LRFI beef cattle . Recentlvia a glucose tolerance test, and had higher levels of resting blood cholesterol and triglycerides than the non-restricted group. While a critical mass of metabolomic data has been collected and published on RFI, there is very little published, large-scale metabolomic data on prenatal diet restriction and their effects on progeny. Indeed, a literature review conducted by us revealed only a few studies that investigated metabolite levels or metabolite changes in the progeny of livestock or other animals that were fed restricted or enhanced diets. For example, While metabolomics allows one to explore downstream changes associated with physiological perturbations or phenotypic differences, transcriptomics is the next upstream step controlling metabolic and phenotypic differences that is amendable to large-scale quantification. Transcriptomics has been widely used in livestock studies , and utiAHSG], growth hormone receptor [GHR] and inhibin, beta A [INHBA]), were negatively correlated with RFI, and genes associated with fibrinolysis, coagulation, and development and regulation of cell differentiation , member2 [SERPINI2], superoxide dismutase 3, extracellular [SOD3], and glutathione S-transferase mu 1 [GSTM1]), were positively associated with RFI. Although all genes explained variation in RFI phenotype, different subsets of these genes explained more variation for high or low RFI groups, identifying the fact that genes may act differently at the ends of the RFI spectrum seen in cattle populations. Altogether, selective gene expression explained 32.2% of the variance in RFI across all animals in the study, and explained 76.3 and 79.5% of the variation in RFI in low and high RFI groups, respectively.Studies examining gene expression in relation to RFI are extensive, and the biological information they have generated has moved past the discovery stage. For example, expression values of selected genes are now being assessed to predict RFI phenotype. Longissimus dorsi muscle of LRFI compared to HRFI cattle, including peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) [PPARGC1A], upregulated in HRFI cattle, and uncoupling protein 3 [UCP3], upregulated in LRFI cattle. They also utilized a diet (high forage versus low forage) by RFI (high versus low) interaction to identify expression differences in ADP/ATP translocase T1 [ANT1] and cyto c oxidase II [COX II]. Both the A significant proportion in the variance of RFI is predicted to involve pathways important to mitochondrial function . Kelly eHBB], myxovirus resistance one interferon-inducible protein p78 [MX1], ISG15 ubiquitin-like modifier [ISG15], hect domain and RLD 6 [HERC6], and interferon-induced protein 44 [IFI44] have shown differential expression patterns in the liver between HRFI and LRFI heifers  and oxidative stress , L-carnitine, butyrylcarnitine, and propionyl-L-carnitine), in agreement with the results of studies where LRFI cattle showed potential differences in immune response and stress, and whole body chemical protein level compared to their HRFI counterparts , as well as insulin receptor [INSR], have been identified in at least one or several studies examining effects of prenatal nutrition in cattle , peroxisome proliferator activated receptor gamma [PPARG] (associated with adipocyte differentiation), and protocadherin 19 [PCDH19] (associated with immune function)  . Diniz eLongissimus thoracis muscle; mRNA abundance of MEF2A was higher in moderate prenatal diet-LRFI bulls compared to other three groups . With these two publications we start to get a glimpse on how cattle selected for divergent RFI react, or can be stoic to, environmental stressors. For example, LRFI bulls might be predisposed to responding to the differential prenatal diet in muscle tissue, but not in reproductive tissue. Alternatively, the timing of the prenatal nutritional insult may have had a greater effect upon muscle growth than testis development, and LRFI cattle might be poised to take greater advantage of more abundant prenatal nutritional resources than HRFI cattle.To date only two published articles have evaluated the impact of the interaction between both prenatal diet and RFI on gene expression. Epigenomics offers a layer of genomic regulation that can link environmental influences to transcriptomic changes, and ultimately differences in metabolites and phenotype. Epigenetics is defined as the study of heritable changes in gene expression, resulting from alterations in chromatin structure but not alterations in the DNA sequence . The epiIGF2 DMR2, IGF2/H19 ICR (intergenic control region), and IGF2R DMR2, between parental LRFI and HRFI heifer and steer calves. All three DMRs were hyper-methylated in the liver of LRFI steers. As well, IGF2/H19 ICR was hyper-methylated in the Longissimus dorsi muscle of LRFI steers at birth, providing some evidence that consistent hyper-methylation of these DMRs may underlie some variation in RFI.As RFI is moderately heritable , examiniIGF2  to identify gene-tissue pairs associated with production traits (https://cgtex.roslin.ed.ac.uk/). cGTEx was developed to help facilitate the functional understanding of genetic variants in the cattle genome. Considerable efforts are also being placed on the systematic measurement and organization of gene expression and other functional information such as single-cell atlases, areas of conserved genomic sequence across species, and the development of tools to test gene and genetic variant functionality, by the FAANG Consortium  in cattle have been performed to identify metabolomic and several classes of transcriptomic QTLs , and epigenetic information such as non-coding RNA QTLs, while genome wide methylation and histone modification data is becoming more readily available. Omic and other functional genetic information are contributing to the understanding of the genetic architecture of a phenotypic trait through its use to subset DNA variants that are more likely to be related to causal variants of trait variability. These subsets can then be used in GWAS and genomic prediction to explain greater amounts of heritability and have greater efficacy of use across populations . RNA-Seqn traits . This lansortium . These eMaximizing the efficiency and growth potential of beef cattle requires not only genetic selection  but also adequate nutrition throughout all stages of gestation in the developing fetuses. Although both RFI and maternal nutrition during gestation can affect progeny traits, our understanding of the biological mechanisms by which this occurs is still at a broad physiological level. Exploring the interaction between the genetic potential for RFI and an environmental stimulus such as prenatal nutrition will help the scientific community determine how selection for RFI affects the physiology of the gestating cow and her offspring, and how they both may be more or less resistant to environmental influences. An improved understanding of the influences of RFI, prenatal nutrition and their interaction, on progeny traits can be achieved with the help of multiple omics technologies, especially if differences in gross physical phenotypes are subtle, or are manifested later in life. Gene and pathway information generated by exposing high or low RFI cattle to different environments will also help identify those pathways or phenotypes that may be co-selected with low RFI, and that may negatively affect other production traits. Integration of data generated by omics technologies shows a great deal of promise to help animal scientists understand the molecular and biological regulatory mechanisms underlying any important physiological trait in cattle, and will contribute to the improvement of genetic merit cattle and the efficiency by which animal breeders can perform genetic selection.


Arabidopsis thaliana have provided insight into the molecular mechanisms that regulate the initiation, patterning, and maintenance of the cambium. In this review, the roles of intercellular signalling via mobile transcription factors, peptide–receptor modules, and phytohormones are described. Crosstalk between these regulatory pathways is becoming increasingly apparent, yet the underlying mechanisms are not fully understood. Future study of the interaction between multiple independently identified regulators, as well as the functions of their orthologues in trees, will deepen our understanding of radial growth in plants.The development of secondary vascular tissue enhances the transport capacity and mechanical strength of plant bodies, while contributing a huge proportion of the world’s biomass in the form of wood. Cell divisions in the cambium, which constitutes the vascular meristem, provide progenitors from which conductive xylem and phloem are derived. The cambium is a somewhat unusual stem cell population in two respects, making it an interesting subject for developmental research. Firstly, it arises post-germination, and thus represents a model for understanding stem cell initiation beyond embryogenesis. Secondly, xylem and phloem differentiate on opposing sides of cambial stem cells, making them bifacial in nature. Recent discoveries in  We review the literature describing the molecular mechanisms by which the vascular cambium is initiated and maintained in Arabidopsis. Plants exhibit an extraordinary capacity for growth and developmental plasticity, owing to their ability to maintain populations of continuously dividing stem cells in tissues called meristems. Two indeterminate meristems are established during embryogenesis. These are the root apical meristem (RAM), which gives rise to the subterranean root system, and the shoot apical meristem (SAM), from which all aerial tissues are derived. The elongation of plants along the apical–basal axis that results from RAM and SAM activity is termed primary growth. In addition, plants develop a variety of post-embryonic meristems, from which axillary branches, floral organs, and lateral roots are derived . As the Secondary growth arises from tightly controlled cell divisions in post-embryonic meristems known as the vascular and cork cambia. The vascular cambium, which is the focus of this review, gives rise to a network of interconnected transport cells and their supporting tissues, which span the entire primary plant body and its lateral organs. Uniquely among plant meristems, the vascular cambium harbours a single, bifacial stem cell in each radial file, which divides periclinally  to drive the development of distinct specialized tissues on opposing sides .Xylem tissue is composed of conductive vessel elements that become hollow and vertically connected following programmed cell death  contributes to radial growth by producing protective tissue , D, E. IWhile secondary growth is evidently important for plant development, the position of cambial meristems deep within internal tissues has hampered efforts to study their activity. Nevertheless, advances in plant genetics, tissue processing, and microscopy have recently accelerated vascular development research  and LOG4, generating a cytokinin gradient that peaks in the xylem-adjacent procambium (DNA-BINDING ONE FINGER2.1 (DOF2.1), which drives procambial cell division alongside its two homologues, TMO6 and DOF6 (ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 6 (AHP6), a repressor of cytokinin signalling, is cell autonomously induced by LHW–TMO5 which act downstream of auxin signalling to maintain the primary xylem in a non-dividing state  transcription factors were found to be necessary for the establishment of primary vasculature in Arabidopsis embryos  proteins, which promote the division of protophloem sieve elements in young roots  . In a wild-type stem, 5–8 fascicles are derived from procambial cells below the SAM and arranged around a central pith , as is t sheath) . The preFollowing the initiation of secondary growth, cell to cell communication remains integral for maintaining the activity of the vascular cambium. Coordinated regulation of cambial cell division and differentiation requires integration of multiple external signals, including those from the extracellular matrix and adjacent cell layers . The molPXY has two close homologues in Arabidopsis, PXY-LIKE 1 (PXL1) and PXL2 (Peptide perception by transmembrane receptor(-like) kinases (RK/RLK) is a key signalling mechanism underpinning vascular cambium activity. The ligand–receptor pair comprising TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTOR (TDIF) and PHLOEM INTERCALATED WITH XYLEM (PXY), also known as TDIF RECEPTOR (TDR), is an extensively studied and multifunctional signalling module in this context . As partPXY is expressed in vascular tissues throughout the plant body, including in leaf veins, inflorescence stems, hypocotyls, and root steles. This expression specifically localizes to the xylem side of the vascular cambium (CLAVATA3/ENDOSPERM SURROUNDING REGION 41 (CLE41), CLE42, and CLE44  promoter triggered disorganized vascular development  showed reduced expression of the xylem development marker, IRX3  transcription factor genes, WOX4 and WOX14  and WOX14 were identified as positive regulators of procambium activity in Arabidopsis roots  resulted in a loss of the characteristic phloem arc and reduced expansion of fascicles along the radial axis  enzymes, required for cytokinin biosynthesis  and ERL2  expression of ER  and NST3, were up-regulated in er erl1 mutants  . Followipxy single or pxf triple mutants, hypocotyl vascular tissue developed in a disorganized manner, as exemplified by the occurrence of non-periclinal cell divisions  in a pxf background resulted in non-additive enhancement of organizational defects, this is indicative of a synergistic interaction between the two receptor families.In addition to their individual characterization, understanding the crosstalk between RLK-mediated signalling pathways is an important focus of ongoing vascular development research . ER was ERL1 and ERL2 was up-regulated in pxf er hypocotyls, suggesting that PXf and ER may ordinally interact to suppress ERL gene expression  to regulate cambial proliferation, while simultaneously inhibiting xylem fibre development through suppression of NAC transcription factors  and ARR15 expression  have been employed as models for understanding wood formation owing to their relatively rapid growth and availability of genomic resources  reportedly have organ-specific roles in the Arabidopsis stem and root. Nevertheless, key signalling components, such as PXY–TDIF and cytokinin, function similarly across the vascular system and may therefore represent conserved organizers and drivers of secondary growth. Indeed, there is increasing evidence that these components also regulate wood formation in divergent plant lineages. Tree species in the genus 4 peaks in the developing xylem of Populus trichocarpa (Pinus densiflora GA20-oxidase (PdGA20ox1) under constitutive or xylem-specific promoters triggered increased xylem width and cell number in hybrid poplar , are also implicated in wood formation. For instance, GA signalling is crucial for triggering the xylem expansion phase of secondary growth of Arabidopsis hypocotyls (Populus. In cryosections of hybrid aspen stems (Populus tremula×tremuloides), a gradient of IAA (a major bioactive auxin) was detected in developing vascular tissue, with a peak in the cambium and decreasing concentration on either side , the Populus orthologue of Arabidopsis REV, was up-regulated following the transition of stems to secondary growth, while expression of an miRNA-resistant form of PRE resulted in aberrant vascular patterning, including polarity defects and ectopic cambium initiation  to either the cambium or the phloem  are specifically expressed in secondary phloem , followed by in situ hybridization, revealed restricted expression of RsHB8, RsWOX4a, and RsPXY to a subset of root cambial cell layers  family. As ethylene positively regulates secondary growth  receptors to promote phloem initiation  of the world’s biomass, with stems and tree trunks alone thought to contribute 70% of this . While tJPE gratefully acknowledges support from the Biotechnology and Biological Sciences Research Council (Grant: BB/V008129/1).