Wake-like skin patterning and neural activity during octopus sleep.

While sleeping, many vertebrate groups alternate between at least two sleep stages: rapid eye movement and slow wave sleep1-4, in part characterized by wake-like and synchronous brain activity, respectively. Here we delineate neural and behavioural correlates of two stages of sleep in octopuses, marine invertebrates that evolutionarily diverged from vertebrates roughly 550 million years ago (ref. 5) and have independently evolved large brains and behavioural sophistication. 'Quiet' sleep in octopuses is rhythmically interrupted by approximately 60-s bouts of pronounced body movements and rapid changes in skin patterning and texture6. We show that these bouts are homeostatically regulated, rapidly reversible and come with increased arousal threshold, representing a distinct 'active' sleep stage. Computational analysis of active sleep skin patterning reveals diverse dynamics through a set of patterns conserved across octopuses and strongly resembling those seen while awake. High-density electrophysiological recordings from the central brain reveal that the local field potential (LFP) activity during active sleep resembles that of waking. LFP activity differs across brain regions, with the strongest activity during active sleep seen in the superior frontal and vertical lobes, anatomically connected regions associated with learning and memory function7-10. During quiet sleep, these regions are relatively silent but generate LFP oscillations resembling mammalian sleep spindles11,12 in frequency and duration. The range of similarities with vertebrates indicates that aspects of two-stage sleep in octopuses may represent convergent features of complex cognition.

Insulin regulates neurovascular coupling through astrocytes.

Mice with insulin receptor (IR)-deficient astrocytes (GFAP-IR knockout [KO] mice) show blunted responses to insulin and reduced brain glucose uptake, whereas IR-deficient astrocytes show disturbed mitochondrial responses to glucose. While exploring the functional impact of disturbed mitochondrial function in astrocytes, we observed that GFAP-IR KO mice show uncoupling of brain blood flow with glucose uptake. Since IR-deficient astrocytes show higher levels of reactive oxidant species (ROS), this leads to stimulation of hypoxia-inducible factor-1α and, consequently, of the vascular endothelial growth factor angiogenic pathway. Indeed, GFAP-IR KO mice show disturbed brain vascularity and blood flow that is normalized by treatment with the antioxidant N-acetylcysteine (NAC). NAC ameliorated high ROS levels, normalized angiogenic signaling and mitochondrial function in IR-deficient astrocytes, and normalized neurovascular coupling in GFAP-IR KO mice. Our results indicate that by modulating glucose uptake and angiogenesis, insulin receptors in astrocytes participate in neurovascular coupling.

Generation of Periventricular Reactive Astrocytes Overexpressing Aquaporin 4 Is Stimulated by Mesenchymal Stem Cell Therapy.

Aquaporin-4 (AQP4) plays a crucial role in brain water circulation and is considered a therapeutic target in hydrocephalus. Congenital hydrocephalus is associated with a reaction of astrocytes in the periventricular white matter both in experimental models and human cases. A previous report showed that bone marrow-derived mesenchymal stem cells (BM-MSCs) transplanted into the lateral ventricles of hyh mice exhibiting severe congenital hydrocephalus are attracted by the periventricular astrocyte reaction, and the cerebral tissue displays recovery. The present investigation aimed to test the effect of BM-MSC treatment on astrocyte reaction formation. BM-MSCs were injected into the lateral ventricles of four-day-old hyh mice, and the periventricular reaction was detected two weeks later. A protein expression analysis of the cerebral tissue differentiated the BM-MSC-treated mice from the controls and revealed effects on neural development. In in vivo and in vitro experiments, BM-MSCs stimulated the generation of periventricular reactive astrocytes overexpressing AQP4 and its regulatory protein kinase D-interacting substrate of 220 kDa (Kidins220). In the cerebral tissue, mRNA overexpression of nerve growth factor (NGF), vascular endothelial growth factor (VEGF), hypoxia-inducible factor-1 (HIF1α), and transforming growth factor beta 1 (TGFß1) could be related to the regulation of the astrocyte reaction and AQP4 expression. In conclusion, BM-MSC treatment in hydrocephalus can stimulate a key developmental process such as the periventricular astrocyte reaction, where AQP4 overexpression could be implicated in tissue recovery.

Kidins220 deficiency causes ventriculomegaly via SNX27-retromer-dependent AQP4 degradation.

Several psychiatric, neurologic and neurodegenerative disorders present increased brain ventricles volume, being hydrocephalus the disease with the major manifestation of ventriculomegaly caused by the accumulation of high amounts of cerebrospinal fluid (CSF). The molecules and pathomechanisms underlying cerebral ventricular enlargement are widely unknown. Kinase D interacting substrate of 220 kDa (KIDINS220) gene has been recently associated with schizophrenia and with a novel syndrome characterized by spastic paraplegia, intellectual disability, nystagmus and obesity (SINO syndrome), diseases frequently occurring with ventriculomegaly. Here we show that Kidins220, a transmembrane protein effector of various key neuronal signalling pathways, is a critical regulator of CSF homeostasis. We observe that both KIDINS220 and the water channel aquaporin-4 (AQP4) are markedly downregulated at the ventricular ependymal lining of idiopathic normal pressure hydrocephalus (iNPH) patients. We also find that Kidins220 deficient mice develop ventriculomegaly accompanied by water dyshomeostasis and loss of AQP4 in the brain ventricular ependymal layer and astrocytes. Kidins220 is a known cargo of the SNX27-retromer, a complex that redirects endocytosed plasma membrane proteins (cargos) back to the cell surface, thus avoiding their targeting to lysosomes for degradation. Mechanistically, we show that AQP4 is a novel cargo of the SNX27-retromer and that Kidins220 deficiency promotes a striking and unexpected downregulation of the SNX27-retromer that results in AQP4 lysosomal degradation. Accordingly, SNX27 silencing decreases AQP4 levels in wild-type astrocytes whereas SNX27 overexpression restores AQP4 content in Kidins220 deficient astrocytes. Together our data suggest that the KIDINS220-SNX27-retromer-AQP4 pathway is involved in human ventriculomegaly and open novel therapeutic perspectives.

CDCA7 finely tunes cytoskeleton dynamics to promote lymphoma migration and invasion.

Metastases, the major cause of death from cancer, require cells' acquisition of the ability to migrate and involve multiple steps, including local tumor cell invasion and basement membrane penetration. Certain lymphoid tumors are highly metastatic, but the mechanisms of invasion by lymphoma cells are poorly understood. We recently showed that CDCA7, a protein induced by MYC, is overexpressed in lymphoid tumors and that its knockdown decreases lymphoid tumor growth without inhibiting the proliferation of normal cells. Here we show that CDCA7 is critical for invasion and migration of lymphoma cells. Indeed, CDCA7 knockdown in lymphoma cells limited tumor cell invasion in matrigel-coated transwell plates and tumor invasion of neighboring tissues in a mouse xenograft model and in a zebrafish model of cell invasion. CDCA7 silencing markedly inhibited lymphoma cell migration on fibronectin without modifying cell adhesion to this protein. Instead, CDCA7 knockdown markedly disrupted the precise dynamic reorganization of actomyosin and tubulin cytoskeletons required for efficient migration. In particular, CDCA7 silencing impaired tubulin and actomyosin cytoskeleton polarization, increased filamentous actin formation, and induced myosin activation. Of note, inhibitors of actin polymerization, myosin II, or ROCK reestablished the migration capacity of CDCA7-silenced lymphoma cells. Given the critical role of CDCA7 in lymphoma-genesis and invasion, therapies aimed at inhibiting its expression or activity might provide significant control of lymphoma growth, invasion, and metastatic dissemination.

Cyclic nature of the REM sleep-like state in the cuttlefish Sepia officinalis.

Sleep is a state of immobility characterized by three key criteria: an increased threshold of arousal, rapid reversal to an alert state and evidence of homeostatic 'rebound sleep' in which there is an increase in the time spent in this quiescent state following sleep deprivation. Common European cuttlefish, Sepia officinalis, show states of quiescence during which they meet the last two of these three criteria, yet also show spontaneous bursts of arm and eye movements that accompany rapid changes in chromatophore patterns in the skin. Here, we report that this rapid eye movement sleep-like (REMS-like) state is cyclic in nature. Iterations of the REMS-like state last 2.42±0.22 min (mean±s.e.m.) and alternate with 34.01±1.49 min of the quiescent sleep-like state for durations lasting 176.89±36.71 min. We found clear evidence that this REMS-like state (i) occurs in animals younger than previously reported; (ii) follows an ultradian pattern; (iii) includes intermittent dynamic chromatophore patterning, representing fragments of normal patterning seen in the waking state for a wide range of signaling and camouflage; and (iv) shows variability in the intensity of expression of these skin patterns between and within individuals. These data suggest that cephalopods, which are mollusks with an elaborate brain and complex behavior, possess a sleep-like state that resembles behaviorally the vertebrate REM sleep state, although the exact nature and mechanism of this form of sleep may differ from that of vertebrates.

MAZ induces MYB expression during the exit from quiescence via the E2F site in the MYB promoter.

Most E2F-binding sites repress transcription through the recruitment of Retinoblastoma (RB) family members until the end of the G1 cell-cycle phase. Although the MYB promoter contains an E2F-binding site, its transcription is activated shortly after the exit from quiescence, before RB family members inactivation, by unknown mechanisms. We had previously uncovered a nuclear factor distinct from E2F, Myb-sp, whose DNA-binding site overlapped the E2F element and had hypothesized that this factor might overcome the transcriptional repression of MYB by E2F-RB family members. We have purified Myb-sp and discovered that Myc-associated zinc finger proteins (MAZ) are major components. We show that various MAZ isoforms are present in Myb-sp and activate transcription via the MYB-E2F element. Moreover, while forced RB or p130 expression repressed the activity of a luciferase reporter driven by the MYB-E2F element, co-expression of MAZ proteins not only reverted repression, but also activated transcription. Finally, we show that MAZ binds the MYB promoter in vivo, that its binding site is critical for MYB transactivation, and that MAZ knockdown inhibits MYB expression during the exit from quiescence. Together, these data indicate that MAZ is essential to bypass MYB promoter repression by RB family members and to induce MYB expression.

CDCA7 is a critical mediator of lymphomagenesis that selectively regulates anchorage-independent growth.

Tumor formation involves the acquisition of numerous capacities along the progression from a normal cell into a malignant cell, including limitless proliferation (immortalization) and anchorage-independent growth, a capacity that correlates extremely well with tumorigenesis. Great efforts have been made to uncover genes involved in tumor formation, but most genes identified participate in processes related to cell proliferation. Accordingly, therapies targeting these genes also affect the proliferation of normal cells. To identify potential targets for therapeutic intervention more specific to tumor cells, we looked for genes implicated in the acquisition of anchorage-independent growth and in vivo tumorigenesis capacity. A transcriptomic analysis identified CDCA7 as a candidate gene. Indeed, CDCA7 protein was upregulated in Burkitt's lymphoma cell lines and human tumor biopsy specimens relative to control cell lines and tissues, respectively. CDCA7 levels were also markedly elevated in numerous T and B-lymphoid tumor cell lines. While CDCA7 was not required for anchorage-dependent growth of normal fibroblasts or non-malignant lymphocytes, it was essential but not sufficient for anchorage-independent growth of lymphoid tumor cells and for lymphomagenesis. These data suggest that therapies aimed at inhibiting CDCA7 expression or function might significantly decrease the growth of lymphoid tumors.

Eyes Wide Shut: the impact of dim-light vision on neural investment in marine teleosts.

Understanding how organismal design evolves in response to environmental challenges is a central goal of evolutionary biology. In particular, assessing the extent to which environmental requirements drive general design features among distantly related groups is a major research question. The visual system is a critical sensory apparatus that evolves in response to changing light regimes. In vertebrates, the optic tectum is the primary visual processing centre of the brain and yet it is unclear how or whether this structure evolves while lineages adapt to changes in photic environment. On one hand, dim-light adaptation is associated with larger eyes and enhanced light-gathering power that could require larger information processing capacity. On the other hand, dim-light vision may evolve to maximize light sensitivity at the cost of acuity and colour sensitivity, which could require less processing power. Here, we use X-ray microtomography and phylogenetic comparative methods to examine the relationships between diel activity pattern, optic morphology, trophic guild and investment in the optic tectum across the largest radiation of vertebrates-teleost fishes. We find that despite driving the evolution of larger eyes, enhancement of the capacity for dim-light vision generally is accompanied by a decrease in investment in the optic tectum. These findings underscore the importance of considering diel activity patterns in comparative studies and demonstrate how vision plays a role in brain evolution, illuminating common design principles of the vertebrate visual system.

Protein kinase D activity controls endothelial nitric oxide synthesis.

Vascular endothelial growth factor (VEGF) regulates key functions of the endothelium, such as angiogenesis or vessel repair in processes involving endothelial nitric oxide synthase (eNOS) activation. One of the effector kinases that become activated in endothelial cells upon VEGF treatment is protein kinase D (PKD). Here, we show that PKD phosphorylates eNOS, leading to its activation and a concomitant increase in NO synthesis. Using mass spectrometry, we show that the purified active kinase specifically phosphorylates recombinant eNOS on Ser1179. Treatment of endothelial cells with VEGF or phorbol 12,13-dibutyrate (PDBu) activates PKD and increases eNOS Ser1179 phosphorylation. In addition, pharmacological inhibition of PKD and gene silencing of both PKD1 and PKD2 abrogate VEGF signaling, resulting in a clear diminished migration of endothelial cells in a wound healing assay. Finally, inhibition of PKD in mice results in an almost complete disappearance of the VEGF-induced vasodilatation, as monitored through determination of the diameter of the carotid artery. Hence, our data indicate that PKD is a new regulatory kinase of eNOS in endothelial cells whose activity orchestrates mammalian vascular tone.

Kidins220 accumulates with tau in human Alzheimer's disease and related models: modulation of its calpain-processing by GSK3ß/PP1 imbalance.

Failures in neurotrophic support and signalling play key roles in Alzheimer's disease (AD) pathogenesis. We previously demonstrated that downregulation of the neurotrophin effector Kinase D interacting substrate (Kidins220) by excitotoxicity and cerebral ischaemia contributed to neuronal death. This downregulation, triggered through overactivation of N-methyl-D-aspartate receptors (NMDARs), involved proteolysis of Kidins220 by calpain and transcriptional inhibition. As excitotoxicity is at the basis of AD aetiology, we hypothesized that Kidins220 might also be downregulated in this disease. Unexpectedly, Kidins220 is augmented in necropsies from AD patients where it accumulates with hyperphosphorylated tau. This increase correlates with enhanced Kidins220 resistance to calpain processing but no higher gene transcription. Using AD brain necropsies, glycogen synthase kinase 3-ß (GSK3ß)-transgenic mice and cell models of AD-related neurodegeneration, we show that GSK3ß phosphorylation decreases Kidins220 susceptibility to calpain proteolysis, while protein phosphatase 1 (PP1) action has the opposite effect. As altered activities of GSK3ß and phosphatases are involved in tau aggregation and constitute hallmarks in AD, a GSK3ß/PP1 imbalance may also contribute to Kidins220 decreased clearance, accumulation and hampered neurotrophin signalling from early stages of the disease pathogenesis. These results encourage searches for mutations in Kidins220 gene and their possible associations to dementias. Finally, our data support a model where the effects of excitotoxicity drastically differ when occurring in cerebral ischaemia versus progressively sustained toxicity along AD progression. The striking differences in Kidins220 stability resulting from chronic versus acute brain damage may also have important implications for the therapeutic intervention of neurodegenerative disorders.

Impact on prognosis of the regional distribution of MGMT methylation with respect to the CpG island methylator phenotype and age in glioma patients.

Clinical and molecular prognostic factors in gliomas include age, IDH mutation, the glioma CpG island methylator phenotype (G-CIMP+) and promoter methylation of the O(6)-methylguanine DNA-methyltransferase (MGMT) gene. Among these markers, a predictive value was reported in glioblastomas (GBM) for MGMT promoter methylation, in particular in elderly GBM patients. In this study, methylation data from 46 glioma samples with the Illumina 450K platform were obtained and extended using external data to include a total of 247 glioma samples. Methylation analysis of the whole MGMT gene with this platform revealed two strongly survival-associated CpG regions within the promoter and the gene body, which were confirmed in a reported dataset of high grade-gliomas. Methylation at the promoter (CpG 25, cg12981137 and the prognostic model MGMT-STP27) and at the gene body CpG 165 (cg07933035), were significantly associated with better overall survival, and strongly correlated with G-CIMP+ status. In this series, the prognostic value of MGMT methylation at the promoter was not observed in G-CIMP- cases, although around 50 % of them were MGMT-methylated. These results were also obtained in an homogeneously-treated series of chemoradiated G-CIMP- GBMs analyzed by MSP and qMSP, and confirmed in a reported pyrosequencing-analyzed series of gliomas. Interestingly, in contrast to the MGMT promoter, gene body methylation was of prognostic value in G-CIMP-patients older than 65 years. Our study highlights the relevance of the prognostic value of the different regions of methylation throughout the MGMT gene that could be affected by specific G-CIMP profiles and age groups.

Neuronal Galectin-4 is required for axon growth and for the organization of axonal membrane L1 delivery and clustering.

Axon membrane glycoproteins are essential for neuronal differentiation, although the mechanisms underlying their polarized sorting and organization are poorly understood. We describe here that galectin-4 (Gal-4), a lectin highly expressed in gastrointestinal tissues and involved in epithelial glycoprotein transport, is expressed by hippocampal and cortical neurons where it is sorted to discrete segments of the axonal membrane in a microtubule- and sulfatide-dependent manner. Gal-4 knockdown retards axon growth, an effect that can be rescued by recombinant Gal-4 addition. This Gal-4 reduction, as inhibition of sulfatide synthesis does, lowers the presence and clustered organization of axon growth-promoting molecule NCAM L1 at the axon membrane. Furthermore, we find that Gal-4 interacts with L1 by specifically binding to LacNAc branch ends of L1 N-glycans. Impairing the maturation of these N-glycans precludes Gal-4/L1 association resulting in a failure of L1 membrane cluster organization. In all, Gal-4 sorts to axon plasma membrane segments by binding to sulfatide-containing microtubule-associated carriers and being bivalent, it organizes the transport of L1, and likely other axonal glycoproteins, by attaching them to the carriers through their LacNAc termini. This mechanism would underlie L1 functional organization on the plasma membrane, required for proper axon growth.

Biomarker testing strategies in non-small cell lung cancer in the real-world setting: analysis of methods in the Prospective Central Lung Cancer Biomarker Registry (LungPath) from the Spanish Society of Pathology (SEAP).

AIMS: The aim of this study is to extend the analysis of the Lung Cancer Biomarker Testing Registry (LungPath), by analysing the techniques used in the determination of epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), c-ros oncogene 1 (ROS1) and programmed death ligand-1 (PD-L1) for the diagnostic of patients with advanced non-small-cell lung cancer (NSCLC). METHODS: Information of the technique used for the determination of EGFR, ALK, ROS1 and PD-L1 was recorded from March 2018 to January 2019 from 44 centres, but only 34 centres matched with the 38 centres previously analysed, allowing to analyse the techniques used in 8970 matched determinations of EGFR, ALK, ROS1 and PD-L1. Therefore, a by-centre analysis studied the level of implementation of the techniques in the 44 centres, while a by-determination analysis made it possible to assess the overall frequency of the techniques used on the 9134 matched samples. RESULTS: By-centre analysis showed that only 46.5% and 25.6% of the centres used reflex strategies for ALK and ROS1 determination, respectively. By-determination analysis showed that 94.4% of EGFR determinations were performed by PCR, 80.7% of ALK determinations were performed by IHC with clone D5F3, while 55.7% of ROS1 determinations were performed by IHC with clone D4D6. 22C3 were the PD-L1 clone more used (43.5%) followed by SP263 clone (31.1%). CONCLUSIONS: The real-world evidence obtained from LungPath shows the effort of Spanish hospitals in performing biomarker determination in NSCLC with different methodologies despite that next-generation sequencing (NGS) utilisation in the year of the analysis was low. Biomarker determination results could be optimised with the incorporation of sequencing methods such as NGS in pathology departments.

Kidins220 sets the threshold for survival of neural stem cells and progenitors to sustain adult neurogenesis.

In the adult mammalian brain, neural stem cells (NSCs) located in highly restricted niches sustain the generation of new neurons that integrate into existing circuits. A reduction in adult neurogenesis is linked to ageing and neurodegeneration, whereas dysregulation of proliferation and survival of NSCs have been hypothesized to be at the origin of glioma. Thus, unravelling the molecular underpinnings of the regulated activation that NSCs must undergo to proliferate and generate new progeny is of considerable relevance. Current research has identified cues promoting or restraining NSCs activation. Yet, whether NSCs depend on external signals to survive or if intrinsic factors establish a threshold for sustaining their viability remains elusive, even if this knowledge could involve potential for devising novel therapeutic strategies. Kidins220 (Kinase D-interacting substrate of 220 kDa) is an essential effector of crucial pathways for neuronal survival and differentiation. It is dramatically altered in cancer and in neurological and neurodegenerative disorders, emerging as a regulatory molecule with important functions in human disease. Herein, we discover severe neurogenic deficits and hippocampal-based spatial memory defects accompanied by increased neuroblast death and high loss of newly formed neurons in Kidins220 deficient mice. Mechanistically, we demonstrate that Kidins220-dependent activation of AKT in response to EGF restraints GSK3 activity preventing NSCs apoptosis. We also show that NSCs with Kidins220 can survive with lower concentrations of EGF than the ones lacking this molecule. Hence, Kidins220 levels set a molecular threshold for survival in response to mitogens, allowing adult NSCs growth and expansion. Our study identifies Kidins220 as a key player for sensing the availability of growth factors to sustain adult neurogenesis, uncovering a molecular link that may help paving the way towards neurorepair.

PKD phosphorylation and COP9/Signalosome modulate intracellular Spry2 protein stability.

Spry2 is a molecular modulator of tyrosine kinase receptor signaling pathways that has cancer-type-specific effects. Mammalian Spry2 protein undergoes tyrosine and serine phosphorylation in response to growth factor stimulation. Spry2 expression is distinctly altered in various cancer types. Inhibition of the proteasome functionality results in reduced intracellular Spry2 degradation. Using in vitro and in vivo assays, we show that protein kinase D (PKD) phosphorylates Spry2 at serine 112 and interacts in vivo with the C-terminal half of this protein. Importantly, missense mutation of Ser112 decreases the rate of Spry2 intracellular protein degradation. Either knocking down the expression of all three mammalian PKD isoforms or blocking their kinase activity with a specific inhibitor contributes to the stabilization of Spry2 wild-type protein. Downregulation of CSN3, a component of the COP9/Signalosome that binds PKD, significantly increases the half-life of Spry2 wild-type protein but does not affect the stability of a Spry2 after mutating Ser112 to the non-phosphorylatable residue alanine. Our data demonstrate that both PKD and the COP9/Signalosome play a significant role in control of Spry2 intracellular stability and support the consideration of the PKD/COP9 complex as a potential therapeutic target in tumors where Spry2 expression is reduced.

The neuronal protein Kidins220/ARMS associates with ICAM-3 and other uropod components and regulates T-cell motility.

Kinase D interacting substrate of 220 kDa (Kidins220), also known as ankyrin repeat-rich membrane spanning (ARMS), is a protein that is mainly expressed in brain and neural cells where its function is only starting to be characterized. Here, we show that Kidins220/ARMS is also expressed in T lymphocytes where it is highly concentrated at the uropod of polarized T cells. In this cellular model, Kidins220/ARMS colocalizes with typical uropod T-cell molecules and coimmunoprecipitates with ICAM-3. Furthermore, Kidins220/ARMS associates with raft domains at the uropod and coimmunoprecipitates with caveolin-1, a molecule we show here to be also expressed in T cells. Importantly, induction of morphological polarization in primary T lymphocytes and Jurkat cells enhances Kidins220/ARMS colocalization with ICAM-3. Conversely, disruption of cell polarity provokes Kidins220/ARMS redistribution from the uropod to other cellular regions and drastically impairs its association with ICAM-3 in a protein kinase C-dependent manner. Finally, Kidins220/ARMS knockdown in human polarized T-cell lines promotes both basal and stromal cell-derived factor-1α-induced directed migration, identifying a novel function for this molecule. Altogether, our findings show that Kidins220/ARMS is a novel component of the uropod involved in the regulation of T-cell motility, an essential process for the immune response.

Molecular phylogenetics of squirrelfishes and soldierfishes (Teleostei: Beryciformes: Holocentridae): reconciling more than 100 years of taxonomic confusion.

Squirrelfishes and soldierfishes (Holocentridae) are among the most conspicuous species in the nocturnal reef fish community. However, there is no clear consensus regarding their evolutionary relationships, which is reflected in a complicated taxonomic history. We collected DNA sequence data from multiple single copy nuclear genes and one mitochondrial gene sampled from over fifty percent of the recognized holocentrid species and infer the first species-level phylogeny of the Holocentridae. Our results strongly support the monophyly of the clades Myripristinae (soldierfishes) and Holocentrinae (squirrelfishes). The molecular phylogenies differ with regard to previous hypotheses of relationships within the Myriprisitinae, resolving a clade of cryptic reef associated and deep water non-reef dwelling lineages (Corniger+Plectrypops+Ostichthys) that is the sister lineage to a monophyletic Myripristis. Within Holocentrinae, Neoniphon and Sargocentron are strongly supported as paraphyletic, while Holocentrus is nested within Sargocentron. Using Bayesian ancestral state reconstruction methods, we demonstrate the taxonomically diagnostic characters for Neoniphon and Sargocentron likely represent character states with a complex evolutionary history that is not reflective of shared common ancestry. We propose a new classification for Holocentrinae, recognizing four lineages that are treated as genera: Sargocentron Fowler, 1904, Holocentrus Scopoli, 1777, Flameo Jordan and Evermann, 1898, and Neoniphon Castelnau, 1875.