Engrailed transcription factors direct excitatory cerebellar neuron diversity and survival.

The excitatory neurons of the three cerebellar nuclei (eCN) form the primary output for the cerebellum. The medial eCN (eCNm) were recently divided into molecularly defined subdomains in the adult, however how they are established during development is not known. We define molecular subdomains of the embryonic eCNm using scRNA-seq and spatial expression analysis, showing they evolve during embryogenesis to prefigure the adult. Furthermore, the medial eCN are transcriptionally divergent from the other nuclei by E14.5. We previously showed that loss of the homeobox genes En1 and En2 leads to loss of approximately half of embryonic eCNm. We demonstrate that mutation of En1/2 in embryonic eCNm results in death of specific posterior eCNm molecular subdomains and down regulation of TBR2 (EOMES) in an anterior embryonic subdomain, as well as reduced synaptic gene expression. We further reveal a similar function for EN1/2 in mediating TBR2 expression, neuron differentiation and survival in the other excitatory neurons (granule and unipolar brush cells). Thus, our work defines embryonic eCNm molecular diversity and reveals conserved roles for EN1/2 in the cerebellar excitatory neuron lineage.

A collection of genetic mouse lines and related tools for inducible and reversible intersectional mis-expression.

Thanks to many advances in genetic manipulation, mouse models have become very powerful in their ability to interrogate biological processes. In order to precisely target expression of a gene of interest to particular cell types, intersectional genetic approaches using two promoter/enhancers unique to a cell type are ideal. Within these methodologies, variants that add temporal control of gene expression are the most powerful. We describe the development, validation and application of an intersectional approach that involves three transgenes, requiring the intersection of two promoter/enhancers to target gene expression to precise cell types. Furthermore, the approach uses available lines expressing tTA/rTA to control the timing of gene expression based on whether doxycycline is absent or present, respectively. We also show that the approach can be extended to other animal models, using chicken embryos. We generated three mouse lines targeted at the Tigre (Igs7) locus with TRE-loxP-tdTomato-loxP upstream of three genes (p21, DTA and Ctgf), and combined them with Cre and tTA/rtTA lines that target expression to the cerebellum and limbs. Our tools will facilitate unraveling biological questions in multiple fields and organisms.

Lateral cerebellum is preferentially sensitive to high sonic hedgehog signaling and medulloblastoma formation.

The main cell of origin of the Sonic hedgehog (SHH) subgroup of medulloblastoma (MB) is granule cell precursors (GCPs), a SHH-dependent transient amplifying population in the developing cerebellum. SHH-MBs can be further subdivided based on molecular and clinical parameters, as well as location because SHH-MBs occur preferentially in the lateral cerebellum (hemispheres). Our analysis of adult patient data suggests that tumors with Smoothened (SMO) mutations form more specifically in the hemispheres than those with Patched 1 (PTCH1) mutations. Using sporadic mouse models of SHH-MB with the two mutations commonly seen in adult MB, constitutive activation of Smo (SmoM2) or loss-of-Ptch1, we found that regardless of timing of induction or type of mutation, tumors developed primarily in the hemispheres, with SmoM2-mutants indeed showing a stronger specificity. We further uncovered that GCPs in the hemispheres are more susceptible to high-level SHH signaling compared with GCPs in the medial cerebellum (vermis), as more SmoM2 or Ptch1-mutant hemisphere cells remain undifferentiated and show increased tumorigenicity when transplanted. Finally, we identified location-specific GCP gene-expression profiles, and found that deletion of the genes most highly expressed in the hemispheres (Nr2f2) or vermis (Engrailed1) showed opposing effects on GCP differentiation. Our studies thus provide insights into intrinsic differences within GCPs that impact on SHH-MB progression.

Substance Use in Healthcare Workers: Importance of Stress Perception, Smoking Temptation, Social Support, and Humor.

BACKGROUND: Research indicates healthcare workers' personal substance use may affect quality of care. Investigating factors that correlate with coping through substance use may provide insight into improving quality care. OBJECTIVES: This study aims to examine potential correlates of coping through substance use among healthcare workers, with a particular focus on humor, social support, stress perception, and smoking temptation. METHOD: Participants, recruited from healthcare facilities, anonymously completed a 30-minute questionnaire of psychometrically valid measurements. RESULTS: In a sample of primarily female (75.7%), age 20-39 (74.8%), floor staff (i.e., doctors, nurses, technicians/assistants; 61.2%), perceived stress [ß = .036, t(98) = 2.55, p = .012], smoking temptation [ß = .036, t(98) = 2.21, p = .030], and coping through humor [ß = .163, t(98) = 2.033, p = .045] were significant correlates of the coping through substance use, with all relationships positively co-varying. Social support at work did not predict coping through substance use [ß = -.032, t(98) = -.814, p > .05]. Furthermore, negative affect/situation smoking temptation was associated with increased coping through substance use [ß = .246, t(99) = 2.859, p = .005] and habit/craving temptation was associated with decreased coping through substance use [ß = -.260, t(99) = -2.201, p = .030; however, positive affect/social temptation was not [ß = .054, t(99) = -.553, p > .05]. Conclusions/Importance: These findings suggest that coping with humor may relate to coping through substance use, while social support at work is either unrelated to coping through substance use in this sample or may not be adequately assessed with the measure used. Consistent with the literature, negative affect/situation was associated with increased coping through substance use. However, habit/craving was negatively predictive. Further research should explore the variables related to coping through substance use among healthcare workers.

Cerebellar output neurons impair non-motor behaviors by altering development of extracerebellar connectivity.

The capacity of the brain to compensate for insults during development depends on the type of cell loss, whereas the consequences of genetic mutations in the same neurons are difficult to predict. We reveal powerful compensation from outside the cerebellum when the excitatory cerebellar output neurons are ablated embryonically and demonstrate that the minimum requirement for these neurons is for motor coordination and not learning and social behaviors. In contrast, loss of the homeobox transcription factors Engrailed1/2 (EN1/2) in the cerebellar excitatory lineage leads to additional deficits in adult learning and spatial working memory, despite half of the excitatory output neurons being intact. Diffusion MRI indicates increased thalamo-cortico-striatal connectivity in En1/2 mutants, showing that the remaining excitatory neurons lacking En1/2 exert adverse effects on extracerebellar circuits regulating motor learning and select non-motor behaviors. Thus, an absence of cerebellar output neurons is less disruptive than having cerebellar genetic mutations.

The Engrailed homeobox genes determine the different foliation patterns in the vermis and hemispheres of the mammalian cerebellum.

Little is known about the genetic pathways and cellular processes responsible for regional differences in cerebellum foliation, which interestingly are accompanied by regionally distinct afferent circuitry. We have identified the Engrailed (En) homeobox genes as being crucial to producing the distinct medial vermis and lateral hemisphere foliation patterns in mammalian cerebella. By producing a series of temporal conditional mutants in En1 and/or En2, we demonstrate that both En genes are required to ensure that folia exclusive to the vermis or hemispheres form in the appropriate mediolateral position. Furthermore, En1/En2 continue to regulate foliation after embryonic day 14, at which time Fgf8 isthmic organizer activity is complete and the major output cells of the cerebellar cortex have been specified. Changes in spatially restricted gene expression occur prior to foliation in mutants, and foliation is altered from the onset and is accompanied by changes in the thickness of the layer of proliferating granule cell precursors. In addition, the positioning and timing of fissure formation are altered. Thus, the En genes represent a new class of genes that are fundamental to patterning cerebellum foliation throughout the mediolateral axis and that act late in development.

Engrailed transcription factors direct excitatory cerebellar neuron diversity and survival.

The excitatory neurons of the three cerebellar nuclei (eCN) form the primary output for the cerebellar circuit. The medial eCN (eCNm) were recently divided into molecularly defined subdomains in the adult, however how they are established during development is not known. We define molecular subdomains of the eCNm using scRNA-seq and spatial expression analysis and show they evolve during embryogenesis to resemble the adult. Furthermore, the eCNm is transcriptionally divergent from the rest of the eCN by E14.5. We previously showed that loss of the homeobox genes En1 and En2 leads to death of a subset of embryonic eCNm. We demonstrate that mutation of En1/2 in embryonic eCNm results in cell death of specific posterior eCNm molecular subdomains and loss of TBR2 (EOMES) expression in an anterior subdomain, as well as reduced synaptic gene expression. We further reveal a similar function for EN1/2 in mediating TBR2 expression, neuron differentiation and survival in the two other cerebellar excitatory neuron types. Thus, our work defines embryonic eCNm molecular diversity and reveals conserved roles for EN1/2 in the cerebellar excitatory neuron lineage.

KMT2D suppresses Sonic hedgehog-driven medulloblastoma progression and metastasis.

The major cause of treatment failure and mortality among medulloblastoma patients is metastasis intracranially or along the spinal cord. The molecular mechanisms driving tumor metastasis in Sonic hedgehog-driven medulloblastoma (SHH-MB) patients, however, remain largely unknown. In this study we define a tumor suppressive role of KMT2D (MLL2), a gene frequently mutated in the most metastatic ß-subtype. Strikingly, genetic mouse models of SHH-MB demonstrate that heterozygous loss of Kmt2d in conjunction with activation of the SHH pathway causes highly penetrant disease with decreased survival, increased hindbrain invasion and spinal cord metastasis. Loss of Kmt2d attenuates neural differentiation and shifts the transcriptional/chromatin landscape of primary and metastatic tumors toward a decrease in differentiation genes and tumor suppressors and an increase in genes/pathways implicated in advanced stage cancer and metastasis (TGFß, Notch, Atoh1, Sox2, and Myc). Thus, secondary heterozygous KMT2D mutations likely have prognostic value for identifying SHH-MB patients prone to develop metastasis.

Single Position Lateral Lumbar Interbody Fusion With Posterior Instrumentation Utilizing Computer Navigation and Robotic Assistance: Retrospective case review and surgical technique considerations.

STUDY DESIGN: Retrospective cohort study. OBJECTIVE: To determine safety and short-term outcomes of single-position lateral lumbar interbody fusion (LLIF) with bilateral posterior instrumentation and robotic assistance. The article also describes surgical technique considerations for the procedure. METHODS: 20 patients underwent single-position LLIF with posterior instrumentation and robotic assistance. The patients were followed for a minimum of 3 months post-operatively. RESULTS: Average operative time was 211 ± 34 minutes, average blood loss was 51.25 ± 17 cc's, and average length of stay was 1.4 ± .75 days. There were no intraoperative complications, readmissions, revision surgeries, and no incidence of hardware malposition. Significant improvement in pain and ODI scores was noted at 3 month follow up. CONCLUSIONS: The study demonstrated safety and short-term clinical efficacy of minimally invasive single-position lateral lumbar interbody fusion with bilateral posterior instrumentation utilizing robotic assistance and navigation. There are certain surgical technique considerations that must be followed to ensure optimal surgical workflow and predictable outcomes.

Therapeutic Efficacy of Rhemercise: A Novel Mindfulness Technique in Patients With Opioid Use Disorder.

Objective: Rhemercise is a novel mindfulness technique used to prevent relapse in opioid use disorder (OUD). Rhemercise is a quantifiable and intentional slow-breathing technique that could increase subjective well-being, which helps to prevent relapse in OUD by reducing craving, negative affect, and visceral reactivity. The objective of this study was to assess the efficacy of rhemercise as an adjunctive therapy in patients with OUD undergoing detoxification.Methods: This was a hospital-based, open-label, prospective, and exploratory study conducted between June 2018 and June 2019 that included 126 male inpatients admitted for detoxification of OUD. Patients with OUD diagnosed according to ICD-10 criteria who were aged 18-65 years were included in the study. Patients with other psychiatric disorders were excluded. Participants were divided into 2 groups: group A (n = 63) comprised patients receiving treatment as usual + rhemercise, and group B (n = 63) received treatment as usual only. Assessment tools included the Clinical Opiate Withdrawal Scale, Brief Pain Inventory, and Subjective Well-Being Inventory.Results: Various domains of the Subjective Well-Being Inventory (general well-being-positive affect [P = .02], confidence in coping [P = .007], inadequate mental mastery [P = .002]) improved significantly among OUD patients who received rhemercise treatment compared to treatment as usual.Conclusion: Rhemercise promoted general well-being and positive affect and decreased the opioid withdrawal symptoms, thereby potentially reducing the overall risk for relapse. Future studies are warranted with rhemercise to validate these promising findings.

Injury-induced ASCL1 expression orchestrates a transitory cell state required for repair of the neonatal cerebellum.

To understand repair processes, it is critical to identify the molecular foundations underlying progenitor diversity and plasticity. Upon injury to the neonatal cerebellum, a normally gliogenic nestin-expressing progenitor (NEP) in the Bergmann glia layer (BgL) undergoes adaptive reprograming to restore granule cell production. However, the cellular states and genes regulating the NEP fate switch are unknown. Using single-cell RNA sequencing and fate mapping, we defined molecular subtypes of NEPs and their lineages under homeostasis and repair. NEPs contain two major subtypes: Hopx+ astrogliogenic and Ascl1+ neurogenic NEPs that are further subdivided based on their location, lineage, and differentiation status. Upon injury, an Ascl1+ transitory cellular state arises from Hopx+ BgL-NEPs. Furthermore, mutational analysis revealed that induction of Ascl1 is required for adaptive reprogramming by orchestrating a glial-to-neural switch in vivo following injury. Thus, we provide molecular and cellular insights into context-dependent progenitor plasticity and repair mechanisms in the brain.

Exceptional fossil assemblages confirm the existence of complex Early Triassic ecosystems during the early Spathian.

The mass extinction characterizing the Permian/Triassic boundary (PTB; ~ 252 Ma) corresponds to a major faunal shift between the Palaeozoic and the Modern evolutionary fauna. The temporal, spatial, environmental, and ecological dynamics of the associated biotic recovery remain highly debated, partly due to the scarce, or poorly-known, Early Triassic fossil record. Recently, an exceptionally complex ecosystem dated from immediately after the Smithian/Spathian boundary (~ 3 myr after the PTB) was reported: the Paris Biota (Idaho, USA). However, the spatiotemporal representativeness of this unique assemblage remained questionable as it was hitherto only reported from a single site. Here we describe three new exceptionally diverse assemblages of the same age as the Paris Biota, and a fourth younger one. They are located in Idaho and Nevada, and are taxonomic subsets of the Paris Biota. We show that the latter covered a region-wide area and persisted at least partially throughout the Spathian. The presence of a well-established marine fauna such as the Paris Biota, as soon as the early Spathian, indicates that the post-PTB biotic recovery and the installation of complex ecosystems probably took place earlier than often assumed, at least at a regional scale.

Patterning of ventral telencephalon requires positive function of Gli transcription factors.

The ability of neuroepithelial cells to generate a diverse array of neurons is influenced by locally secreted signals. In the spinal cord, Sonic Hedgehog (Shh) is known to induce distinct cell fates in a concentration-dependent manner by regulating the activities of the three Gli transcription factors in neural precursors. However, whether Gli-mediated Shh signaling is also required to induce different cell types in the ventral telencephalon has been controversial. In particular, loss of Shh has little effect on dorsoventral patterning of the telencephalon when Gli3 is also removed. Furthermore, no ventral telencephalic phenotypes have been found in individual Gli mutants. To address this issue, we first characterized Shh-responding ventral telencephalic progenitors between E9.5 and E12.5 and found that they produce neurons migrating to different layers of the cortex. We also discovered a loss of Nkx2.1 and Nkx6.2 expression in two subgroups of progenitors in embryos lacking major Gli activators. Finally, we analyzed the telencephalic phenotypes of embryos lacking all Gli genes and found that the ventral telencephalon was highly disorganized with intermingling of distinct neuronal cell types. Together, these studies unravel a role for Gli transcription factors in mediating Shh signaling to control specification, differentiation and positioning of ventral telencephalic neurons.

Engrailed homeobox genes determine the organization of Purkinje cell sagittal stripe gene expression in the adult cerebellum.

Underlying the seemingly uniform cellular composition of the adult mammalian cerebellum (Cb) are striking parasagittal stripes of gene expression along the medial-lateral (ML) axis that are organized with respect to the lobules that divide the Cb along the anterior-posterior (AP) axis. Although there is a clear correlation between the organization of gene expression stripes and Cb activity patterns, little is known about the genetic pathways that determine the intrinsic stripe molecular code. Here we establish that ML molecular code patterning is highly dependent on two homeobox transcription factors, Engrailed1 (En1) and En2, both of which are also required for patterning the lobules. Gene expression analysis of an allelic series of En1/2 mutant mice that have an intact Purkinje cell layer revealed severe patterning defects using three known components of the ML molecular code and a new marker of Hsp25 negative stripes (Neurofilament heavy chain, Nfh). Importantly, the complementary expression of ZebrinII/PhospholipaseC beta4 and Hsp25/Nfh changes in unison in each mutant. Furthermore, each En gene has unique as well as overlapping functions in patterning the ML molecular code and each En protein has dominant functions in different AP domains (subsets of lobules). Remarkably, in En1/2 mutants with almost normal foliation, ML molecular code patterning is severely disrupted. Thus, independent mechanisms that use En1/2 must pattern foliation and spatial gene expression separately. Our studies reveal that En1/2 are fundamental components of the genetic pathways that pattern the two intersecting coordinate systems of the Cb, morphological divisions and the molecular code.

All mouse ventral spinal cord patterning by hedgehog is Gli dependent and involves an activator function of Gli3.

An important question is how the gradient of Hedgehog is interpreted by cells at the level of the Gli transcription factors. The full range of Gli activity and its dependence on Hh have not been determined, although the Gli2 activator and Gli3 repressor have been implicated. Using the spinal cord as a model system, we demonstrate that Gli3 can transduce Hedgehog signaling as an activator. All expression of the Hh target gene Gli1 is dependent on both Gli2 and Gli3. Unlike Gli2, however, Gli3 requires endogenous Gli1 for induction of floor plate and V3 interneurons. Strikingly, embryos lacking all Gli function develop motor neurons and three ventral interneuron subtypes, similar to embryos lacking Hh signaling and Gli3. Therefore, in the spinal cord all Hh signaling is Gli dependent. Furthermore, a combination of Gli2 and Gli3 is required to regulate motor neuron development and spatial patterning of ventral spinal cord progenitors.

Genetic deletion of genes in the cerebellar rhombic lip lineage can stimulate compensation through adaptive reprogramming of ventricular zone-derived progenitors.

BACKGROUND: The cerebellum is a foliated posterior brain structure involved in coordination of motor movements and cognition. The cerebellum undergoes rapid growth postnataly due to Sonic Hedgehog (SHH) signaling-dependent proliferation of ATOH1+ granule cell precursors (GCPs) in the external granule cell layer (EGL), a key step for generating cerebellar foliation and the correct number of granule cells. Due to its late development, the cerebellum is particularly vulnerable to injury from preterm birth and stress around birth. We recently uncovered an intrinsic capacity of the developing cerebellum to replenish ablated GCPs via adaptive reprogramming of Nestin-expressing progenitors (NEPs). However, whether this compensation mechanism occurs in mouse mutants affecting the developing cerebellum and could lead to mis-interpretation of phenotypes was not known. METHODS: We used two different approaches to remove the main SHH signaling activator GLI2 in GCPs: 1) Our mosaic mutant analysis with spatial and temporal control of recombination (MASTR) technique to delete Gli2 in a small subset of GCPs; 2) An Atoh1-Cre transgene to delete Gli2 in most of the EGL. Genetic Inducible Fate Mapping (GIFM) and live imaging were used to analyze the behavior of NEPs after Gli2 deletion. RESULTS: Mosaic analysis demonstrated that SHH-GLI2 signaling is critical for generating the correct pool of granule cells by maintaining GCPs in an undifferentiated proliferative state and promoting their survival. Despite this, inactivation of GLI2 in a large proportion of GCPs in the embryo did not lead to the expected dramatic reduction in the size of the adult cerebellum. GIFM uncovered that NEPs do indeed replenish GCPs in Gli2 conditional mutants, and then expand and partially restore the production of granule cells. Furthermore, the SHH signaling-dependent NEP compensation requires Gli2, demonstrating that the activator side of the pathway is involved. CONCLUSION: We demonstrate that a mouse conditional mutation that results in loss of SHH signaling in GCPs is not sufficient to induce long term severe cerebellum hypoplasia. The ability of the neonatal cerebellum to regenerate after loss of cells via a response by NEPs must therefore be considered when interpreting the phenotypes of Atoh1-Cre conditional mutants affecting GCPs.

Cerebellar nuclei excitatory neurons regulate developmental scaling of presynaptic Purkinje cell number and organ growth.

For neural systems to function effectively, the numbers of each cell type must be proportioned properly during development. We found that conditional knockout of the mouse homeobox genes En1 and En2 in the excitatory cerebellar nuclei neurons (eCN) leads to reduced postnatal growth of the cerebellar cortex. A subset of medial and intermediate eCN are lost in the mutants, with an associated cell non-autonomous loss of their presynaptic partner Purkinje cells by birth leading to proportional scaling down of neuron production in the postnatal cerebellar cortex. Genetic killing of embryonic eCN throughout the cerebellum also leads to loss of Purkinje cells and reduced postnatal growth but throughout the cerebellar cortex. Thus, the eCN play a key role in scaling the size of the cerebellum by influencing the survival of their Purkinje cell partners, which in turn regulate production of granule cells and interneurons via the amount of sonic hedgehog secreted.

The value of orthodontics: Do parents' willingness to pay values reflect the IOTN?

INTRODUCTION: Given the limited evidence about the benefits of orthodontic treatment, many health care systems have rationed access to orthodontic care with the Index of Orthodontic Treatment Need (IOTN) being one tool used to attempt to allocate resources based on need. However, it is not clear whether patient and public valuations of different levels of need (as described by the IOTN) reflect the resource allocation decisions. The aim of this project was therefore to determine the values parents placed on correction of malocclusions at different IOTN levels using the willingness to pay (WTP) technique. METHOD: 401 parents of children attending hospital-based orthodontic clinics in the North of England were recruited to complete a questionnaire eliciting WTP for the correction of seven malocclusions with different IOTN scores. In addition demographic and orthodontic history characteristics were collected. Results were analysed with appropriate pairwise significance tests and regression. RESULTS: A significant difference in WTP was noted between all the possible pairs of malocclusions with the exception of overjets with moderate versus great need of treatments. At moderate levels, correction of crowding was valued less than overjet but this was reversed at great need levels. Very little of the variance in WTP was explained by the variables collected. When looking at factors affecting percentage difference between values for different pairs of malocclusions, in general, no factors predicted the magnitude of difference. CONCLUSION: Median valuations for correction of malocclusions vary significantly for different levels of need (as judged by IOTN), with increasing levels of need generating higher values. However, there was a limited effect of demographic or orthodontic characteristics on the magnitude of percentage difference in values for correcting malocclusions different levels of need.

Altered paracrine signaling from the injured knee joint impairs postnatal long bone growth.

Regulation of organ growth is a poorly understood process. In the long bones, the growth plates (GPs) drive elongation by generating a scaffold progressively replaced by bone. Although studies have focused on intrinsic GP regulation, classic and recent experiments suggest that local signals also modulate GP function. We devised a genetic mouse model to study extrinsic long bone growth modulation, in which injury is specifically induced in the left hindlimb, such that the right hindlimb serves as an internal control. Remarkably, when only mesenchyme cells surrounding postnatal GPs were killed, left bone growth was nevertheless reduced. GP signaling was impaired by altered paracrine signals from the knee joint, including activation of the injury response and, in neonates, dampened IGF1 production. Importantly, only the combined prevention of both responses rescued neonatal growth. Thus, we identified signals from the knee joint that modulate bone growth and could underlie establishment of body proportions.

A Review of the Clinical Utility of Intravascular Ultrasound and Optical Coherence Tomography in the Assessment and Treatment of Coronary Artery Disease.

Coronary artery disease remains one of the leading causes of morbidity and mortality in the United States. As a medical society, we continue to search for ways to better treat coronary artery disease and prevent acute coronary syndrome (ACS). As it stands, only statins and antiplatelet agents have been proven to significantly reduce the occurrence of ACS. A histopathological understanding of the pathogenesis of ACS has provided insight into the importance of plaque morphology. Therefore, it has been proposed that increasing the ability to detect true vulnerable, "at-risk" lesions, would foster the use of percutaneous coronary intervention as a means for the prevention of ACS. There are now several different imaging modalities to help cardiologists stratify plaque stability. These include, but are not limited to, angioscopy, magnetic resonance angiography, intravascular ultrasound (IVUS), optical coherence tomography (OCT), and near-infrared fluorescence. To date, the most studied and frequently used in clinical trials are IVUS and OCT. Following a brief background discussion of IVUS and OCT, we will objectively evaluate each modality's ability to detect specific morphological characteristics. This article will also discuss IVUS and OCT's clinical utility with regard to proper stent placement and follow-up after percutaneous coronary interventions.