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Context The use of osteopathic manipulative medicine (OMM) continues to decline in medical practice, despite an increasing number of osteopathic physicians. Objective This pilot study was designed to determine if a brochure created to increase knowledge about osteopathic medicine and OMM was read by patients, reviewed as being helpful, needed modifications and increased patient understanding of and willingness to receive OMM in preparation for a large scale trial that will assess this in both the hospital and ambulatory settings. Methods The study was performed using an educational brochure and 2 closed questionnaires. Twenty-seven patients of either inpatient or observation status aged 18 and above with English literacy were enrolled. Participants first completed a pre-questionnaire with questions regarding understanding of OMM and willingness to receive treatment. They then read the provided educational brochure, which contained a checkbox to verify the material was read in its entirety. Participants completed a post-questionnaire with similar questions. The results were analyzed with Wilcoxon signed rank test with 95% confidence to observe any changes in pre- and post-questionnaire responses. Results Of the participants, 48.1% provided verification that they read the brochure. A significant increase in patient willingness to receive OMM as part of their treatment regimen was observed for those who read the brochure (P=.008 ). No significant change was seen for those who didn’t read the brochure (P=.26). Additionally, 100% of participants indicated that the brochure was helpful, and 100% of participants indicated a better understanding of OMM. Cost remained a significant barrier to accepting or pursuing OMM treatment. Conclusion This pilot study demonstrated a statistically significant improvement in willingness to receive treatment after reviewing the designed brochure. It also identified a need to convey information regarding cost of OMM treatment to patients and a need to better emphasize the checkbox located within the brochure for verification purposes. The brochure and study design proved feasible and will provide the foundation for a larger scale trial looking to assess if a patient educational handout improves understanding of OMM and willingness to receive treatment in the hospital and ambulatory settings.

Autism spectrum condition or ‘autism’ is associated with numerous monogenic and polygenic genetic risk factors including the polygenic 16p11.2 microdeletion. A central question is what neural cells are affected. To systematically investigate we analysed single cell transcriptomes from gestational week (GW) 8-26 human foetal prefrontal cortex and identified a subset of interneurons (INs) first appearing at GW23 with enriched expression of a disproportionately large fraction of risk factor transcripts. This suggests the hypothesis that these INs are disproportionately vulnerable to mutations causing autism. We investigated this in a rat model of the 16p11.2 microdeletion. We found no change in the numbers or position of either excitatory or inhibitory neurons in the somatosensory cortex or CA1 of 16p11.2+/- rats but found that CA1 Sst INs were hyperexcitable with an enlarged axon initial segment, which was not the case for CA1 pyramidal cells. This study prompts deeper investigation of IN development as a convergent target for autism genetic risk factors.

The interaction between cell surface heparan sulphate and diffusible ligands such as FGFs is of vital importance for downstream signaling, however, there are few techniques that can be used to investigate this binding event. The ligand and carbohydrate engagement (LACE) assay is a powerful tool which can be used to probe the molecular interaction between heparan sulphate and diffusible ligands and can detect changes in binding that may occur following genetic or pharmacological intervention. In this protocol we describe an FGF17:FGFR1 LACE assay performed on embryonic mouse brain tissue. We also describe the method we have used to quantify changes in fluorescent LACE signal in response to altered HS sulphation.

Organotypic slice culture is a powerful technique for exploring the embryonic development of the mammalian brain. In this protocol we describe a basic slice culture technique we have used for two sets of experiments: axon guidance transplant assays and bead culture assays.

Thalamocortical axons (TCAs) cross several tissues on their journey to the cortex. Mechanisms must be in place along the route to ensure they connect with their targets in an orderly fashion. The ventral telencephalon acts as an instructive tissue, but the importance of the diencephalon in TCA mapping is unknown. We report that disruption of diencephalic development by Pax6 deletion results in a thalamocortical projection containing mapping errors. We used conditional mutagenesis to test whether these errors are due to the disruption of pioneer projections from prethalamus to thalamus and found that, although this correlates with abnormal TCA fasciculation, it does not induce topographical errors. To test whether the thalamus contains navigational cues for TCAs, we used slice culture transplants and gene expression studies. We found the thalamic environment is instructive for TCA navigation and that the molecular cues netrin 1 and semaphorin 3a are likely to be involved. Our findings indicate that the correct topographic mapping of TCAs onto the cortex requires the order to be established from the earliest stages of their growth by molecular cues in the thalamus itself., Summary: The diencephalon plays a role in the correct organization of thalamocortical axons. The thalamic environment is instructive for their correct medial-lateral position, while prethalamic pioneer axons help to avoid premature fasciculation.

Heparan sulfate (HS) is a cell surface and extracellular matrix carbohydrate extensively modified by differential sulfation. HS interacts physically with canonical fibroblast growth factor (FGF) proteins that signal through the extracellular signal regulated kinase (ERK)/mitogen activated protein kinase (MAPK) pathway. At the embryonic mouse telencephalic midline, FGF/ERK signaling drives astroglial precursor somal translocation from the ventricular zone of the corticoseptal boundary (CSB) to the induseum griseum (IG), producing a focus of Slit2-expressing astroglial guidepost cells essential for interhemispheric corpus callosum (CC) axon navigation. Here, we investigated the cell and molecular function of a specific form of HS sulfation, 2-O HS sulfation catalyzed by the enzyme Hs2st, in midline astroglial development and in regulating FGF protein levels and interaction with HS. Hs2st−/− embryos of either sex exhibit a grossly enlarged IG due to precocious astroglial translocation and conditional Hs2st mutagenesis and ex vivo culture experiments show that Hs2st is not required cell autonomously by CC axons or by the IG astroglial cell lineage, but rather acts non-cell autonomously to suppress the transmission of translocation signals to astroglial precursors. Rescue of the Hs2st−/− astroglial translocation phenotype by pharmacologically inhibiting FGF signaling shows that the normal role of Hs2st is to suppress FGF-mediated astroglial translocation. We demonstrate a selective action of Hs2st on FGF protein by showing that Hs2st (but not Hs6st1) normally suppresses the levels of Fgf17 protein in the CSB region in vivo and use a biochemical assay to show that Hs2st (but not Hs6st1) facilitates a physical interaction between the Fgf17 protein and HS. SIGNIFICANCE STATEMENT We report a novel non-cell-autonomous mechanism regulating cell signaling in developing brain. Using the developing mouse telencephalic midline as an exemplar, we show that the specific sulfation modification of the cell surface and extracellular carbohydrate heparan sulfate (HS) performed by Hs2st suppresses the supply of translocation signals to astroglial precursors by a non-cell-autonomous mechanism. We further show that Hs2st modification selectively facilitates a physical interaction between Fgf17 and HS and suppresses Fgf17 protein levels in vivo, strongly suggesting that Hs2st acts selectively on Fgf17 signaling. HS interacts with many signaling proteins potentially encoding numerous selective interactions important in development and disease, so this class of mechanism may apply more broadly to other biological systems.

During embryogenesis, the pallial–subpallial boundary (PSB) divides the two main progenitor domains in the telencephalon: the pallium, the major source of excitatory neurons, and the subpallium, the major source of inhibitory neurons. The PSB is formed at the molecular interface between the pallial (high Pax6+) and subpallial (high Gsx2+) ventricular zone (VZ) compartments. Initially, the PSB contains cells that express both Pax6 and Gsx2, but during later stages of development this boundary is largely refined into two separate compartments. In this study we examined the developmental mechanisms underlying PSB boundary formation and the postnatal consequences of conditional loss ofPax6function at the PSB on neuronal fate in the amygdala and olfactory bulb, two targets of PSB-derived migratory populations. Our cell fate and time-lapse imaging analyses reveal that the sorting of Pax6+ and Gsx2+ progenitors during embryogenesis is the result of a combination of changes in gene expression and cell movements. Interestingly, we find that in addition to giving rise to inhibitory neurons in the amygdala and olfactory bulb, Gsx2+ progenitors generate a subpopulation of amygdala excitatory neurons. Consistent with this finding, targeted conditional ablation ofPax6in Gsx2+ progenitors results in discrete local embryonic patterning defects that are linked to changes in the generation of subsets of postnatal excitatory and inhibitory neurons in the amygdala and inhibitory neurons in the olfactory bulb. Thus, in PSB progenitors,Pax6plays an important role in the generation of multiple subtypes of neurons that contribute to the amygdala and olfactory bulb.

Background In mouse embryos, the Pax6 transcription factor is expressed in the progenitors of thalamic neurons but not in thalamic neurons themselves. Its null-mutation causes early mis-patterning of thalamic progenitors. It is known that thalamic neurons generated by Pax6−/− progenitors do not develop their normal connections with the cortex, but it is not clear why. We investigated the extent to which defects intrinsic to the thalamus are responsible. Results We first confirmed that, in constitutive Pax6−/− mutants, the axons of thalamic neurons fail to enter the telencephalon and, instead, many of them take an abnormal path to the hypothalamus, whose expression of Slits would normally repel them. We found that thalamic neurons show reduced expression of the Slit receptor Robo2 in Pax6−/− mutants, which might enhance the ability of their axons to enter the hypothalamus. Remarkably, however, in chimeras comprising a mixture of Pax6−/− and Pax6+/+ cells, Pax6−/− thalamic neurons are able to generate axons that exit the diencephalon, take normal trajectories through the telencephalon and avoid the hypothalamus. This occurs despite abnormalities in their molecular patterning (they express Nkx2.2, unlike normal thalamic neurons) and their reduced expression of Robo2. In conditional mutants, acute deletion of Pax6 from the forebrain at the time when thalamic axons are starting to grow does not prevent the development of the thalamocortical tract, suggesting that earlier extra-thalamic patterning and /or morphological defects are the main cause of thalamocortical tract failure in Pax6−/− constitutive mutants. Conclusions Our results indicate that Pax6 is required by thalamic progenitors for the normal molecular patterning of the thalamic neurons that they generate but thalamic neurons do not need normal Pax6-dependent patterning to become competent to grow axons that can be guided appropriately.

The role of programmed cell death 4 (PDCD4) in tumor biology is context-dependent. PDCD4 is described as a tumor suppressor, but its coexpression with protein arginine methyltransferase 5 (PRMT5) promotes accelerated tumor growth. Here, we report that PDCD4 is methylated during nutrient deprivation. Methylation occurs because of increased stability of PDCD4 protein as well as increased activity of PRMT5 toward PDCD4. During nutrient deprivation, levels of methylated PDCD4 promote cell viability, which is dependent on an enhanced interaction with eIF4A. Upon recovery from nutrient deprivation, levels of methylated PDCD4 are regulated by phosphorylation, which controls both the localization and stability of methylated PDCD4. This study reveals that, in response to particular environmental cues, the role of PDCD4 is up-regulated and is advantageous for cell viability. These findings suggest that the methylated form of PDCD4 promotes tumor viability during nutrient deprivation, ultimately allowing the tumor to grow more aggressively.

The corpus callosum (CC) connects the left and right cerebral hemispheres in mammals and its development requires intercellular communication at the telencephalic midline mediated by signaling proteins. Heparan sulfate (HS) is a sulfated polysaccharide that decorates cell surface and extracellular matrix proteins and regulates the biological activity of numerous signaling proteins via sugar-protein interactions. HS is subject to regulated enzymatic sulfation and desulfation and an attractive, although not proven, hypothesis is that the biological activity of HS is regulated by a sugar sulfate code. Mutant mouse embryos lacking the heparan sulfotransferases Hs2st or Hs6st1 have severe CC phenotypes and form Probst bundles of noncrossing axons flanking large tangles of midline glial processes. Here, we identify a precocious accumulation of Sox9-expressing glial cells in the indusium griseum region and a corresponding depletion at the glial wedge associated with the formation of Probst bundles along the rostrocaudal axis in both mutants. Molecularly, we found a surprising hyperactivation of Erk signaling in Hs2st(-/-) (2-fold) and Hs6st1(-/-) (6-fold) embryonic telencephalon that was most striking at the midline, where Erk signaling is lowest in wild-types, and a 2-fold increase in Fgf8 protein levels in Hs6st1(-/-) embryos that could underpin Erk hyperactivation and excessive glial movement to the indusium griseum. The tightly linked Hs6st1(-/-) CC glial and axonal phenotypes can be rescued by genetic or pharmacological suppression of Fgf8/Erk axis components. Overall, our data fit a model in which Hs2st and Hs6st1 normally generate conditions conducive to CC development by generating an HS-containing environment that keeps Erk signaling in check.