Nuclear receptors are a family of transcription factors with structurally conserved ligand binding domains that regulate their activity. Despite intensive efforts to identify ligands, most nuclear receptors are still "orphans." Here, we demonstrate that the ligand binding pocket of the Drosophila nuclear receptor E75 contains a heme prosthetic group. E75 absorption spectra, resistance to denaturants, and effects of site-directed mutagenesis indicate a single, coordinately bound heme molecule. A correlation between the levels of E75 expression and the levels of available heme suggest a possible role as a heme sensor. The oxidation state of the heme iron also determines whether E75 can interact with its heterodimer partner DHR3, suggesting an additional role as a redox sensor. Further, the E75-DHR3 interaction is also regulated by the binding of NO or CO to the heme center, suggesting that E75 may also function as a diatomic gas sensor. Possible mechanisms and roles for these interactions are discussed.

Neocortical projection neurons exhibit layer-specific molecular profiles and axonal connections. Here we show that the molecular identities of early-born subplate and deep-layer neurons are not acquired solely during generation or shortly thereafter but undergo progressive postmitotic refinement mediated by SOX5. Fezf2 and Bcl11b, transiently expressed in all subtypes of newly postmigratory early-born neurons, are subsequently downregulated in layer 6 and subplate neurons, thereby establishing their layer 5-enriched postnatal patterns. In Sox5-null mice, this downregulation is disrupted, and layer 6 and subplate neurons maintain an immature differentiation state, abnormally expressing these genes postnatally. Consistent with this disruption, SOX5 binds and represses a conserved enhancer near Fezf2. The Sox5-null neocortex exhibits failed preplate partition and laminar inversion of early-born neurons, loss of layer 5 subcerebral axons, and misrouting of subplate and layer 6 corticothalamic axons to the hypothalamus. Thus, SOX5 postmitotically regulates the migration, postmigratory differentiation, and subcortical projections of subplate and deep-layer neurons.neocortex development ͉ postmitotic mechanisms ͉ pyramidal neurons ͉ Sox genes ͉ transcriptional enhancer T he neocortex is composed of six distinct layers, each containing a unique subset of projection (pyramidal) neurons with specific molecular profiles and axonal connectivities (1-3). Subcortical axons arise solely from deep-layer (L5 and L6) neurons, whereas upper-layer (L2-L4) neurons project intracortically. The first projection neurons, born from progenitors in the ventricular zone (VZ) (4, 5), migrate radially and settle within the preplate (PP), where they form the nascent cortical plate (CP) (6). Incoming CP neurons, which split the PP into the marginal zone (MZ) and subplate (SP), are generated sequentially so that early-born neurons occupy the deep layers and later-born neurons migrate past older neurons to settle in more superficial layers. The molecular mechanisms that regulate the laminar position and identity of projection neurons are being unraveled (3, 7). Previous studies suggested that neurons are specified at the time of their birth (2, 8). However, the extent to which postmitotic events contribute to their laminar and molecular identity remains an open question.Fezf2 (Fezl or Zfp312) and Bcl11b (Ctip2) encode transcription factors enriched in and necessary for the development of L5 neurons (9-12). Here we show that the L5-enriched postnatal expression patterns of Fezf2 and Bcl11b are not acquired solely during neuronal generation but rather are the result of progressive refinement during postmigratory differentiation mediated by SOX5 (L-SOX5), a transcription factor that has been shown to regulate chondrogenesis, oligodendrogenesis, and the sequential generation of cortical neurons (13)(14)(15)(16). In this study, we show that Sox5 postmitotically controls the laminar positioning, molecular differentiation, and layer-specific pattern o...

The corticospinal (CS) tract is involved in controlling discrete voluntary skilled movements in mammals. The CS tract arises exclusively from layer (L) 5 projection neurons of the cerebral cortex, and its formation requires L5 activity of Fezf2 (Fezl, Zfp312). How this L5-specific pattern of Fezf2 expression and CS axonal connectivity is established with such remarkable fidelity had remained elusive. Here we show that the transcription factor TBR1 directly binds the Fezf2 locus and represses its activity in L6 corticothalamic projection neurons to restrict the origin of the CS tract to L5. In Tbr1 null mutants, CS axons ectopically originate from L6 neurons in a Fezf2-dependent manner. Consistently, misexpression of Tbr1 in L5 CS neurons suppresses Fezf2 expression and effectively abolishes the CS tract. Taken together, our findings show that TBR1 is a direct transcriptional repressor of Fezf2 and a negative regulator of CS tract formation that restricts the laminar origin of CS axons specifically to L5.neocortex | pyramidal neuron | axon guidance | transcriptional repression S patial specificity of axonal connections is one of the most important prerequisites for normal development (1-3). In mammals, this is especially crucial for axons of the corticospinal (CS) system (4-7). Development of the CS tract is an intricate process that involves the molecular specification of CS neurons and axon pathfinding. All long-range subcortical axons projecting to the brainstem and spinal cord, including those that form the CS tract, originate exclusively from layer (L) 5 projection (pyramidal) neurons of the cerebral cortex (8-11). Projection neurons in other cortical layers give rise to axons that project within the cortex (L2-4) or to the thalamus (L6). How this highly conserved laminar pattern of projections is formed with such perfect accuracy remains elusive.Previous work revealed that the transcription factor FEZF2 (FEZL, ZFP312) is highly enriched in L5 CS neurons and is critical to the development of the CS tract (12-14). Inactivation of Fezf2 disrupts formation of the CS tract (12-14), whereas misexpression of Fezf2 in upper layer projection neurons induces ectopic subcortical projections (13). These findings indicate that Fezf2 transcription is tightly regulated during development, and that the integrity of normal Fezf2 expression is critical to proper development of the CS tract. Interestingly, Fezf2 is transiently expressed in L6 neurons during early embryonic development, where its transcription is directly repressed by SOX5, thereby establishing a high-in-L5, low-in-L6 postnatal pattern (15, 16). Paradoxically, in Sox5 null mutants, the number of axon projections reaching the brainstem and spinal cord is severely reduced despite increased cortical Fezf2 expression (15). This suggests that Sox5 is required for the formation of these connections independent of its regulation of Fezf2. Furthermore, SOX5 is normally expressed in L5 Fezf2-expressing CS neurons (15). Therefore, the down-regulation of Fezf2 in L6 neu...

SUMMARY Fragile X syndrome (FXS), the leading monogenic cause of intellectual disability and autism, results from loss of function of the RNA-binding protein FMRP. Here we show that FMRP regulates the translation of neuronal nitric oxide synthase 1 (NOS1) in the developing human neocortex. Whereas NOS1 mRNA is ubiquitously expressed, NOS1 protein is transiently co-expressed with FMRP during early synaptogenesis in layer- and region-specific subpopulations of pyramidal neurons. These include mid-fetal layer 5 subcortically projecting neurons arranged into alternating columns in the prospective Broca’s area and orofacial motor cortex. Human NOS1 translation is activated by FMRP via interactions with coding region binding motifs absent from mouse Nos1 mRNA, which is expressed in mouse pyramidal neurons, but not efficiently translated. Correspondingly, neocortical NOS1 protein levels are severely reduced in developing human FXS cases but not FMRP-deficient mice. Thus, alterations in FMRP post-transcriptional regulation of NOS1 in developing neocortical circuits may contribute to cognitive dysfunction in FXS.

BackgroundMagnetic resonance-guided high intensity focused ultrasound (MR-HIFU) has recently emerged as an effective treatment option for painful bone metastases. We describe here the first experience with volumetric MR-HIFU for palliative treatment of painful bone metastases and evaluate the technique on three levels: technical feasibility, safety, and initial effectiveness.MethodsIn this observational cohort study, 11 consecutive patients (7 male and 4 female; median age, 60 years; age range, 53–86 years) underwent 13 treatments for 12 bone metastases. All patients exhibited persistent metastatic bone pain refractory to the standard of care. Patients were asked to rate their worst pain on an 11-point pain scale before treatment, 3 days after treatment, and 1 month after treatment. Complications were monitored. All data were prospectively recorded in the context of routine clinical care. Response was defined as a ≥2-point decrease in pain at the treated site without increase in analgesic intake. Baseline pain scores were compared to pain scores at 3 days and 1 month using the Wilcoxon signed-rank test. For reporting, the STROBE guidelines were followed.ResultsNo treatment-related major adverse events were observed. At 3 days after volumetric MR-HIFU ablation, pain scores decreased significantly (p = 0.045) and response was observed in a 6/11 (55%) patients. At 1-month follow-up, which was available for nine patients, pain scores decreased significantly compared to baseline (p = 0.028) and 6/9 patients obtained pain response (overall response rate 67% (95% confidence interval (CI) 35%–88%)).ConclusionsThis is the first study reporting on the volumetric MR-HIFU ablation for painful bone metastases. No major treatment-related adverse events were observed during follow-up. The results of our study showed that volumetric MR-HIFU ablation for painful bone metastases is technically feasible and can induce pain relief in patients with metastatic bone pain refractory to the standard of care. Future research should be aimed at standardization of the treatment procedures and treatment of larger numbers of patients to assess treatment effectiveness and comparison to the standard of care.