Species-dependent posttranscriptional regulation of NOS1 by FMRP in the developing cerebral cortex.

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 translation of neuronal nitric oxide synthase 1 (NOS1) in the developing human neocortex. Whereas NOS1 mRNA is widely expressed, NOS1 protein is transiently coexpressed with FMRP during early synaptogenesis in layer- and region-specific pyramidal neurons. These include midfetal 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 posttranscriptional regulation of NOS1 in developing neocortical circuits may contribute to cognitive dysfunction in FXS.

Postmitotic accumulation of histone variant H3.3 in new cortical neurons establishes neuronal chromatin, transcriptome, and identity.

Histone variants, which can be expressed outside of S-phase and deposited DNA synthesis-independently, provide long-term histone replacement in postmitotic cells, including neurons. Beyond replenishment, histone variants also play active roles in gene regulation by modulating chromatin states or enabling nucleosome turnover. Here, we uncover crucial roles for the histone H3 variant H3.3 in neuronal development. We find that newborn cortical excitatory neurons, which have only just completed replication-coupled deposition of canonical H3.1 and H3.2, substantially accumulate H3.3 immediately postmitosis. Codeletion of H3.3-encoding genes H3f3a and H3f3b from newly postmitotic neurons abrogates H3.3 accumulation, markedly alters the histone posttranslational modification landscape, and causes widespread disruptions to the establishment of the neuronal transcriptome. These changes coincide with developmental phenotypes in neuronal identities and axon projections. Thus, preexisting, replication-dependent histones are insufficient for establishing neuronal chromatin and transcriptome; de novo H3.3 is required. Stage-dependent deletion of H3f3a and H3f3b from 1) cycling neural progenitor cells, 2) neurons immediately postmitosis, or 3) several days later, reveals the first postmitotic days to be a critical window for de novo H3.3. After H3.3 accumulation within this developmental window, codeletion of H3f3a and H3f3b does not lead to immediate H3.3 loss, but causes progressive H3.3 depletion over several months without widespread transcriptional disruptions or cellular phenotypes. Our study thus uncovers key developmental roles for de novo H3.3 in establishing neuronal chromatin, transcriptome, identity, and connectivity immediately postmitosis that are distinct from its role in maintaining total histone H3 levels over the neuronal lifespan.

Chromatin remodeler Arid1a regulates subplate neuron identity and wiring of cortical connectivity.

Loss-of-function mutations in chromatin remodeler gene ARID1A are a cause of Coffin-Siris syndrome, a developmental disorder characterized by dysgenesis of corpus callosum. Here, we characterize Arid1a function during cortical development and find unexpectedly selective roles for Arid1a in subplate neurons (SPNs). SPNs, strategically positioned at the interface of cortical gray and white matter, orchestrate multiple developmental processes indispensable for neural circuit wiring. We find that pancortical deletion of Arid1a leads to extensive mistargeting of intracortical axons and agenesis of corpus callosum. Sparse Arid1a deletion, however, does not autonomously misroute callosal axons, implicating noncell-autonomous Arid1a functions in axon guidance. Supporting this possibility, the ascending axons of thalamocortical neurons, which are not autonomously affected by cortical Arid1a deletion, are also disrupted in their pathfinding into cortex and innervation of whisker barrels. Coincident with these miswiring phenotypes, which are reminiscent of subplate ablation, we unbiasedly find a selective loss of SPN gene expression following Arid1a deletion. In addition, multiple characteristics of SPNs crucial to their wiring functions, including subplate organization, subplate axon-thalamocortical axon cofasciculation ("handshake"), and extracellular matrix, are severely disrupted. To empirically test Arid1a sufficiency in subplate, we generate a cortical plate deletion of Arid1a that spares SPNs. In this model, subplate Arid1a expression is sufficient for subplate organization, subplate axon-thalamocortical axon cofasciculation, and subplate extracellular matrix. Consistent with these wiring functions, subplate Arid1a sufficiently enables normal callosum formation, thalamocortical axon targeting, and whisker barrel development. Thus, Arid1a is a multifunctional regulator of subplate-dependent guidance mechanisms essential to cortical circuit wiring.

TBR1 directly represses Fezf2 to control the laminar origin and development of the corticospinal tract.

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.

SOX5 postmitotically regulates migration, postmigratory differentiation, and projections of subplate and deep-layer neocortical neurons.

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.

Symmetric neural progenitor divisions require chromatin-mediated homologous recombination DNA repair by Ino80.

Chromatin regulates spatiotemporal gene expression during neurodevelopment, but it also mediates DNA damage repair essential to proliferating neural progenitor cells (NPCs). Here, we uncover molecularly dissociable roles for nucleosome remodeler Ino80 in chromatin-mediated transcriptional regulation and genome maintenance in corticogenesis. We find that conditional Ino80 deletion from cortical NPCs impairs DNA double-strand break (DSB) repair, triggering p53-dependent apoptosis and microcephaly. Using an in vivo DSB repair pathway assay, we find that Ino80 is selectively required for homologous recombination (HR) DNA repair, which is mechanistically distinct from Ino80 function in YY1-associated transcription. Unexpectedly, sensitivity to loss of Ino80-mediated HR is dependent on NPC division mode: Ino80 deletion leads to unrepaired DNA breaks and apoptosis in symmetric NPC-NPC divisions, but not in asymmetric neurogenic divisions. This division mode dependence is phenocopied following conditional deletion of HR gene Brca2. Thus, distinct modes of NPC division have divergent requirements for Ino80-dependent HR DNA repair.

Robust elimination of genome-damaged cells safeguards against brain somatic aneuploidy following Knl1 deletion.

The brain is a genomic mosaic shaped by cellular responses to genome damage. Here, we manipulate somatic genome stability by conditional Knl1 deletion from embryonic mouse brain. KNL1 mutations cause microcephaly and KNL1 mediates the spindle assembly checkpoint, a safeguard against chromosome missegregation and aneuploidy. We find that following Knl1 deletion, segregation errors in mitotic neural progenitor cells give rise to DNA damage on the missegregated chromosomes. This triggers rapid p53 activation and robust apoptotic and microglial phagocytic responses that extensively eliminate cells with somatic genome damage, thus causing microcephaly. By leaving only karyotypically normal progenitors to continue dividing, these mechanisms provide a second safeguard against brain somatic aneuploidy. Without Knl1 or p53-dependent safeguards, genome-damaged cells are not cleared, alleviating microcephaly, but paradoxically leading to total pre-weaning lethality. Thus, mitotic genome damage activates robust responses to eliminate somatic mutant cells, which if left unpurged, can impact brain and organismal fitness.

Increased expression of insulin-like growth factor in intestinal lengthening by mechanical force in rats.

INTRODUCTION: A segment of the jejunum could double its length by the application of an axial mechanical force. We hypothesize that this growth is correlated with an increased expression of insulin-like growth factor (IGF-I) in the jejunum. METHODS: Adult Sprague-Dawley rats underwent the isolation of a 1.5-cm segment of the jejunum. The isolated jejunal segment was either lengthened using mechanical force or left alone for 3 weeks. The jejunal segments were analyzed by quantitative polymerase chain reaction and immunofluorescence for the expression of IGF-I. RESULTS: Whereas jejunal segments that underwent isolation alone did not change their length, isolated jejunal segments that were stretched by applying a gradual mechanical force doubled their initial length. Both groups increased their muscular thickness 5 folds as compared to the normal jejunum. The mRNA level of IGF-I in the lengthened jejunum was 6 folds higher than that in the normal jejunum, but the IGF-I mRNA level in the isolated jejunum without mechanical lengthening was unchanged. By immunofluorescence, the increased IGF-I expression in the lengthened jejunum was localized to the intestinal smooth muscle cells. CONCLUSIONS: Insulin-like growth factor I may be an important signal induced by the applied axial force that mediates longitudinal intestinal growth.

Distension enterogenesis: increasing the size and function of small intestine.

INTRODUCTION: The purpose of this study is to evaluate the feasibility of using saline infusion to lengthen small bowel while preserving intestinal enzymatic function. METHODS: Male Sprague-Dawley rats had a 3-cm jejunal segment taken out of continuity. A catheter was inserted in the proximal end, and the distal end was oversewn. Continuous infusion of saline into the isolated jejunal segment was started 2 weeks postoperatively. Segments were harvested 1 week later. Segment weights and lengths were measured preoperatively and at the time of harvest. Histology of harvested segments was performed. Alkaline phosphatase (ALP) and lactase assays were performed. Comparisons were made with normal jejunum from control animals. RESULTS: A 32% increase in length was achieved with saline distension of small intestine. The segment weight to length ratio was significantly increased by saline distension; however, the total protein-to-weight ratio was unchanged. Specific activities of ALP and lactase were not affected by saline distension. Because of the increased length and weight of the distended jejunal segments, total segment activities for both enzymes were significantly increased. CONCLUSIONS: Saline infusion appears to be a viable method for increasing small intestinal length without compromising enzymatic function. This phenomenon may provide a new method for the treatment of patients with short bowel syndrome in the future, and further study is warranted.

Sustainability of mechanically lengthened bowel in rats.

INTRODUCTION: It has been shown that the length of an intestinal segment may be doubled by applying gradual mechanical stretching. This study evaluated whether the lengthened intestinal segment retained the structure and function after the stretching device was removed. METHODS: A 1.5-cm jejunal segment was separated from intestinal continuity in 20 rats. After advancing a screw into the isolated jejunal segment by 5 mm 3 times a week until it was stretched by 3 cm, the screw was removed. Three weeks later, the jejunal segments were retrieved for analyses. Comparisons were made between the lengthened jejunal segments. RESULTS: The jejunal segment doubled its length after gradual stretching and retained this length 3 weeks after the screw removal (3.1 +/- 0.8 vs 3.2 +/- 0.4 cm, P > .05). The villous height, the muscular thickness, and the total alkaline phosphatase and lactase activities of the stretched jejunal segments were also unchanged 3 weeks after the screw removal. CONCLUSIONS: Mechanical force induced the sustained lengthening of isolated jejunal segments in rats. The histologic and enzymatic alterations also persisted 3 weeks after the mechanical force was removed. This phenomenon may provide a novel method for the treatment of short bowel syndrome.

The Drosophila nuclear receptor e75 contains heme and is gas responsive.

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.

Quantitative evolutionary design of nutrient processing: glucose.

Quantitative evolutionary design involves the numerical relationships, evolved through natural selection, of biological capacities to each other and to natural loads. Here we study the relation of nutrient-processing capacities of the intestine and of organs beyond it (such as liver and kidneys) to each other and to natural loads of nutrients normally consumed. To control experimentally the rate of nutrient delivery to organs beyond the intestine, we administered nutrients directly into the veins of rats by the method of total parenteral nutrition (TPN). Control rats consuming the TPN solution by mouth ingested glucose at 42 mmol/day and processed it completely, as gauged by negligible appearance of glucose in urine and feces. Experimental rats receiving TPN were able to process infused glucose completely at rates up to 92 mmol/day. At higher infusion rates, they were unable to process further glucose, as gauged by rises in serum and urinary glucose levels and serum osmolality. At the highest infusion rates, they exhibited diuresis, dehydration, and both decreased weight gain and survival. These symptoms closely resemble the human diabetic condition known as nonketotic hypertonicity. Thus, a rat's body has a safety factor of 2.2 (=92/42) for glucose processing: it can process glucose at a rate 2.2 times its voluntary intake. This safety factor represents apparent excess capacity that may have evolved to process other nutrients converted into glucose, to minimize the risk of loads swamping capacities, to handle suddenly increased nutrient requirements, or to effect rapid mobilization of glucose.

Loads, capacities and safety factors of maltase and the glucose transporter SGLT1 in mouse intestinal brush border.

Safety factors are defined as ratios of biological capacities to prevailing natural loads. We measured the safety factor of the mouse intestinal brush-border hydrolase maltase in series with the glucose transporter SGLT1, for comparison with previous studies of sucrase and lactase. Dietary maltose loads increased 4-fold from virgin to lactating mice. As in previous studies of intestinal adaptive regulation, that increase in load without change in dietary composition resulted in an increase in maltase and SGLT1 capacities mediated non-specifically by an increase in intestinal mass, without change in maltase or SGLT1 activities per milligram of tissue. Maltase and SGLT1 capacities increased only sublinearly with load during lactation, such that safety factors decreased with load: from 6.5 to 2.4 for maltase, and from 1.1 to 0.5 for SGLT1. The apparently high safety factor for maltase may be related to the multiple natural substrates hydrolysed by the multiple sites of maltase activity. The apparently low safety factor for SGLT1 is made possible by the contribution of hindgut fermentation to carbohydrate digestion. SGLT1 activity is paradoxically higher for mice consuming sucrose than for mice consuming maltose, despite maltose hydrolysis yielding double the glucose load yielded by sucrose hydrolysis, and despite glucose constituting the load upon SGLT1.

Adrenal cortical cell transplantation.

PURPOSE: The adrenal cortex is a critical component of the hypothalamic-pituitary-adrenal/gonadal axis that coordinates the stress response and maintains homeostasis. The authors hypothesize that adrenal cortical cells can be transplanted in adrenal insufficiency states to regenerate the adrenal cortex. METHODS: Murine adrenal glands were dissociated into adrenal cortical cells. Cells cultured in a collagen matrix were transplanted under the renal capsule. The implants were retrieved 1, 2, 4, and 8 weeks later. Total RNA was extracted from the retrieved specimens and was analyzed by real-time polymerase chain reaction. RESULTS: All animals survived the surgical procedure. At implant retrieval, a distinct organoid could be visualized under the renal capsule. The expressions of adrenal-specific markers including Sf1, Dax1, Star, Cyp11a, Cyp11b1, and Cyp21 were detectable in the retrieved specimens up to 8 weeks posttransplantation. CONCLUSION: Primary adrenal cortical cells retained their gene expressions after heterotopic transplantation. Ex vivo gene transfer followed by adrenal cortical cell transplantation may lead to curative therapy for patients with adrenal insufficiency.