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Correction After publication of the original article [1] it was realised that there were errors in figures 2a,b,f,g, which arose as a result of preparing figures from data collected and analysed at the same time as the work reported in [2] (Supplementary Figure 1 of [2]). An updated Fig. 2 is included with this Correction.

Background During the development of the mammalian cerebral cortex, newborn postmitotic projection neurons are born from local neural stem cells and must undergo radial migration so as to position themselves appropriately to form functional neural circuits. The zinc finger transcriptional repressor Rp58 (also known as Znf238 or Zbtb18) is critical for coordinating corticogenesis, but its underlying molecular mechanism remains to be better characterised. Findings Here, we demonstrate that the co-expression of Rp58 and the cyclin dependent kinase inhibitor (CDKI) p27kip1 is important for E14.5-born cortical neurons to coordinate cell cycle exit and initiate their radial migration. Notably, we find that the impaired radial positioning of Rp58-deficient cortical neurons within the embryonic (E17.5) mouse cortex, as well as their multipolar to bipolar transition from the intermediate zone to the cortical plate can be restored by forced expression of p27kip1 in concert with suppression of Rnd2, a downstream target gene of Rp58. Furthermore, the restorative effects of p27kip1 and Rnd2 abrogation are reminiscent of suppressing RhoA signalling in Rp58-deficient cells. Conclusions Our findings demonstrate functional interplay between a transcriptional regulator and a CDKI to mediate neuroprogenitor cell cycle exit, as well as to promote radial migration through a molecular mechanism consistent with suppression of RhoA signalling. Electronic supplementary material The online version of this article (doi:10.1186/s13064-017-0084-3) contains supplementary material, which is available to authorized users.

The effects of Rp58 siRNA treatment together with expression constructs for p27kip1 and p27kip1ck- on progenitors. (A) There was a significant interaction between non-surface (SVZ) divisions in treated cells, identified as mitoses marked by pHH3 expression away from the ventricular surface (F3,9 = 7, p 600 cells counted from 3 to 4 independent brains per condition). (G-J) Representative photomicrographs of GFP and Tbr2 immunostained sections from each treatment, as indicated. (K) The proportion of Tbr2-expressing intermediate progenitors was significantly affected between treatment groups (F3,11 = 6.5, p = 0.00858, One-way ANOVA, >600 cells counted from 3 to 4 independent brains per condition). Values in graphs represent mean ± SEM. Scale bars represent 50 μm. (PDF 193 kb)

Forced expression of p27kip1 enhances radial migration and alters the morphological characteristics of embryonic cortical neurons. (A-B) Forced expression of p27kip1 leads to enhanced radial migration, observed as a significant increase in the proportion of cells arriving in the CP (F2,12 = 7.245, p = 0.0085; Two-way ANOVA followed by Bonferroni’s posthoc t-test; *p 285 neurons per condition). Scale bars represent 100 μm (B) and 20 μm (F, I) respectively. (PDF 331 kb)

p27kip1 is a potent mediator of cell cycle exit and radial migration within the embryonic cortex. In utero electroporation of a control (GFP only), p27kip1 or p27kip1(ck-) construct into E14.5 embryonic cortices collected 36 h after surgery for analysis. (A-B) Sections were immunostained for Ki67 and counted to find that forced expression of p27kip1 but not p27kip1(ck-) led to a significant suppression in Ki67 co-staining (F2,6 = 53, p = 0.0002). (C-D) Sections were immunostained for pHH3, a marker of cell mitosis to find that forced expression of p27kip1 but not p27kip1(ck-) led to a significant suppression in pHH3 co-staining (F2,6 = 56, p = 0.0001). (E) Overexpression of p27kip1 or p27kip1ck- alone did not influence Pax6-immunoreactive radial glial progenitors (F2,7 = 0.35, p = 0.7171, One-way ANOVA, >600 cells counted from 3 to 4 independent brains per condition). (F) Treatment with Rp58 siRNAs led to a significant reduction in the proportion of Tbr2-expressing intermediate progenitors which could not be augmented by co-delivery of p27kip1 or p27kip1ck- expression constructs (F2,7 = 11, p = 0.0072, One-way ANOVA, >600 cells counted from 3 to 4 independent brains per condition). (G) The distribution of GFP-labelled cells is significantly affected by treatment with p27kip1 or p27kip1(ck-). Forced expression of p27kip1 or p27kip1(ck-) led to a significant increase in the proportion of treated cells arriving within the upper IZ (F8,30 = 18, p

A summary diagram highlighting the combinatorial functions for Rp58 and p27kip1 on cell cycle exit and radial migration during the development of the mouse embryonic cerebral cortex. In this scheme, the regulation of neuroprogenitor cell cycle exit and neurogenesis is mediated by Rp58. Newborn postmitotic cells within the E14.5 embryonic cortex undergo cell cycle exit and express p27kip1 and Neurog2. The timing of p27kip1 expression and Neurog2 expression is influenced by Rp58. Neurog2 stimulates the expression of Rp58 which induces a feedback loop to temper Neurog2 expression levels, as well as to abrogate Rnd2 expression in migrating cortical neurons. Meanwhile, p27kip1 stabilises Neurog2 protein levels to specify glutamatergic neuron identity as well as promote radial migration. In the context of Rp58 deficiency, cortical cells lose their capacity to transit from the IZ to the CP owing to their failure to undergo MP to BP transition (B). Neither forced expression of p27kip1 nor Rnd2 RNAi alone was capable of restoring the capacity of Rp58-deficient cells to migrate into the CP (C-D). However, the defective migration of Rp58-deficient cells was significantly augmented by the combination of p27kip1 overexpression and Rnd2 RNAi (F). This restorative capacity is reminiscent of Rp58-deficient cells co-treated with RhoA(N19), a dominant-negative form which suppresses RhoA signalling (see Fig. 4). (PDF 123Â kb)

Steady state levels of p27kip1 are not significantly altered in Rp58(+/−) and Rp58(−/−) mutant mice compared to Rp58(+/+) wildtype littermate controls. (A) Western blotting of mouse brain lysates from E14.5 mouse embryonic cortices of wildtype, heterozygote and homozygote mutant Rp58-knockout mice. Quantification was conducted on biological triplicates. (B) There was no significant difference in the steady state levels of immunoblotted p27kip1 signal (F2,7 = 0.26, p = 0.77, One-way ANOVA). (C) Analysis of p27kip1 immunofluorescence signal (red) in E14.5-electroporated, GFP-expressing cortical cells collected 36 h after surgical manipulation. Boxed inserts are representative of individual cells analysed from the IZ (C′) and VZ/SVZ (C″). (D) Raw images were analysed using ImageJ software to measure the average intensity of p27kip1 signal in GFP-labelled, IZ cells, with values in Arbitrary Units defined as the signal intensity of an IZ cell divided by the average intensity of 20 CP cells which are negative for GFP immunostaining, and multiplied by 100. There was no significant interaction between treatment groups (F2,222 = 2.9, p = 0.0557, One-way ANOVA, >75 cells counted from 3 independent brains per condition). (E) The average intensity of p27kip1 signal in GFP-labelled, VZ/SVZ cells, with values in Arbitrary Units defined as the signal intensity of an IZ cell divided by the average intensity of unelectroporated CP cells, and multiplied by 100. The intensity of p27kip1-immunofluorescence signal within VZ/SVZ cells was significantly reduced upon knockdown, while overexpression did not have a significant effect (F2,222 = 95, p 75 cells counted from 3 independent brains per condition). Values in (B), (D) and (E) represent mean ± SEM. Scale bars represent 100 μm (C) and 20 μm (C″) respectively. (PDF 208 kb)

During brain development, many factors influence the assembly and final positioning of cortical neurons, and this process is essential for proper circuit formation and normal brain function. Among many important extrinsic factors that guide the maturation of embryonic cortical neurons, the secreted neurotransmitter GABA has been proposed to influence both their migratory behaviour and their terminal differentiation. The full extent of the short-term and long-term changes in brain patterning and function caused by modulators of the GABA system is not known. In this study, we specifically investigated whether diazepam, a commonly used benzodiazepine that modulates the GABAA receptor, alters neuronal positioning in vivo, and whether this can lead to lasting effects on brain function. We found that fetal exposure to diazepam did not change cell positioning within the embryonic day (E)14.5 mouse cerebral cortex, but significantly altered neuron positioning within the E18.5 cortex. In adult mice, diazepam treatment affected the distribution of cortical interneurons that express parvalbumin or calretinin, and also led to a decrease in the numbers of calretinin-expressing interneurons. In addition, we observed that neonatal exposure to diazepam altered the sensitivity of mice to a proconvulsant challenge. Therefore, exposure of the fetal brain to benzodiazepines has consequences for the positioning of neurons and cortical network excitability.

Copy number variations to chromosome 21 (HSA21) cause intellectual disability and Down Syndrome, but our understanding of the HSA21 genetic factors which contribute to fetal brain development remains incomplete. Here, we focussed on the neurodevelopmental functions for EURL (also known as C21ORF91, Refseq Gene ID:54149), a protein-coding gene at the centromeric boundary of the Down Syndrome Critical Region (DSCR) of HSA21. We report that EURL is expressed during human and mouse cerebral cortex development, and we report that alterations to EURL mRNA levels within the human brain underlie Down Syndrome. Our gene perturbation studies in mice demonstrate that disruptions to Eurl impair progenitor proliferation and neuronal differentiation. Also, we find that disruptions to Eurl impair the long-term positioning and dendritic spine densities of cortical projection neurons. We provide evidence that EURL interacts with the coiled-coil domain-containing protein CCDC85B so as to modulate β-catenin levels in cells. Further, we utilised a fluorescent reporter (8xTOPFLASHd2EGFP) to demonstrate that disruptions to Eurl alter β-catenin signalling in vitro as well as in vivo. Together, these studies highlight EURL as an important new player in neuronal development that is likely to impact on the neuropathogenesis of HSA21-related disorders including Down Syndrome.

Summary Disruptions to neuronal mRNA translation are hypothesized to underlie human neurodevelopmental syndromes. Notably, the mRNA translation re-initiation factor DENR is a regulator of eukaryotic translation and cell growth, but its mammalian functions are unknown. Here, we report that Denr influences the migration of murine cerebral cortical neurons in vivo with its binding partner Mcts1, whereas perturbations to Denr impair the long-term positioning, dendritic arborization, and dendritic spine characteristics of postnatal projection neurons. We characterized de novo missense mutations in DENR (p.C37Y and p.P121L) detected in two unrelated human subjects diagnosed with brain developmental disorder to find that each variant impairs the function of DENR in mRNA translation re-initiation and disrupts the migration and terminal branching of cortical neurons in different ways. Thus, our findings link human brain disorders to impaired mRNA translation re-initiation through perturbations in DENR (OMIM: 604550) function in neurons., Graphical Abstract, Highlights • DENR is an important player in mammalian neurodevelopment • DENR controls radial migration through its binding partner MCTS1 • Mutations in DENR influence neuronal positioning and neurodifferentiation • This study links impaired mRNA translation re-initiation to neurological disorders, Haas et al. report that the expression of the mRNA translation re-initiation factor DENR is important for the radial positioning, dendritic branching, and dendritic spine properties of developing cerebral cortex neurons. Characterization of disease-associated mutations in DENR links impaired mRNA translation re-initiation to human neurological disorders.

Background The development of neural circuits within the embryonic cerebral cortex relies on the timely production of neurons, their positioning within the embryonic cerebral cortex as well as their terminal differentiation and dendritic spine connectivity. The RhoA GTPases Rnd2 and Rnd3 are important for neurogenesis and cell migration within the embryonic cortex (Nat Commun 4:1635, 2013), and we recently identified the BTB/POZ domain-containing Adaptor for Cul3-mediated RhoA Degradation family member Bacurd2 (also known as Tnfaip1) as an interacting partner to Rnd2 for the migration of embryonic mouse cortical neurons (Neural Dev 10:9, 2015). Findings We have extended this work and report that Bacurd1/Kctd13 and Bacurd2/Tnfaip1 are interacting partners to Rnd2 and Rnd3 in vitro. Given that these genes are expressed during cortical development, we performed a series of in utero electroporation studies in mice and found that disruptions to Bacurd1/Kctd13 or Bacurd2/Tnfaip1 expression impair the long-term positioning of E14.5-born cortical neurons within the postnatal (P17) mouse cerebral cortex. We also find that forced expression of Bacurd1/Kctd13 and Bacurd2/Tnfaip1 alters the branching and dendritic spine properties of layer II/III projection neurons. Conclusions We identify Bacurd1/Kctd13 and Bacurd2/Tnfaip1 as interacting partners to Rnd proteins which influence the development of cortical neurons. Their neurodevelopmental functions are likely to be relevant to human brain development and disease. Electronic supplementary material The online version of this article (doi:10.1186/s13064-016-0062-1) contains supplementary material, which is available to authorized users.

In the present study the relationship between tissue damage and changed electro-physiological properties of Dogiel type II myenteric neurons within the first 24 hours after induction of inflammation with trinitrobenzene sulfonate (TNBS) in the guinea-pig ileum was investigated. Treatment with TNBS causes damage to the mucosa, inflammatory responses in the mucosa and enteric ganglia and changes in myenteric neuron properties. Thus we hypothesise that the physiological changes in the myenteric neurons could be due to damage to their mucosal processes or inflammation in the vicinity of cell bodies or the processes. We found an association between hyperexcitability of myenteric Dogiel type II neurons and damage to the mucosa and its innervation at 3 and 24 h, times when there was also an inflammatory reaction. The lack of hyperexcitability in neurons from control tissues in which axons projecting to the mucosa were severed suggests that inflammation may be an important contributing factor to the neuronal hyperexcitability at the acute stage of inflammation. Despite mucosal repair and re-innervation of the mucosa before 7 days after induction of inflammation, neuronal hyperexcitability persists. Although the mechanisms underlying neuronal hyperexcitability at the acute stage of inflammation might be different from those underlying long-term changes in the absence of active inflammation in the ganglia, the persistent changes in neuronal excitability may contribute to post-inflammatory gut dysfunctions.

The definition of the nerve cell types of the myenteric plexus of the mouse small intestine has become important, as more researchers turn to the use of mice with genetic mutations to analyze roles of specific genes and their products in enteric nervous system function and to investigate animal models of disease. We have used a suite of antibodies to define neurons by their shapes, sizes, and neurochemistry in the myenteric plexus. Anti-Hu antibodies were used to reveal all nerve cells, and the major subpopulations were defined in relation to the Hu-positive neurons. Morphological Type II neurons, revealed by anti-neurofilament and anti-calcitonin gene-related peptide antibodies, represented 26% of neurons. The axons of the Type II neurons projected through the circular muscle and submucosa to the mucosa. The cell bodies were immunoreactive for choline acetyltransferase (ChAT), and their terminals were immunoreactive for vesicular acetylcholine transporter (VAChT). Nitric oxide synthase (NOS) occurred in 29% of nerve cells. Most were also immunoreactive for vasoactive intestinal peptide, but they were not tachykinin (TK)-immunoreactive, and only 10% were ChAT-immunoreactive. Numerous NOS terminals occurred in the circular muscle. We deduced that 90% of NOS neurons were inhibitory motor neurons to the muscle (26% of all neurons) and 10% (3% of all neurons) were interneurons. Calretinin immunoreactivity was found in a high proportion of neurons (52%). Many of these had TK immunoreactivity. Small calretinin neurons were identified as excitatory neurons to the longitudinal muscle (about 20% of neurons, with ChAT/calretinin/+/- TK chemical coding). Excitatory neurons to the circular muscle (about 10% of neurons) had the same coding. Calretinin immunoreactivity also occurred in a proportion of Type II neurons. Thus, over 90% of neurons in the myenteric plexus of the mouse small intestine can be currently identified by their neurochemistry and shape.

Background During fetal brain development in mammals, newborn neurons undergo cell migration to reach their appropriate positions and form functional circuits. We previously reported that the atypical RhoA GTPase Rnd2 promotes the radial migration of mouse cerebral cortical neurons (Nature 455(7209):114–8, 2008; Neuron 69(6):1069–84, 2011), but its downstream signalling pathway is not well understood. Results We have identified BTB-domain containing adaptor for Cul3-mediated RhoA degradation 2 (Bacurd2) as a novel interacting partner to Rnd2, which promotes radial migration within the developing cerebral cortex. We find that Bacurd2 binds Rnd2 at its C-terminus, and this interaction is critical to its cell migration function. We show that forced expression or knockdown of Bacurd2 impairs neuronal migration within the embryonic cortex and alters the morphology of immature neurons. Our in vivo cellular analysis reveals that Bacurd2 influences the multipolar-to-bipolar transition of radially migrating neurons in a cell autonomous fashion. When we addressed the potential signalling relationship between Bacurd2 and Rnd2 using a Bacurd2-Rnd2 chimeric construct, our results suggest that Bacurd2 and Rnd2 could interact to promote radial migration within the embryonic cortex. Conclusions Our studies demonstrate that Bacurd2 is a novel player in neuronal development and influences radial migration within the embryonic cerebral cortex. Electronic supplementary material The online version of this article (doi:10.1186/s13064-015-0032-z) contains supplementary material, which is available to authorized users.

The microtubule cytoskeleton is critical for the generation and maturation of neurons in the developing mammalian nervous system. We have previously shown that mutations in the β-tubulin gene TUBB5 cause microcephaly with structural brain abnormalities in humans. While it is known that TUBB5 is necessary for the proper generation and migration of neurons, little is understood of the role it plays in neuronal differentiation and connectivity. Here, we report that perturbations to TUBB5 disrupt the morphology of cortical neurons, their neuronal complexity, axonal outgrowth, as well as the density and shape of dendritic spines in the postnatal murine cortex. The features we describe are consistent with defects in synaptic signaling. Cellular-based assays have revealed that TUBB5 substitutions have the capacity to alter the dynamic properties and polymerization rates of the microtubule cytoskeleton. Together, our studies show that TUBB5 is essential for neuronal differentiation and dendritic spine formation in vivo, providing insight into the underlying cellular pathology associated with TUBB5 disease states.

The impact of aberrant centrosomes and/or spindles on asymmetric cell division in embryonic development indicates the tight regulation of bipolar spindle formation and positioning that is required for mitotic progression and cell fate determination. WD40-repeat protein 62 (WDR62) was recently identified as a spindle pole protein linked to the neurodevelopmental defect of microcephaly but its roles in mitosis have not been defined. We report here that the in utero electroporation of neuroprogenitor cells with WDR62 siRNAs induced their cell cycle exit and reduced their proliferative capacity. In cultured cells, we demonstrated cell-cycle-dependent accumulation of WDR62 at the spindle pole during mitotic entry that persisted until metaphase–anaphase transition. Utilizing siRNA depletion, we revealed WDR62 function in stabilizing the mitotic spindle specifically during metaphase. WDR62 loss resulted in spindle orientation defects, decreased the integrity of centrosomes displaced from the spindle pole and delayed mitotic progression. Additionally, we revealed JNK phosphorylation of WDR62 is required for maintaining metaphase spindle organization during mitosis. Our study provides the first functional characterization of WDR62 and has revealed requirements for JNK/WDR62 signaling in mitotic spindle regulation that may be involved in coordinating neurogenesis.

Gene-targeted knockout technologies are invaluable tools for understanding the functions of genes in vivo. CRISPR/Cas9 system of RNA-guided genome editing is revolutionizing genetics research in a wide spectrum of organisms. Here, we combined CRISPR with in vivo electroporation in the chicken embryo to efficiently target the transcription factor PAX7 in tissues of the developing embryo. This approach generated mosaic genetic mutations within a wild-type cellular background. This series of proof-of-principle experiments indicate that in vivo CRISPR-mediated cell genome engineering is an effective method to achieve gene loss-of-function in the tissues of the chicken embryo and it completes the growing genetic toolbox to study the molecular mechanisms regulating development in this important animal model.

Supplementary Methods related to Figure S2 [19, 27]. (DOCX 14.2Â kb)

Immunostaining for Nestin reveals radial glial fibres within sections of electroporated (E14.5 + 72 h) mouse brains. Nestin immunostaining indicates that the radial glial scaffolds within the cortices of each treatment group are not qualitatively different. Scale bar represents 50 μm. (PDF 6040 kb)

Immunostaining for Nestin reveals radial glial fibres within sections of electroporated (E14.5 + 72 h) mouse brains. Nestin immunostaining indicates that the radial glial scaffolds within the cortices of each treatment group are not qualitatively different. Scale bar represents 50 μm. (PDF 6040 kb)

BMP/RA-inducible neural-specific protein 1 (Brinp1) is highly conserved in vertebrates, and continuously expressed in the neocortex, hippocampus, olfactory bulb and cerebellum from mid-embryonic development through to adulthood. Brinp1 knock-out (Brinp1 −/−) mice were generated by Cre-recombinase-mediated removal of the third exon of Brinp1. Knock-out mice were characterised by behavioural phenotyping, immunohistochemistry and expression analysis of the developing and adult brain. Absence of Brinp1 during development results in a behavioural phenotype resembling autism spectrum disorder (ASD), in which knock-out mice show reduced sociability and changes in vocalisation capacity. In addition, Brinp1 −/− mice exhibit hyper-locomotor activity, have impaired short-term memory, and exhibit poor reproductive success. Brinp1 −/− mice show increased density of parvalbumin-expressing interneurons in the adult mouse brain. Brinp1 −/− mice do not show signs of altered neural precursor proliferation or increased apoptosis during late embryonic brain development. The expression of the related neuronal migration genes Astn1 and Astn2 is increased in the brains of Brinp1 −/− mice, suggesting that they may ameliorate the effects of Brinp1 loss. Brinp1 plays an important role in normal brain development and function by influencing neuronal distribution within the cortex. The increased cortical PV-positive interneuron density and altered behaviour of Brinp1 −/− mice resemble features of a subset of human neurological disorders; namely autism spectrum disorder (ASD) and the hyperactivity aspect of attention deficit hyperactivity disorder (ADHD).

Supplementary Methods related to Figure S2 [19, 27]. (DOCX 14.2Â kb)