PSmad3+/Olig2- expression defines a subpopulation of gfap-GFP+/Sox9+ neural progenitors and radial glia-like cells in mouse dentate gyrus through embryonic and postnatal development.

In mouse dentate gyrus, radial glia-like cells (RGLs) persist throughout life and play a critical role in the generation of granule neurons. A large body of evidence has shown that the combinatorial expression of transcription factors (TFs) defines cell types in the developing central nervous system (CNS). As yet, the identification of specific TFs that exclusively define RGLs in the developing mouse dentate gyrus (DG) remains elusive. Here we show that phospho-Smad3 (PSmad3) is expressed in a subpopulation of neural progenitors in the DG. During embryonic stage (E14-15), PSmad3 was predominantly expressed in gfap-GFP-positive (GFP+)/Sox2+ progenitors located at the lower dentate notch (LDN). As the development proceeds (E16-17), the vast majority of PSmad3+ cells were GFP+/Sox2+/Prox1low+/Ki67+ proliferative progenitors that eventually differentiated into granule neurons. During postnatal stage (P1-P6) PSmad3 expression was observed in GFP+ progenitors and astrocytes. Subsequently, at P14-P60, PSmad3 expression was found both in GFP+ RGLs in the subgranular zone (SGZ) and astrocytes in the molecular layer (ML) and hilus. Notably, PSmad3+ SGZ cells did not express proliferation markers such as PCNA and phospho-vimentin, suggesting that they are predominantly quiescent from P14 onwards. Significantly PSmad3+/GFP+ astrocytes, but not SGZ cells, co-expressed Olig2 and S100ß. Together, PSmad3+/Olig2- expression serves as an exclusive marker for a specific subpopulation of GFP+ neural progenitors and RGLs in the mouse DG during both embryonic and postnatal period.

SHH and Notch regulate SOX9+ progenitors to govern arcuate POMC neurogenesis.

Pro-opiomelanocortin (POMC)-expressing neurons in the hypothalamic arcuate nucleus (ARC) play key roles in feeding and energy homoeostasis, hence their development is of great research interest. As the process of neurogenesis is accompanied by changes in adhesion, polarity, and migration that resemble aspects of epithelial-to-mesenchymal transitions (EMTs), we have characterised the expression and regulation within the prospective ARC of transcription factors with context-dependent abilities to regulate aspects of EMT. Informed by pseudotime meta-analysis of recent scRNA-seq data, we use immunohistochemistry and multiplex in situ hybridisation to show that SOX2, SRY-Box transcription factor 9 (SOX9), PROX1, Islet1 (ISL1), and SOX11 are sequentially expressed over the course of POMC neurogenesis in the embryonic chick. Through pharmacological studies ex vivo, we demonstrate that while inhibiting either sonic hedgehog (SHH) or Notch signalling reduces the number of SOX9+ neural progenitor cells, these treatments lead, respectively, to lesser and greater numbers of differentiating ISL1+/POMC+ neurons. These results are consistent with a model in which SHH promotes the formation of SOX9+ progenitors, and Notch acts to limit their differentiation. Both pathways are also required to maintain normal levels of proliferation and to suppress apoptosis. Together our findings demonstrate that hypothalamic neurogenesis is accompanied by dynamic expression of transcription factors (TFs) that mediate EMTs, and that SHH and Notch signalling converge to regulate hypothalamic cellular homoeostasis.

Single-cell analysis of early chick hypothalamic development reveals that hypothalamic cells are induced from prethalamic-like progenitors.

The hypothalamus regulates many innate behaviors, but its development remains poorly understood. Here, we used single-cell RNA sequencing (RNA-seq) and hybridization chain reaction (HCR) to profile multiple stages of early hypothalamic development in the chick. Hypothalamic neuroepithelial cells are initially induced from prethalamic-like cells. Two distinct hypothalamic progenitor populations then emerge and give rise to tuberal and mammillary/paraventricular hypothalamic cells. At later stages, the regional organization of the chick and mouse hypothalamus is highly similar. We identify selective markers for major subdivisions of the developing chick hypothalamus and many previously uncharacterized candidate regulators of hypothalamic induction, regionalization, and neurogenesis. As proof of concept for the power of the dataset, we demonstrate that prethalamus-derived follistatin inhibits hypothalamic induction. This study clarifies the organization of the nascent hypothalamus and identifies molecular mechanisms that may control its induction and subsequent development.

Involvement of the neuronal phosphotyrosine signal adaptor N-Shc in kainic acid-induced epileptiform activity.

BDNF-TrkB signaling is implicated in experimental seizures and epilepsy. However, the downstream signaling involved in the epileptiform activity caused by TrkB receptor activation is still unknown. The aim of the present study was to determine whether TrkB-mediated N-Shc signal transduction was involved in kainic acid (KA)-induced epileptiform activity. We investigated KA-induced behavioral seizures, epileptiform activities and neuronal cell loss in hippocampus between N-Shc deficient and control mice. There was a significant reduction in seizure severity and the frequency of epileptiform discharges in N-Shc deficient mice, as compared with wild-type and C57BL/6 mice. KA-induced neuronal cell loss in the CA3 of hippocampus was also inhibited in N-Shc deficient mice. This study demonstrates that the activation of N-Shc signaling pathway contributes to an acute KA-induced epileptiform activity and neuronal cell loss in the hippocampus. We propose that the N-Shc-mediated signaling pathway could provide a potential target for the novel therapeutic approaches of epilepsy.

ProNodal acts via FGFR3 to govern duration of Shh expression in the prechordal mesoderm.

The secreted glycoprotein sonic hedgehog (Shh) is expressed in the prechordal mesoderm, where it plays a crucial role in induction and patterning of the ventral forebrain. Currently little is known about how Shh is regulated in prechordal tissue. Here we show that in the embryonic chick, Shh is expressed transiently in prechordal mesoderm, and is governed by unprocessed Nodal. Exposure of prechordal mesoderm microcultures to Nodal-conditioned medium, the Nodal inhibitor CerS, or to an ALK4/5/7 inhibitor reveals that Nodal is required to maintain both Shh and Gsc expression, but whereas Gsc is largely maintained through canonical signalling, Nodal signals through a non-canonical route to maintain Shh. Further, Shh expression can be maintained by a recombinant Nodal cleavage mutant, proNodal, but not by purified mature Nodal. A number of lines of evidence suggest that proNodal acts via FGFR3. ProNodal and FGFR3 co-immunoprecipitate and proNodal increases FGFR3 tyrosine phosphorylation. In microcultures, soluble FGFR3 abolishes Shh without affecting Gsc expression. Further, prechordal mesoderm cells in which Fgfr3 expression is reduced by Fgfr3 siRNA fail to bind to proNodal. Finally, targeted electroporation of Fgfr3 siRNA to prechordal mesoderm in vivo results in premature Shh downregulation without affecting Gsc. We report an inverse correlation between proNodal-FGFR3 signalling and pSmad1/5/8, and show that proNodal-FGFR3 signalling antagonises BMP-mediated pSmad1/5/8 signalling, which is poised to downregulate Shh. Our studies suggest that proNodal/FGFR3 signalling governs Shh duration by repressing canonical BMP signalling, and that local BMPs rapidly silence Shh once endogenous Nodal-FGFR3 signalling is downregulated.

Mdm20 stimulates polyQ aggregation via inhibiting autophagy through Akt-Ser473 phosphorylation.

Mdm20 is an auxiliary subunit of the NatB complex, which includes Nat5, the catalytic subunit for protein N-terminal acetylation. The NatB complex catalyzes N-acetylation during de novo protein synthesis initiation; however, recent evidence from yeast suggests that NatB also affects post-translational modification of tropomyosin, which is involved in intracellular sorting of aggregated proteins. We hypothesized that an acetylation complex such as NatB may contribute to protein clearance and/or proteostasis in mammalian cells. Using a poly glutamine (polyQ) aggregation system, we examined whether the NatB complex or its components affect protein aggregation in rat primary cultured hippocampal neurons and HEK293 cells. The number of polyQ aggregates increased in Mdm20 over-expressing (OE) cells, but not in Nat5-OE cells. Conversely, in Mdm20 knockdown (KD) cells, but not in Nat5-KD cells, polyQ aggregation was significantly reduced. Although Mdm20 directly associates with Nat5, the overall cellular localization of the two proteins was slightly distinct, and Mdm20 apparently co-localized with the polyQ aggregates. Furthermore, in Mdm20-KD cells, a punctate appearance of LC3 was evident, suggesting the induction of autophagy. Consistent with this notion, phosphorylation of Akt, most notably at Ser473, was greatly reduced in Mdm20-KD cells. These results demonstrate that Mdm20, the so-called auxiliary subunit of the translation-coupled protein N-acetylation complex, contributes to protein clearance and/or aggregate formation by affecting the phosphorylation level of Akt indepenently from the function of Nat5.

Wild-type but not mutant SOD1 transgenic astrocytes promote the efficient generation of motor neuron progenitors from mouse embryonic stem cells.

BACKGROUND: The efficient derivation of mature (Hb9+) motor neurons from embryonic stem cells is a sought-after goal in the understanding, and potential treatment, of motor neuron diseases. Conditions that promote the robust generation of motor neuron progenitors from embryonic stem cells and that promote the survival of differentiated motor neurons ex vivo are likely, therefore, to be critical in future biological/therapeutic/screening approaches. Previous studies have shown that astrocytes have a protective effect on differentiated motor neurons (in vivo and ex vivo), but it remains unclear whether astrocytes also play a beneficial role in the support of motor neuron progenitors. Here we explore the effect of murine astrocyte-conditioned medium on monolayer cultures of mouse embryonic stem cell-derived motor neuron progenitors. RESULTS: Our data show that wild-type astrocyte-conditioned medium significantly increases the number of Olig2+/Hb9- progenitors, which subsequently differentiate into Hb9+/Islet1+ post-mitotic motor neurons. Intriguingly, while astrocyte-conditioned medium derived from mice transgenic for wild-type human SOD1 mimics the effect of wild-type astrocytes, conditioned medium derived from astrocytes carrying an amyotrophic lateral sclerosis-related SOD1-G93A mutation shows no such beneficial effect. The effect of astrocyte-conditioned medium, moreover, is specific to motor neurons: we find that interneurons generated from mouse embryonic stem cells are unaffected by conditioned media from any type of astrocyte. CONCLUSIONS: Our study indicates that conditioned medium derived from wild type astrocytes enhances the efficient generation of motor neurons from mouse embryonic stem cells by enhancing motor neuron progenitors. In contrast, conditioned medium from SOD1-G93A astrocytes does not show a similar enhancing effect.

Direct and indirect roles of Fgf3 and Fgf10 in innervation and vascularisation of the vertebrate hypothalamic neurohypophysis.

The neurohypophysis is a crucial component of the hypothalamo-pituitary axis, serving as the site of release of hypothalamic neurohormones into a plexus of hypophyseal capillaries. The growth of hypothalamic axons and capillaries to the forming neurohypophysis in embryogenesis is therefore crucial to future adult homeostasis. Using ex vivo analyses in chick and in vivo analyses in mutant and transgenic zebrafish, we show that Fgf10 and Fgf3 secreted from the forming neurohypophysis exert direct guidance effects on hypothalamic neurosecretory axons. Simultaneously, they promote hypophyseal vascularisation, exerting early direct effects on endothelial cells that are subsequently complemented by indirect effects. Together, our studies suggest a model for the integrated neurohemal wiring of the hypothalamo-neurohypophyseal axis.

Spatio-temporal expression pattern of the NatB complex, Nat5/Mdm20 in the developing mouse brain: implications for co-operative versus non-co-operative actions of Mdm20 and Nat5.

The NatB complex, Nat5/Mdm20 acetyltransferase mediates N-acetylation to control cell cycle progression and actin dynamics in yeast. As yet, little is known about the expression pattern of Mdm20 and Nat5 in multi-cellular organisms. Here we show that Mdm20 is highly expressed in mouse embryonic brain. At E11.5, Mdm20 was widely expressed in both neural progenitors and early differentiating neurons, whereas Nat5 was expressed in Sox1/3+/Mdm20+ neural progenitors. By E14.5, both Mdm20 and Nat5 were downregulated in most ventricular zone neural progenitors, whereas both proteins were found in differentiating neurons and co-expression was maintained at E18.5 in derivatives of these cells, such as midbrain dopaminergic (DA) neurons and septal neurons. These data suggest that Nat5/Mdm20 complex-mediated acetylation may play a role in the proliferation and differentiation of neural progenitors. Intriguingly, our data also showed that Mdm20 is not always co-expressed with Nat5 in all differentiated neurons, for example deep cerebellar neurons. Moreover, detailed examination of the subcellular localization of Mdm20 and Nat5 in cultured Nat5+/Mdm20+ midbrain DA neurons revealed that Mdm20 is also not necessarily co-localized with Nat5 within neurons. Given that Nat5 is only a known member of Nat family protein that interacts with Mdm20, our data imply that Mdm20 may function either with an unidentified Nat protein partner(s) or possibly in a Nat-independent manner.

FGF-dependent midline-derived progenitor cells in hypothalamic infundibular development.

The infundibulum links the nervous and endocrine systems, serving as a crucial integrating centre for body homeostasis. Here we describe that the chick infundibulum derives from two subsets of anterior ventral midline cells. One set remains at the ventral midline and forms the posterior-ventral infundibulum. A second set migrates laterally, forming a collar around the midline. We show that collar cells are composed of Fgf3(+) SOX3(+) proliferating progenitors, the induction of which is SHH dependent, but the maintenance of which requires FGF signalling. Collar cells proliferate late into embryogenesis, can generate neurospheres that passage extensively, and differentiate to distinct fates, including hypothalamic neuronal fates and Fgf10(+) anterior-dorsal infundibular cells. Together, our study shows that a subset of anterior floor plate-like cells gives rise to Fgf3(+) SOX3(+) progenitor cells, demonstrates a dual origin of infundibular cells and reveals a crucial role for FGF signalling in governing extended infundibular growth.

F3/contactin and TAG1 play antagonistic roles in the regulation of sonic hedgehog-induced cerebellar granule neuron progenitor proliferation.

Modulation of the sonic hedgehog (SHH) pathway is a crucial factor in cerebellar morphogenesis. Stimulation of granule neuron progenitor (GNP) proliferation is a central function of SHH signalling, but how this is controlled locally is not understood. We show that two sequentially expressed members of the contactin (CNTN) family of adhesion molecules, TAG1 and F3, act antagonistically to control SHH-induced proliferation: F3 suppresses SHH-induced GNP proliferation and induces differentiation, whereas TAG1 antagonises F3. Production of GNPs in TAG1-null mice is delayed and reduced. F3 and TAG1 colocalise on GNPs with the related L1-like adhesion molecule NrCAM, and F3 fails to suppress the SHH-induced proliferation of NrCAM-deficient GNPs. We show that F3 and SHH both primarily affect a group of intermediate GNPs (IPs), which, though actively dividing, also express molecules associated with differentiation, including ß-tubulin III (TuJ1) and TAG1. In vivo, intermediate progenitors form a discrete layer in the middle of the external germinal layer (mEGL), while F3 becomes expressed on the axons of postmitotic granule neurons as they leave the inner EGL (iEGL). We propose, therefore, that F3 acts as a localised signal in the iEGL that induces SHH-stimulated cells in the overlying mEGL to exit cell cycle and differentiate. By contrast, expression of TAG1 on GNPs antagonises this signal in the mEGL, preventing premature differentiation and sustaining GNP expansion in a paracrine fashion. Together, these findings indicate that CNTN and L1-like proteins play a significant role in modulating SHH-induced neuronal precursor proliferation.

Temporal progression of hypothalamic patterning by a dual action of BMP.

In the developing chick hypothalamus, Shh and BMPs are expressed in a spatially overlapping, but temporally consecutive, manner. Here, we demonstrate how the temporal integration of Shh and BMP signalling leads to the late acquisition of Pax7 expression in hypothalamic progenitor cells. Our studies reveal a requirement for a dual action of BMPs: first, the inhibition of GliA function through Gli3 upregulation; and second, activation of a Smad5-dependent BMP pathway. Previous studies have shown a requirement for spatial antagonism of Shh and BMPs in early CNS patterning; here, we propose that neural pattern elaboration can be achieved through a versatile temporal antagonism between Shh and BMPs.

Regional morphogenesis in the hypothalamus: a BMP-Tbx2 pathway coordinates fate and proliferation through Shh downregulation.

A central challenge in embryonic development is to understand how growth and pattern are coordinated to direct emerging new territories during morphogenesis. Here, we report on a signaling cascade that links cell proliferation and fate, promoting formation of a distinct progenitor domain within the developing chick hypothalamus. We show that the downregulation of Shh in floor plate-like cells in the forebrain governs their progression to a distinctive, proliferating hypothalamic progenitor domain. Shh downregulation occurs via a local BMP-Tbx2 pathway, Tbx2 acting to repress Shh expression. We show in vivo and in vitro that forced maintenance of Shh in hypothalamic progenitors prevents their normal morphogenesis, leading to maintenance of the Shh receptor, ptc, and preventing progression to an Emx2(+)-proliferative progenitor state. Our data identify a molecular pathway for the downregulation of Shh via a BMP-Tbx2 pathway and provide a mechanism for expansion of a discrete progenitor domain within the developing forebrain.

A robust system for RNA interference in the chicken using a modified microRNA operon.

RNA interference (RNAi) provides an effective method to silence gene expression and investigate gene function. However, RNAi tools for the chicken embryo have largely been adapted from vectors designed for mammalian cells. Here we present plasmid and retroviral RNAi vectors specifically designed for optimal gene silencing in chicken cells. The vectors use a chicken U6 promoter to express RNAs modelled on microRNA30, which are embedded within chicken microRNA operon sequences to ensure optimal Drosha and Dicer processing of transcripts. The chicken U6 promoter works significantly better than promoters of mammalian origin and in combination with a microRNA operon expression cassette (MOEC), achieves up to 90% silencing of target genes. By using a MOEC, we show that it is also possible to simultaneously silence two genes with a single vector. The vectors express either RFP or GFP markers, allowing simple in vivo tracking of vector delivery. Using these plasmids, we demonstrate effective silencing of Pax3, Pax6, Nkx2.1, Nkx2.2, Notch1 and Shh in discrete regions of the chicken embryonic nervous system. The efficiency and ease of use of this RNAi system paves the way for large-scale genetic screens in the chicken embryo.

Mutual dependence of murine fetal cutaneous regeneration and peripheral nerve regeneration.

Mammalian fetal cutaneous wounds made at certain developmental stages show complete regeneration. It is reported that wound healing in both adult and fetal skin is disrupted by denervation. Furthermore, fetal cutaneous regeneration has unique aspects such as epidermal wrinkle texture regeneration and dermal regeneration that depend on developmental stage. Therefore, we have examined the relationship of fetal cutaneous regeneration with denervation. We made cutaneous wounds on fetal mice at various developmental time points including embryonic days (E)13, E15, and E17, and compared the regenerating patterns of peripheral nerves in the skin. We found that when the fetuses are wounded at an early stage of development, peripheral nerves regenerate quicker than at later stages of development when peripheral nerve regeneration is delayed. Next, we denervated the intercostal nerves and made wounds at the denervated sites on E13 and E15. We found that epidermal wrinkling and dermal regeneration were disrupted by denervation. These findings indicate that components of fetal cutaneous regeneration and peripheral nerve regeneration are mutually dependent.

Directed differentiation of neural cells to hypothalamic dopaminergic neurons.

Hypothalamic neurons play a key role in homeostasis, yet little is known about their differentiation. Here, we demonstrate that Shh and Bmp7 from the adjacent prechordal mesoderm govern hypothalamic neural fate, their sequential action controlling hypothalamic dopaminergic neuron generation in a Six3-dependent manner. Our data suggest a temporal distinction in the requirement for the two signals. Shh acts early to specify dopaminergic neurotransmitter phenotype. Subsequently, Bmp7 acts on cells that are ventralised by Shh, establishing aspects of hypothalamic regional identity in late-differentiating/postmitotic cells. The concerted actions of Shh and Bmp7 can direct mouse embryonic stem cell-derived neural progenitor cells to a hypothalamic dopaminergic fate ex vivo.

Neural cell adhesion molecule L1 is required for fasciculation and routing of thalamocortical fibres and corticothalamic fibres.

To examine the role of neural cell adhesion molecule L1 in thalamocortical projections, we analysed L1 deficient (L1-/y) mice. Immunohistochemistry of pleiotrophin/HB-GAM, a marker for thalamocortical axons and axonal tracing experiments showed that thalamocortical axons were abnormally and highly fasciculated when they pass through the developing internal capsule. Within the cortex, however, their course was more diffuse. The corticofugal fibres immunoreactive for TAG-1 were also more strongly fasciculated and their number was decreased in L1-/y mice. Furthermore, no TAG-1-positive corticofugal axons reached the dorsal thalamus. These data suggest that L1 plays an important role in the fasciculation and routing of axons connecting between the thalamus and the cortex.

Receptor-like protein tyrosine phosphatase zeta/RPTP beta is expressed on tangentially aligned neurons in early mouse neocortex.

Protein tyrosine phosphatase zeta (PTPzeta)/RPTPbeta is a chondroitin sulfate proteoglycan predominantly expressed in the brain. In this study, we examined immunohistochemical localisation of PTPzeta in the mouse telencephalon from embryonic day 9.5 (E9.5) to E15.5. During E10.5-E12.5, immunoreactivities for PTPzeta are specifically observed on the tangentially aligned neurons at the preplate (PP) of the neocortex, as well as on the neurons at the mantle layer (ML) of the ganglionic eminences (GEs). Likewise, neurons immunoreactive for CR50, a marker for Cajal-Retzius neurons, are aligned from the ML of the ganglionic eminences to the PP of the neocortex and co-express PTPzeta. During E13.5-E15.5, PTPzeta-positive neurons are present at the subplate (SP) as well as at the marginal zone (MZ) of the neocortex. These results indicate that PTPzeta is a useful marker for early-generated neocortical neurons in mice: Cajal-Retzius neurons as well as the subplate neurons.

Coordinate expression of beta1 integrins and their regulator, TGF beta2 at the floor plate of the medulla oblongata is correlated with the crossing of the fibers of olivocerebellar projection in mice.

During embryonic day 11 (E11) to E16, contact-dependent interacting molecules beta1 integrins and their putative regulator TGF beta2 are coordinately expressed at the floor plate in the caudal part of mouse myelencephalon. Their expression disappears at E18. Consistent with the peak of their expression (E13-E16), olivocerebellar fibers primarily cross the floor plate. These data indicate that spatiotemporal expression of beta1 integrins and TGF beta2 is correlated with the crossing of olivocerebellar fibers.