CNKSR2, a downstream mediator of retinoic acid signaling, modulates the Ras/Raf/MEK pathway to regulate patterning and invagination of the chick forebrain roof plate.

During embryonic development, the forebrain roof plate undergoes invagination, leading to separation of the cerebral hemispheres. Any defects in this process, in humans, lead to middle interhemispheric holoprosencephaly (MIH-HPE). In this study, we have identified a previously unreported downstream mediator of retinoic acid (RA) signaling, CNKSR2, which is expressed in the forebrain roof plate in the chick embryo. Knockdown of CNKSR2 affects invagination, cell proliferation and patterning of the roof plate, similar to the phenotypes observed upon inhibition of RA signaling. We further demonstrate that CNKSR2 functions by modulating the Ras/Raf/MEK signaling. This appears to be crucial for patterning of the forebrain roof plate and its subsequent invagination, leading to the formation of the cerebral hemispheres. Thus, a set of novel molecular players have been identified that regulate the morphogenesis of the avian forebrain.

Multi-length scale strengthening and cytocompatibility of ultra high molecular weight polyethylene bio-composites by functionalized carbon nanotube and hydroxyapatite reinforcement.

The mechanical properties, such as hardness and elastic modulus, of ultra-high molecular weight polyethylene (UHMWPE) composites for acetabular cup liner are improved by adding hydroxyapatite (HAp) and carbon nanotubes (CNT). However, the weak adhesion of HAp (H) and CNT (C) with UHMWPE (U) limits the enhancement of mechanical properties. Thus, the surface of these reinforcements is silane-treated to improve the adhesion with polymer via Si-O and C=O bonds, as evidenced from spectroscopy techniques. An increased dispersion and interfacial adhesion of functionalized HAp (fH) and CNT (fC) with the polymer matrix is confirmed by nearly two-fold increased reinforcement fraction (Rf: 0.55) of U-10 wt% fHAp-2 wt.% fCNT (U10fH2fC) in comparison to U-10 wt% HAp-2 wt.% CNT (U10H2C). Additionally, Voronoi Tessellation (VT) on SEM micrographs of U10H2C and U10fH2fC revealed the dispersion of functionalized CNTs in U10fH2fC with a center-to-center distance of 0.076 µm, which is 74% higher for unfunctionalized CNT in U10H2C. The multilength scale strengthening of the UHMWPE matrix is confirmed from atomic level modification via functionalization of fillers which effectively adhered to the polymer chain on a micro-scale level. A uniform distribution of CNTs rendered increased crystallinity (+28%) of U10fH2fC, which in turn resulted in significant improvement in bulk mechanical properties (18%, 49%, and 12% increased hardness (148.1 MPa), elastic modulus (3.51 GPa) and tensile elastic modulus (219.8 MPa), respectively) in comparison to that of U10H2C. Functionalized-HAp/CNT reinforced UHMWPE composites maintained its cytocompatibility in the MTT test and fluorescence microscopy, affirming their potential employment as acetabular cup liners for hip joint arthroplasty.

Retinoic acid signaling regulates proliferation and lamina formation in the developing chick optic tectum.

The retinotectal system has been extensively studied for investigating the mechanism(s) for topographic map formation. The optic tectum, which is composed of multiple laminae, is the major retino recipient structure in the developing avian brain. Laminar development of the tectum results from cell proliferation, differentiation and migration, coordinated in strict temporal and spatial patterns. However, the molecular mechanisms that orchestrate these complex developmental events, have not been fully elucidated. In this study, we have identified the presence of differential retinoic acid (RA) signaling along the rostro-caudal and dorsoventral axis of the tectum. We show for the first time that loss of RA signaling in the anterior optic tectum, leads to an increase in cell proliferation and gross changes in the morphology manifested as defects in lamination. Detailed analysis points to delayed migration of cells as the plausible cause for the defects in lamina formation. Thus, we conclude that in the optic tectum, RA signaling is involved in maintaining cell proliferation and in regulating the formation of the tectal laminae.

Forebrain roof plate morphogenesis and hippocampus development in the chick embryo.

The forebrain roof plate undergoes dramatic morphogenetic changes to invaginate, and this leads to formation of the two cerebral hemispheres. While many genetic factors are known to regulate this process, the mechanism of forebrain roof plate invagination remains unknown. In a recent study we have identified retinoic acid as a signal from the dorsal mesenchyme that regulates the invagination of the roof plate. This has brought into focus the importance of the interaction between the dorsal mesenchyme and the underlying roof plate. One of the structures derived from the dorso-medial forebrain after roof plate invagination is the hippocampus. While the functions of the hippocampus are conserved between birds and mammals, there are distinct structural differences. We have studied hippocampus development in the chick embryo and uncovered several similarities and differences between the process in mammals and birds. This study has also lent support to one of the prevalent models of structural homology between the avian and mammalian hippocampus. In this review, we have underscored the importance of the chick embryo as a model for studying forebrain roof plate morphogenesis and hippocampus development.

MicroRNA-19b restricts Wnt7b to the hem, which induces aspects of hippocampus development in the avian forebrain.

The functions of the hippocampus are conserved between birds and mammals; however, it is not known whether similar mechanisms are responsible for its development in these two classes. In mammals, hippocampus development is known to be regulated by the hem organizer. Here, we have identified that, in birds, Wnt7b secreted from the hem is sufficient for inducing the expression of hippocampal markers. Furthermore, we have demonstrated that a microRNA, miR-19b, which is selectively excluded from the hem region, is necessary and sufficient for restricting the expression of Wnt7b to the hem. This study suggests that the role of the Wnt signal emanating from the hem is conserved between birds and mammals, and that a microRNA-based mechanism is crucial for determining the position of the hippocampus.

Zika virus E protein alters the properties of human fetal neural stem cells by modulating microRNA circuitry.

Zika virus (ZV) infects neural stem cells (NSCs) and causes quiescence in NSCs, reducing the pool of brain cells, leading to microcephaly. Despite conscientious efforts, the molecular mechanisms for ZV-mediated effects on NSCs lack clarity. This study aimed to explore the underlying mechanisms for ZV-mediated induction of quiescence in the primary cultures of human fetal neural stem cells (fNSCs). We demonstrate that expression of ZV envelope (E) protein displays maximum quiescence in human fNSCs by accumulating cells in the G0/G1 phase of the cell cycle as compared to other non-structural proteins, viz. NS2A, NS4A and NS4B. E protein induces immature differentiation by induction of pro-neuronal genes in proliferating fNSCs, induces apoptosis in differentiating fNSCs 3 days post differentiation, and disrupts migration of cells from differentiating neurospheres. In utero electroporation of mouse brain with E protein shows drastic downregulation of proliferating cells in ventricular and subventricular zone regions. Global microRNA sequencing suggests that E protein modulates miRNA circuitry. Among differentially expressed miRNAs, we found 14 upregulated and 11 downregulated miRNAs. Mir-204-3p and mir-1273g-3p directly regulate NOTCH2 and PAX3 expression, respectively, by binding to their 3'UTR. Bioinformatic analysis using GO analysis for the targets of differentially expressed miRNAs revealed enrichment of cell cycle and developmental processes. Furthermore, WNT, CCKR, PDGF, EGF, p53, and NOTCH signaling pathways were among the top enriched pathways. Thus, our study provides evidence for the involvement of ZV E protein and novel insights into the molecular mechanism through identification of miRNA circuitry. Art work depicting the effect of Zika virus E protein on human fetal neural stem cells.

Perturbation of canonical and non-canonical BMP signaling affects migration, polarity and dendritogenesis of mouse cortical neurons.

Bone morphogenetic protein (BMP) signaling has been implicated in the regulation of patterning of the forebrain and as a regulator of neurogenesis and gliogenesis in the mammalian cortex. However, its role in other aspects of cortical development in vivo remains unexplored. We hypothesized that BMP signaling might regulate additional processes during the development of cortical neurons after observing active BMP signaling in a spatiotemporally dynamic pattern in the mouse cortex. Our investigation revealed that BMP signaling specifically regulates the migration, polarity and the dendritic morphology of upper layer cortical neurons born at E15.5. On further dissection of the role of canonical and non-canonical BMP signaling in each of these processes, we found that migration of these neurons is regulated by both pathways. Their polarity, however, appears to be affected more strongly by canonical BMP signaling, whereas dendritic branch formation appears to be somewhat more strongly affected by LIMK-mediated non-canonical BMP signaling.

Identification of novel candidate regulators of retinotectal map formation through transcriptional profiling of the chick optic tectum.

Information from the retina is carried along the visual pathway with accuracy and spatial conservation as a result of topographically mapped axonal connections. The optic tectum in the midbrain is the primary region to which retinal ganglion cells project their axons in the chick. The two primary axes of the retina project independently onto the tectum using different sets of guidance cues to give rise to the retinotectal map. Specificity of the map is determined by attractive or repulsive interactions between molecular tags that are distributed in gradients in the retina and the tectum. Despite several studies, knowledge of the retinotectal guidance molecules is far from being complete. We screened for all molecules that are expressed differentially along the anterior-posterior and medial-lateral axes of the chick tectum using microarray based transcriptional profiling and identified several novel candidate retinotectal guidance molecules. Two such genes, encoding Wnt5a and Raldh2, the synthesizing enzymes for retinoic acid, were further analyzed for their function as putative regulators of retinotectal map formation. Wnt5a and retinoic acid were found to exhibit differential effects on the growth of axons from retinal explants derived from different quadrants of the retina. This screen also yielded a large number of genes expressed in a lamina-specific manner in the tectum, which may have other roles in tectal development. J. Comp. Neurol. 525:459-477, 2017. © 2016 Wiley Periodicals, Inc.

Mouse bone marrow stromal cells differentiate to neuron-like cells upon inhibition of BMP signaling.

Bone marrow stromal cells (BMSCs) are a source of autologous stem cells that have the potential for undergoing differentiation into multiple cell types including neurons. Although the neuronal differentiation of mesenchymal stem cells has been studied for a long time, the molecular players involved are still not defined. Here we report that the genetic deletion of two members of the bone morphogenetic protein (Bmp) family, Bmp2 and Bmp4 in mouse BMSCs causes their differentiation into cells with neuron-like morphology. Surprisingly these cells expressed certain markers characteristic of both neuronal and glial cells. Based on this observation, we inhibited BMP signaling in mouse BMSCs through a brief exposure to Noggin protein which also led to their differentiation into cells expressing both neuronal and glial markers. Such cells seem to have the potential for further differentiation into subtypes of neuronal and glial cells and thus could be utilized for cell-based therapeutic applications.

Roof plate mediated morphogenesis of the forebrain: New players join the game.

The roof plate is a crucial signaling center located at the dorsal midline of the developing central nervous system (CNS) along its rostro-caudal axis. By virtue of secreting multiple signaling molecules, it regulates diverse processes such as specification of dorsal fate, proliferation and axon guidance. In the forebrain, the roof plate is not only involved in patterning but is also involved in the division of the single forebrain vesicle into the two cerebral hemispheres, the failure of which leads to certain forms of holoprosencephaly. Although several molecular players such as Fgfs, BMPs, Wnts and Shh have been identified as crucial regulators of development of the forebrain, little is known about how they interact to bring about the morphological changes associated with the division of the forebrain vesicle into the cerebral hemispheres. Recent studies have now identified the dorsal mesenchyme as an additional source of signaling cues, which is likely to influence the division of the forebrain vesicle into cerebral hemispheres. In this review, we discuss the current understanding about the molecular mechanisms of roof plate mediated patterning and morphogenesis of the forebrain including some recently identified factors that influence this process and also highlight the gaps in our knowledge that remain.

Retinoic acid signaling regulates development of the dorsal forebrain midline and the choroid plexus in the chick.

The developing forebrain roof plate (RP) contains a transient signaling center, perturbations in which have been linked to holoprosencephaly (HPE). Here, we describe a novel domain of retinoic acid (RA) signaling that is specific to the chick RP and demonstrate that RA signaling is sufficient for inducing characteristics of the RP in ectopic locations. We further demonstrate that, unlike what has been observed in the mouse, RA signaling is essential for invagination of the RP in chick, failure of which leads to an HPE-like phenotype. In addition, we found that RA exerts a negative influence on choroid plexus differentiation. Thus, our findings identify RA as a novel regulator of chick forebrain RP development.

Defining structural homology between the mammalian and avian hippocampus through conserved gene expression patterns observed in the chick embryo.

The mammalian hippocampus, a center of neurogenesis in the adult brain, is involved in critical functions such as learning and memory processing. Although there is an overall functional conservation between birds and mammals in the hippocampal region of the brain, there are several morphological differences. A few different models have been proposed for identifying regional and structural homology between the avian and mammalian hippocampus however a consensus is yet to be reached. In this study we have systematically and comprehensively characterized the developing chicken hippocampus at the molecular level. We have identified the time window of neurogenesis and apoptosis during hippocampal development as well as the likely origin and migration path of neurons of the ventral v-shaped region of chick hippocampus. In addition to this we have identified several genes with expression patterns that are conserved between the hippocampus of chicken and mice. Our study provides molecular data that partially supports one of the models reported in literature for structural homology between the avian and mammalian hippocampus. Functional characterization of the genes found in this study to be specifically expressed in the developing chicken hippocampus is likely to provide valuable information on the mechanisms regulating hippocampus development of birds and perhaps could be extrapolated to mammalian hippocampus development as well.

Cross-regulatory interactions between Fgf8 and Shh in the avian frontonasal prominence.

The frontonasal prominence of the developing avian embryo contains an organizing center, defined by juxtaposition of the Sonic hedgehog (Shh) and Fibroblast growth factor 8 (Fgf8) expression domains. This molecular interface presages any detectable growth of the frontonasal prominence, and experiments involving transplantation of this boundary epithelium have demonstrated it is a source of dorsal-ventral and rostral-caudal patterning information for the neural crest-derived mesenchyme of the upper beak. We explored the ontogeny of this organizing center by mapping the expression domains of both genes and their receptors and downstream targets. We tested the extent to which Shh and Fgf8 regulate each other's expression in this frontonasal organizer by either blocking or ectopically activating these pathways. Our experiments revealed mutual antagonism between the two molecules, which aids in establishing and maintaining a molecular boundary that subsequently influences patterning and growth of the middle and upper face.

Retinoic acid regulates the expression of dorsoventral topographic guidance molecules in the chick retina.

Asymmetric expression of several genes in the early eye anlagen is required for the dorsoventral (DV) and anteroposterior (AP) patterning of the retina. Some of these early patterning genes play a role in determining the graded expression of molecules that are needed to form the retinotectal map. The polarized expression of retinoic acid synthesizing and degrading enzymes along the DV axis in the retina leads to several zones of varied retinoic acid (RA) activity. This is suggestive of RA playing a role in DV patterning of the retina. A dominant-negative form of the retinoic acid receptor alpha (DNhRARalpha) was expressed in the chick retina to block RA activity. RA signaling was found to play a role in regulating the expression of EphB2, EphB3 and ephrin B2, three molecules whose graded expression in the retina along the DV axis is important for establishing the correct retinotectal map. Blocking RA signaling by misexpression of a RA degrading enzyme, Cyp26A1 recapitulated some but not all the effects of DNhRARalpha. It also was found that Vax, a ventrally expressed transcription factor that regulates the expression of the EphB and ephrin B molecules, functions upstream of, or in parallel to, RA. Expression of DNhRARalpha led to increased levels of RA-synthesizing enzymes and loss of RA-degrading enzymes. Activation of such compensatory mechanisms when RA activity is blocked suggests that RA homeostasis is very strictly regulated in the retina.

Drosophila pipe protein activity in the ovary and the embryonic salivary gland does not require heparan sulfate glycosaminoglycans.

The Drosophila pipe gene encodes ten related proteins that exhibit amino acid sequence similarity to vertebrate heparan sulfate 2-O-sulfotransferase. One of the Pipe isoforms, which is expressed in the ventral follicular epithelium, is a key determinant of embryonic dorsoventral polarity, suggesting that Pipe-mediated sulfation of a heparan sulfate proteoglycan provides a spatial cue for dorsoventral axis formation. We used several approaches to investigate this possibility in the work described here. We determined the nucleotide alterations in 11 different pipe alleles. Ten of the mutations specifically affect the pipe isoform that is expressed in the ovary. Among these ten mutations, two alter an amino acid in the putative binding site for 3'-phosphoadenosine 5'-phosphosulfate, the universal sulfate donor. Using Alcian Blue, a histochemical stain that detects sulfated glycans, we observed a novel, pipe-dependent macromolecule in the embryonic salivary glands. Genes known to participate in the formation of heparan sulfate in Drosophila are not required for the production of this material. To investigate whether a heparan sulfate proteoglycan is involved in pipe function in dorsoventral patterning, we generated females carrying follicle cell clones mutant for heparan sulfate synthesis-related genes. Embryos from follicles with mutant clones did not exhibit a dorsalized phenotype. Taken together, our data provide evidence that Pipe acts as a sulfotransferase, but argue against the hypothesis that the target of Pipe is a heparan sulfate glycosaminoglycan.

The rod photoreceptor pattern is set at the optic vesicle stage and requires spatially restricted cVax expression.

How and when positional identities in the neural retina are established have been addressed primarily with respect to the topographic projections of retinal ganglion cells onto their targets in the brain. Although retinotectal map formation is a prominent manifestation of retinal patterning, it is not the only one. Photoreceptor subtypes are arranged in distinct, species-specific patterns. The mechanisms used to establish photoreceptor patterns have been relatively unexplored at the mechanistic level. We performed ablations of the eye anlage in chickens and found that removal of the anterior or dorsal optic vesicle caused loss of the area centralis, which is a rod-free central area of the retina, and severely disorganized other aspects of the rod pattern. These observations indicate that the anteroposterior and dorsoventral distribution of rods is determined by the optic vesicle stage. To investigate the molecular mechanisms involved, the rod distribution was analyzed after viral misexpression of several patterning genes that were previously shown to be important in positional specification of retinal ganglion cells. Ectopic expression of FoxG1, SOHo1,or GH6 transcription factors expressed in the anterior optic vesicle and/or optic cup, respectively, did not affect the rod pattern. This pattern therefore appears to be specified by an activity acting before, or in parallel with, these factors. In contrast, misexpression of the ventrally restricted transcription factor, cVax, severely disturbed the rod pattern.