Medulloblastoma (MB) is the most common malignant brain tumor in children and is stratified into three major subgroups. The Sonic hedgehog (SHH) subgroup represents ~30% of all MB cases and has significant survival disparity depending upon TP53 status. Here, we describe the first zebrafish model of SHH MB using CRISPR to mutate ptch1, the primary genetic driver in human SHH MB. These tumors rapidly arise adjacent to the valvula cerebelli and resemble human SHH MB by histology and comparative genomics. In addition, ptch1-deficient MB tumors with loss of tp53 have aggressive tumor histology and significantly worse survival outcomes, comparable to human patients. The simplicity and scalability of the ptch1 MB model makes it highly amenable to CRISPR-based genome editing screens to identify genes required for SHH MB tumor formation in vivo, and here we identify the grk3 kinase as one such target.

In early vertebrate development, organizer regions-groups of cells that signal to and thereby influence neighboring cells by secreted morphogens-play pivotal roles in the establishment and maintenance of cell identities within defined tissue territories. The midbrain-hindbrain organizer drives regionalization of neural tissue into midbrain and hindbrain territories with fibroblast growth factor 8 (FGF8) acting as a key morphogen. This organizer has been extensively studied in chicken, mouse, and zebrafish. Here, we demonstrate the enrichment of FGF8-expressing cells from human pluripotent stem cells (hPSCs), cultured as attached embryoid bodies using antibodies that recognize \"Similar Expression to Fgf\" (SEF) and Frizzled proteins. The arrangement of cells in embryoid body subsets of these cultures and the gene expression profile of the FGF8-expressing population show certain similarities to the midbrain-hindbrain organizer in animal models. In the embryonic chick brain, the enriched cell population induces formation of midbrain structures, consistent with FGF8-organizing capability.

The process that partitions the nascent vertebrate central nervous system into forebrain, midbrain, hindbrain, and spinal cord after neural induction is of fundamental interest in developmental biology, and is known to be dependent on Wnt/β-catenin signaling at multiple steps. Neural induction specifies neural ectoderm with forebrain character that is subsequently posteriorized by graded Wnt signaling: embryological and mutant analyses have shown that progressively higher levels of Wnt signaling induce progressively more posterior fates. However, the mechanistic link between Wnt signaling and the molecular subdivision of the neural ectoderm into distinct domains in the anteroposterior (AP) axis is still not clear. To better understand how Wnt mediates neural AP patterning, we performed a temporal dissection of neural patterning in response to manipulations of Wnt signaling in zebrafish. We show that Wnt-mediated neural patterning in zebrafish can be divided into three phases: (I) a primary AP patterning phase, which occurs during gastrulation, (II) a mes/r1 (mesencephalon-rhombomere 1) specification and refinement phase, which occurs immediately after gastrulation, and (III) a midbrain-hindbrain boundary (MHB) morphogenesis phase, which occurs during segmentation stages. A major outcome of these Wnt signaling phases is the specification of the major compartment divisions of the developing brain: first the MHB, then the diencephalic-mesencephalic boundary (DMB). The specification of these lineage divisions depends upon the dynamic changes of gene transcription in response to Wnt signaling, which we show primarily involves transcriptional repression or indirect activation. We show that otx2b is directly repressed by Wnt signaling during primary AP patterning, but becomes resistant to Wnt-mediated repression during late gastrulation. Also during late gastrulation, Wnt signaling becomes both necessary and sufficient for expression of wnt8b, en2a, and her5 in mes/r1. We suggest that the change in otx2b response to Wnt regulation enables a transition to the mes/r1 phase of Wnt-mediated patterning, as it ensures that Wnts expressed in the midbrain and MHB do not suppress midbrain identity, and consequently reinforce formation of the DMB. These findings integrate important temporal elements into our spatial understanding of Wnt-mediated neural patterning and may serve as an important basis for a better understanding of neural patterning defects that have implications in human health.

In early vertebrate embryos, the dorsal ectoderm is induced by the axial mesendoderm to form the neural plate, which is given competence to form neural cells by soxB1 genes. Subsequently, neurogenesis proceeds in proneural clusters that are generated by a gene network involving proneural genes and Notch signaling. However, what occurs between early neural induction and the later initiation of neurogenesis has not been fully revealed. In the present study, we demonstrated that during gastrulation, the expression of the Oct4-related PouV gene pou5f3 (also called pou2), which is widely observed at earlier stages, was rapidly localized to an array of isolated spotted domains, each of which coincided with individual proneural clusters. Two-color in situ hybridization confirmed that each pou5f3-expressing domain included a proneural cluster. Further analysis demonstrated that anterior pou5f3 domains straddled the boundaries between rhombomere 1 (r1) and r2, whereas posterior domains were included in r4. The effects of forced expression of an inducible negative dominant-interfering pou5f3 gene suggested that pou5f3 activated early proneural genes, such as neurog1 and ebf2, and also soxB1, but repressed the late proneural genes atoh1a and ascl1b. Furthermore, pou5f3 was considered to repress her4.1, a Notch-dependent Hairy/E(spl) gene involved in lateral inhibition in proneural clusters. These results suggest that pou5f3 promotes early neurogenesis in proneural clusters, but negatively regulates later neurogenesis. Suppression of pou5f3 also altered the expression of other her genes, including her3, her5, and her9, further supporting a role for pou5f3 in neurogenesis. In vitro reporter assays in P19 cells showed that pou5f3 was repressed by neurog1, but activated by Notch signaling. These findings together demonstrate the importance of the pou5f3-mediated gene regulatory network in neural development in vertebrate embryos.

Fibroblast growth factor (Fgf) signaling regulates many processes during development. In most cases, one tissue layer secretes an Fgf ligand that binds and activates an Fgf receptor (Fgfr) expressed by a neighboring tissue. Although studies have identified the roles of specific Fgf ligands during development, less is known about the requirements for the receptors. We have generated null mutations in each of the five fgfr genes in zebrafish. Considering the diverse requirements for Fgf signaling throughout development, and that null mutations in the mouse Fgfr1 and Fgfr2 genes are embryonic lethal, it was surprising that all zebrafish homozygous mutants are viable and fertile, with no discernable embryonic defect. Instead, we find that multiple receptors are involved in coordinating most Fgf-dependent developmental processes. For example, mutations in the ligand fgf8a cause loss of the midbrain-hindbrain boundary, whereas, in the fgfr mutants, this phenotype is seen only in embryos that are triple mutant for fgfr1a;fgfr1b;fgfr2, but not in any single or double mutant combinations. We show that this apparent fgfr redundancy is also seen during the development of several other tissues, including posterior mesoderm, pectoral fins, viscerocranium, and neurocranium. These data are an essential step toward defining the specific Fgfrs that function with particular Fgf ligands to regulate important developmental processes in zebrafish.

The present study initiates our investigation regarding the role of calb2a and calb2b genes that are expressed in the central nervous system, including the multiple tissues during early embryonic development of zebrafish. In this study, we have adopted individual and combined morpholino-mediated inactivation approach to investigate the functions of calb2a and calb2b in early development of the zebrafish. We have found that calb2a and calb2b morpholino alone failed to generate an obvious phenotype; however, morphological inspection in early developmental stages of calb2a and calb2b combined knockdown morphants show abnormal neural plate folding in midbrain-hindbrain region. In addition to this, combinatorial loss of these mRNA leads to severe hydrocephalus, axial curvature defect, and yolk sac edema in later developmental stages. Also, the combined knockdown of calb2a and calb2b are found to be associated with an impaired touchdown and swimming performance in the zebrafish. Co-injection of the calb2a and calb2b morpholino oligonucleotide cocktail with human CALB2 mRNA leads to the rescue of the strong phenotype. This study provided the first comprehensive analyses of the zebrafish Calb2a and Calb2b proteins; we have found that Calb2a and Calb2b are highly conserved across vertebrate species and originated from the same ancestral gene long back in the evolution. Homology modeling and docking with the similar structure and Ca2+ binding sites for both proteins provide the evidence that both the proteins may have similar function and one can compensate for the loss of other. Collectively, these findings confirm the unique and essential functions of calb2a and calb2b genes in the early development of the zebrafish.

Brd2 is a member of the bromodomain-extraterminal domain (BET) family of proteins and functions as an acetyl-histone-directed transcriptional co-regulator and recruitment scaffold in chromatin modification complexes affecting signal-dependent transcription. While Brd2 acts as a protooncogene in mammalian blood, developmental studies link it to regulation of neuronal apoptosis and epilepsy, and complete knockout of the gene is invariably embryonic lethal. In Drosophila, the Brd2 homolog acts as a maternal effect factor necessary for segment formation and identity and proper expression of homeotic loci, including Ultrabithorax and engrailed. To test the various roles attributed to Brd2 in a single developmental system representing a non-mammalian vertebrate, we conducted a phenotypic characterization of Brd2a deficient zebrafish embryos produced by morpholino knockdown and corroborated by Crispr-Cas9 disruption and small molecule inhibitor treatments. brd2aMO morphants exhibit reduced hindbrain with an ill-defined midbrain-hindbrain boundary (MHB) region; irregular notochord, neural tube, and somites; and abnormalities in ventral trunk and ventral nerve cord interneuron positioning. Using whole mount TUNEL and confocal microscopy, we uncover a significant decrease, then a dramatic increase, of p53-independent cell death at the start and end of segmentation, respectively. In contrast, using qualitative and quantitative analyses of BrdU incorporation, phosphohistone H3-tagging, and flow cytometry, we detect little effect of Brd2a knockdown on overall proliferation levels in embryos. RNA in situ hybridization shows reduced or absent expression of homeobox gene eng2a and paired box gene pax2a, in the hindbrain domain of the MHB region, and an overabundance of pax2a-positive kidney progenitors, in knockdowns. Together, these results suggest an evolutionarily conserved role for Brd2 in the proper formation and/or patterning of segmented tissues, including the vertebrate CNS, where it acts as a bi-modal regulator of apoptosis, and is necessary, directly or indirectly, for proper expression of genes that pattern the MHB and/or regulate differentiation in the anterior hindbrain.

Stem cell treatments for neurodegenerative diseases are expected to reach clinical trials soon. Most of the approaches currently under development involve transplantation of immature progenitors that subsequently undergo phenotypic and functional maturation in vivo, and predicting the long-term graft outcome already at the progenitor stage remains a challenge. Here, we took an unbiased approach to identify predictive markers expressed in dopamine neuron progenitors that correlate with graft outcome in an animal model of Parkinson\'s disease through gene expression analysis of >30 batches of grafted human embryonic stem cell (hESC)-derived progenitors. We found that many of the commonly used markers did not accurately predict in vivo subtype-specific maturation. Instead, we identified a specific set of markers associated with the caudal midbrain that correlate with high dopaminergic yield after transplantation in vivo. Using these markers, we developed a good manufacturing practice (GMP) differentiation protocol for highly efficient and reproducible production of transplantable dopamine progenitors from hESCs.

MicroRNAs (miRNAs) are a class of small non-coding RNA molecules with wide-ranging and subtle effects on protein production. Their activity during the development of the cerebellum provides a valuable exemplar of how non-coding molecules may assist the development and function of the central nervous system and drive neurodevelopmental disorders. Three distinct aspects of miRNA contribution to early cerebellar development will here be reviewed. Aspects are the establishment of the cerebellar anlage, the generation and maturation of at least two principal cell types of the developing cerebellar microcircuit, and the etiology and early progression of autism spectrum disorder. It will be argued here that the autism spectrum is an adept model to explore miRNA impact on the cognitive and affective processes that descend from the developing cerebellum.

The midbrain-hindbrain boundary (MHB) is one of the key organizing centers of the vertebrate central nervous system (CNS). Its patterning is governed by a well-described gene regulatory network (GRN) involving several transcription factors, namely, pax, gbx, en, and otx, together with signaling molecules of the Wnt and Fgf families. Here, we describe the onset of these markers in Oryzias latipes (medaka) early brain development in comparison to previously known zebrafish expression patterns. Moreover, we show for the first time that vox, a member of the vent gene family, is expressed in the developing neural tube similarly to CNS markers. Overexpression of vox leads to profound changes in the gene expression patterns of individual components of MHB-specific GRN, most notably of fgf8, a crucial organizer molecule of MHB. Our data suggest that genes from the vent family, in addition to their crucial role in body axis formation, may play a role in regionalization of vertebrate CNS.