Midbrain Hindbrain Research Articles

Knockdown of FoxO3a induces increased neuronal apoptosis during embryonic development in zebrafish

Neuronal apoptosis sculpts the developing brain, and nearly all identified classes of neurons seem to be produced “in excess” during development. FoxO transcription factors regulate apoptosis in vitro in deprived of neurotrophins. It is unknown if FoxO3a is involved in the development of neurons. Here, we report a role of FoxO3a during neuronal development in zebrafish. By using in situ hybridization, we revealed that FoxO3a transcripts in zebrafish were gradually confined to regions of the central nervous system during embryonic development, including the forebrain, midbrain, midbrain–hindbrain boundary and hindbrain. By using FoxO3a morpholino antisense oligonucleotides, we observed that FoxO3a loss-of-function led to neural developmental defects, including increased neural apoptosis as detected by acridine orange and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling. These defects could be partially rescued by the injection of FoxO3a mRNA. In this study, we found that FoxO3a loss-of-function resulted in the decreased expression of neuronal markers as determined by in situ hybridization and relative quantitative real-time PCR. Furthermore, the activation of FoxO3a was required for the maintenance of neuron survival but not necessary for the induction of neurogenesis. Our results indicated that FoxO3a might be essential for the maintenance of neural development in zebrafish. Therefore, this work provides novel evidence of FoxO3a in the embryonic neurodevelopment from zebrafish to other mammals.

Sagging and swelling of the midbrain suggest spontaneous intracranial hypotension rather than a malformation

Sir, In a recent Brain review article, Barkovich et al. (2009) proposed a new classification for midbrain–hindbrain malformations that takes into account a vast amount of neuroimaging data, as well as results of research in developmental biology and molecular genetics. Based on these findings, the authors also envisage the existence of some malformation that is still not completely understood and for which the pathogenetic mechanism is inferred from biological and genetic studies in animal models. This is the case in a malformation secondary to early anteroposterior transformation at the diencephalic–mesencephalic junction, resulting in shortening and thickening of the midbrain with midline mesencephalic cleft (labelled in the article: Group 1, A.1.c. Gain of diencephalon and loss of midbrain). This malformation is shown in Fig. 4. The magnetic resonance images presented in the figure, however, are identical to those observed in a subgroup of the most severe cases of spontaneous intracranial hypotension, reported in the literature and examined with diffusion studies by our group (Savoiardo et al. , 2007). Spontaneous intracranial hypotension, usually due to …

Migrating Brain Cells Stick Together

Migrating Brain Cells Stick Together

Evidence for a link among cognition, language and emotion in cerebellar malformations

We compared the neurobehavioral profiles of children with Joubert syndrome (JS participants), a rare autosomal recessive condition characterized on magnetic resonance imaging (MRI) by hypoplasia of the cerebellar vermis and midbrain–hindbrain malformations, and children with malformations confined to the cerebellar vermis and one or both hemispheres (Cerebellar malformations – CM participants). We aimed at investigating the influence of anatomo-clinical similarities (vermian malformation) and differences (intact cerebellar hemispheres vs sparing of the pons, respectively) with respect to cognitive, linguistic and emotional development, assuming as a reference framework the Cerebellar Cognitive Affective Syndrome (CCAS). Results show that severe to moderate mental retardation is infrequent in JS children, while it is present in more than half the sample of CM children. Affect development was generally preserved in JS, in high-functioning CM individuals and also in some of the CM children with moderate mental retardation, which raised questions as to the role of a cerebellar vermis lesion in determining affect disorders. Further, cognitive and linguistic profiles on both intellectual and neuropsychological evaluations provided evidence for distinct patterns of peaks and valleys in the two groups, with JS children being significantly more impaired in language and verbal working memory and CM individuals showing a significant impairment of executive functions and emotional development. The overall evidence provides support for an important role of cerebellar structures per se in shaping emotional, cognitive and linguistic development, when vermian lesions are associated to cerebellar hemispheric lesions. Cerebellar vermis and brainstem lesions instead appear to have a major impact on motor-related skills, including oro-motor abilities and verbal working memory.

13-P044 Sip1/Zfhx1b is a regulator of Wnt–β-catenin signaling during early midbrain and anterior hindbrain development

13-P044 Sip1/Zfhx1b is a regulator of Wnt–β-catenin signaling during early midbrain and anterior hindbrain development

13-P013 Notch signalling regulates formation of the midbrain–hindbrain boundary

13-P013 Notch signalling regulates formation of the midbrain–hindbrain boundary

21-P010 FGF-signaling in the neurogenesis of the developing midbrain–hindbrain region

21-P010 FGF-signaling in the neurogenesis of the developing midbrain–hindbrain region

Intraflagellar transport protein 172 is essential for primary cilia formation and plays a vital role in patterning the mammalian brain

IFT172, also known as Selective Lim-domain Binding protein (SLB), is a component of the intraflagellar transport (IFT) complex. In order to evaluate the biological role of the Ift172 gene, we generated a loss-of-function mutation in the mouse. The resulting Slb mutant embryos die between E12.5 and 13.0, and exhibit severe cranio-facial malformations, failure to close the cranial neural tube, holoprosencephaly, heart edema and extensive hemorrhages. Cilia outgrowth in cells of the neuroepithelium is initiated but the axonemes are severely truncated and do not contain visible microtubules. Morphological and molecular analyses revealed a global brain-patterning defect along the dorsal–ventral (DV) and anterior–posterior (AP) axes. We demonstrate that Ift172 gene function is required for early regulation of Fgf8 at the midbrain–hindbrain boundary and maintenance of the isthmic organizer. In addition, Ift172 is required for proper function of the embryonic node, the early embryonic organizer and for formation of the head organizing center (the anterior mesendoderm, or AME). We propose a model suggesting that forebrain and mid–hindbrain growth and AP patterning depends on the early function of Ift172 at gastrulation. Our data suggest that the formation and function of the node and AME in the mouse embryo relies on an indispensable role of Ift172 in cilia morphogenesis and cilia-mediated signaling.

Formation of the zebrafish midbrain–hindbrain boundary constriction requires laminin-dependent basal constriction

The midbrain–hindbrain boundary (MHB) is a highly conserved fold in the vertebrate embryonic brain. We have termed the deepest point of this fold the MHB constriction (MHBC) and have begun to define the mechanisms by which it develops. In the zebrafish, the MHBC is formed soon after neural tube closure, concomitant with inflation of the brain ventricles. The MHBC is unusual, as it forms by bending the basal side of the neuroepithelium. At single cell resolution, we show that zebrafish MHBC formation involves two steps. The first is a shortening of MHB cells to approximately 75% of the length of surrounding cells. The second is basal constriction, and apical expansion, of a small group of cells that contribute to the MHBC. In the absence of inflated brain ventricles, basal constriction still occurs, indicating that the MHBC is not formed as a passive consequence of ventricle inflation. In laminin mutants, basal constriction does not occur, indicating an active role for the basement membrane in this process. Apical expansion also fails to occur in laminin mutants, suggesting that apical expansion may be dependent on basal constriction. This study demonstrates laminin-dependent basal constriction as a previously undescribed molecular mechanism for brain morphogenesis.

Transcription of fgf8 is regulated by activating and repressive cis-elements at the midbrain–hindbrain boundary in zebrafish embryos

Fgf8 is expressed in the isthmic region of the developing brain, serving an organizing function in vertebrate embryos. We previously identified S4.2 downstream to the zebrafish fgf8 gene as a regulatory region that drives transcription in the anterior hindbrain. Here, we investigated the mechanism of fgf8 regulation by the S4.2 region during development. Reporter analyses in embryos revealed that S4.2 closely recapitulates fgf8 expression in the anteriormost hindbrain during somitogenesis. This region contains a sequence highly conserved in fgf8 of diverse vertebrates. Further analyses of S4.2 revealed a 342-bp core region composed of three subregions (#2, #3, and #4). Regions #3 and #4 drove expression broadly in the brain from the midbrain to r5 of the hindbrain, whereas a 28-bp sequence in #2 repressed ectopic expression in the midbrain and in r2 to r5. The enhancer function of S4.2 was absent in pax2a mutant embryos, while it was activated ectopically by pax2a misexpression in the hindbrain. We identified two sites in the core region that are bound by Pax2a in vitro and in vivo, the disruption of which abrogated the S4.2 activity. Thus, fgf8 expression in the anteriormost hindbrain involves activation and repression, with Pax2a as a pivotal regulator.

Analysis of the midbrain–hindbrain boundary cell fate using a boundary cell‐specific Cre‐mouse strain

We describe here a transgenic mouse line MHB-Cre, which expresses Cre recombinase in a group of cells at the midbrain-hindbrain boundary. Using this mouse line, we studied the contribution of the boundary cells to distinct brain areas during development. Initially, the MHB-Cre expression coincides with that of Cdh22 and p21 around the Otx2 expression border in a narrow population of cells with reduced proliferative activity. Consistent with their location on both sides of the Otx2 expression border, the Cre expressing boundary cells contribute both to midbrain as well as hindbrain. However, the majority of recombinant cells remain close to the mid- and hindbrain border, suggesting very limited cell mixing within these brain compartments during development. Interestingly, dorsocaudally oriented fibers of the midbrain dopaminergic neurons follow the path marked by the boundary cells.

Genotypes and phenotypes of Joubert syndrome and related disorders

Joubert syndrome is an autosomal recessive condition characterized by hypotonia, ataxia, psychomotor delay and variable occurrence of oculomotor apraxia and neonatal breathing abnormalities. The neuroradiological hallmark of JS is a complex midbrain–hindbrain malformation known as the “molar tooth sign” (MTS), originating from the association of cerebellar vermis hypo-/aplasia, horizontally-oriented and thickened superior cerebellar peduncles and a deepened interpeduncular fossa. A group of pleiotropic conditions, termed “Joubert syndrome related disorders” (JSRDs), present the pathognomonic clinical and neuroradiological features of JS associated with the variable involvement of other organs and systems, mainly the eyes and kidneys. Genetic heterogeneity mirrors the clinical heterogeneity of JSRDs, with several genes identified over the last few years. By reviewing all molecular screenings of JSRD patients published so far and evaluating genotype–phenotype correlates, we propose an algorithm for molecular diagnosis of these conditions. We also discuss the emerging clinical and genetic overlap between JSRDs and a growing number of distinct syndromes that share a common pathogenetic mechanism that is the loss of normal function of the primary cilium and its apparatus.

Dynamic change in the expression of developmental genes in the ascidian central nervous system: Revisit to the tripartite model and the origin of the midbrain–hindbrain boundary region

Comparative studies on expression patterns of developmental genes along the anterior–posterior axis of the embryonic central nervous system (CNS) between vertebrates and ascidians led to the notion of “tripartite organization,” a common ground plan of the CNS, consisting of the anterior, central and posterior regions expressing Otx, Pax2/5/8 and Hox genes, respectively. In ascidians, however, descriptions and interpretations about expression of the developmental genes regarded as region specific have become not necessarily consistent. To address this issue, we examined detailed expression of key developmental genes for the ascidian CNS, including Otx, Pax2/5/8a, En, Fgf8/17/18, Dmbx, Lhx3 and Hox genes, in the CNS around the junction of the trunk and tail of three different tailbud-stage embryos of Ciona intestinalis, employing double-fluorescence in situ hybridization, followed by staining with DAPI to precisely locate expressing cells for each gene. Based on these observations, we have constructed detailed gene expression maps of the region at the tailbud stages. Our analysis shows that expression of several genes regarded as markers for specific domains in the ascidian CNS changes dynamically within a relatively short period. This motivates us to revisit to the tripartite ground plan and the origin of the midbrain–hindbrain boundary (MHB) region.

Cloning, expression, and functional characterization of zebrafish Mist1

The basic helix–loop–helix (bHLH) protein Mist1 is an important exocrine pancreas transcriptional factor expressed in the acinar cells of mammals. In the present study, we cloned the homologous Mist1 cDNA encoding a predicted protein of 184 amino acids in zebrafish. The typical bHLH domain of zebrafish Mist1 shares high identity with that of its orthologs in mouse, rat, and human. Expression analysis revealed that Mist1 maternal transcripts are distinct in the very beginning of embryogenesis and that endogenous Mist1 is chronologically expressed in polster, hatching gland, hindbrain and appears exclusively in the pancreas from 72 hpf onward. Knockdown of Mist1 conditionally causes mild morphological defects in embryos. In MO-treated embryos, midbrain–hindbrain boundary is missing and exocrine pancreas is significantly reduced and disorganized. These results suggest that Mist1 functions in an evolutionary conserved way as a key transcriptional regulator specific for exocrine pancreas development in zebrafish.

CREB activity modulates neural cell proliferation, midbrain–hindbrain organization and patterning in zebrafish

Neural stem/progenitor cells (NPCs) self-renew and differentiate, generating neuronal and non-neuronal (glial) cell lineages. Although a number of factors, including transcription factors, have been shown to be important in the regulation of NPC proliferation and differentiation, the precise molecular networks remain to be identified. The cAMP Response Element-Binding protein (CREB) is a transcription factor important for neuronal survival, differentiation and plasticity. Recent work suggests that CREB activation, via serine phosphorylation in the kinase inducible domain, is important for neurogenesis in the adult rodent brain. We sought to further investigate CREB function in neurogenesis, using the zebrafish ( Danio rerio). Structural and functional analysis of the zebrafish CREB orthologue showed high conservation with mammalian CREB. Activated (phosphorylated) CREB (pCREB) was localised to all known proliferation zones in the adult zebrafish brain, including actively cycling cells. Furthermore, we found that modulating CREB activity during early zebrafish development caused significant defects in neural proliferation, midbrain–hindbrain organization and body patterning. These findings reveal broader and stage-specific physiological roles of CREB function during vertebrate neural development and proliferation.

Gene expression analysis of the hedgehog signaling cascade in the chick midbrain and spinal cord

The signaling molecule Sonic Hedgehog (SHH) plays a critical role in patterning the ventral midbrain of vertebrates. Our recent studies have established that the requirement for Hedgehog (HH) signaling in the chick midbrain is modulated spatially and temporally in a complex manner across the midbrain anlage. Unfortunately, the patterns of expression of downstream regulators that might modulate the HH signal in the midbrain are not currently known. To fill this gap, we have examined across time, the expression pattern of 14 genes that function in the HH signaling cascade in the midbrain and spinal cord. Our results suggest that SHH expression in the axial mesendoderm begins before the expression of known HH receptors/HH-binding proteins (e.g., PTC1, PTC2, HHIP, BOC, MEGALIN). In the midbrain, PTC and GLI genes are expressed and then eliminated very early from the ventral midline. However, they exhibit high and persistent expression in the midbrain region circumscribing the SHH source. Intriguingly, multiple HH-binding proteins (BOC, MEGALIN) and HH effectors (GLI1-3, SMO, SUFU, DZIP) are expressed in the dorsal midbrain and the midbrain-hindbrain boundary. Finally, we report for the first time that IHH is expressed in intermediate regions of the spinal cord, where its expression does not overlap with that of SHH.

The serotonergic phenotype is acquired by converging genetic mechanisms within the zebrafish central nervous system

To gain knowledge about the developmental origin of serotonergic precursors and the regulatory cascades of serotonergic differentiation in vertebrates, we determined the spatiotemporal expression profile of the Ets-domain transcription factor-encoding gene pet1 in developing and adult zebrafish. We show that it is an early, specific marker of raphe serotonergic neurons, but not of other serotonergic populations. We then use pet1 expression together with tracing techniques to demonstrate that serotonergic neurons of rhombomeres (r) 1-2 largely originate from a progenitor pool at the midbrain-hindbrain boundary. Furthermore, by combining expression analyses of pet1 and the raphe tryptophan hydroxylase (Tph2) with rhombomere identity markers, we show that anterior and posterior hindbrain clusters of serotonergic precursors are separated by r3, rather than r4 as in other vertebrates. Our findings establish the origin of r1-2 serotonergic precursors, and strengthen the evidence for molecular, ontogenic and phylogenic heterogeneities among the vertebrate brain serotonergic cell populations.

Mutant analyses reveal different functions of fgfr1 in medaka and zebrafish despite conserved ligand–receptor relationships

Medaka ( Oryzias latipes) is a small freshwater teleost that provides an excellent developmental genetic model complementary to zebrafish. Our recent mutagenesis screening using medaka identified headfish ( hdf) which is characterized by the absence of trunk and tail structures with nearly normal head including the midbrain–hindbrain boundary (MHB). Positional-candidate cloning revealed that the hdf mutation causes a functionally null form of Fgfr1. The fgfr1 hdf is thus the first fgf receptor mutant in fish. Although FGF signaling has been implicated in mesoderm induction, mesoderm is induced normally in the fgfr1 hdf mutant, but subsequently, mutant embryos fail to maintain the mesoderm, leading to defects in mesoderm derivatives, especially in trunk and tail. Furthermore, we found that morpholino knockdown of medaka fgf8 resulted in a phenotype identical to the fgfr1 hdf mutant, suggesting that like its mouse counterpart, Fgf8 is a major ligand for Fgfr1 in medaka early embryogenesis. Intriguingly, Fgf8 and Fgfr1 in zebrafish are also suggested to form a major ligand–receptor pair, but their function is much diverged, as the zebrafish fgfr1 morphant and zebrafish fgf8 mutant acerebellar ( ace) only fail to develop the MHB, but develop nearly unaffected trunk and tail. These results provide evidence that teleost fish have evolved divergent functions of Fgf8–Fgfr1 while maintaining the ligand–receptor relationships. Comparative analysis using different fish is thus invaluable for shedding light on evolutionary diversification of gene function.

Expression of Plxdc2/TEM7R in the developing nervous system of the mouse

Plexin-domain containing 2 ( Plxdc2) is a relatively uncharacterised transmembrane protein with an area of nidogen homology and a plexin repeat (PSI domain) in its extracellular region. Here, we describe Plxdc2 expression in the embryonic mouse, with particular emphasis on the developing central nervous system. Using light microscopy and optical projection tomography (OPT), we analyse RNA in situ hybridization patterns and expression of two reporter genes, β-geo (a fusion of β-galactosidase to neomycin phosphotransferase) and placental alkaline phosphatase (PLAP) in a Plxdc2 gene trap mouse line (KST37; [Leighton, P.A., Mitchell, K.J., Goodrich, L.V., Lu, X., Pinson, K., Scherz, P., Skarnes, W.C., Tessier-Lavigne, M., 2001. Defining brain wiring patterns and mechanisms through gene trapping in mice. Nature 410, 174–179]). At mid-embryonic stages (E9.5–E11.5) Plxdc2-βgeo expression is prominent in a number of patterning centres of the brain, including the cortical hem, midbrain–hindbrain boundary and the midbrain floorplate. Plxdc2 is expressed in other tissues, most notably the limbs, lung buds and developing heart, as well as the spinal cord and dorsal root ganglia. At E15.5, expression is apparent in a large number of discrete nuclei and structures throughout the brain, including the glial wedge and derivatives of the cortical hem. Plxdc2-βgeo expression is particularly strong in the developing Purkinje cell layer, especially in the posterior half of the cerebellum. The PLAP marker is expressed in a number of axonal tracts, including the posterior commissure, mammillotegmental tract and cerebellar peduncle. We compare Plxdc2-βgeo expression in the embryonic brain with the much more restricted expression of the related gene Plxdc1 and with members of the Wnt family ( Wnt3a, Wnt5a and Wnt8b) that show a striking overlap with Plxdc2 expression in certain areas.

Analysis of the midbrain–hindbrain boundary cell fate using a boundary cell-specific Cre-mouse strain

Analysis of the midbrain–hindbrain boundary cell fate using a boundary cell-specific Cre-mouse strain