Cerebellar granular neuron progenitors exit their germinative niche via BarH-like1 activity mediated partly by inhibition of T-Cell Factor.

Cerebellar granule neuron progenitors (GNP) originate from the upper rhombic lip (URL), a germinative niche whose developmental defects produce human diseases. T-Cell Factors (TCF) responsiveness and Notch dependence are hallmarks of self-renewal in neural stem cells. TCF activity together with transcripts coding for proneural genes repressors hairy and enhancer of split (hes/hey), are detected in the URL. However, their functions and regulatory modes are undeciphered. Here we established amphibian as a pertinent model to study vertebrate URL development. Amphibians long-lived URL is Tcf active, while the External Granular layer (EGL) is non-proliferative and expresses hes4/5 genes. Using functional and transcriptomic approaches, we show that Tcf activity is necessary for URL emergence and maintenance. We establish that the transcription factor Barhl1 controls GNP exit from the URL acting partly through direct Tcf inhibition. Identification of Barhl1 target genes argues that besides Tcf, Barhl1 inhibits transcription of hes5 genes independently of Notch signaling. Observations in amniotes suggest a conserved role of a Barhl in maintenance of the URL/EGL via coregulation of TCF and hes/hey genes.

T-Cell Factors as Transcriptional Inhibitors: Activities and Regulations in Vertebrate Head Development.

Since its first discovery in the late 90s, Wnt canonical signaling has been demonstrated to affect a large variety of neural developmental processes, including, but not limited to, embryonic axis formation, neural proliferation, fate determination, and maintenance of neural stem cells. For decades, studies have focused on the mechanisms controlling the activity of ß-catenin, the sole mediator of Wnt transcriptional response. More recently, the spotlight of research is directed towards the last cascade component, the T-cell factor (TCF)/Lymphoid-Enhancer binding Factor (LEF), and more specifically, the TCF/LEF-mediated switch from transcriptional activation to repression, which in both embryonic blastomeres and mouse embryonic stem cells pushes the balance from pluri/multipotency towards differentiation. It has been long known that Groucho/Transducin-Like Enhancer of split (Gro/TLE) is the main co-repressor partner of TCF/LEF. More recently, other TCF/LEF-interacting partners have been identified, including the pro-neural BarH-Like 2 (BARHL2), which belongs to the evolutionary highly conserved family of homeodomain-containing transcription factors. This review describes the activities and regulatory modes of TCF/LEF as transcriptional repressors, with a specific focus on the functions of Barhl2 in vertebrate brain development. Specific attention is given to the transcriptional events leading to formation of the Organizer, as well as the roles and regulations of Wnt/ß-catenin pathway in growth of the caudal forebrain. We present TCF/LEF activities in both embryonic and neural stem cells and discuss how alterations of this pathway could lead to tumors.

Mcl1 protein levels and Caspase-7 executioner protease control axial organizer cells survival.

BACKGROUND: Organizing centers are groups of specialized cells that secrete morphogens, thereby influencing development of their neighboring territories. Apoptosis is a form of programmed cell death reported to limit the size of organizers. Little is known about the identity of intracellular signals driving organizer cell death. Here we investigated in Xenopus the role of both the anti-apoptotic protein Myeloid-cell-leukemia 1 (Mcl1) and the cysteine proteases Caspase-3 and Caspase-7 in formation of the axial organizing center-the notochord-that derives from the Spemann organizer, and participates in the induction and patterning of the neuroepithelium. RESULTS: We confirm a role for apoptosis in establishing the axial organizer in early neurula. We show that the expression pattern of mcl1 is coherent with a role for this gene in early notochord development. Using loss of function approaches, we demonstrate that Mcl1 depletion decreases neuroepithelium width and increases notochord cells apoptosis, a process that relies on Caspase-7, and not on Caspase-3, activity. Our data provide evidence that Mcl1 protein levels physiologically control notochord cells' survival and that Caspase-7 is the executioner protease in this developmental process. CONCLUSIONS: Our study reveals new functions for Mcl1 and Caspase-7 in formation of the axial signalling center.

Barhl2 maintains T cell factors as repressors and thereby switches off the Wnt/ß-Catenin response driving Spemann organizer formation.

A hallmark of Wnt/ß-Catenin signaling is the extreme diversity of its transcriptional response, which varies depending on the cell and developmental context. What controls this diversity is poorly understood. In all cases, the switch from transcriptional repression to activation depends on a nuclear increase in ß-Catenin, which detaches the transcription factor T cell factor 7 like 1 (Tcf7l1) bound to Groucho (Gro) transcriptional co-repressors from its DNA-binding sites and transiently converts Tcf7/Lymphoid enhancer binding factor 1 (Lef1) into a transcriptional activator. One of the earliest and evolutionarily conserved functions of Wnt/ß-Catenin signaling is the induction of the blastopore lip organizer. Here, we demonstrate that the evolutionarily conserved BarH-like homeobox-2 (Barhl2) protein stabilizes the Tcf7l1-Gro complex and maintains the repressed expression of Tcf target genes by a mechanism that depends on histone deacetylase 1 (Hdac-1) activity. In this way, Barhl2 switches off the Wnt/ß-Catenin-dependent early transcriptional response, thereby limiting the formation of the organizer in time and/or space. This study reveals a novel nuclear inhibitory mechanism of Wnt/Tcf signaling that switches off organizer fate determination.

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo.

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.

An Evolutionarily Conserved Network Mediates Development of the zona limitans intrathalamica, a Sonic Hedgehog-Secreting Caudal Forebrain Signaling Center.

Recent studies revealed new insights into the development of a unique caudal forebrain-signaling center: the zona limitans intrathalamica (zli). The zli is the last brain signaling center to form and the first forebrain compartment to be established. It is the only part of the dorsal neural tube expressing the morphogen Sonic Hedgehog (Shh) whose activity participates in the survival, growth and patterning of neuronal progenitor subpopulations within the thalamic complex. Here, we review the gene regulatory network of transcription factors and cis-regulatory elements that underlies formation of a shh-expressing delimitated domain in the anterior brain. We discuss evidence that this network predates the origin of chordates. We highlight the contribution of Shh, Wnt and Notch signaling to zli development and discuss implications for the fact that the morphogen Shh relies on primary cilia for signal transduction. The network that underlies zli development also contributes to thalamus induction, and to its patterning once the zli has been set up. We present an overview of the brain malformations possibly associated with developmental defects in this gene regulatory network (GRN).

Early development of the neural plate: new roles for apoptosis and for one of its main effectors caspase-3.

Despite its tremendous complexity, the vertebrate nervous system emerges from a homogenous layer of neuroepithelial cells, the neural plate. Its formation relies on the time- and space-controlled progression of developmental programs. Apoptosis is a biological process that removes superfluous and potentially dangerous cells and is implemented through the activation of a molecular pathway conserved during evolution. Apoptosis and an unconventional function of one of its main effectors, caspase-3, contribute to the patterning and growth of the neuroepithelium. Little is known about the intrinsic and extrinsic cues controlling activities of the apoptotic machinery during development. The BarH-like (Barhl) proteins are homeodomain-containing transcription factors. The observations in Caenorhabditis elegans, Xenopus, and mice document that Barhl proteins act in cell survival and as cell type-specific regulators of a caspase-3 function that limits neural progenitor proliferation. In this review, we discuss the roles and regulatory modes of the apoptotic machinery in the development of the neural plate. We focus on the Barhl2, the Sonic Hedgehog, and the Wnt pathways and their activities in neural progenitor survival and proliferation.

The conserved barH-like homeobox-2 gene barhl2 acts downstream of orthodentricle-2 and together with iroquois-3 in establishment of the caudal forebrain signaling center induced by Sonic Hedgehog.

In this study, we investigated the gene regulatory network that governs formation of the Zona limitans intrathalamica (ZLI), a signaling center that secretes Sonic Hedgehog (Shh) to control the growth and regionalization of the caudal forebrain. Using loss- and gain-of-function, explants and grafting experiments in amphibians, we demonstrate that barhl2 acts downstream of otx2 and together with the iroquois (irx)-3 gene in establishment of the ZLI compartment initiated by Shh influence. We find that the presumptive (pre)-ZLI domain expresses barhl2, otx2 and irx3, whereas the thalamus territory caudally bordering the pre-ZLI expresses barhl2, otx2 and irx1/2 and early on irx3. We demonstrate that Barhl2 activity is required for determination of the ZLI and thalamus fates and that within the p2 alar plate the ratio of Irx3 to Irx1/2 contributes to ZLI specification and size determination. We show that when continuously exposed to Shh, neuroepithelial cells coexpressing barhl2, otx2 and irx3 acquire two characteristics of the ZLI compartment-the competence to express shh and the ability to segregate from anterior neural plate cells. In contrast, neuroepithelial cells expressing barhl2, otx2 and irx1/2, are not competent to express shh. Noteworthy in explants, under Shh influence, ZLI-like cells segregate from thalamic-like cells. Our study establishes that Barhl2 activity plays a key role in p2 alar plate patterning, specifically ZLI formation, and provides new insights on establishment of the signaling center of the caudal forebrain.

Barhl2 limits growth of the diencephalic primordium through Caspase3 inhibition of beta-catenin activation.

Little is known about the respective contributions of cell proliferation and cell death to the control of vertebrate forebrain growth. The homeodomain protein barhl2 is expressed in the diencephalons of Xenopus, zebrafish, and mouse embryos, and we previously showed that Barhl2 overexpression in Xenopus neuroepithelial cells induces Caspase3-dependent apoptosis. Here, barhl2 is shown to act as a brake on diencephalic proliferation through an unconventional function of Caspase3. Depletion of Barhl2 or Caspase3 causes an increase in diencephalic cell number, a disruption of the neuroepithelium architecture, and an increase in Wnt activity. Surprisingly, these changes are not caused by decreased apoptosis but instead, are because of an increase in the amount and activation of ß-catenin, which stimulates excessive neuroepithelial cell proliferation and induces defects in ß-catenin intracellular localization and an up-regulation of axin2 and cyclinD1, two downstream targets of ß-catenin/T-cell factor/lymphoïd enhancer factor signaling. Using two different sets of complementation experiments, we showed that, in the developing diencephalon, Caspase3 acts downstream of Barhl2 in limiting neuroepithelial cell proliferation by inhibiting ß-catenin activation. Our data argue that Bar homeodomain proteins share a conserved function as cell type-specific regulators of Caspase3 activities.