During vertebrate neurulation, the embryonic ectoderm is patterned into lineage progenitors for neural plate, neural crest, placodes and epidermis. Here, we use Xenopus laevis embryos to analyze the spatial and temporal transcriptome of distinct ectodermal domains in the course of neurulation, during the establishment of cell lineages. In order to define the transcriptome of small groups of cells from a single germ layer and to retain spatial information, dorsal and ventral ectoderm was subdivided along the anterior-posterior and medial-lateral axes by microdissections. Principal component analysis on the transcriptomes of these ectoderm fragments primarily identifies embryonic axes and temporal dynamics. This provides a genetic code to define positional information of any ectoderm sample along the anterior-posterior and dorsal-ventral axes directly from its transcriptome. In parallel, we use nonnegative matrix factorization to predict enhanced gene expression maps onto early and mid-neurula embryos, and specific signatures for each ectoderm area. The clustering of spatial and temporal datasets allowed detection of multiple biologically relevant groups (e.g., Wnt signaling, neural crest development, sensory placode specification, ciliogenesis, germ layer specification). We provide an interactive network interface, EctoMap, for exploring synexpression relationships among genes expressed in the neurula, and suggest several strategies to use this comprehensive dataset to address questions in developmental biology as well as stem cell or cancer research.

SUMMARYContinuous neurogenesis in the adult nervous system requires a delicate balance between proliferation and differentiation. Although Wnt/b-catenin and Hedgehog signalling pathways are thought to share a mitogenic function in adult neural stem/progenitor cells, it remains unclear how they interact in this process. Adult amphibians produce retinal neurons from a pool of neural stem cells localised in the ciliary marginal zone (CMZ). Surprisingly, we found that perturbations of the Wnt and Hedgehog pathways result in opposite proliferative outcomes of neural stem/progenitor cells in the CMZ. Additionally, our study revealed that Wnt and Hedgehog morphogens are produced in mutually exclusive territories of the post-embryonic retina. Using genetic and pharmacological tools, we found that the Wnt and Hedgehog pathways exhibit reciprocal inhibition. Our data suggest that Sfrp-1 and Gli3 contribute to this negative cross-regulation. Altogether, our results reveal an unexpected antagonistic interplay of Wnt and Hedgehog signals that may tightly regulate the extent of neural stem/progenitor cell proliferation in the Xenopus retina. and a transgenic male. The latter was selected beforehand as having a single transgene insertion site (as inferred by mendelian ratios in its progeny) in order to ensure homogeneous levels of GFP expression in the offspring. Construction of the LEF1-VP16 and LEF1-EnR transgenesis vectors has been described previously (Denayer et al., 2008) and transgenic X. tropicalis lines (LEF1-VP16Tg and LEF1-EnRTg) were generated as described (Sekkali et al., 2008). These constructs are fused with the dexamethasone-responsive hormone-binding domain of the human glucocorticoid receptor (GR). Expression constructs and morpholinospCS2- TCF3-VP16GR and pCS2-dnTCF3-GR (de Croze et al., 2011), pCS2-Ihh-CD2 [previously called Bhh (Locker et al., 2006)], pCS2-Smo-M2 , pCS2-cyclinA2 and pCS2-cdk2 (Decembrini et al., 2006) and pCS2-GFP (a gift from David Turner, University of Michigan, Ann Arbor, USA) were described previously. pCS2-Shh-CD2 and pCS2-Dhh-CD2 (previously called Chh) were generated by subcloning the N-terminal coding regions (devoid of the C-terminal cleavage product) of Shh and Dhh cDNAs (Ekker et al., 1995) Microinjection and in vivo DNA lipofectionCapped mRNAs encoding TCF3-VP16GR and GFP were transcribed from pCS2 plasmids after NotI digestion using the mMessage mMachine SP6 Kit (Ambion). Then, 400 pg of each mRNA was injected into two blastomeres of four-cell stage embryos. Gli3, Sfrp-1 or standard control morpholino oligonucleotides (Gene Tools) were injected into one blastomere at the one-cell stage (30 ng). Their efficacy was tested by analysing in vivo GFP fluorescence following co-injection of a chimeric GFP construct fused downstream of the morpholino-complementary sequence (supplementary material Fig. S1).Lipofection experiments were performed by cotransfecting the indicated pCS2 constructs together with pCS2-GFP at stage 18 into the presumptive region of the retina, as previously...

The adult frog retina retains a reservoir of active neural stem cells that contribute to continuous eye growth throughout life. We found that Yap, a downstream effector of the Hippo pathway, is specifically expressed in these stem cells. Yap knock-down leads to an accelerated S-phase and an abnormal progression of DNA replication, a phenotype likely mediated by upregulation of c-Myc. This is associated with an increased occurrence of DNA damage and eventually p53-p21 pathway-mediated cell death. Finally, we identified PKNOX1, a transcription factor involved in the maintenance of genomic stability, as a functional and physical interactant of YAP. Altogether, we propose that YAP is required in adult retinal stem cells to regulate the temporal firing of replication origins and quality control of replicated DNA. Our data reinforce the view that specific mechanisms dedicated to S-phase control are at work in stem cells to protect them from genomic instability.DOI: http://dx.doi.org/10.7554/eLife.08488.001

The retina of fish and amphibian contains genuine neural stem cells located at the most peripheral edge of the ciliary marginal zone (CMZ). However, their cell-of-origin as well as the mechanisms that sustain their maintenance during development are presently unknown. We identified Hes4 (previously named XHairy2), a gene encoding a bHLH-O transcriptional repressor, as a stem cell-specific marker of the Xenopus CMZ that is positively regulated by the canonical Wnt pathway and negatively by Hedgehog signaling. We found that during retinogenesis, Hes4 labels a small territory, located first at the pigmented epithelium (RPE)/neural retina (NR) border and later in the retinal margin, that likely gives rise to adult retinal stem cells. We next addressed whether Hes4 might impart this cell subpopulation with retinal stem cell features: inhibited RPE or NR differentiation programs, continuous proliferation, and slow cell cycle speed. We could indeed show that Hes4 overexpression cell autonomously prevents retinal precursor cells from commitment toward retinal fates and maintains them in a proliferative state. Besides, our data highlight for the first time that Hes4 may also constitute a crucial regulator of cell cycle kinetics. Hes4 gain of function indeed significantly slows down cell division, mainly through the lengthening of G1 phase. As a whole, we propose that Hes4 maintains particular stemness features in a cellular cohort dedicated to constitute the adult retinal stem cell pool, by keeping it in an undifferentiated and slowly proliferative state along embryonic retinogenesis. Stem Cells 2012;30:2784–2795

ABSTRACT

Observations of knock-out mice suggest that breathing at birth requires correct development of a specific hindbrain territory corresponding to rhombomeres (r) 3 and 4. Focusing on this territory, we examined the development of a neuronal rhythm generator in the chick embryo. We show that rhythmic activity in r4 is inducible after developmental stage 10 through interaction with r3. Although the nature of this interaction remains obscure, we find that the expression of Krox20, a segmentation gene responsible for specifying r3 and r5, is sufficient to endow other rhombomeres with the capacity to induce rhythmic activity in r4. Induction is robust, because it can be reproduced with r2 and r6 instead of r4 and with any hindbrain territory that normally expresses Krox20 (r3, r5) or can be forced to do so (r1, r4). Interestingly, the interaction between r4 and r3/r5 that results in rhythm production can only take place through the anterior border of r4, revealing a heretofore unsuspected polarity in individual rhombomeres. The r4 rhythm generator appears to be homologous to a murine respiratory parafacial neuronal system developing in r4 under the control of Krox20 and Hoxa1. These results identify a late role for Krox20 at the onset of neurogenesis.

A hallmark of Wnt/β-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 β-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/β-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/β-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.

Neural crest (NC) specification comprises an early phase, initiating immature NC progenitors formation at neural plate stage, and a later phase at neural fold stage, resulting in a functional premigratory NC that is able to delaminate and migrate. We found that the NC gene regulatory network triggers upregulation of (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4) during this late specification phase. As shown in previous studies, PFKFB4 controls AKT signaling in gastrulas and glycolysis rate in adult cells. Here, we focus on PFKFB4 function in NC during and after neurulation, using time-controlled or hypomorph depletions We find that PFKFB4 is essential both for specification of functional premigratory NC and for its migration. PFKFB4-depleted embryos fail to activate and late NC specifiers, and exhibit severe migration defects resulting in craniofacial defects. AKT signaling mediates PFKFB4 function in NC late specification, whereas both AKT signaling and glycolysis regulate migration. These findings highlight novel and essential roles of PFKFB4 activity in later stages of NC development that are wired into the NC gene regulatory network.