Gene regulatory logic for reading the Sonic Hedgehog signaling gradient in the vertebrate neural tube.

Secreted signals, known as morphogens, provide the positional information that organizes gene expression and cellular differentiation in many developing tissues. In the vertebrate neural tube, Sonic Hedgehog (Shh) acts as a morphogen to control the pattern of neuronal subtype specification. Using an in vivo reporter of Shh signaling, mouse genetics, and systems modeling, we show that a spatially and temporally changing gradient of Shh signaling is interpreted by the regulatory logic of a downstream transcriptional network. The design of the network, which links three transcription factors to Shh signaling, is responsible for differential spatial and temporal gene expression. In addition, the network renders cells insensitive to fluctuations in signaling and confers hysteresis--memory of the signal. Our findings reveal that morphogen interpretation is an emergent property of the architecture of a transcriptional network that provides robustness and reliability to tissue patterning.

Combination of blockade of endothelin signalling and compensation of IGF1 expression protects the retina from degeneration.

Retinitis pigmentosa (RP) and macular dystrophy (MD) cause severe retinal dysfunction, affecting 1 in 4000 people worldwide. This disease is currently assumed to be intractable, because effective therapeutic methods have not been established, regardless of genetic or sporadic traits. Here, we examined a RP mouse model in which the Prominin-1 (Prom1) gene was deficient and investigated the molecular events occurring at the outset of retinal dysfunction. We extracted the Prom1-deficient retina subjected to light exposure for a short time, conducted single-cell expression profiling, and compared the gene expression with and without stimuli. We identified the cells and genes whose expression levels change directly in response to light stimuli. Among the genes altered by light stimulation, Igf1 was decreased in rod photoreceptor cells and astrocytes under the light-stimulated condition. Consistently, the insulin-like growth factor (IGF) signal was weakened in light-stimulated photoreceptor cells. The recovery of Igf1 expression with the adeno-associated virus (AAV) prevented photoreceptor cell death, and its treatment in combination with the endothelin receptor antagonist led to the blockade of abnormal glial activation and the promotion of glycolysis, thereby resulting in the improvement of retinal functions, as assayed by electroretinography. We additionally demonstrated that the attenuation of mammalian/mechanistic target of rapamycin (mTOR), which mediates IGF signalling, leads to complications in maintaining retinal homeostasis. Together, we propose that combinatorial manipulation of distinct mechanisms is useful for the maintenance of the retinal condition.

Rspo1 and Rspo3 are required for sensory lineage neural crest formation in mouse embryos.

BACKGROUND: R-spondins (Rspos) are secreted proteins that modulate Wnt/ß-catenin signaling. At the early stages of spinal cord development, Wnts (Wnt1, Wnt3a) and Rspos (Rspo1, Rspo3) are co-expressed in the roof plate, suggesting that Rspos are involved in development of dorsal spinal cord and neural crest cells in cooperation with Wnt ligands. RESULTS: Here, we found that Rspo1 and Rspo3, as well as Wnt1 and Wnt3a, maintained roof-plate-specific expression until late embryonic stages. Rspo1- and Rspo3-double-knock-out (dKO) embryos partially exhibited the phenotype of Wnt1 and Wnt3a dKO embryos. While the number of Ngn2-positive sensory lineage neural crest cells is reduced in Rspo-dKO embryos, development of dorsal spinal cord, including its size and dorso-ventral patterning in early development, elongation of the roof plate, and proliferation of ependymal cells, proceeded normally. Consistent with these slight defects, Wnt/ß-catenin signaling was not obviously changed in developing spinal cord of dKO embryos. CONCLUSIONS: Our results show that Rspo1 and Rspo3 are dispensable for most developmental processes involving roof plate-derived Wnt ligands, except for specification of a subtype of neural crest cells. Thus, Rspos may modulate Wnt/ß-catenin signaling in a context-dependent manner.

Regulation of progenitor cell survival by a novel chromatin remodeling factor during neural tube development.

During development, progenitor cell survival is essential for proper tissue functions, but the underlying mechanisms are not fully understood. Here we show that ERCC6L2, a member of the Snf2 family of helicase-like proteins, plays an essential role in the survival of developing chick neural cells. ERCC6L2 expression is induced by the Sonic Hedgehog (Shh) signaling molecule by a mechanism similar to that of the known Shh target genes Ptch1 and Gli1. ERCC6L2 blocks programmed cell death induced by Shh inhibition and this inhibition is independent of neural tube patterning. ERCC6L2 knockdown by siRNA resulted in the aberrant appearance of apoptotic cells. Furthermore, ERCC6L2 cooperates with the Shh signal and plays an essential role in the induction of the anti-apoptotic factor Bcl-2. Taken together, ERCC6L2 acts as a key factor in ensuring the survival of neural progenitor cells.

Manipulation of Signal Gradient and Transcription Factors Recapitulates: Multiple Hypothalamic Identities.

During development, the hypothalamus emerges from the ventral diencephalon and is regionalized into several distinct functional domains. Each domain is characterized by a different combination of transcription factors, including Nkx2.1, Nkx2.2, Pax6, and Rx, which are expressed in the presumptive hypothalamus and its surrounding regions, and play critical roles in defining each area. Here, we recapitulated the molecular networks formed by the gradient of Sonic Hedgehog (Shh) and the aforementioned transcription factors. Using combinatorial experimental systems of directed neural differentiation of mouse embryonic stem (ES) cells, as well as a reporter mouse line and gene overexpression in chick embryos, we deciphered the regulation of transcription factors by different Shh signal intensities. We then used CRISPR/Cas9 mutagenesis to demonstrate the mutual repression between Nkx2.1 and Nkx2.2 in a cell-autonomous manner; however, they induce each other in a non-cell-autonomous manner. Moreover, Rx resides upstream of all these transcription factors and determines the location of the hypothalamic region. Our findings suggest that Shh signaling and its downstream transcription network are required for hypothalamic regionalization and establishment.

Ectodermal factor restricts mesoderm differentiation by inhibiting p53.

During gastrulation of the amphibian embryo, specification of the three germ layers, endo-, ecto-, and mesoderm, is regulated by maternal and zygotic mechanisms. Although it is known that mesoderm specification requires the cooperation between TGF-beta signaling and p53 activity and requires maternal factors, essential zygotic factors have been elusive. Here, we report that the Zn-finger protein XFDL156 is an ectodermal, zygotic factor that suppresses mesodermal differentiation. XFDL156 overexpression suppresses mesodermal markers, and its depletion induces aberrant mesodermal differentiation in the presumptive ectoderm. Furthermore, we find that XFDL156 and its mammalian homologs interact with the C-terminal regulatory region of p53, thereby inhibiting p53 target gene induction and mesodermal differentiation. Thus, XFDL156 actively restricts mesodermal differentiation in the presumptive ectoderm by controlling the spatiotemporal responsiveness to p53.

Sox14 is essential for initiation of neuronal differentiation in the chick spinal cord.

BACKGROUND: The neural tube comprises several different types of progenitors and postmitotic neurons that co-ordinately act with each other to play integrated functions. Its development consists of two phases: proliferation of progenitor cells and differentiation into postmitotic neurons. How progenitor cells differentiate into each corresponding neuron is an important question for understanding the mechanisms of neuronal development. RESULTS: Here we introduce one of the Sox transcription factors, Sox14, which plays an essential role in the promotion of neuronal differentiation. Sox14 belongs to the SoxB2 subclass and its expression starts in the progenitor regions before neuronal differentiation is initiated at the trunk level of the neural tube. After neuronal differentiation is initiated, Sox14 expression gradually becomes confined to the V2a region of the neural tube, where Chx10 is co-expressed. Overexpression of Sox14 restricts progenitor cell proliferation. Conversely, the blockade of Sox14 expression by the RNAi strategy inhibits V2a neuron differentiation and causes expansion of the progenitor domain. We further found that Sox14 acted as a transcriptional activator. CONCLUSIONS: Sox14 acts as a modulator of cell proliferation and is essential for initiation of neuronal differentiation in the chick neural tube.

Prominin-1 deletion results in spermatogenic impairment, sperm morphological defects, and infertility in mice.

Purpose: Spermatogenesis is a complex process orchestrated by several essential genes. Prominin-1 (Prom1/PROM1) is a gene that is expressed in the testis but with a poorly understood role in spermatogenesis. Methods: We used Prom1 knockout (Prom1 KO) mice to assess the role of Prom1 in spermatogenesis. To this end, we performed immunohistochemistry, immunofluorescence, western blotting, ß-galactosidase staining, and apoptosis assay. Additionally, we analyzed the morphology of sperm and assessed litter sizes. Results: We observed that PROM1 is localized to the dividing spermatocytes in seminiferous epithelial cells, sperm, and columnar epithelium in the epididymis. In the Prom1 KO testis, an aberrant increase in apoptotic cells and a decrease in proliferating seminiferous epithelial cells were observed. Cellular FLICE-like inhibitory protein (c-FLIP) and extracellular signal-regulated kinase 1/2 (ERK1/2) expression were also significantly decreased in Prom1 KO testis. In addition, a significantly increased number of epididymal spermatozoa with abnormal morphology and less motility was found in Prom1 KO mice. Conclusions: PROM1 maintains spermatogenic cell proliferation and survival via c-FLIP expression in the testis. It is also involved in sperm motility and fertilization potential. The mechanism underlying the effect of Prom1 on sperm morphology and motility remains to be identified.

GPR17 is an essential regulator for the temporal adaptation of sonic hedgehog signalling in neural tube development.

Dorsal-ventral pattern formation of the neural tube is regulated by temporal and spatial activities of extracellular signalling molecules. Sonic hedgehog (Shh) assigns ventral neural subtypes via activation of the Gli transcription factors. Shh activity in the neural progenitor cells changes dynamically during differentiation, but the mechanisms regulating this dynamicity are not fully understood. Here, we show that temporal change of intracellular cAMP levels confers the temporal Shh signal, and the purinergic G-protein-coupled receptor GPR17 plays an essential role in this regulation. GPR17 is highly expressed in the ventral progenitor regions of the neural tube and acts as a negative regulator of the Shh signal in chick embryos. Although the activation of the GPR17-related signal inhibits ventral identity, perturbation of Gpr17 expression leads to aberrant expansion of ventral neural domains. Notably, perturbation of Gpr17 expression partially inhibits the negative feedback of Gli activity. Moreover, GPR17 increases cAMP activity, suggesting that it exerts its function by inhibiting the processing of Gli3 protein. GPR17 also negatively regulates Shh signalling in neural cells differentiated from mouse embryonic stem cells, suggesting that GPR17 function is conserved among different organisms. Our results demonstrate that GPR17 is a novel negative regulator of Shh signalling in a wide range of cellular contexts.

The Rx transcription factor is required for determination of the retinal lineage and regulates the timing of neuronal differentiation.

Understanding the molecular mechanisms leading to retinal development is of great interest for both basic scientific and clinical applications. Several signaling molecules and transcription factors involved in retinal development have been isolated and analyzed; however, determining the direct impact of the loss of a specific molecule is problematic, due to difficulties in identifying the corresponding cellular lineages in different individuals. Here, we conducted genome-wide expression analysis with embryonic stem (ES) cells devoid of the Rx gene, which encodes one of several homeobox transcription factors essential for retinal development. We performed three-dimensional differentiation of wild-type and mutant cells and compared their gene-expression profiles. The mutant tissue failed to differentiate into the retinal lineage and exhibited precocious expression of genes characteristic of neuronal cells. Together, these results suggest that Rx expression is an important biomarker of the retinal lineage and that it helps regulates appropriate differentiation stages.

Neural induction: Historical views and application to pluripotent stem cells.

Embryonic stem (ES) cells are a useful experimental material to recapitulate the differentiation steps of early embryos, which are usually invisible and inaccessible from outside of the body, especially in mammals. ES cells have greatly facilitated the analyses of gene expression profiles and cell characteristics. In addition, understanding the mechanisms during neural differentiation is important for clinical purposes, such as developing new therapeutic methods or regenerative medicine. As neurons have very limited regenerative ability, neurodegenerative diseases are usually intractable, and patients suffer from the disease throughout their lifetimes. The functional cells generated from ES cells in vitro could replace degenerative areas by transplantation. In this review, we will first demonstrate the historical views and widely accepted concepts regarding the molecular mechanisms of neural induction and positional information to produce the specific types of neurons in model animals. Next, we will describe how these concepts have recently been applied to the research in the establishment of the methodology of neural differentiation from mammalian ES cells. Finally, we will focus on examples of the applications of differentiation systems to clinical purposes. Overall, the discussion will focus on how historical developmental studies are applied to state-of-the-art stem cell research.

Autosomal-Dominant Corneal Endothelial Dystrophies CHED1 and PPCD1 Are Allelic Disorders Caused by Non-coding Mutations in the Promoter of OVOL2.

Congenital hereditary endothelial dystrophy 1 (CHED1) and posterior polymorphous corneal dystrophy 1 (PPCD1) are autosomal-dominant corneal endothelial dystrophies that have been genetically mapped to overlapping loci on the short arm of chromosome 20. We combined genetic and genomic approaches to identify the cause of disease in extensive pedigrees comprising over 100 affected individuals. After exclusion of pathogenic coding, splice-site, and copy-number variations, a parallel approach using targeted and whole-genome sequencing facilitated the identification of pathogenic variants in a conserved region of the OVOL2 proximal promoter sequence in the index families (c.-339_361dup for CHED1 and c.-370T>C for PPCD1). Direct sequencing of the OVOL2 promoter in other unrelated affected individuals identified two additional mutations within the conserved proximal promoter sequence (c.-274T>G and c.-307T>C). OVOL2 encodes ovo-like zinc finger 2, a C2H2 zinc-finger transcription factor that regulates mesenchymal-to-epithelial transition and acts as a direct transcriptional repressor of the established PPCD-associated gene ZEB1. Interestingly, we did not detect OVOL2 expression in the normal corneal endothelium. Our in vitro data demonstrate that all four mutated OVOL2 promoters exhibited more transcriptional activity than the corresponding wild-type promoter, and we postulate that the mutations identified create cryptic cis-acting regulatory sequence binding sites that drive aberrant OVOL2 expression during endothelial cell development. Our data establish CHED1 and PPCD1 as allelic conditions and show that CHED1 represents the extreme of what can be considered a disease spectrum. They also implicate transcriptional dysregulation of OVOL2 as a common cause of dominantly inherited corneal endothelial dystrophies.

The small leucine-rich repeat secreted protein Asporin induces eyes in Xenopus embryos through the IGF signalling pathway.

Small leucine-rich repeat proteoglycan (SLRP) family proteins play important roles in a number of biological events. Here, we demonstrate that the SLRP family member Asporin (ASPN) plays a crucial role in the early stages of eye development in Xenopus embryos. During embryogenesis, ASPN is broadly expressed in the neuroectoderm of the embryo. Overexpression of ASPN causes the induction of ectopic eyes. By contrast, blocking ASPN function with a morpholino oligonucleotide (ASPN-MO) inhibits eye formation, indicating that ASPN is an essential factor for eye development. Detailed molecular analyses revealed that ASPN interacts with insulin growth factor receptor (IGFR) and is essential for activating the IGF receptor-mediated intracellular signalling pathway. Moreover, ASPN perturbed the Wnt, BMP and Activin signalling pathways, suggesting that ASPN thereby creates a favourable environment in which the IGF signal can dominate. ASPN is thus a novel secreted molecule essential for eye induction through the coordination of multiple signalling pathways.

Distinct Sonic Hedgehog signaling dynamics specify floor plate and ventral neuronal progenitors in the vertebrate neural tube.

The secreted ligand Sonic Hedgehog (Shh) organizes the pattern of cellular differentiation in the ventral neural tube. For the five neuronal subtypes, increasing levels and durations of Shh signaling direct progenitors to progressively more ventral identities. Here we demonstrate that this mode of action is not applicable to the generation of the most ventral cell type, the nonneuronal floor plate (FP). In chick and mouse embryos, FP specification involves a biphasic response to Shh signaling that controls the dynamic expression of key transcription factors. During gastrulation and early somitogenesis, FP induction depends on high levels of Shh signaling. Subsequently, however, prospective FP cells become refractory to Shh signaling, and this is a prerequisite for the elaboration of their identity. This prompts a revision to the model of graded Shh signaling in the neural tube, and provides insight into how the dynamics of morphogen signaling are deployed to extend the patterning capacity of a single ligand. In addition, we provide evidence supporting a common scheme for FP specification by Shh signaling that reconciles mechanisms of FP development in teleosts and amniotes.

Integration of signals along orthogonal axes of the vertebrate neural tube controls progenitor competence and increases cell diversity.

A relatively small number of signals are responsible for the variety and pattern of cell types generated in developing embryos. In part this is achieved by exploiting differences in the concentration or duration of signaling to increase cellular diversity. In addition, however, changes in cellular competence-temporal shifts in the response of cells to a signal-contribute to the array of cell types generated. Here we investigate how these two mechanisms are combined in the vertebrate neural tube to increase the range of cell types and deliver spatial control over their location. We provide evidence that FGF signaling emanating from the posterior of the embryo controls a change in competence of neural progenitors to Shh and BMP, the two morphogens that are responsible for patterning the ventral and dorsal regions of the neural tube, respectively. Newly generated neural progenitors are exposed to FGF signaling, and this maintains the expression of the Nk1-class transcription factor Nkx1.2. Ventrally, this acts in combination with the Shh-induced transcription factor FoxA2 to specify floor plate cells and dorsally in combination with BMP signaling to induce neural crest cells. As development progresses, the intersection of FGF with BMP and Shh signals is interrupted by axis elongation, resulting in the loss of Nkx1.2 expression and allowing the induction of ventral and dorsal interneuron progenitors by Shh and BMP signaling to supervene. Hence a similar mechanism increases cell type diversity at both dorsal and ventral poles of the neural tube. Together these data reveal that tissue morphogenesis produces changes in the coincidence of signals acting along orthogonal axes of the neural tube and this is used to define spatial and temporal transitions in the competence of cells to interpret morphogen signaling.

Intrinsic transition of embryonic stem-cell differentiation into neural progenitors.

The neural fate is generally considered to be the intrinsic direction of embryonic stem (ES) cell differentiation. However, little is known about the intracellular mechanism that leads undifferentiated cells to adopt the neural fate in the absence of extrinsic inductive signals. Here we show that the zinc-finger nuclear protein Zfp521 is essential and sufficient for driving the intrinsic neural differentiation of mouse ES cells. In the absence of the neural differentiation inhibitor BMP4, strong Zfp521 expression is intrinsically induced in differentiating ES cells. Forced expression of Zfp521 enables the neural conversion of ES cells even in the presence of BMP4. Conversely, in differentiation culture, Zfp521-depleted ES cells do not undergo neural conversion but tend to halt at the epiblast state. Zfp521 directly activates early neural genes by working with the co-activator p300. Thus, the transition of ES cell differentiation from the epiblast state into neuroectodermal progenitors specifically depends on the cell-intrinsic expression and activator function of Zfp521.

An inducible transgene expression system for zebrafish and chick.

We have generated an inducible system to control the timing of transgene expression in zebrafish and chick. An estrogen receptor variant (ERT2) fused to the GAL4 transcriptional activator rapidly and robustly activates transcription within 3 hours of treatment with the drug 4-hydroxy-tamoxifen (4-OHT) in tissue culture and transgenic zebrafish. We have generated a broadly expressed inducible ERT2-GAL4 zebrafish line using the ubiquitin (ubi) enhancer. In addition, use of ERT2-GAL4 in conjunction with tissue-specific enhancers enables the control of transgene expression in both space and time. This spatial restriction and the ability to sustain forced expression are important advantages over the currently used heat-shock promoters. Moreover, in contrast to currently available TET and LexA systems, which require separate constructs with their own unique recognition sequences, ERT2-GAL4 is compatible with the growing stock of UAS lines being generated in the community. We also applied the same inducible system to the chick embryo and find that it is fully functional, suggesting that this strategy is generally applicable.

Sonic Hedgehog dependent phosphorylation by CK1α and GRK2 is required for ciliary accumulation and activation of smoothened.

Hedgehog (Hh) signaling regulates embryonic development and adult tissue homeostasis through the GPCR-like protein Smoothened (Smo), but how vertebrate Smo is activated remains poorly understood. In Drosophila, Hh dependent phosphorylation activates Smo. Whether this is also the case in vertebrates is unclear, owing to the marked sequence divergence between vertebrate and Drosophila Smo (dSmo) and the involvement of primary cilia in vertebrate Hh signaling. Here we demonstrate that mammalian Smo (mSmo) is activated through multi-site phosphorylation of its carboxyl-terminal tail by CK1α and GRK2. Phosphorylation of mSmo induces its active conformation and simultaneously promotes its ciliary accumulation. We demonstrate that graded Hh signals induce increasing levels of mSmo phosphorylation that fine-tune its ciliary localization, conformation, and activity. We show that mSmo phosphorylation is induced by its agonists and oncogenic mutations but is blocked by its antagonist cyclopamine, and efficient mSmo phosphorylation depends on the kinesin-II ciliary motor. Furthermore, we provide evidence that Hh signaling recruits CK1α to initiate mSmo phosphorylation, and phosphorylation further increases the binding of CK1α and GRK2 to mSmo, forming a positive feedback loop that amplifies and/or sustains mSmo phosphorylation. Hence, despite divergence in their primary sequences and their subcellular trafficking, mSmo and dSmo employ analogous mechanisms for their activation.

Dynamic assignment and maintenance of positional identity in the ventral neural tube by the morphogen sonic hedgehog.

Morphogens are secreted signalling molecules that act in a graded manner to control the pattern of cellular differentiation in developing tissues. An example is Sonic hedgehog (Shh), which acts in several developing vertebrate tissues, including the central nervous system, to provide positional information during embryonic patterning. Here we address how Shh signalling assigns the positional identities of distinct neuronal subtype progenitors throughout the ventral neural tube. Assays of intracellular signal transduction and gene expression indicate that the duration as well as level of signalling is critical for morphogen interpretation. Progenitors of the ventral neuronal subtypes are established sequentially, with progressively more ventral identities requiring correspondingly higher levels and longer periods of Shh signalling. Moreover, cells remain sensitive to changes in Shh signalling for an extended time, reverting to antecedent identities if signalling levels fall below a threshold. Thus, the duration of signalling is important not only for the assignment but also for the refinement and maintenance of positional identity. Together the data suggest a dynamic model for ventral neural tube patterning in which positional information corresponds to the time integral of Shh signalling. This suggests an alternative to conventional models of morphogen action that rely solely on the level of signalling.

Acquisition of neural fate by combination of BMP blockade and chromatin modification.

Neural induction is a process where naive cells are converted into committed cells with neural characteristics, and it occurs at the earliest step during embryogenesis. Although the signaling molecules and chromatin remodeling for neural induction have been identified, the mutual relationships between these molecules are yet to be fully understood. By taking advantage of the neural differentiation system of mouse embryonic stem (ES) cells, we discovered that the BMP signal regulates the expression of several polycomb repressor complex (PRC) component genes. We particularly focused on Polyhomeotic Homolog 1 (Phc1) and established Phc1-knockout (Phc1-KO) ES cells. We found that Phc1-KO failed to acquire the neural fate, and the cells remained in pluripotent or primitive non-neural states. Chromatin accessibility analysis suggests that Phc1 is essential for chromatin packing. Aberrant upregulation of the BMP signal was confirmed in the Phc1 homozygotic mutant embryos. Taken together, Phc1 is required for neural differentiation through epigenetic modification.