The secreted BMP antagonist ERFE is required for the development of a functional circulatory system in Xenopus.

The hormone Erythroferrone (ERFE) is a member of the C1q/TNF-related protein family that regulates iron homeostasis through the suppression of hamp. In a gain of function screen in Xenopus embryos, we identified ERFE as a potent secondary axis-inducing agent. Experiments in Xenopus embryos and ectodermal explants revealed that ERFE functions as a selective inhibitor of the BMP pathway and the conserved C1q domain is not required for this activity. Inhibition occurs at the extracelluar level, through the interaction of ERFE with the BMP ligand. During early Xenopus embryogenesis, erfe is first expressed in the ventral blood islands where initial erythropoiesis occurs and later in circulating blood cells. ERFE knockdown does not alter the expression of etv.2, aplnr and flt1 in tailbud stage embryos indicating endothelial cell specification is independent of ERFE. However, in tadpole embryos, defects of the vascular network and primitive blood circulation are observed as well as edema formation. RNAseq analysis of ERFE morphant embryos also revealed the inhibition of gja4 indicating disruption of dorsal aorta formation.

Retinoic acid-induced expression of Hnf1b and Fzd4 is required for pancreas development in Xenopus laevis.

Retinoic acid (RA) is required for pancreas specification in Xenopus and other vertebrates. However, the gene network that is directly induced by RA signalling in this context remains to be defined. By RNA sequencing of in vitro-generated pancreatic explants, we identified the genes encoding the transcription factor Hnf1ß and the Wnt-receptor Fzd4/Fzd4s as direct RA target genes. Functional analyses of Hnf1b and Fzd4/Fzd4s in programmed pancreatic explants and whole embryos revealed their requirement for pancreatic progenitor formation and differentiation. Thus, Hnf1ß and Fzd4/Fzd4s appear to be involved in pre-patterning events of the embryonic endoderm that allow pancreas formation in Xenopus.

DNA Methylation-Mediated Modulation of Endocytosis as Potential Mechanism for Synaptic Function Regulation in Murine Inhibitory Cortical Interneurons.

The balance of excitation and inhibition is essential for cortical information processing, relying on the tight orchestration of the underlying subcellular processes. Dynamic transcriptional control by DNA methylation, catalyzed by DNA methyltransferases (DNMTs), and DNA demethylation, achieved by ten-eleven translocation (TET)-dependent mechanisms, is proposed to regulate synaptic function in the adult brain with implications for learning and memory. However, focus so far is laid on excitatory neurons. Given the crucial role of inhibitory cortical interneurons in cortical information processing and in disease, deciphering the cellular and molecular mechanisms of GABAergic transmission is fundamental. The emerging relevance of DNMT and TET-mediated functions for synaptic regulation irrevocably raises the question for the targeted subcellular processes and mechanisms. In this study, we analyzed the role dynamic DNA methylation has in regulating cortical interneuron function. We found that DNMT1 and TET1/TET3 contrarily modulate clathrin-mediated endocytosis. Moreover, we provide evidence that DNMT1 influences synaptic vesicle replenishment and GABAergic transmission, presumably through the DNA methylation-dependent transcriptional control over endocytosis-related genes. The relevance of our findings is supported by human brain sample analysis, pointing to a potential implication of DNA methylation-dependent endocytosis regulation in the pathophysiology of temporal lobe epilepsy, a disease characterized by disturbed synaptic transmission.

DMRT5, DMRT3, and EMX2 Cooperatively Repress Gsx2 at the Pallium-Subpallium Boundary to Maintain Cortical Identity in Dorsal Telencephalic Progenitors.

Specification of dorsoventral regional identity in progenitors of the developing telencephalon is a first pivotal step in the development of the cerebral cortex and basal ganglia. Previously, we demonstrated that the two zinc finger doublesex and mab-3 related (Dmrt) genes, Dmrt5 (Dmrta2) and Dmrt3, which are coexpressed in high caudomedial to low rostrolateral gradients in the cerebral cortical primordium, are separately needed for normal formation of the cortical hem, hippocampus, and caudomedial neocortex. We have now addressed the role of Dmrt3 and Dmrt5 in controlling dorsoventral division of the telencephalon in mice of either sex by comparing the phenotypes of single knock-out (KO) with double KO embryos and by misexpressing Dmrt5 in the ventral telencephalon. We find that DMRT3 and DMRT5 act as critical regulators of progenitor cell dorsoventral identity by repressing ventralizing regulators. Early ventral fate transcriptional regulators expressed in the dorsal lateral ganglionic eminence, such as Gsx2, are upregulated in the dorsal telencephalon of Dmrt3;Dmrt5 double KO embryos and downregulated when ventral telencephalic progenitors express ectopic Dmrt5 Conditional overexpression of Dmrt5 throughout the telencephalon produces gene expression and structural defects that are highly consistent with reduced GSX2 activity. Further, Emx2;Dmrt5 double KO embryos show a phenotype similar to Dmrt3;Dmrt5 double KO embryos, and both DMRT3, DMRT5 and the homeobox transcription factor EMX2 bind to a ventral telencephalon-specific enhancer in the Gsx2 locus. Together, our findings uncover cooperative functions of DMRT3, DMRT5, and EMX2 in dividing dorsal from ventral in the telencephalon.SIGNIFICANCE STATEMENT We identified the DMRT3 and DMRT5 zinc finger transcription factors as novel regulators of dorsoventral patterning in the telencephalon. Our data indicate that they have overlapping functions and compensate for one another. The double, but not the single, knock-out produces a dorsal telencephalon that is ventralized, and olfactory bulb tissue takes over most remaining cortex. Conversely, overexpressing Dmrt5 throughout the telencephalon causes expanded expression of dorsal gene determinants and smaller olfactory bulbs. Furthermore, we show that the homeobox transcription factor EMX2 that is coexpressed with DMRT3 and DMRT5 in cortical progenitors cooperates with them to maintain dorsoventral patterning in the telencephalon. Our study suggests that DMRT3/5 function with EMX2 in positioning the pallial-subpallial boundary by antagonizing the ventral homeobox transcription factor GSX2.

Prdm12 specifies V1 interneurons through cross-repressive interactions with Dbx1 and Nkx6 genes in Xenopus.

V1 interneurons are inhibitory neurons that play an essential role in vertebrate locomotion. The molecular mechanisms underlying their genesis remain, however, largely undefined. Here, we show that the transcription factor Prdm12 is selectively expressed in p1 progenitors of the hindbrain and spinal cord in the frog embryo, and that a similar restricted expression profile is observed in the nerve cord of other vertebrates as well as of the cephalochordate amphioxus. Using frog, chick and mice, we analyzed the regulation of Prdm12 and found that its expression in the caudal neural tube is dependent on retinoic acid and Pax6, and that it is restricted to p1 progenitors, due to the repressive action of Dbx1 and Nkx6-1/2 expressed in the adjacent p0 and p2 domains. Functional studies in the frog, including genome-wide identification of its targets by RNA-seq and ChIP-Seq, reveal that vertebrate Prdm12 proteins act as a general determinant of V1 cell fate, at least in part, by directly repressing Dbx1 and Nkx6 genes. This probably occurs by recruiting the methyltransferase G9a, an activity that is not displayed by the amphioxus Prdm12 protein. Together, these findings indicate that Prdm12 promotes V1 interneurons through cross-repressive interactions with Dbx1 and Nkx6 genes, and suggest that this function might have only been acquired after the split of the vertebrate and cephalochordate lineages.

The DNA Methyltransferase 1 (DNMT1) Controls the Shape and Dynamics of Migrating POA-Derived Interneurons Fated for the Murine Cerebral Cortex.

The proliferative niches in the subpallium generate a rich cellular variety fated for diverse telencephalic regions. The embryonic preoptic area (POA) represents one of these domains giving rise to the pool of cortical GABAergic interneurons and glial cells, in addition to striatal and residual POA cells. The migration from sites of origin within the subpallium to the distant targets like the cerebral cortex, accomplished by the adoption and maintenance of a particular migratory morphology, is a critical step during interneuron development. To identify factors orchestrating this process, we performed single-cell transcriptome analysis and detected Dnmt1 expression in murine migratory GABAergic POA-derived cells. Deletion of Dnmt1 in postmitotic immature cells of the POA caused defective migration and severely diminished adult cortical interneuron numbers. We found that DNA methyltransferase 1 (DNMT1) preserves the migratory shape in part through negative regulation of Pak6, which stimulates neuritogenesis at postmigratory stages. Our data underline the importance of DNMT1 for the migration of POA-derived cells including cortical interneurons.

Controlled levels of canonical Wnt signaling are required for neural crest migration.

Canonical Wnt signaling plays a dominant role in the development of the neural crest (NC), a highly migratory cell population that generates a vast array of cell types. Canonical Wnt signaling is required for NC induction as well as differentiation, however its role in NC migration remains largely unknown. Analyzing nuclear localization of ß-catenin as readout for canonical Wnt activity, we detect nuclear ß-catenin in premigratory but not migratory Xenopus NC cells suggesting that canonical Wnt activity has to decrease to basal levels to enable NC migration. To define a possible function of canonical Wnt signaling in Xenopus NC migration, canonical Wnt signaling was modulated at different time points after NC induction. This was accomplished using either chemical modulators affecting ß-catenin stability or inducible glucocorticoid fusion constructs of Lef/Tcf transcription factors. In vivo analysis of NC migration by whole mount in situ hybridization demonstrates that ectopic activation of canonical Wnt signaling inhibits cranial NC migration. Further, NC transplantation experiments confirm that this effect is tissue-autonomous. In addition, live-cell imaging in combination with biophysical data analysis of explanted NC cells confirms the in vivo findings and demonstrates that modulation of canonical Wnt signaling affects the ability of NC cells to perform single cell migration. Thus, our data support the hypothesis that canonical Wnt signaling needs to be tightly controlled to enable migration of NC cells.

A novel role for Celf1 in vegetal RNA localization during Xenopus oogenesis.

The localization of certain mRNAs to the vegetal cortex of Xenopus oocytes is of crucial importance for germ cell development and early embryonic patterning. Vegetal RNA localization is mediated by cis-acting RNA localization elements (LE). Several proteins assemble on the RNA LE and direct transport to the vegetal cortex. Although a number of localization RNP components have been identified, their full composition is unknown. In an RNA affinity purification approach, using the dead end 1 (dnd1) RNA LE, we identified Xenopus Celf1 as a novel component of vegetal localization RNP complexes. Celf1 is part of an RNP complex together with known vegetal localization factors and shows specific interactions with LEs from several but not all vegetally localizing RNAs. Immunostaining experiments reveal co-localization of Celf1 with vegetally localizing RNA and with known localization factors. Inhibition of Celf1 protein binding by localization element mutagenesis as well as Celf1 overexpression interfere with vegetal RNA localization. These results argue for a role of Celf1 in vegetal RNA localization during Xenopus oogenesis.

The Prdm13 histone methyltransferase encoding gene is a Ptf1a-Rbpj downstream target that suppresses glutamatergic and promotes GABAergic neuronal fate in the dorsal neural tube.

The basic helix-loop-helix (bHLH) transcriptional activator Ptf1a determines inhibitory GABAergic over excitatory glutamatergic neuronal cell fate in progenitors of the vertebrate dorsal spinal cord, cerebellum and retina. In an in situ hybridization expression survey of PR domain containing genes encoding putative chromatin-remodeling zinc finger transcription factors in Xenopus embryos, we identified Prdm13 as a histone methyltransferase belonging to the Ptf1a synexpression group. Gain and loss of Ptf1a function analyses in both frog and mice indicates that Prdm13 is positively regulated by Ptf1a and likely constitutes a direct transcriptional target. We also showed that this regulation requires the formation of the Ptf1a-Rbp-j complex. Prdm13 knockdown in Xenopus embryos and in Ptf1a overexpressing ectodermal explants lead to an upregulation of Tlx3/Hox11L2, which specifies a glutamatergic lineage and a reduction of the GABAergic neuronal marker Pax2. It also leads to an upregulation of Prdm13 transcription, suggesting an autonegative regulation. Conversely, in animal caps, Prdm13 blocks the ability of the bHLH factor Neurog2 to activate Tlx3. Additional gain of function experiments in the chick neural tube confirm that Prdm13 suppresses Tlx3(+)/glutamatergic and induces Pax2(+)/GABAergic neuronal fate. Thus, Prdm13 is a novel crucial component of the Ptf1a regulatory pathway that, by modulating the transcriptional activity of bHLH factors such as Neurog2, controls the balance between GABAergic and glutamatergic neuronal fate in the dorsal and caudal part of the vertebrate neural tube.

Scratch2 modulates neurogenesis and cell migration through antagonism of bHLH proteins in the developing neocortex.

Scratch genes (Scrt) are neural-specific zinc-finger transcription factors (TFs) with an unknown function in the developing brain. Here, we show that, in addition to the reported expression of mammalian Scrt2 in postmitotic differentiating and mature neurons in the developing and early postnatal brain, Scrt2 is also localized in subsets of mitotic and neurogenic radial glial (RGP) and intermediate (IP) progenitors, as well as in their descendants-postmitotic IPs and differentiating neurons at the border subventricular/intermediate zone. Conditional activation of transgenic Scrt2 in cortical progenitors in mice promotes neuronal differentiation by favoring the direct mode of neurogenesis of RGPs at the onset of neurogenesis, at the expense of IP generation. Neuronal amplification via indirect IP neurogenesis is thereby extenuated, leading to a mild postnatal reduction of cortical thickness. Forced in vivo overexpression of Scrt2 suppressed the generation of IPs from RGPs and caused a delay in the radial migration of upper layer neurons toward the cortical plate. Mechanistically, our results indicate that Scrt2 negatively regulates the transcriptional activation of the basic helix loop helix TFs Ngn2/NeuroD1 on E-box containing common target genes, including Rnd2, a well-known major effector for migrational defects in developing cortex. Altogether, these findings reveal a modulatory role of Scrt2 protein in cortical neurogenesis and neuronal migration.

Homeoprotein hhex-induced conversion of intestinal to ventral pancreatic precursors results in the formation of giant pancreata in Xenopus embryos.

Liver and ventral pancreas develop from neighboring territories within the endoderm of gastrulae. ventral pancreatic precursor 1 (vpp1) is a marker gene that is differentially expressed in a cell population within the dorsal endoderm in a pattern partially overlapping with that of hematopoietically expressed homeobox (hhex) during gastrulation. In tail bud embryos, vpp1 expression specifically demarcates two ventral pancreatic buds, whereas hhex expression is mainly restricted to the liver diverticulum. Ectopic expression of a critical dose of hhex led to a greatly enlarged vpp1-positive domain and, subsequently, to the formation of giant ventral pancreata, putatively by conversion of intestinal to ventral pancreatic precursor cells. Conversely, antisense morpholino oligonucleotide-mediated knockdown of hhex resulted in a down-regulation of vpp1 expression and a specific loss of the ventral pancreas. Furthermore, titration of hhex with a dexamethasone-inducible hhex-VP16GR fusion construct suggested that endogenous hhex activity during gastrulation is essential for the formation of ventral pancreatic progenitor cells. These observations suggest that, beyond its role in liver development, hhex controls specification of a vpp1-positive endodermal cell population during gastrulation that is required for the formation of the ventral pancreas.

NumbL is essential for Xenopus primary neurogenesis.

BACKGROUND: Members of the vertebrate Numb family of cell fate determinants serve multiple functions throughout early embryogenesis, including an essential role in the development of the nervous system. The Numb proteins interact with various partner proteins and correspondingly participate in multiple cellular activities, including inhibition of the Notch pathway. RESULTS: Here, we describe the expression characteristics of Numb and Numblike (NumbL) during Xenopus development and characterize the function of NumbL during primary neurogenesis. NumbL, in contrast to Numb, is expressed in the territories of primary neurogenesis and is positively regulated by the Neurogenin family of proneural transcription factors. Knockdown of NumbL afforded a complete loss of primary neurons and did not lead to an increase in Notch signaling in the open neural plate. Furthermore, we provide evidence that interaction of NumbL with the AP-2 complex is required for NumbL function during primary neurogenesis. CONCLUSION: We demonstrate an essential role of NumbL during Xenopus primary neurogenesis and provide evidence for a Notch-independent function of NumbL in this context.

Elr-type proteins protect Xenopus Dead end mRNA from miR-18-mediated clearance in the soma.

Segregation of the future germ line defines a crucial cell fate decision during animal development. In Xenopus, germ cells are specified by inheritance of vegetally localized maternal determinants, including a group of specific mRNAs. Here, we show that the vegetal localization elements (LE) of Xenopus Dead end (XDE) and of several other germ-line-specific, vegetally localized transcripts mediate germ cell-specific stabilization and somatic clearance of microinjected reporter mRNA in Xenopus embryos. The part of XDE-LE critical for somatic RNA clearance exhibits homology to zebrafish nanos1 and appears to be targeted by Xenopus miR-18 for somatic mRNA clearance. Xenopus Elr-type proteins of the vegetal localization complex can alleviate somatic RNA clearance of microinjected XDE-LE and endogenous XDE mRNA. ElrB1 synergizes with Xenopus Dead end protein in the stabilization of XDE-LE mRNA. Taken together, our findings unveil a functional link of vegetal mRNA localization and the protection of germ-line mRNAs from somatic clearance.

A novel function for KIF13B in germ cell migration.

Primordial germ cell (PGC) development in Xenopus embryos relies on localised maternal determinants. We report on the identification and functional characterisation of such one novel activity, a germ plasm associated mRNA encoding for the Xenopus version of a kinesin termed KIF13B. Modulations of xKIF13B function result in germ cell mismigration and in reduced numbers of such cells. PGCs explanted from Xenopus embryos form bleb-like protrusions enriched in PIP3. Knockdown of xKIF13B results in inhibition of blebbing and PIP3 accumulation. Interference with PIP3 synthesis leads to PGC mismigration in vivo and in vitro. We propose that xKIF13B function is linked to polarized accumulation of PIP3 and directional migration of the PGCs in Xenopus embryos.

Functional dissection of the RNA signal sequence responsible for vegetal localization of XGrip2.1 mRNA in Xenopus oocytes.

Grip2.1 is a conserved PDZ-domain protein with a function in the context of primordial germ cell development and migration in Xenopus embryos. Its mRNA is maternally supplied and found to be associated with the germ plasm, located at the tip of the vegetal cortex in Xenopus oocytes. Here, we demonstrate that the 3'-UTR of XGrip2.1 contains a 211 nucleotide RNA signal sequence that promotes localization to the mitochondrial cloud via the early localization pathway upon injection into stage I oocytes. The same element is also capable of using the late transport pathway if injected into stage III/IV oocytes. In vitro protein interaction studies reveal binding to ElrA/B, Vg1RBP and VgRBP60, proteins that have previously been associated with the vegetal localization machinery. Mutational interference with Vg1RBP and VgRBP60 binding severely reduces early and late localization activity. Selective interference with Vg1RBP binding significantly reduces late localization while having only a mild effect on localization to the mitochondrial cloud, indicating that the signal sequences and protein machinery required for early and late pathway localization though overlapping are not identical.

Identification of vegetal RNA-localization elements in Xenopus oocytes.

Localized mRNAs have been identified in a large variety of cell types where they contribute to the establishment of cell asymmetries and can function as cell fate determinants. In Xenopus, RNAs that localize to the vegetal cortex during oogenesis function in early embryonic patterning as well as in the development of primordial germ cells. Based on their temporal and spatial localization patterns, vegetally localizing RNAs are referred to as either early-pathway RNAs which transiently localize in the mitochondrial cloud, or as late-pathway RNAs. Vegetal RNA-localization is driven by cis-acting signal sequences that, in most cases, were found to reside in the 3'-UTRs and which are recognized by trans-acting localization factors. Here we describe the methods of how vegetal RNA-localization elements can be identified by injection of fluorescently-labeled or tagged RNAs.

XsFRP5 modulates endodermal organogenesis in Xenopus laevis.

Canonical Wnt signalling is known to be involved in the regulation of differentiation and proliferation in the context of endodermal organogenesis. Wnt mediated beta-catenin activation is understood to be modulated by secreted Frizzled-related proteins, such as XsFRP5, which is dynamically expressed in the prospective liver/ventral pancreatic precursor cells during late neurula stages, becoming liver specific at tailbud stages and shifting to the posterior stomach/anterior duodenum territory during tadpole stages of Xenopus embryogenesis. These expression characteristics prompted us to analyse the function of XsFRP5 in the context of endodermal organogenesis. We demonstrate that XsFRP5 can form a complex with and inhibit a multitude of different Wnt ligands, including both canonical and non-canonical ones. Knockdown of XsFRP5 results in transient pancreatic hypoplasia as well as in an enlargement of the stomach. In VegT-injected animal cap explants, XsFRP5 can induce expression of exocrine but not endocrine pancreatic marker genes. Both, its expression characteristics as well as its interactions with XsFRP5, define Wnt2b as a putative target for XsFRP5 in vivo. Knockdown of Wnt2b results in a hypoplastic stomach as well as in hypoplasia of the pancreas. On the basis of these findings we propose that XsFRP5 exerts an early regulatory function in the specification of the ventral pancreas, as well as a late function in controlling stomach size via inhibition of Wnt signalling.

DNA Methyltransferase 1 (DNMT1) Function Is Implicated in the Age-Related Loss of Cortical Interneurons.

Increased life expectancy in modern society comes at the cost of age-associated disabilities and diseases. Aged brains not only show reduced excitability and plasticity, but also a decline in inhibition. Age-associated defects in inhibitory circuits likely contribute to cognitive decline and age-related disorders. Molecular mechanisms that exert epigenetic control of gene expression contribute to age-associated neuronal impairments. Both DNA methylation, mediated by DNA methyltransferases (DNMTs), and histone modifications maintain neuronal function throughout lifespan. Here we provide evidence that DNMT1 function is implicated in the age-related loss of cortical inhibitory interneurons. Dnmt1 deletion in parvalbumin-positive interneurons attenuates their age-related decline in the cerebral cortex. Moreover, conditional Dnmt1-deficient mice show improved somatomotor performance and reduced aging-associated transcriptional changes. A decline in the proteostasis network, responsible for the proper degradation and removal of defective proteins, is implicated in age- and disease-related neurodegeneration. Our data suggest that DNMT1 acts indirectly on interneuron survival in aged mice by modulating the proteostasis network during life-time.

Generation of transgenic frogs.

The possibility of generating transgenic animals is of obvious advantage for the analysis of gene function in development and disease. One of the established vertebrate model systems in developmental biology is the amphibian Xenopus laevis. Different techniques have been successfully applied to create Xenopus transgenics; in this chapter, the so-called meganuclease method is described. This technique is not only technically simple, but also comparably efficient and applicable to both Xenopus laevis and Xenopus tropicalis. The commercially available endonuclease I-SceI (meganuclease) mediates the integration of foreign DNA into the frog genome after coinjection into fertilized eggs. Tissue-specific gene expression, as well as germline transmission, has been observed.

Prdm12 Directs Nociceptive Sensory Neuron Development by Regulating the Expression of the NGF Receptor TrkA.

In humans, many cases of congenital insensitivity to pain (CIP) are caused by mutations of components of the NGF/TrkA signaling pathway, which is required for survival and specification of nociceptors and plays a major role in pain processing. Mutations in PRDM12 have been identified in CIP patients that indicate a putative role for this transcriptional regulator in pain sensing. Here, we show that Prdm12 expression is restricted to developing and adult nociceptors and that its genetic ablation compromises their viability and maturation. Mechanistically, we find that Prdm12 is required for the initiation and maintenance of the expression of TrkA by acting as a modulator of Neurogenin1/2 transcription factor activity, in frogs, mice, and humans. Altogether, our results identify Prdm12 as an evolutionarily conserved key regulator of nociceptor specification and as an actionable target for new pain therapeutics.