A novel function for KIF13B in germ cell migration

AbstractPrimordial 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

Ptf1a triggers GABAergic neuronal cell fates in the retina

International audienceBACKGROUND: In recent years, considerable knowledge has been gained on the molecular mechanisms underlying retinal cell fate specification. However, hitherto studies focused primarily on the six major retinal cell classes (five types of neurons of one type of glial cell), and paid little attention to the specification of different neuronal subtypes within the same cell class. In particular, the molecular machinery governing the specification of the two most abundant neurotransmitter phenotypes in the retina, GABAergic and glutamatergic, is largely unknown. In the spinal cord and cerebellum, the transcription factor Ptf1a is essential for GABAergic neuron production. In the mouse retina, Ptf1a has been shown to be involved in horizontal and most amacrine neurons differentiation. RESULTS: In this study, we examined the distribution of neurotransmitter subtypes following Ptf1a gain and loss of function in the Xenopus retina. We found cell-autonomous dramatic switches between GABAergic and glutamatergic neuron production, concomitant with profound defects in the genesis of amacrine and horizontal cells, which are mainly GABAergic. Therefore, we investigated whether Ptf1a promotes the fate of these two cell types or acts directly as a GABAergic subtype determination factor. In ectodermal explant assays, Ptf1a was found to be a potent inducer of the GABAergic subtype. Moreover, clonal analysis in the retina revealed that Ptf1a overexpression leads to an increased ratio of GABAergic subtypes among the whole amacrine and horizontal cell population, highlighting its instructive capacity to promote this specific subtype of inhibitory neurons. Finally, we also found that within bipolar cells, which are typically glutamatergic interneurons, Ptf1a is able to trigger a GABAergic fate. CONCLUSION: Altogether, our results reveal for the first time in the retina a major player in the GABAergic versus glutamatergic cell specification genetic pathway

DMRT5, DMRT3, and EMX2 Cooperatively Repress 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

Programming Pluripotent Precursor Cells Derived from Xenopus Embryos to Generate Specific Tissues and Organs

Xenopus embryos provide a rich source of pluripotent cells that can be differentiated into functional organs. Since the molecular principles of vertebrate organogenesis appear to be conserved between Xenopus and mammals, this system can provide useful guidelines for the directional manipulation of human embryonic stem cells. Pluripotent Xenopus cells can be easily isolated from the animal pole of blastula stage Xenopus embryos. These so called “animal cap” cells represent prospective ectodermal cells, but give rise to endodermal, mesodermal and neuro-ectodermal derivatives if treated with the appropriate factors. These factors include evolutionary conserved modulators of the key developmental signal transduction pathways that can be supplied either by mRNA microinjection or direct application of recombinant proteins. This relatively simple system has added to our understanding of pancreas, liver, kidney, eye and heart development. In particular, recent studies have used animal cap cells to generate ectopic eyes and hearts, setting the stage for future work aimed at programming pluripotent cells for regenerative medicine

XPak3 promotes cell cycle withdrawal during primary neurogenesis in Xenopus laevis

We have isolated the Xenopus p21-activated kinase 3 (XPak3) by virtue of its expression in the territory of primary neurogenesis in the developing embryo. XPak3, but not the other Pak variants, responds positively to X-Ngnr-1 and negatively to X-Notch-1. A constitutively active form of XPak3, generated by fusing a myristylation signal to the N-terminus (XPak3-myr), induces early cell cycle arrest at high concentrations, while ectopic expression of low amounts induces premature neuronal differentiation. Conversely, XPak3 loss of function achieved by use of an antisense morpholino oligonucleotide increases cell proliferation and inhibits neuronal differentiation; this phenotype is rescued by co-injection of XPak3-myr. We conclude that XPak3 is a novel member of the proneural pathway, functioning downstream of neurogenin to withdraw neuronally programmed cells from the mitotic cell cycle, thus allowing for their differentiation

XsFRP5 modulates endodermal organogenesis in Xenopus laevis

AbstractCanonical Wnt signalling is known to be involved in the regulation of differentiation and proliferation in the context of endodermal organogenesis. Wnt mediated β-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

Vax1, a novel homeobox-containing gene, directs development of the basal forebrain and visual system

The novel homeobox-containing gene Vax1, a member of the Emx/Not gene family, is specifically expressed in the developing basal forebrain and optic nerve. Here, we show that Vax1 is essential for normal development of these structures. Mice carrying a targeted mutation of Vax1 show dysgenesis of the optic nerve, coloboma, defects in the basal telencephalon, and lobar holoprosencephaly. With the help of molecular markers we determined that in the developing visual system, the absence of Vax1 results in a proximal expansion of the activity of Pax6 and Rx. This observation suggests that Vax1 may interfere negatively with the expression of Pax6 and Rx. In reciprocal gain-of-function experiments, injection of Xvax1 mRNA or Shh into Xenopus embryos primarily affects the brain at the level of the eye primordium. Consistent with the loss-of-function results, the injection of Xvax1 results in a down-regulation of Rx. Similarly, Shh injection expands the Vax1 and Pax2 territory at the expense of the Pax6 and Rx region. On the basis of these results, we propose a model for a molecular cascade involved in the establishment of structures of the visual system

mPER1-mediated nuclear export of mCRY1/2 is an important element in establishing circadian rhythm

Receptor-mediated nucleocytoplasmic transport of clock proteins is an important, conserved element of the core mechanism for circadian rhythmicity. A systematic analysis of the nuclear export characteristics for the different murine period (mPER) and cryptochrome (mCRY) proteins using Xenopus oocytes as an experimental system demonstrates that all three mPER proteins, but neither mCRY1 nor mCRY2, are exported if injected individually. However, nuclear injection of heterodimeric complexes that contain combinations of mPER and mCRY proteins shows that mPER1 serves as an export adaptor for mCRY1 and mCRY2. Functional analysis of dominant-negative mPER1 variants designed either to sequester mPER3 to the cytoplasm or to inhibit nuclear export of mCRY1/2 in synchronized, stably transfected fibroblasts suggests that mPER1-mediated export of mCRY1/2 defines an important new element of the core clock machinery in vertebrates

Migratory and adhesive properties of Xenopus laevis primordial germ cells in vitro

Summary The directional migration of primordial germ cells (PGCs) to the site of gonad formation is an advantageous model system to study cell motility. The embryonic development of PGCs has been investigated in different animal species, including mice, zebrafish, Xenopus and Drosophila. In this study we focus on the physical properties of Xenopus laevis PGCs during their transition from the passive to the active migratory state. Pre-migratory PGCs from Xenopus laevis embryos at developmental stages 17–19 to be compared with migratory PGCs from stages 28–30 were isolated and characterized in respect to motility and adhesive properties. Using single-cell force spectroscopy, we observed a decline in adhesiveness of PGCs upon reaching the migratory state, as defined by decreased attachment to extracellular matrix components like fibronectin, and a reduced adhesion to somatic endodermal cells. Data obtained from qPCR analysis with isolated PGCs reveal that down-regulation of E-cadherin might contribute to this weakening of cell-cell adhesion. Interestingly, however, using an in vitro migration assay, we found that movement of X. laevis PGCs can also occur independently of specific interactions with their neighboring cells. The reduction of cellular adhesion during PGC development is accompanied by enhanced cellular motility, as reflected in increased formation of bleb-like protrusions and inferred from electric cell-substrate impedance sensing (ECIS) as well as time-lapse image analysis. Temporal alterations in cell shape, including contraction and expansion of the cellular body, reveal a higher degree of cellular dynamics for the migratory PGCs in vitro