Cornelia Donow

The POU Factor Oct-25 Regulates the Xvent-2B Gene and Counteracts Terminal Differentiation in Xenopus Embryos

The Xvent-2B promoter is regulated by a BMP-2/4-induced transcription complex comprising Smad signal transducers and specific transcription factors. Using a yeast one-hybrid screen we have found that Oct-25, a Xenopus POU domain protein related to mammalian Oct-3/4, binds as an additional factor to the Xvent-2B promoter. This interaction was further confirmed by both in vitro and in vivo analyses. The Oct-25 gene is mainly transcribed during blastula and gastrula stages in the newly forming ectodermal and mesodermal germ layers. Luciferase reporter gene assay demonstrated that Oct-25 stimulates transcription of the Xvent-2B gene. This stimulation depends on the Oct-25 binding site and the bone morphogenetic protein-responsive element. Furthermore, Oct-25 interacts in vitro with components of the Xvent-2B transcription complex, like Smad1/4 and Xvent-2. Overexpression of Oct-25 results in anterior/posterior truncations and lack of differentiation for neuroectoderm- and mesoderm-derived tissues including blood cells. This effect is consistent with an evolutionarily conserved role of class V POU factors in the maintenance of an undifferentiated cell state. In Xenopus, the molecular mechanism underlying this process might be coupled to the expression of Xvent proteins.

POU-V factors antagonize maternal VegT activity and β-Catenin signaling in Xenopus embryos

VegT and β‐Catenin are key players in the hierarchy of factors that are required for induction and patterning of mesendoderm in Xenopus embryogenesis. By descending the genetic cascades, cells lose their pluripotent status and are determined to differentiate into distinct tissues. Mammalian Oct‐3/4, a POU factor of subclass V (POU‐V), is required for the maintenance of pluripotency of embryonic stem cells. However, its molecular function within the early embryo is yet poorly understood. We here show that the two maternal Xenopus POU‐V factors, Oct‐60 and Oct‐25, inhibit transcription of genes activated by VegT and β‐Catenin. Maternal POU‐V factors and maternal VegT show an opposite distribution along the animal/vegetal axis. Oct‐25, VegT and Tcf3 interact with each other and form repression complexes on promoters of VegT and β‐Catenin target genes. We suggest that POU‐V factors antagonize primary inducers to allow germ layer specification in a temporally and spatially coordinated manner.

Characterization of Danio rerio Nanog and functional comparison to Xenopus Vents.

Nanog is a homeodomain transcription factor associated with the acquisition of pluripotency. Genome analyses of lower and higher vertebrates revealed that the existence of Nanog is restricted to gnathostomata but absent from agnatha and invertebrates. To elucidate the function of Nanog in nonmammalia, we identified the Danio rerio ortholog of Nanog and characterized its role in gain and loss of function experiments. We found Nanog to be crucial for survival of early zebrafish embryos, because depletion of Nanog led to gastrulation defects with subsequent lethality. Mouse Nanog overexpression could rescue these defects. Vice versa, zebrafish Nanog was found to promote proliferation and to inhibit differentiation of mouse embryonic stem cells in the absence of leukemia inhibitory factor. These findings indicate functional conservation of Nanog from teleost fishes to mammals. However, Nanog was lost in the genome of the anurans Xenopus laevis and Xenopus tropicalis. Phylogenetic analysis revealed that deletion probably occurred in a common anuran ancestor along with chromosomal translocations. The closest homologs of Nanog in Xenopus are the Vent proteins. We, therefore, investigated whether the Xvent genes might substitute for Nanog function in Xenopus. Although we found some similarities in phenotypes after overexpression and in the regulation of several marker genes, Xvent1/2 and Nanog cannot substitute each other. Depletion of Nanog in zebrafish cannot be rescued by ectopic expression of Xvent, and Xvent depletion in Xenopus cannot be overcome by ectopic expression of zebrafish Nanog.

FoxN3 is required for craniofacial and eye development of Xenopus laevis

A functional knockdown of FoxN3, a member of subclass N of fork head/winged helix transcription factors in Xenopus laevis, leads to an abnormal formation of the jaw cartilage, absence or malformation of distinct cranial nerves, and reduced size of the eye. While the eye phenotype is due to an increased rate of apoptosis, the cellular basis of the jaw phenotype is more complex. The upper and lower jaw cartilages are derivatives of a subset of cranial neural crest cells, which migrate into the first pharyngeal arch. Histological analysis of FoxN3‐depleted embryos reveals severe deformation and false positioning of infrarostral, Meckels, and palatoquadrate cartilages, structural elements derived from the first pharyngeal arch, and of the ceratohyale, which derives from the second pharyngeal arch. The derivatives of the third and fourth pharyngeal arches are less affected. FoxN3 is not required for early neural crest migration. Defects in jaw formation rather arise by failure of differentiation than by positional effects of crest migration. By GST‐pulldown analysis, we have identified two different members of histone deacetylase complexes (HDAC), xSin3 and xRPD3, as putative interaction partners of FoxN3, suggesting that FoxN3 regulates craniofacial and eye development by recruiting HDAC. Developmental Dynamics 236:226–239, 2007.

XBP1 forms a regulatory loop with BMP-4 and suppresses mesodermal and neural differentiation in Xenopus embryos

The active form of the Xenopus X-box binding protein 1 (xXBP1) partially synergizes and partially antagonizes with BMP-4 signaling. xXBP1 overexpression inhibits mesoderm differentiation and formation of neural tissues. A functional knockdown promotes differentiation of lateral and dorsal mesoderm but not of ventral mesoderm and of neuroectoderm. We show that the active form of xXBP1 in gastrula and early neurula stage embryos is generated by removal of exon 4 and not by an endoribonuclease activity in the endoplasmic reticulum. The N-terminal region of xXBP1 which contains the basic leucine-zipper also contains a nuclear localization signal and both, the N-terminal as well as the C-terminal regions are required for xXBP1 function. The effects of xXBP1 are in part correlated to a regulatory loop between xXBP1 and BMP-4. xXBP1 and BMP-4 stimulate mutually the transcription of each other, but xXBP1 inhibits the BMP-4 target gene, Xvent-2. Both, in vitro and in vivo assays demonstrate that xXBP1 interacts with BMP-4 and Xvent-2B promoters. GST-pulldown assays reveal that xXBP1 can interact with c-Jun, the transcriptional co-activator p300 and with the BMP-4 responsive Smad1. On the other hand, xXBP1 also binds to the inhibitory Smads, Smad6 and Smad7, that can act as transcriptional co-repressors. Based on these data, we conclude that xXBP1 might function as an inhibitor of mesodermal and neural tissue formation by acting either as transcriptional activator or as repressor. This dual activity depends upon binding of co-factors being involved in the formation of distinct transcription complexes.

The FoxP subclass in Xenopus laevis development

We have investigated the sequences and the expression patterns of different members of the Xenopus laevis FoxP gene subfamily during embryogenesis. Low stringency hybridisation of a tadpole cDNA library with an xlFoxP2 fragment led to the isolation of several splice variants of xlFoxP1, xlFoxP2 and xlFoxP4. These variants do not only differ by utilisation of different leader exons, but also by alternative usage of coding exons thereby leading to functional alterations. For xlFoxP1b, we show that insertion of an additional exon disrupts binding to the co-repressor C-terminal binding protein1. Temporal and spatial expression patterns of xlFoxP2 and xlFoxP4 were analysed by RT-PCR and by whole mount in situ hybridisation. xlFoxP2 transcripts are detected from mid-gastrula to late tadpole stages and are found to be localised to pronephros, branchial arches and distinct structures of the hind-, mid- and forebrain, including the ciliary marginal zone of the retina. xlFoxP4 RNA is already present in early cleavage stage embryos and accumulates from midblastula until the end of embryogenesis. Localised expression is found within the anterior neural fold, in the mid- and hindbrain, in the branchial arches as well as in the pancreas.

FoxO genes are dispensable during gastrulation but required for late embryogenesis in Xenopus laevis

Forkhead box (Fox) transcription factors of subclass O are involved in cell survival, proliferation, apoptosis, cell metabolism and prevention of oxidative stress. FoxO genes are highly conserved throughout evolution and their functions were analyzed in several vertebrate and invertebrate organisms. We here report on the identification of FoxO4 and FoxO6 genes in Xenopus laevis and analyze their expression patterns in comparison with the previously described FoxO1 and FoxO3 genes. We demonstrate significant differences in their temporal and spatial expression during embryogenesis and in their relative expression within adult tissues. Overexpression of FoxO1, FoxO4 or FoxO6 results in severe gastrulation defects, while overexpression of FoxO3 reveals this defect only in a constitutively active form containing mutations of Akt-1 target sites. Injections of FoxO antisense morpholino oligonucleotides (MO) did not influence gastrulation, but, later onwards, the embryos showed a delay of development, severe body axis reduction and, finally, a high rate of lethality. Injection of FoxO4MO leads to specific defects in eye formation, neural crest migration and heart development, the latter being accompanied by loss of myocardin expression. Our observations suggest that FoxO genes in X. laevis are dispensable until blastopore closure but are required for tissue differentiation and organogenesis.

Two Hoxc6 transcripts are differentially expressed and regulate primary neurogenesis in Xenopus laevis.

Hox genes are key players in defining positional information along the main body axis of vertebrate embryos. In Xenopus laevis, Hoxc6 was the first homeobox gene isolated. It encodes two isoforms. We analyzed in detail their spatial and temporal expression pattern during early development. One major expression domain of both isoforms is the spinal cord portion of the neural tube. Within the spinal cord and its populations of primary neurons, Hox genes have been found to play a crucial role for defining positional information. Here we report that a loss‐of‐function of either one of the Hoxc6 products does not affect neural induction, the expression of general neural markers is not modified. However, Hoxc6 does widely affect the formation of primary neurons within the developing neural tissue. Manipulations of Hoxc6 expression severly changes the expression of the neuronal markers N‐tubulin and Islet‐1. Formation of primary neurons and formation of cranial nerves are affected. Hence, Hoxc6 functions are not restricted to the expected role in anterior‐posterior pattern formation, but they also regulate N‐tubulin, thereby having an effect on the initial formation of primary neurons in Xenopus laevis embryos. Developmental Dynamics 238:755–765, 2009.

Vertical Signalling Involves Transmission of Hox Information from Gastrula Mesoderm to Neurectoderm

Development and patterning of neural tissue in the vertebrate embryo involves a set of molecules and processes whose relationships are not fully understood. Classical embryology revealed a remarkable phenomenon known as vertical signalling, a gastrulation stage mechanism that copies anterior-posterior positional information from mesoderm to prospective neural tissue. Vertical signalling mediates unambiguous copying of complex information from one tissue layer to another. In this study, we report an investigation of this process in recombinates of mesoderm and ectoderm from gastrulae of Xenopus laevis. Our results show that copying of positional information involves non cell autonomous autoregulation of particular Hox genes whose expression is copied from mesoderm to neurectoderm in the gastrula. Furthermore, this information sharing mechanism involves unconventional translocation of the homeoproteins themselves. This conserved primitive mechanism has been known for three decades but has only recently been put into any developmental context. It provides a simple, robust way to pattern the neurectoderm using the Hox pattern already present in the mesoderm during gastrulation. We suggest that this mechanism was selected during evolution to enable unambiguous copying of rather complex information from cell to cell and that it is a key part of the original ancestral mechanism mediating axial patterning by the highly conserved Hox genes.

Resting cells rely on the DNA helicase component MCM2 to build cilia

Abstract Minichromosome maintenance (MCM) proteins facilitate replication by licensing origins and unwinding the DNA double strand. Interestingly, the number of MCM hexamers greatly exceeds the number of firing origins suggesting additional roles of MCMs. Here we show a hitherto unanticipated function of MCM2 in cilia formation in human cells and zebrafish that is uncoupled from replication. Zebrafish depleted of MCM2 develop ciliopathy-phenotypes including microcephaly and aberrant heart looping due to malformed cilia. In non-cycling human fibroblasts, loss of MCM2 promotes transcription of a subset of genes, which cause cilia shortening and centriole overduplication. Chromatin immunoprecipitation experiments show that MCM2 binds to transcription start sites of cilia inhibiting genes. We propose that such binding may block RNA polymerase II-mediated transcription. Depletion of a second MCM (MCM7), which functions in complex with MCM2 during its canonical functions, reveals an overlapping cilia-deficiency phenotype likely unconnected to replication, although MCM7 appears to regulate a distinct subset of genes and pathways. Our data suggests that MCM2 and 7 exert a role in ciliogenesis in post-mitotic tissues.