Stephan A. Wacker

A green to red photoconvertible protein as an analyzing tool for early vertebrate development

Lineage labeling is one of the most important techniques in developmental biology. Most recently, a set of photoactivatable fluorescent proteins originating from marine cnidarians became available. Here, we introduce the application of the green to red photoconvertible protein EosFP as a novel technique to analyze early vertebrate development. Both injection of EosFP mRNA and purified, recombinant EosFP followed by a light‐driven green to red conversion allow lineage labeling in virtually any temporal and spatial dimension during embryonic development for at least 2 weeks. Specific staining of cells from nonsurface layers is greatly facilitated by light‐driven conversion of EosFP compared with traditional methods. Therefore, green to red photoactivatable proteins promise to be a powerful tool with the potential to satisfy the increasing demand for methods enabling detailed phenotypical analyses after manipulations of morphogenetic events, gene expression, or signal transduction. Developmental Dynamics 236:473–480, 2007.

RITA, a novel modulator of Notch signalling, acts via nuclear export of RBP‐J

The evolutionarily conserved Notch signal transduction pathway regulates fundamental cellular processes during embryonic development and in the adult. Ligand binding induces presenilin‐dependent cleavage of the receptor and a subsequent nuclear translocation of the Notch intracellular domain (NICD). In the nucleus, NICD binds to the recombination signal sequence‐binding protein J (RBP‐J)/CBF‐1 transcription factor to induce expression of Notch target genes. Here, we report the identification and functional characterization of RBP‐J interacting and tubulin associated (RITA) (C12ORF52) as a novel RBP‐J/CBF‐1‐interacting protein. RITA is a highly conserved 36 kDa protein that, most interestingly, binds to tubulin in the cytoplasm and shuttles rapidly between cytoplasm and nucleus. This shuttling RITA exports RBP‐J/CBF‐1 from the nucleus. Functionally, we show that RITA can reverse a Notch‐induced loss of primary neurogenesis in Xenopus laevis. Furthermore, RITA is able to downregulate Notch‐mediated transcription. Thus, we propose that RITA acts as a negative modulator of the Notch signalling pathway, controlling the level of nuclear RBP‐J/CBF‐1, where its amounts are limiting.

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.

Review: Time–space translation regulates trunk axial patterning in the early vertebrate embryo

Here, we review a recently discovered developmental mechanism. Anterior-posterior positional information for the vertebrate trunk is generated by sequential interactions between a timer in the early non-organiser mesoderm and the Spemann organiser. The timer is characterised by temporally colinear activation of a series of Hox genes in the early ventral and lateral mesoderm (i.e., the non-organiser mesoderm) of the Xenopus gastrula. This early Hox gene expression is transient, unless it is stabilised by signals from the Spemann organiser. The non-organiser mesoderm (NOM) and the Spemann organiser undergo timed interactions during gastrulation which lead to the formation of an anterior-posterior axis and stable Hox gene expression. When separated from each other, neither non-organiser mesoderm nor the Spemann organiser is able to induce anterior-posterior pattern formation of the trunk. We present a model describing that NOM acquires transiently stable hox codes and spatial colinearity after involution into the gastrula and that convergence and extension then continually bring new cells from the NOM within the range of organiser signals that cause transfer of the mesodermal pattern to a stable pattern in neurectoderm and thereby create patterned axial structures. In doing so, the age of the non-organiser mesoderm, but not the age of the organiser, defines positional values along the anterior-posterior axis. We postulate that the temporal information from the non-organiser mesoderm is linked to mesodermal Hox expression. The role of the organiser was investigated further and this turns out to be only the induction of neural tissue. Apparently, development of a stable axial hox pattern requires neural hox patterning.

Control of Gastrula Cell Motility by the Goosecoid/Mix.1/ Siamois Network: Basic Patterns and Paradoxical Effects

In the vegetal half of the Xenopus gastrula, cell populations differ with respect to migration on fibronectin substratum. We show that the paired‐class homeodomain transcription factors Goosecoid (Gsc), Mix.1, and Siamois (Sia) are involved in the modulation of migration velocity and cell polarity. Mix.1 is expressed in the whole vegetal half and serves as a competence factor that is necessary, but not sufficient, for rapid cell migration and polarization. In the head mesoderm, Gsc and Sia are coexpressed with Mix.1, promoting rapid cell migration and polarization. Ectopic expression of Gsc and Sia in both vegetal and ventral regions often generates paradoxical effects; if a factor activates a certain motility trait in one region, it inhibits it in the other. Migration velocity and cell polarity are regulated independently. Fast and efficiently migrating multipolar cells and slow‐moving polarized cells can be obtained by ectopic expression of these transcription factors in different combinations. Developmental Dynamics 237:1307‐1320, 2008.

The role of the Spemann organizer in anterior-posterior patterning of the trunk

The formation of the vertebrate body axis during gastrulation strongly depends on a dorsal signaling centre, the Spemann organizer as it is called in amphibians. This organizer affects embryonic development by self-differentiation, regulation of morphogenesis and secretion of inducing signals. Whereas many molecular signals and mechanisms of the organizer have been clarified, its function in anterior-posterior pattern formation remains unclear. We dissected the organizer functions by generally blocking organizer formation and then restoring a single function. In experiments using a dominant inhibitory BMP receptor construct (tBr) we find evidence that neural activation by antagonism of the BMP pathway is the organizer function that enables the establishment of a detailed anterior-posterior pattern along the trunk. Conversely, the exclusive inhibition of neural activation by expressing a constitutive active BMP receptor (hAlk-6) in the ectoderm prohibits the establishment of an anterior-posterior pattern, even though the organizer itself is still intact. Thus, apart from the formerly described separation into a head and a trunk/tail organizer, the organizer does not deliver positional information for anterior-posterior patterning. Rather, by inducing neurectoderm, it makes ectodermal cells competent to receive patterning signals from the non-organizer mesoderm and thereby enable the formation of a complete and stable AP pattern along the trunk.

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.

Time Space Translation: A Hox Mechanism for Vertebrate A-P Patterning

The vertebrate A-P axis is a time axis. The head is made first and more and more posterior levels are made at later and later stages. This is different to the situation in most other animals, for example, in Drosophila. Central to this timing is Hox temporal collinearity (see below). This occurs rarely in the animal kingdom but is characteristic of vertebrates and is used to generate the primary axial Hox pattern using time space translation and to integrate successive derived patterns (see below). This is thus a different situation than in Drosophila, where the primary pattern guiding Hox spatial collinearity is generated externally, by the gap and segmentation genes.

Reversal of xenopus OCT25 function by disruption of the pou domain structure

Xenopus Oct25 is a POU family subclass V (POU-V) transcription factor with a distinct domain structure. To investigate the contribution of different domains to the function of Oct25, we have performed gain of function analyses. Deletions of the N- or C-terminal regions and of the Hox domain (except its nuclear localization signal) result in mutants being indistinguishable from the wild type protein in the suppression of genes promoting germ layer formation. Deletion of the complete POU domain generates a mutant that has no effect on embryogenesis. However, disruption of the α-helical structures in the POU domain, even by a single amino acid mutation, causes reversal of protein function. Overexpression of such mutants leads to dorsalization of embryos and formation of secondary axial structures. The underlying mechanism is an enhanced transcription of genes coding for antagonists of the ligands for ventralizing bone morphogenetic protein and Wnt pathways. Corresponding deletion mutants of Xenopus Oct60, Oct91, or mouse Oct4 also exhibit such a dominant-negative effect. Therefore, our results reveal that the integrity of the POU domain is crucial for the function of POU-V transcription factors in the regulation of genes that promote germ layer formation.