Pescadillo is required for Xenopus laevis eye development and neural crest migration.

Pescadillo is a multifunctional, nuclear protein involved in rRNA precursor processing, ribosomal assembly, and transcriptional regulation. Pescadillo has been assigned important functions in embryonic development and tumor formation. We previously identified pescadillo as a potential downstream target of non-canonical Wnt-4 signaling. Here we have investigated for the first time the function of the Xenopus laevis homolog of pescadillo during early embryogenesis on a molecular level. Loss of function analysis indicates that pescadillo is required for eye development and neural crest migration. BrdU incorporation and TUNEL assays indicate that a loss of pescadillo function affects proliferation and triggers apoptosis through a p53-mediated mechanism. Furthermore, pescadillo affects the expression of early eye-specific marker genes, likely independent of its function in regulating proliferation and apoptosis, and in addition migration of cranial neural crest cells. Our data indicate that pescadillo has multiple important functions during X. laevis development and that its function is highly conserved among different species.

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, Meckel's, 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.

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.

Temporal and spatial expression patterns of FoxN genes in Xenopus laevis embryos.

Using RT-PCR and in situ hybridisation, we have analysed the temporal and spatial expression patterns of Xenopus Fox genes of subclass N. By screening cDNA libraries and by RT-PCR using embryonic RNA and primers derived from EST analyses, we could isolate FoxN2, FoxN4, FoxN5 and different isoforms of FoxN3. FoxN2 and FoxN3 transcripts were found during all developmental stages including early cleavage and tailbud stages. FoxN5 transcripts were only present at early cleavage stages, while FoxN4 expression began after midblastula transition. Spatial expression of FoxN2 was first detected in the early eye field and later, in the branchial arches, the vagal ganglion and in the developing retina. FoxN3 transcripts were found within the animal cap. In post-gastrula embryos, neural crest cells and the early eye field showed strong expression of FoxN3. At late tadpole stages, the branchial arches were stained. FoxN4 was expressed in the early eye field and later in the developing retina cells, the nephrostomes of the pronephric kidney and in the midbrain. A ubiquitous expression of FoxN5 was found in early cleavage stage embryos.

The Fox gene family in Xenopus laevis:FoxI2, FoxM1 and FoxP1 in early development.

We here describe the sequences and expression patterns of three new Fox (fork head box) transcription factors in Xenopus laevis embryos. xlFoxI2, another member of subclass I, is maternally transcribed. Zygotic transcripts are first detected during neurulation and become localised to the dorsal part of epibranchial placodes. xlFoxM1 like xlFoxP1 are the first members of subclasses M and P described in Xenopus. Both genes are maternally expressed and transcripts are found during early cleavage stages in the animal blastomeres. xlFoxM1 is strongly upregulated during neurula stages and transcripts are localised in the neuroectoderm. Later, expression is found in the spinal cord, the rhombencephalon, the retina and in the branchial arches. xlFoxP1 is activated during organogenesis and is mainly expressed in the brain, head mesenchyme and in the splanchnic layer of the lateral plate mesoderm.

The FoxO-subclass in Xenopus laevis development.

Transcription factors of the Fox (fork head box) family are involved in cellular specification and determination processes. Here, we report on the isolation and first characterisation of two members of the FoxO subclass in Xenopus laevis, xFoxO1 and xFoxO3. These sequences exhibit 68% (67%) and 69% (70%) identity to their mouse (human) orthologues, respectively. Serine and threonine residues, which are phosphorylated upon insulin signalling, are evolutionarily conserved from frogs to mammals. xFoxO1 and xFoxO3 genes are maternally transcribed, but transcripts disappear during early cleavage stages. Zygotic transcription of both genes starts at the late neurula stages and transcripts accumulate at the end of organogenesis. While maternal transcripts of both genes are found within the animal half of the early embryo, zygotic transcripts show distinct patterns. xFoxO1 expression is observed in the pronephros, within head mesenchyme in front of the eye, within the branchial arches and in the liver primordium. At the late neurula, xFoxO3 is found to be specifically expressed in the anterior neural plate and in neural crest cells. Later, expression of xFoxO3 is observed in a variety of organs and tissues, like the head, the branchial arches and the somites.