Phylogenetic Distribution of Intron Positions in Alpha-Amylase Genes of Bilateria Suggests Numerous Gains and Losses

Most eukaryotes have at least some genes interrupted by introns. While it is well accepted that introns were already present at moderate density in the last eukaryote common ancestor, the conspicuous diversity of intron density among genomes suggests a complex evolutionary history, with marked differences between phyla. The question of the rates of intron gains and loss in the course of evolution and factors influencing them remains controversial. We have investigated a single gene family, alpha-amylase, in 55 species covering a variety of animal phyla. Comparison of intron positions across phyla suggests a complex history, with a likely ancestral intronless gene undergoing frequent intron loss and gain, leading to extant intron/exon structures that are highly variable, even among species from the same phylum. Because introns are known to play no regulatory role in this gene and there is no alternative splicing, the structural differences may be interpreted more easily: intron positions, sizes, losses or gains may be more likely related to factors linked to splicing mechanisms and requirements, and to recognition of introns and exons, or to more extrinsic factors, such as life cycle and population size. We have shown that intron losses outnumbered gains in recent periods, but that “resets” of intron positions occurred at the origin of several phyla, including vertebrates. Rates of gain and loss appear to be positively correlated. No phase preference was found. We also found evidence for parallel gains and for intron sliding. Presence of introns at given positions was correlated to a strong protosplice consensus sequence AG/G, which was much weaker in the absence of intron. In contrast, recent intron insertions were not associated with a specific sequence. In animal Amy genes, population size and generation time seem to have played only minor roles in shaping gene structures

Reiterative AP2a activity controls sequential steps in the neural crest gene regulatory network

The neural crest (NC) emerges from combinatorial inductive events occurring within its progenitor domain, the neural border (NB). Several transcription factors act early at the NB, but the initiating molecular events remain elusive. Recent data from basal vertebrates suggest that ap2 might have been critical for NC emergence; however, the role of AP2 factors at the NB remains unclear. We show here that AP2a initiates NB patterning and is sufficient to elicit a NB-like pattern in neuralized ectoderm. In contrast, the other early regulators do not participate in ap2a initiation at the NB, but cooperate to further establish a robust NB pattern. The NC regulatory network uses a multistep cascade of secreted inducers and transcription factors, first at the NB and then within the NC progenitors. Here we report that AP2a acts at two distinct steps of this cascade. As the earliest known NB specifier, AP2a mediates Wnt signals to initiate the NB and activate pax3; as a NC specifier, AP2a regulates further NC development independent of and downstream of NB patterning. Our findings reconcile conflicting observations from various vertebrate organisms. AP2a provides a paradigm for the reiterated use of multifunctional molecules, thereby facilitating emergence of the NC in vertebrates

Fanca deficiency is associated with alterations in osteoclastogenesis that are rescued by TNFα

Abstract Background Hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, which includes bone-forming and bone-resorbing cells, i.e., osteoblasts (OBs) and osteoclasts (OCs). OBs originate from mesenchymal progenitors, while OCs are derived from HSCs. Self-renewal, proliferation and differentiation of HSCs are under the control of regulatory signals generated by OBs and OCs within the BM niche. Consequently, OBs and OCs control both bone physiology and hematopoiesis. Since the human developmental and bone marrow failure genetic syndrome fanconi anemia (FA) presents with skeletal abnormalities, osteoporosis and HSC impairment, we wanted to test the hypothesis that the main pathological abnormalities of FA could be related to a defect in OC physiology and/or in bone homeostasis. Results We revealed here that the intrinsic differentiation of OCs from a Fanca −/− mouse is impaired in vitro due to overactivation of the p53–p21 axis and defects in NF-kB signaling. The OC differentiation abnormalities observed in vitro were rescued by treating Fanca −/− cells with the p53 inhibitor pifithrin-α, by treatment with the proinflammatory cytokine TNFα or by coculturing them with Fanca-proficient or Fanca-deficient osteoblastic cells. Conclusions Overall, our results highlight an unappreciated role of Fanca in OC differentiation that is potentially circumvented in vivo by the presence of OBs and TNFα in the BM niche

The Pax3 and Pax7 paralogs cooperate in neural and neural crest patterning using distinct molecular mechanisms, in Xenopus laevis embryos.

International audiencePax3 and Pax7 paralogous genes have functionally diverged in vertebrate evolution, creating opportunity for a new distribution of roles between the two genes and the evolution of novel functions. Here we focus on the regulation and function of Pax7 in the brain and neural crest of amphibian embryos, which display a different pax7 expression pattern, compared to the other vertebrates already described. Pax7 expression is restricted to the midbrain, hindbrain and anterior spinal cord, and Pax7 activity is important for maintaining the fates of these regions, by restricting otx2 expression anteriorly. In contrast, pax3 displays broader expression along the entire neuraxis and Pax3 function is important for posterior brain patterning without acting on otx2 expression. Moreover, while both genes are essential for neural crest patterning, we show that they do so using two distinct mechanisms: Pax3 acts within the ectoderm which will be induced into neural crest, while Pax7 is essential for the inducing activity of the paraxial mesoderm towards the prospective neural crest

Fanconi anemia A protein participates in nucleolar homeostasis maintenance and ribosome biogenesis

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Protein tyrosine phosphatase 4A3 (PTP4A3) is required for Xenopus laevis cranial neural crest migration in vivo.

Uveal melanoma is the most common intraocular malignancy in adults, representing between about 4% and 5% of all melanomas. High expression levels of Protein Tyrosine Phosphatase 4A3, a dual phosphatase, is highly predictive of metastasis development and PTP4A3 overexpression in uveal melanoma cells increases their in vitro migration and in vivo invasiveness. Melanocytes, including uveal melanocytes, are derived from the neural crest during embryonic development. We therefore suggested that PTP4A3 function in uveal melanoma metastasis may be related to an embryonic role during neural crest cell migration. We show that PTP4A3 plays a role in cephalic neural crest development in Xenopus laevis. PTP4A3 loss of function resulted in a reduction of neural crest territory, whilst gain of function experiments increased neural crest territory. Isochronic graft experiments demonstrated that PTP4A3-depleted neural crest explants are unable to migrate in host embryos. Pharmacological inhibition of PTP4A3 on dissected neural crest cells significantly reduced their migration velocity in vitro. Our results demonstrate that PTP4A3 is required for cephalic neural crest migration in vivo during embryonic development

Pax3 and Zic1 trigger the early neural crest gene regulatory network by the direct activation of multiple key neural crest specifiers

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Depletion of xlPTP4A3 inhibits both migration of NCC and uveal melanoma cells.

<p>A) Isotopic and isochronic grafts of xlPTP4A3-depleted NC explants into host embryos showed a delayed NCC migration (b-b’) when compared to the control histone 2b-GFP NC explants (a-a’). B) Timelapse videomicroscopy of stage 17-derived NCC treated with a pharmacological inhibitor of PTP4A3, a rhodanine derivative (PRL-3 inhibitor I), shows that inhibition of PTP4A3 increases the average of pausing NC cells and strongly decreases the migration velocity of the treated cells (relative to that of vehicle treated cells). The result is representative of three independent experiments and the average of counted cells is 60 cells per condition (***p<0,001). C) Timelapse videomicroscopy of uveal melanoma OCM1 cells stably expressing EGFP-PTP4A3, EGFP-PTP4A3(C104S) or EGFP on collagen I shows that inhibition of PTP4A3, using PRL-3 Inhibitor I, decreases specifically the migration velocity of the PTP4A3 expressing cells (relative to the velocity of untreated and control cells). The result is representative of three independent experiments and the average of counted cells is 50 cells per condition (***p<0,001).</p

Depletion of xlPTP4A3 reduces NCC territory.

<p>Control 5MM-MO or xlPTP4A3-MO were injected at the four-cell stage in the animal pole region and embryos were cultured until stage 22. A) Morpholino sequence targeting xlPTP4A3. B) xlPTP4A3-MO specifically knocked down the translation of xlPTP4A3 <i>in </i><i>vitro</i> using the TNT® Coupled Reticulocyte Lysate System. C) xlPTP4A3 depleted embryos showed blockade of NCC territory (a-a’) and can be rescued by co-injection of mouse mPTP4A3 (c-c’), as shown by whole-mount <i>in </i><i>situ</i> hybridization on xlTWIST. The injected side was monitored using the co-injection of nuclear-targeted β-galactosidase (red dotted signal). Injection of the control 5MM-MO does not affect the NCCs territory, as in the control noninjected embryos (b-b’). D) Quantitative results of relative phenotype (**p<0,01). E) The phenotype quantification was determined as the length of the NC migratory streams showing that the migratory streams are shorter in the injected side than in the uninjected side (n=15) (***p<0,001). F) Immunodetection of cleaved caspase 3 on xlPTP4A3-MO injected embryos. Two phenotypes are observed: one of weaker apoptosis in the injected side compared to the uninjected side (a-a’) and one of no differential apoptosis between the two sides (b-b’).</p