Sustained production of ROS triggers compensatory proliferation and is required for regeneration to proceed.

A major issue in regenerative medicine is the role of injury in promoting cell plasticity. Here we explore the function of reactive oxygen species (ROS) induced through lesions in adult zebrafish. We show that ROS production, following adult fin amputation, is tightly regulated in time and space for at least 24 hours, whereas ROS production remains transient (2 hours) in mere wound healing. In regenerative tissue, ROS signaling triggers two distinct parallel pathways: one pathway is responsible for apoptosis, and the other pathway is responsible for JNK activation. Both events are involved in the compensatory proliferation of stump epidermal cells and are necessary for the progression of regeneration. Both events impact the Wnt, SDF1 and IGF pathways, while apoptosis only impacts progenitor marker expression. These results implicate oxidative stress in regeneration and provide new insights into the differences between healing and regeneration.

Developmental role of zebrafish protease-activated receptor 1 (PAR1) in the cardio-vascular system.

Thrombin receptor, F2R or PAR1 is a G-protein coupled receptor, located in the membrane of endothelial cells. It has been initially found to transduce signals in hemostasis, but recently also known to act in cancer and in vascular development. Mouse embryos lacking PAR1 function die from hemorrhages with varying frequency at midgestation. We have performed a survey of potential PAR1 homologs in the zebrafish genome and identified a teleost ortholog of mammalian PAR1. Knockdown of par1 function in zebrafish embryos demonstrates a requirement for Par1 in cardio-vascular development. Furthermore, we show that function of Par1 requires the presence of a phylogenetically conserved proteolytic cleavage site and a second intracellular domain. Altogether our results demonstrate a high degree of conservation of PAR1 proteins in the vertebrate lineage in respect to amino acid sequence as well as protein function.

Implication of type 3 deiodinase induction in zebrafish fin regeneration.

Thyroid hormones are critical determinants of cellular differentiation. We used the zebrafish model to evaluate the involvement of thyroid hormones in regeneration processes after caudal fin amputation. We examined early events following fin amputation, i.e., blastema formation and nerve repair by growth cone formation. Here, we show that the abolition of thyroid gland activity by methimazole treatment had no effect on blastema formation, but slowed growth cone formation of the lateral line. Conversely, the addition of exogenous thyroid hormones enhanced growth cone formation without affecting blastema formation. However, amputation triggered a strong induction in the blastema of type 3 deiodinase mRNA and enzymatic activity, which degrades thyroid hormone (TH). We therefore blocked deiodinase activity with iopanoic acid (IOP) and saw a reduction in blastema formation, suggesting that local degradation of TH is permissive for cell proliferation in the blastema. The effect of IOP on the blastema required endogenous or exogenous TH. Our findings support a model in which local degradation of TH by type 3 deiodinase is permissive for epimorphic regeneration.

Translocator protein (18 kDa) is involved in primitive erythropoiesis in zebrafish.

The translocator protein (18 kDa) (TSPO), also known as peripheral-type benzodiazepine receptor, is directly or indirectly associated with many biological processes. Although extensively characterized, the specific function of TSPO during development remains unclear. It has been reported that TSPO is involved in a variety of mechanisms, including cell proliferation, apoptosis, regulation of mitochondrial functions, cholesterol transport and steroidogenesis, and porphyrin transport and heme synthesis. Although the literature has reported a murine knockout model, the experiment did not generate information because of early lethality. We then used the zebrafish model to address the function of tspo during development. Information about spatiotemporal expression showed that tspo has a maternal and a zygotic contribution which, during somatogenesis, seems to be erythroid restricted to the intermediate cell mass. Genetic and pharmacological approaches used to invalidate Tspo function resulted in embryos with specific erythropoietic cell depletion. Although unexpected, this lack of blood cells is independent of the Tspo cholesterol binding site and reveals a new in vivo key role for Tspo during erythropoiesis.

Pdk1 activity controls proliferation, survival, and growth of developing pancreatic cells.

The formation of adequate masses of endocrine and exocrine pancreatic tissues during embryogenesis is essential to ensure proper nutrition and glucose homeostasis at postnatal stages. We generated mice with pancreas-specific ablation of the 3-phosphoinositide-dependent protein kinase 1 (Pdk1) to investigate how signaling downstream of the phosphatidylinositol-3-OH kinase (PI3K) pathway controls pancreas development. Pdk1-conditional knock-out mice were born with conspicuous pancreas hypoplasia, and within a few weeks, they developed severe hyperglycemia. Our detailed characterization of the mutant embryonic pancreas also revealed distinct temporal, cell type-specific requirements of Pdk1 activity in the control of cell proliferation, cell survival, and cell size during pancreas development. These results thus uncover Pdk1 as a novel, crucial regulator of pancreatic growth during embryogenesis. In addition, we provide evidence that Pdk1 activity is required differently in mature pancreatic cell types, since compensatory proliferation and possible mTORC2 activation occurred in exocrine cells but not in beta cells of the Pdk1-deficient postnatal pancreas.

Fgf and Sdf-1 pathways interact during zebrafish fin regeneration.

The chemokine stromal cell-derived factor-1 (SDF1) was originally identified as a pre-B cell stimulatory factor but has been recently implicated in several other key steps in differentiation and morphogenesis. In addition, SDF1 as well as FGF signalling pathways have recently been shown to be involved in the control of epimorphic regeneration. In this report, we address the question of a possible interaction between the two signalling pathways during adult fin regeneration in zebrafish. Using a combination of pharmaceutical and genetic tools, we show that during epimorphic regeneration, expression of sdf1, as well as of its cognate receptors, cxcr4a, cxcr4b and cxcr7 are controlled by FGF signalling. We further show that, Sdf1a negatively regulates the expression of fgf20a. Together, these results lead us to propose that: 1) the function of Fgf in blastema formation is, at least in part, relayed by the chemokine Sdf1a, and that 2) Sdf1 exerts negative feedback on the Fgf pathway, which contributes to a transient expression of Fgf20a downstream genes at the beginning of regeneration. However this feedback control can be bypassed since the Sdf1 null mutants regenerate their fin, though slower. Very few mutants for the regeneration process were isolated so far, illustrating the difficulty in identifying genes that are indispensable for regeneration. This observation supports the idea that the regeneration process involves a delicate balance between multiple pathways.