| Abstract |
One of the essential features of animal development is that, within individuals of the same species, organs grow to acquire the size and shape necessary for their function. The process of organ growth displays remarkable precision: proportionality between body parts is kept and organ size is reproducibly achieved between individuals with incredible robustness towards environmental and genetic perturbations. Moreover, many species possess incredible regenerative capacity, being able to regrow their organs upon injury, reacquiring their original size and morphology. This precise control over organ size is due to mechanisms that convey tissue size information to the cells and coordinate their behavior, like cell division. The most studied mechanism of organ growth control is based on morphogens, signaling molecules that are secreted from source cells, diffusing to form concentration gradients across a tissue. These molecules activate signaling pathways in target cells, and can modulate cellular behaviors such as proliferation. Due to their graded distribution across tissues, cells experience different levels of morphogen concentration, depending on their position. Thus, downstream signaling dynamics can be influenced by the shape of the morphogen gradient. Some morphogen gradients remain proportional to the growing tissues where they act upon. Such property is called gradient scaling and originates from mechanisms that adjust the gradients’ range to the current tissue size: the shape of the gradient remains invariant to the tissue size. A consequence of scaling is that an increase in morphogen concentration levels happens by the same proportional amount in all relative positions of the tissue. This observation forms the basis of a proposed temporal growth control model: gradient scaling leads to spatially uniform increase in morphogen levels over time, which is then perceived by cells. Once a fold-change in morphogen signaling reaches a certain threshold, cells would then divide. This model thus links gradient scaling with proliferation dynamics. Recently, two BMP signaling gradients were shown to control growth of the zebrafish pectoral fin during development. These gradients scale with fin size during the period between 48 and 78hpf. Scaling is mediated by Smoc1 molecule, that acts as an “expander” to increase the gradients’ range. The gradients’ dynamics are consistent with the temporal growth control model, which was proposed as a mechanism for fin growth. Interestingly, preliminary results from our lab demonstrate that the pectoral fin can regenerate following amputation at 48hpf to match the size of uninjured developing fins. This observation poses a conceptual challenge on the role of a temporal control model in regeneration: distal amputation at 48hpf should result in removal of Smoc1 and affect gradient scaling; yet the fin recovers. If and how does the BMP signaling dynamics change during regeneration are the questions that I aim to address in my PhD thesis. In this study, I first established a laser microdissection protocol that allowed to precisely and reproducibly perform pectoral fin amputations. In order to interfere with the gradient scaling mechanism, I quantified Smoc1 domain in the fin at 48hpf by performing Smoc1 immunostainings combined with confocal microscopy. I show that Smoc1 is mostly located at about 30% length from the distal tip of the fin, which is then taken as the amputation position. With the amputation protocol at hand, I next performed live imaging to characterize fin volume dynamics in both development and regeneration and I show that volumetric growth is different between these conditions. Next, to investigate proliferation contribution to growth dynamics, I performed confocal and light-sheet imaging in FUCCI transgenics and show that proliferation has an important contribution to the different growth dynamics seen in development and regeneration. Given the central role of gradient scaling to the proposed temporal model of growth control used to explain fin growth, I next quantified scaling of BMP signaling gradients in development, regeneration and in Smoc1; Smoc2 mutants. This analysis reveal that gradient scaling occurs during pectoral fin development and regeneration. In contrast, loss of Smoc1 and 2 leads to a large decrease in BMP signaling of one of the gradients. Interestingly, whereas the levels of BMP signaling increase over time during fin development, these decrease during pectoral fin regeneration. This suggests that the current temporal control model would not be applicable to our regeneration scenario, despite the observed gradient scaling dynamics. Finally, I established optogenetic zebrafish transgenics which opens the possibility of manipulating BMP signaling with great spatiotemporal control. Overall, the results in this work contribute to our understanding of the dynamic nature of morphogen signaling in organ growth control. I show that scaling of BMP signaling gradients happens in situations of different growth dynamics, which supports the notion of coupling between gradient dynamics with regulation of tissue size. |