Scales Radi(i)cally Remodel Sensory Axons and Vasculature.

Peripheral axons of sensory neurons innervate skin cells to form a functional sensory organ. In this issue of Developmental Cell, Rasmussen et al. (2018) demonstrate that scale formation is essential for the development and regeneration of zebrafish sensory axons and vasculature.

A regulatory pathway involving retinoic acid and calcineurin demarcates and maintains joint cells and osteoblasts in regenerating fin.

During zebrafish fin regeneration, blastema cells lining the epidermis differentiate into osteoblasts and joint cells to reconstruct the segmented bony rays. We show that osteoblasts and joint cells originate from a common cell lineage, but are committed to different cell fates. Pre-osteoblasts expressing runx2a/b commit to the osteoblast lineage upon expressing sp7, whereas the strong upregulation of hoxa13a correlates with a commitment to a joint cell type. In the distal regenerate, hoxa13a, evx1 and pthlha are sequentially upregulated at regular intervals to define the newly identified presumptive joint cells. Presumptive joint cells mature into joint-forming cells, a distinct cell cluster that maintains the expression of these factors. Analysis of evx1 null mutants reveals that evx1 is acting upstream of pthlha and downstream of or in parallel with hoxa13a Calcineurin activity, potentially through the inhibition of retinoic acid signaling, regulates evx1, pthlha and hoxa13a expression during joint formation. Furthermore, retinoic acid treatment induces osteoblast differentiation in mature joint cells, leading to ectopic bone deposition in joint regions. Overall, our data reveal a novel regulatory pathway essential for joint formation in the regenerating fin.

Regeneration of breeding tubercles on zebrafish pectoral fins requires androgens and two waves of revascularization.

Sexually dimorphic breeding tubercles (BTs) are keratinized epidermal structures that form clusters on the dorsal surface of the anterior rays of zebrafish male pectoral fins. BTs appear during sexual maturation and are maintained through regular shedding and renewal of the keratinized surface. Following pectoral fin amputation, BT clusters regenerate after the initiation of revascularization, but concomitantly with a second wave of angiogenesis. This second wave of regeneration forms a web-like blood vessel network that penetrates the supportive epidermis of BTs. Upon analyzing the effects of sex steroids and their inhibitors, we show that androgens induce and estrogens inhibit BT cluster formation in intact and regenerating pectoral fins. Androgen-induced BT formation in females is accompanied by the formation of a male-like blood vessel network. Treatment of females with both androgens and an angiogenesis inhibitor results in the formation of undersized BT clusters when compared with females treated with androgens alone. Overall, the growth and regeneration of large BTs requires a hormonal stimulus and the presence of an additional blood vessel network that is naturally found in males.

The preparation of Drosophila embryos for live-imaging using the hanging drop protocol.

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

Autophagy promotes caspase-dependent cell death during Drosophila development.

The relationship between autophagic cell death and apoptosis is a poorly understood aspect of programmed cell death (PCD). We have examined this relationship by studying the elimination of an extra-embryonic tissue, known as the amnioserosa (AS), during Drosophila development. The AS becomes autophagic during the final stages of embryogenesis; ultimately, however, the elimination of the AS involves caspase-dependent nuclear fragmentation, tissue dissociation and engulfment by phagocytic macrophages. Mutants that are defective in the activation or execution of caspase-dependent PCD fail to degrade and eliminate the AS but show no abatement in AS autophagy. Sustained autophagy does not, therefore, necessarily result in cell death. Surprisingly, the downregulation of autophagy also results in a persistent AS phenotype and reduced cell death. Conversely, upregulation of autophagy results in caspase-dependent premature AS dissociation. These observations are consistent with the interpretation that autophagy is a prerequisite for caspase-dependent cell death in the AS.