Tissue clearing combined with deep imaging has emerged as a powerful alternative to classical histological techniques. Whereas current techniques have been optimized for imaging selected nonpigmented organs such as the mammalian brain, natural pigmentation remains challenging for most other biological specimens of larger volume. We have developed a fast DEpigmEntation-Plus-Clearing method (DEEP-Clear) that is easily incorporated in existing workflows and combines whole system labeling with a spectrum of detection techniques, ranging from immunohistochemistry to RNA in situ hybridization, labeling of proliferative cells (EdU labeling) and visualization of transgenic markers. With light-sheet imaging of whole animals and detailed confocal studies on pigmented organs, we provide unprecedented insight into eyes, whole nervous systems, and subcellular structures in animal models ranging from worms and squids to axolotls and zebrafish. DEEP-Clear thus paves the way for the exploration of species-rich clades and developmental stages that are largely inaccessible by regular imaging approaches.

Platynereis dumerilii is a marine segmented worm (annelid) with externally fertilized embryos and it can be cultured for the full life cycle in the laboratory. The accessibility of embryos and larvae combined with the breadth of the established molecular and functional techniques has made P. dumerilii an attractive model for studying development, cell lineages, cell type evolution, reproduction, regeneration, the nervous system, and behavior. Traditionally, these worms have been kept in rooms dedicated for their culture. This allows for the regulation of temperature and light cycles, which is critical to synchronizing sexual maturation. However, regulating the conditions of a whole room has limitations, especially if experiments require being able to change culturing conditions. Here we present scalable and flexible culture methods that provide ability to control the environmental conditions, and have a multi-purpose culture space. We provide a closed setup shelving design with proper light conditions necessary for P. dumerilii to mature. We also implemented a standardized method of feeding P. dumerilii cultures with powdered spirulina which relieves the ambiguity associated with using frozen spinach, and helps standardize nutrition conditions across experiments and across different labs. By using these methods, we were able to raise mature P. dumerilii, capable of spawning and producing viable embryos for experimentation and replenishing culture populations. These methods will allow for the further accessibility of P. dumerilii as a model system, and they can be adapted for other aquatic organisms.

40Platynereis dumerilii is a marine segmented worm (annelid) with externally fertilized embryos 41 and it can be cultured for the full life cycle in the laboratory. The accessibility of embryos and 42 larvae combined with the breadth of the established molecular and functional techniques has made 43 P. dumerilii an attractive model for studying development, cell lineages, cell type evolution, 44reproduction, regeneration, the nervous system, and behavior. Traditionally, these worms have 45 been kept in rooms dedicated for their culture. This allows for the regulation of temperature and 46 light cycles, which is critical to synchronizing sexual maturation. However, regulating the 47 conditions of a whole room present limitations, especially if experiments require being able to 48 change culturing conditions. Here we present scalable and flexible culture methods that provide 49 ability to control the environmental conditions, and have a multi-purpose culture space. We 50 provide a closed setup shelving design with proper light conditions necessary for P. dumerilii to 51 mature. We also implemented a standardized method of feeding P. dumerilii cultures with 52 powdered spirulina which relieves the ambiguity associated with using frozen spinach, and helps 53 standardize nutrition conditions across experiments and across different labs. By using these 54 methods, we were able to raise mature P. dumerilii, capable of spawning and producing viable 55 embryos for experimentation and replenishing culture populations. These methods will allow for 56 the further accessibility of P. dumerilii as a model system, and they can be adapted for other aquatic 57 organisms. 58 59 Nereidid worms such as Platynereis have been popular in studies of development and 87 fertilization because of transparent, abundant, and comparatively large eggs and embryos [1,2]. As 88 researchers like Edmund Beecher Wilson did in the late 19th century, many labs today benefit 89 from Platynereis as a model organism for addressing a wide range of biological questions such as 90 cell type evolution, nervous system evo-devo and activity, reproductive periodicity, circalunar 91 cycling, endocrinology, regeneration, post-embryonic segment addition, stem cell biology, 92 fertilization, oocyte maturation, embryonic and larval development [3-16]. The sexual worms 93 broadcast spawn, producing thousands of externally-developing embryos. The embryos are large 94 enough to inject but small (about 160 µm in diameter) and transparent enough to image live and 95 fixed samples. Platynereis has a relatively quick and highly synchronized embryogenesis: it takes 96 only 18 hours from fertilization to hatching as a planktonic trochophore larva in P. dumerilii 97 [2,17]. This allows researchers to study embryonic development over the course of just one day. 98 A number of tools and techniques are already established in P. dumerilii [3] including 99 microinjection [5,12,18], transgenesis and genetic tools [18-21], single cell RNA sequencing [22], 100 behavioral tracking [8,...