Visualizing and Measuring Dendrite Arborization in Drosophila Somatosensory Neurons.

Dendrites of neurons receive synaptic or sensory inputs and are important sites of neuronal computation. The morphological features of dendrites not only are hallmarks of the neuronal type but also largely determine a neuron's function. Thus, dendrite morphogenesis has been a subject of intensive study in neuroscience. Quantification of dendritic morphology, which is required for accurate assessment of phenotypes, can often be a challenging task, especially for complex neurons. Because manual tracing of dendritic branches is labor-intensive and time-consuming, automated or semiautomated methods are required for efficient analysis of a large number of samples. A popular in vivo model system for studying the mechanisms of dendrite morphogenesis is dendritic arborization (da) neurons in the Drosophila larval peripheral nervous system. In this chapter, we introduce methods for visualizing and measuring the dendritic arbors of these neurons. We begin with an introduction of da neurons and an overview of the methods that have been used for measuring da neuron dendrites. We then discuss the techniques and detailed steps of neuron visualization and image acquisition. Finally, we provide example steps for dendrite tracing and measurement.

Transdifferentiation is temporally uncoupled from progenitor pool expansion during hair cell regeneration in the zebrafish inner ear.

Death of mechanosensory hair cells in the inner ear is a common cause of auditory and vestibular impairment in mammals, which have a limited ability to regrow these cells after damage. In contrast, non-mammalian vertebrates, including zebrafish, can robustly regenerate hair cells after severe organ damage. The zebrafish inner ear provides an understudied model system for understanding hair cell regeneration in organs that are highly conserved with their mammalian counterparts. Here, we quantitatively examine hair cell addition during growth and regeneration of the larval zebrafish inner ear. We used a genetically encoded ablation method to induce hair cell death and we observed gradual regeneration with correct spatial patterning over a 2-week period following ablation. Supporting cells, which surround and are a source of new hair cells, divide in response to hair cell ablation, expanding the possible progenitor pool. In parallel, nascent hair cells arise from direct transdifferentiation of progenitor pool cells temporally uncoupled from supporting cell division. These findings reveal a previously unrecognized mechanism of hair cell regeneration with implications for how hair cells may be encouraged to regenerate in the mammalian ear.

Drosophila immune cells transport oxygen through PPO2 protein phase transition.

Insect respiration has long been thought to be solely dependent on an elaborate tracheal system without assistance from the circulatory system or immune cells1,2. Here we describe that Drosophila crystal cells-myeloid-like immune cells called haemocytes-control respiration by oxygenating Prophenoloxidase 2 (PPO2) proteins. Crystal cells direct the movement of haemocytes between the trachea of the larval body wall and the circulation to collect oxygen. Aided by copper and a neutral pH, oxygen is trapped in the crystalline structures of PPO2 in crystal cells. Conversely, PPO2 crystals can be dissolved when carbonic anhydrase lowers the intracellular pH and then reassembled into crystals in cellulo by adhering to the trachea. Physiologically, larvae lacking crystal cells or PPO2, or those expressing a copper-binding mutant of PPO2, display hypoxic responses under normoxic conditions and are susceptible to hypoxia. These hypoxic phenotypes can be rescued by hyperoxia, expression of arthropod haemocyanin or prevention of larval burrowing activity to expose their respiratory organs. Thus, we propose that insect immune cells collaborate with the tracheal system to reserve and transport oxygen through the phase transition of PPO2 crystals, facilitating internal oxygen homeostasis in a process that is comparable to vertebrate respiration.

A DNA replication-independent function of pre-replication complex genes during cell invasion in C. elegans.

Cell invasion is an initiating event during tumor cell metastasis and an essential process during development. A screen of C. elegans orthologs of genes overexpressed in invasive human melanoma cells has identified several components of the conserved DNA pre-replication complex (pre-RC) as positive regulators of anchor cell (AC) invasion. The pre-RC genes function cell-autonomously in the G1-arrested AC to promote invasion, independently of their role in licensing DNA replication origins in proliferating cells. While the helicase activity of the pre-RC is necessary for AC invasion, the downstream acting DNA replication initiation factors are not required. The pre-RC promotes the invasive fate by regulating the expression of extracellular matrix genes and components of the PI3K signaling pathway. Increasing PI3K pathway activity partially suppressed the AC invasion defects caused by pre-RC depletion, suggesting that the PI3K pathway is one critical pre-RC target. We propose that the pre-RC, or a part of it, acts in the postmitotic AC as a transcriptional regulator that facilitates the switch to an invasive phenotype.

The Cellular Innate Immune Response of the Invasive Pest Insect Drosophila suzukii against Pseudomonas entomophila Involves the Release of Extracellular Traps.

Drosophila suzukii is a neobiotic invasive pest that causes extensive damage to fruit crops worldwide. The biological control of this species has been unsuccessful thus far, in part because of its robust cellular innate immune system, including the activity of professional phagocytes known as hemocytes and plasmatocytes. The in vitro cultivation of primary hemocytes isolated from D. suzukii third-instar larvae is a valuable tool for the investigation of hemocyte-derived effector mechanisms against pathogens such as wasp parasitoid larvae, bacteria, fungi and viruses. Here, we describe the morphological characteristics of D. suzukii hemocytes and evaluate early innate immune responses, including extracellular traps released against the entomopathogen Pseudomonas entomophila and lipopolysaccharides. We show for the first time that D. suzukii plasmatocytes cast extracellular traps to combat P. entomophila, along with other cell-mediated reactions, such as phagocytosis and the formation of filopodia.

daf-16/FOXO blocks adult cell fate in Caenorhabditis elegans dauer larvae via lin-41/TRIM71.

Many tissue-specific stem cells maintain the ability to produce multiple cell types during long periods of non-division, or quiescence. FOXO transcription factors promote quiescence and stem cell maintenance, but the mechanisms by which FOXO proteins promote multipotency during quiescence are still emerging. The single FOXO ortholog in C. elegans, daf-16, promotes entry into a quiescent and stress-resistant larval stage called dauer in response to adverse environmental cues. During dauer, stem and progenitor cells maintain or re-establish multipotency to allow normal development to resume after dauer. We find that during dauer, daf-16/FOXO prevents epidermal stem cells (seam cells) from prematurely adopting differentiated, adult characteristics. In particular, dauer larvae that lack daf-16 misexpress collagens that are normally adult-enriched. Using col-19p::gfp as an adult cell fate marker, we find that all major daf-16 isoforms contribute to opposing col-19p::gfp expression during dauer. By contrast, daf-16(0) larvae that undergo non-dauer development do not misexpress col-19p::gfp. Adult cell fate and the timing of col-19p::gfp expression are regulated by the heterochronic gene network, including lin-41 and lin-29. lin-41 encodes an RNA-binding protein orthologous to LIN41/TRIM71 in mammals, and lin-29 encodes a conserved zinc finger transcription factor. In non-dauer development, lin-41 opposes adult cell fate by inhibiting the translation of lin-29, which directly activates col-19 transcription and promotes adult cell fate. We find that during dauer, lin-41 blocks col-19p::gfp expression, but surprisingly, lin-29 is not required in this context. Additionally, daf-16 promotes the expression of lin-41 in dauer larvae. The col-19p::gfp misexpression phenotype observed in dauer larvae with reduced daf-16 requires the downregulation of lin-41, but does not require lin-29. Taken together, this work demonstrates a novel role for daf-16/FOXO as a heterochronic gene that promotes expression of lin-41/TRIM71 to contribute to multipotent cell fate in a quiescent stem cell model.

Pilot RNAi Screen in Drosophila Neural Stem Cell Lineages to Identify Novel Tumor Suppressor Genes Involved in Asymmetric Cell Division.

A connection between compromised asymmetric cell division (ACD) and tumorigenesis was proven some years ago using Drosophila larval brain neural stem cells, called neuroblasts (NBs), as a model system. Since then, we have learned that compromised ACD does not always promote tumorigenesis, as ACD is an extremely well-regulated process in which redundancy substantially overcomes potential ACD failures. Considering this, we have performed a pilot RNAi screen in Drosophila larval brain NB lineages using RasV12 scribble (scrib) mutant clones as a sensitized genetic background, in which ACD is affected but does not cause tumoral growth. First, as a proof of concept, we have tested known ACD regulators in this sensitized background, such as lethal (2) giant larvae and warts. Although the downregulation of these ACD modulators in NB clones does not induce tumorigenesis, their downregulation along with RasV12 scrib does cause tumor-like overgrowth. Based on these results, we have randomly screened 79 RNAi lines detecting 15 potential novel ACD regulators/tumor suppressor genes. We conclude that RasV12 scrib is a good sensitized genetic background in which to identify tumor suppressor genes involved in NB ACD, whose function could otherwise be masked by the high redundancy of the ACD process.

Ecdysone regulates Drosophila wing disc size via a TORC1 dependent mechanism.

Most cells in a developing organ stop proliferating when the organ reaches a correct, final size. The underlying molecular mechanisms are not understood. We find that in Drosophila the hormone ecdysone controls wing disc size. To study how ecdysone affects wing size, we inhibit endogenous ecdysone synthesis and feed larvae exogenous ecdysone in a dose-controlled manner. For any given ecdysone dose, discs stop proliferating at a particular size, with higher doses enabling discs to reach larger sizes. Termination of proliferation coincides with a drop in TORC1, but not Dpp or Yki signaling. Reactivating TORC1 bypasses the termination of proliferation, indicating that TORC1 is a main downstream effector causing proliferation termination at the maximal ecdysone-dependent size. Experimental manipulation of Dpp or Yki signaling can bypass proliferation termination in hinge and notum regions, but not the pouch, suggesting that the mechanisms regulating proliferation termination may be distinct in different disc regions.

Gene regulation of adult skeletogenesis in starfish and modifications during gene network co-option.

The larval skeleton of the echinoderm is believed to have been acquired through co-option of a pre-existing gene regulatory network (GRN); that is, the mechanism for adult skeleton formation in the echinoderm was deployed in early embryogenesis during echinoderm diversification. To explore the evolutionary changes that occurred during co-option, we examined the mechanism for adult skeletogenesis using the starfish Patiria pectinifera. Expression patterns of skeletogenesis-related genes (vegf, vegfr, ets1/2, erg, alx1, ca1, and clect) suggest that adult skeletogenic cells develop from the posterior coelom after the start of feeding. Treatment with inhibitors and gene knockout using transcription activator-like effector nucleases (TALENs) suggest that the feeding-nutrient sensing pathway activates Vegf signaling via target of rapamycin (TOR) activity, leading to the activation of skeletogenic regulatory genes in starfish. In the larval skeletogenesis of sea urchins, the homeobox gene pmar1 activates skeletogenic regulatory genes, but in starfish, localized expression of the pmar1-related genes phbA and phbB was not detected during the adult skeleton formation stage. Based on these data, we provide a model for the adult skeletogenic GRN in the echinoderm and propose that the upstream regulatory system changed from the feeding-TOR-Vegf pathway to a homeobox gene-system during co-option of the skeletogenic GRN.

Genetically encoded cell-death indicators (GEDI) to detect an early irreversible commitment to neurodegeneration.

Cell death is a critical process that occurs normally in health and disease. However, its study is limited due to available technologies that only detect very late stages in the process or specific death mechanisms. Here, we report the development of a family of fluorescent biosensors called genetically encoded death indicators (GEDIs). GEDIs specifically detect an intracellular Ca2+ level that cells achieve early in the cell death process and that marks a stage at which cells are irreversibly committed to die. The time-resolved nature of a GEDI delineates a binary demarcation of cell life and death in real time, reformulating the definition of cell death. We demonstrate that GEDIs acutely and accurately report death of rodent and human neurons in vitro, and show that GEDIs enable an automated imaging platform for single cell detection of neuronal death in vivo in zebrafish larvae. With a quantitative pseudo-ratiometric signal, GEDIs facilitate high-throughput analysis of cell death in time-lapse imaging analysis, providing the necessary resolution and scale to identify early factors leading to cell death in studies of neurodegeneration.

Thyroid hormone deficiency during zebrafish development impairs central nervous system myelination.

Thyroid hormones are messengers that bind to specific nuclear receptors and regulate a wide range of physiological processes in the early stages of vertebrate embryonic development, including neurodevelopment and myelogenesis. We here tested the effects of reduced T3 availability upon the myelination process by treating zebrafish embryos with low concentrations of iopanoic acid (IOP) to block T4 to T3 conversion. Black Gold II staining showed that T3 deficiency reduced the myelin density in the forebrain, midbrain, hindbrain and the spinal cord at 3 and 7 dpf. These observations were confirmed in 3 dpf mbp:egfp transgenic zebrafish, showing that the administration of IOP reduced the fluorescent signal in the brain. T3 rescue treatment restored brain myelination and reversed the changes in myelin-related gene expression induced by IOP exposure. NG2 immunostaining revealed that T3 deficiency reduced the amount of oligodendrocyte precursor cells in 3 dpf IOP-treated larvae. Altogether, the present results show that inhibition of T4 to T3 conversion results in hypomyelination, suggesting that THs are part of the key signaling molecules that control the timing of oligodendrocyte differentiation and myelin synthesis from very early stages of brain development.

FDA-Approved Drug Screening for Compounds That Facilitate Hematopoietic Stem and Progenitor Cells (HSPCs) Expansion in Zebrafish.

Hematopoietic stem cells (HSCs) are a specialized subset of cells with self-renewal and multilineage differentiation potency, which are essential for their function in bone marrow or umbilical cord blood transplantation to treat blood disorders. Expanding the hematopoietic stem and progenitor cells (HSPCs) ex vivo is essential to understand the HSPCs-based therapies potency. Here, we established a screening system in zebrafish by adopting an FDA-approved drug library to identify candidates that could facilitate HSPC expansion. To date, we have screened 171 drugs of 7 categories, including antibacterial, antineoplastic, glucocorticoid, NSAIDS, vitamins, antidepressant, and antipsychotic drugs. We found 21 drugs that contributed to HSPCs expansion, 32 drugs' administration caused HSPCs diminishment and 118 drugs' treatment elicited no effect on HSPCs amplification. Among these drugs, we further investigated the vitamin drugs ergocalciferol and panthenol, taking advantage of their acceptability, limited side-effects, and easy delivery. These two drugs, in particular, efficiently expanded the HSPCs pool in a dose-dependent manner. Their application even mitigated the compromised hematopoiesis in an ikzf1-/- mutant. Taken together, our study implied that the larval zebrafish is a suitable model for drug repurposing of effective molecules (especially those already approved for clinical use) that can facilitate HSPCs expansion.

Xenopus chip for single-egg trapping, in vitro fertilization, development, and tadpole escape.

Xenopus laevis is highly suitable as a toxicology animal model owing to its advantages in embryogenesis research. For toxicological studies, a large number of embryos must be handled simultaneously because they very rapidly develop into the target stages within a short period of time. To efficiently handle the embryos, a convenient embryo housing device is essential for fast and reliable assessment and statistical evaluation of malformation caused by toxicants. Here, we suggest 3D fabrication of single-egg trapping devices in which Xenopus eggs are fertilized in vitro, and the embryos are cultured. We used manual pipetting to insert the Xenopus eggs inside the trapping sites of the chip. By introducing a liquid circulating system, we connected a sperm-mixed solution with the chip to induce in vitro fertilization of the eggs. After the eggs were fertilized, we observed embryo development involving the formation of egg cleavage, blastula, gastrula, and tadpole. After the tadpoles grew inside the chip, we saved their lives by enabling their escape from the chip through reverse flow of the culture medium. The Xenopus chip can serve as an incubator to induce fertilization and monitor normal and abnormal development of the Xenopus from egg to tadpole.

Role of autophagy in midgut stem cells of silkworm Bombyx mori, during larval-pupal metamorphosis.

Autophagy is a critical mechanism for the self-renewal, proliferation, and differentiation of stem cells. Bombyx mori midgut has stem cells that play a role in the regeneration of the larval epithelium in larval stages and the formation of the pupal midgut epithelium during larval-pupal metamorphosis. In this study, the role of the autophagy mechanism in midgut stem cells during the formation of the pupal midgut was investigated. For this purpose, two different doses of autophagy inhibitor chloroquine were administered to B. mori larvae on days 7 and 8 of the fifth larval stage. Morphological changes during the formation process of the pupal epithelium, expression levels of autophagy-related genes Atg8 and Atg12 in stem cells, and the amounts of lysosomal enzyme acid phosphatase were determined after the application. The obtained findings were evaluated in comparison with the control groups. Abnormalities in the formation of the pupal midgut after inhibition of autophagy showed the significance of the autophagy mechanism during this period.

Protocol for quantitative analysis of pulsatile contractions and cell extrusion in epithelial tissues of larval zebrafish.

Cell elimination by extrusion is important for epithelial homeostasis, but knowing when and where cells will extrude has made in vivo studies difficult. Here, we describe a step-by-step protocol for inducing cell extrusion from the larval zebrafish epidermis. We detail how to capture the dynamics of extrusion via time-lapse imaging and describe how existing protocols can be implemented for the analysis of cell shape changes preceding extrusion events and derivation of mechanical measurements associated with these shape changes. For complete details on the use and execution of this protocol, please refer to Atieh et al. (2021).

DNA Damage in Liver Cells of the Tilapia Fish Oreochromis mossambicus Larva Induced by the Insecticide Cyantraniliprole at Sublethal Doses During Chronic Exposure.

Cyantraniliprole can effectively control lepidopteran pests and has been used all over the world. In general, the risk of cyantraniliprole seems low for fish, but the toxicity selectivity among different fish species was not clear. Here, we present the methods for the acute toxicity and chronic effects of cyantraniliprole by using juvenile tilapia (Oreochromis mossambicus). Based on this test, 96 h LC50 of cyantraniliprole to tilapia was 38.0 mg/L. After exposed for 28 days, specific growth rates of the blank control, solution control, and the treatments of 0.037, 0.37 and 3.7 mg/L of cyantraniliprole were 1.14, 0.95, 0.93, 0.82, and 0.70% per day, respectively. The results of micronucleus experiment and single cell gel electrophoresis showed that cyantraniliprole damaged DNA in liver cells of tilapia larvae. Quantitative PCR results showed that cyantraniliprole could induce the upregulation of Rpa 3 that is responsible for the DNA repair. The significant downregulation of Chk 2 gene was related to p53 pathway. It is therefore proposed that cyantraniliprole causes DNA damage in liver cells of tilapia and activates DNA damage and repair pathways.

Histological assessment of developmental cell death in Drosophila pupae.

This protocol describes the embedding and processing of Drosophila pupae in paraffin to monitor tissue changes during development. Although multiple methods are available to evaluate developmental changes in Drosophila embryos, imaging detailed changes during metamorphosis is challenging as the animal is enclosed in the cuticle, rendering it inaccessible to whole mount imaging. Here, we present a protocol that focuses on developmental clearance of the larval salivary glands in Drosophila pupae that can be extended to examine other tissues/stages for similar purposes. For complete details on the use and execution of this protocol, please refer to Velentzas et al. (2018).

Photoacoustic-fluorescence microendoscopy in vivo.

A miniature endoscope capable of imaging multiple tissue contrasts in high resolution is highly attractive, because it can provide complementary and detailed tissue information of internal organs. Here we present a photoacoustic (PA)-fluorescence (FL) endoscope for optical-resolution PA microscopy (PAM) and FL microscopy (FLM). The endoscope with a diameter of 2.8 mm achieves high lateral resolutions of 5.5 and 6.3 µm for PAM and FLM modes, respectively. In vivo imaging of zebrafish larvae and a mouse ear is conducted, and high-quality images are obtained. Additionally, in vivo endoscopic imaging of a rat rectum is demonstrated, showing the endoscopic imaging capability of our endoscope. By providing dual contrasts with high resolution, the endoscope may open up new opportunities for clinical endoscopic imaging applications.

Myomixer is expressed during embryonic and post-larval hyperplasia, muscle regeneration and differentiation of myoblats in rainbow trout (Oncorhynchus mykiss).

In contrast to mice or zebrafish, trout exhibits post-larval muscle growth through hypertrophy and formation of new myofibers (hyperplasia). The muscle fibers are formed by the fusion of mononucleated cells (myoblasts) regulated by several muscle-specific proteins such as Myomaker or Myomixer. In this work, we identified a unique gene encoding a Myomixer protein of 77 amino acids (aa) in the trout genome. Sequence analysis and phylogenetic tree showed moderate conservation of the overall protein sequence across teleost fish (61% of aa identity between trout and zebrafish Myomixer sequences). Nevertheless, the functionally essential motif, AxLyCxL is perfectly conserved in all studied sequences of vertebrates. Using in situ hybridization, we observed that myomixer was highly expressed in the embryonic myotome, particularly in the hyperplasic area. Moreover, myomixer remained readily expressed in white muscle of juvenile (1 and 20 g) although its expression decreased in mature fish. We also showed that myomixer is up-regulated during muscle regeneration and in vitro myoblasts differentiation. Together, these data indicate that myomixer expression is consistently associated with the formation of new myofibers during somitogenesis, post-larval growth and muscle regeneration in trout.