Specificity of time- and dose-dependent morphological endpoints in the fish embryo acute toxicity (FET) test for substances with diverse modes of action: the search for a “fingerprint”

The fish embryo acute toxicity (FET) test with the zebrafish (Danio rerio) embryo according to OECD TG 236 was originally developed as an alternative test method for acute fish toxicity testing according to, e.g., OECD TG 203. Given the versatility of the protocol, however, the FET test has found application beyond acute toxicity testing as a common tool in environmental hazard and risk assessment. Whereas the standard OECD guideline is restricted to four core endpoints (coagulation as well as lack of somite formation, heartbeat, and tail detachment) for simple, rapid assessment of acute toxicity, further endpoints can easily be integrated into the FET test protocol. This has led to the hypothesis that an extended FET test might allow for the identification of different classes of toxicants via a “fingerprint” of morphological observations. To test this hypothesis, the present study investigated a set of 18 compounds with highly diverse modes of action with respect to acute and sublethal endpoints. Especially at higher concentrations, most observations proved toxicant-unspecific. With decreasing concentrations, however, observations declined in number, but gained in specificity. Specific observations may at best be made at test concentrations ≤ EC10. The existence of a “fingerprint” based on morphological observations in the FET is, therefore, highly unlikely in the range of acute toxicity, but cannot be excluded for experiments at sublethal concentrations.

Stress-driven tissue fluidization physically segments vertebrate somites

Shaping functional structures during embryonic development requires both genetic and physical control. During somitogenesis, cell-cell coordination sets up genetic traveling waves in the presomitic mesoderm (PSM) that orchestrate somite formation. While key molecular and genetic aspects of this process are known, the mechanical events required to physically segment somites from the PSM remain unclear. Combining direct mechanical measurements during somite formation, live imaging of cell and tissue structure, and computer simulations, here we show that somites are mechanically sectioned off from the PSM by a large, actomyosin-driven increase in anisotropic stress at the nascent somite-somite boundary. Our results show that this localized increase in stress drives the regional fluidization of the tissue adjacent to the forming somite border, enabling local tissue remodeling and the shaping of the somite. Moreover, we find that active tension fluctuations in the tissue are optimized to mechanically define sharp somite boundaries while minimizing somite morphological defects. Altogether, these results indicate that mechanical changes at the somite-somite border and optimal tension fluctuations in the tissue are essential physical aspects of somite formation.

Paraxial mesoderm organoids model development of human somites

During the development of the vertebrate embryo, segmented structures called somites are periodically formed from the presomitic mesoderm (PSM), and give rise to the vertebral column. While somite formation has been studied in several animal models, it is less clear how well this process is conserved in humans. Recent progress has made it possible to study aspects of human paraxial mesoderm development such as the human segmentation clock in vitro using human pluripotent stem cells (hPSCs), however, somite formation has not been observed in these monolayer cultures. Here, we describe the generation of human paraxial mesoderm (PM) organoids from hPSCs (termed Somitoids), which recapitulate the molecular, morphological and functional features of paraxial mesoderm development, including formation of somite-like structures in vitro. Using a quantitative image-based screen, we identify critical parameters such as initial cell number and signaling modulations that reproducibly yielded somite formation in our organoid system. In addition, using single-cell RNA sequencing and 3D imaging, we show that PM organoids both transcriptionally and morphologically resemble their in vivo counterparts and can be differentiated into somite derivatives. Our organoid system is reproducible and scalable, allowing for the systematic and quantitative analysis of human spinal cord development and disease in vitro.

Characterization and Biotechnological Potential of Intracellular Polyhydroxybutyrate by Stigeoclonium sp. B23 Using Cassava Peel as Carbon Source

The possibility of utilizing lignocellulosic agro-industrial waste products such as cassava peel hydrolysate (CPH) as carbon sources for polyhydroxybutyrate (PHB) biosynthesis and characterization by Amazonian microalga Stigeoclonium sp. B23. was investigated. Cassava peel was hydrolyzed to reducing sugars to obtain increased glucose content with 2.56 ± 0.07 mmol/L. Prior to obtaining PHB, Stigeoclonium sp. B23 was grown in BG-11 for characterization and Z8 media for evaluation of PHB nanoparticles’ cytotoxicity in zebrafish embryos. As results, microalga produced the highest amount of dry weight of PHB with 12.16 ± 1.28 (%) in modified Z8 medium, and PHB nanoparticles exerted some toxicity on zebrafish embryos at concentrations of 6.25–100 µg/mL, increased mortality (<35%) and lethality indicators as lack of somite formation (<25%), non-detachment of tail, and lack of heartbeat (both <15%). Characterization of PHB by scanning electron microscopy (SEM), X-ray diffraction (XRD), differential scanning calorimeter (DSC), and thermogravimetry (TGA) analysis revealed the polymer obtained from CPH cultivation to be morphologically, thermally, physically, and biologically acceptable and promising for its use as a biomaterial and confirmed the structure of the polymer as PHB. The findings revealed that microalgal PHB from Stigeoclonium sp. B23 was a promising and biologically feasible new option with high commercial value, potential for biomaterial applications, and also suggested the use of cassava peel as an alternative renewable resource of carbon for PHB biosynthesis and the non-use of agro-industrial waste and dumping concerns.

Induced reproduction and early development in dourado, Salminus franciscanus Lima & Britski, 2007 (Pisces: Characiformes)

Summary Our study aimed to establish the response of Salminus franciscanus to hypophysation and describe the main morphological events of its embryonic process. Wild fish were captured in São Francisco River and selected broodstock (females: 66.4 ± 11.1 cm and 4.04 ± 2.32 kg; males: 58.3 ± 10.2 cm and 3.62 ± 1.12 kg) were kept at 26.1 ± 0.6°C for induction of final maturation/gamete release via the hypophysation technique. In females, two doses (0.8 and 5.6 mg/kg body weight) of crude pituitary extract of common carp (Cyprinus carpio) were administered with a 14 h interval. For males, a single dose (2.7 mg/kg body weight) of crude pituitary extract was applied at the same time as the females’ second dose. Oocytes and sperm were manually stripped 8 h after a females’ second hormonal dose. Fertilization was carried out using the dry method. Eggs were kept in funnel-type 60 L incubators at 24.3 ± 0.3°C and were analyzed and photographed every 10 min. After hormonal induction, 60% of females and 100% of males reacted positively and no broodstock mortality was recorded. The females released an average of 385.2 ± 78.4 g of oocytes and the fertilization rate observed was 50.4 ± 12.3%. The blastopore closure occurred at 7.5 h, somite formation at 12 h and hatching at 20 h post-fertilization. In general, the results of this study improve the understanding of the reproductive biology of dourado and confirm its potential for fish farming in the neotropical region.

Mouse embryonic stem cells self-organize into trunk-like structures with neural tube and somites

Post-implantation embryogenesis is a highly dynamic process comprising multiple lineage decisions and morphogenetic changes that are inaccessible to deep analysis in vivo. We found that pluripotent mouse embryonic stem cells (mESCs) form aggregates that upon embedding in an extracellular matrix compound induce the formation of highly organized “trunk-like structures” (TLSs) comprising the neural tube and somites. Comparative single-cell RNA sequencing analysis confirmed that this process is highly analogous to mouse development and follows the same stepwise gene-regulatory program. Tbx6 knockout TLSs developed additional neural tubes mirroring the embryonic mutant phenotype, and chemical modulation could induce excess somite formation. TLSs thus reveal an advanced level of self-organization and provide a powerful platform for investigating post-implantation embryogenesis in a dish.

Teratogenic and developmental toxic effects of etridiazole on zebrafish (Danio rerio) embryos

Etridiazole (EDZ), a thiadiazole-containing toxic chemical, is widely used as a fungicide. Regular usage of EDZ may reach and contaminate water bodies, but its adverse effects on aquatic vertebrates have not been well studied. Therefore, the present study aimed to evaluate the harmful effects of EDZ using zebrafish (ZF) (Danio rerio) embryos. ZF embryos were treated with 3.75, 7.5, 15, 30, and 60 mg/L of EDZ. Subsequently, mortality and developmental toxicities were quantified at 24, 48, 72, and 96 h post fertilization (hpf). The results showed that embryo mortality was concentration- and time-dependent. The median lethal concentration (LC50) of EDZ at 96-h was 25.58 ± 1.49 mg/L. Besides, EDZ induced a series of morphological deformities, including abnormal somite formation, abnormal eye pigmentation, abnormal tail morphology, tail kinks, skeletal malformations (lordosis, kyphosis, and scoliosis), and yolk sac edema in a concentration-dependent manner. Among the deformities, the most significant were reduced heartbeat and increased incidence of pericardial edema. The median effective concentration (EC50) of EDZ at 96-h was 17.93 ± 2.22 mg/L and the 96-h teratogenic index (TI) value was 1.52. Taken together, these results indicate that EDZ is a teratogen, and primarily affects the cardiovascular system of ZF.

Somite surface tension buffers imprecise segment lengths to ensure left-right symmetry

The body axis of vertebrate embryos is periodically segmented into bilaterally symmetric pairs of somites. The anteroposterior (AP) length of somites, their position and left-right symmetry are thought to be molecularly determined prior to somite morphogenesis. Here we discover that in zebrafish embryos, initial somite AP lengths and positions are imprecise and consequently many somite pairs form left-right asymmetrically. Strikingly, these imprecisions are not left unchecked and we find that AP lengths adjust within an hour after somite formation, thereby increasing morphological symmetry. We find that AP length adjustments result entirely from changes in somite shape without change in somite volume, with changes in AP length being compensated by corresponding changes in mediolateral length. The AP adjustment mechanism is facilitated by somite surface tension, which we show by comparing in vivo experiments and in vitro single-somite explant cultures with a mechanical model. Length adjustment is inhibited by perturbation of Integrin and Fibronectin, consistent with their involvement in surface tension. In contrast, the adjustment mechanism is unaffected by perturbations to the segmentation clock, thus revealing a distinct process that determines morphological segment lengths. We propose that tissue surface tension provides a general mechanism to adjust shapes and ensure precision and symmetry of tissues in developing embryos.

No Predictable Relationship Between Presacral Vertebral Number and Hes7 Intron Number Across Mammals

In mammals, the number of vertebrae and the somites they derive from is highly limited. Nevertheless, there are some lineages that have an increased number of presacral vertebrae and thus an elongated trunk. This suggests that somitogenesis, the process of somite formation in early development, is altered in these lineages. According the ‘clock and wavefront’ model of somitogenesis, temporal information of somite boundary formation is generated by a traveling wave of cyclic expression of oscillator genes. Hes7 has been suggested to be a key oscillator gene of this molecular segmentation clock. A previous study showed that reducing the number of introns within the Hes7 gene results in a more rapid tempo of Hes7 oscillation and an increased number of presacral vertebrae. Variation in Hes7 intron number could therefore be a potential evolutionary mechanism for varying vertebral number across mammals. In order to test this hypothesis, Hes7 intron number is here compared to presacral vertebral number across a variety of mammals.No significant relationship between both metrics could be detected as their variation across the mammalian phylogeny is fundamentally different. Integrating our data in the previously published mathematical model of Hes7 oscillation confirms the finding that variation in intron number does not predict variation in presacral vertebrae, rendering a direct causal relationship unlikely. However, our data support the previous suggestion that at least two introns are required for Hes7 pace making function of the segmentation clock.