Gastrula

The total RNA of 15 samples was extracted separately by Unizol reagent (Biostar, China) following the manufacturer's instructions, RNA were assessed by electrophoresis in 1% agarose gel and quantified by NanoDrop 1000 spectrophotometer (Thermo Scientific, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, USA). Afterwards, the RNA samples of zygote, blastula, gastrula, limb bud embryo and larva in membrane were mixed equivalently into embryo sample (E), the RNA samples of NI, NIII and NVI were mixed equivalently into nauplius sample (N), the RNA samples of ZI, ZII and ZIII were mixed equivalently into zoea sample (Z), the RNA samples of MI, MII and MIII were mixed equivalently into mysis sample (M) and the RNA samples of postlarvae 1 were considered as postlarvae sample (P). The sample mixture was based on both morphological classification and physiological characters (Figure 1). Samples of zygote, blastula, gastrula, limb bud embryo and larva in membrane were typical stages before hatching. They mixed into E sample and represented the features of embryo in membrane. Similarly, NI, NIII and NVI composed N sample which represented nauplius stage, ZI, ZII and ZIII composed Z sample which represented zoea stage and MI, MII and MIII composed M sample which represented mysis stage. Then the five mixed RNA samples were used for library construction and sequencing.

Ripe animals of the Mediterranean amphioxus species (B. lanceolatum) were collected from Argelès-sur-Mer (France), and gametes were obtained by heat stimulation [13] (link), [14] (link) (note that no specific permits were required for this study). B. lanceolatum embryos at different developmental stages (eight-cell embryos, blastula, gastrula, early neurula, mid-neurula, late neurula, neurula before the mouth opening and larva stages), as well as ripe adults, were frozen in liquid nitrogen. Total RNA was extracted using the RNeasy Plus Mini Kit (QIAGEN) after disrupting and homogenizing the sample with TissueLyser (QIAGEN). A mix of 25 µg of total RNA was used for the cDNA library construction by GATC Biotech SARL.

Wild type adult animals (AB strain) were bred in a recirculating aquaculture system (Techniplast ZebTEC, Buguggiate, VA, Italy), maintained at 28.5 °C in a 14 h light and 10 h dark daily cycle, at the Zebrafish Facility of the University of Brescia, essentially as previously described by Zizioli and colleagues [29 (link)]. Adult male and female fish were mated in the mating box overnight. Freshly spawned eggs were collected the next morning, washed with fresh zebrafish water (0.1 g/L Instant Ocean Sea Salts, 0.1 g/L Sodium Bicarbonate, 0.19 g/L Calcium Sulfate), and maintained at 28 °C in Petri dishes containing fresh fish water in 14–10 h light-dark cycle until exposure to CAB. Embryo staging was done as described by Kimmel and colleagues [30 (link)]. All experiments were conducted exposing embryos to tested solutions at the gastrula stage (4 hpf) up to 48, 72, 96, 120, or 144 hpf, as described in each experiment.

Stage 9 Xenopus laevis blastula, >55 per condition, were microinjected into the blastocoel with different concentrations of purified BMP ligand homodimers or heterodimer: 0.05 pmol in 4.3 nL, 0.15 pmol in 13nL, 0.5 pmol in 40nL, 1.5 pmol in 120nL or with 10mM HCl vehicle control at the same volumes. Dose-response experiments measuring embryo survival were performed to determine the minimal effective dose of BMP ligand homodimers or heterodimers for subsequent biological assays (Fig. 3A). Embryos were permitted to develop until stage NF37, and then blindly scored using the dorso-anterior index (DAI) to determine the extent of ventralization caused by excessive BMP signaling [49 (link), 82 (link)]. In the DAI scoring system, 5 is a normally developed tadpole and increasing ventralization corresponds with a progressively lower score down to 0 for a fully ventralized blastula (Fig. 3) [49 (link)]. Injected Xenopus blastula were compared to uninjected controls. DAI scoring is reported for the 0.15 pmol and 0.5 pmol doses.
An additional 20 embryos per condition were injected and fixed at gastrula stage NF10, bisected, and assayed by in situ hybridization to determine the expansion of ventx1/2 expression, which are well-known direct BMP/SMAD1 target genes as described previously [82 (link), 83 (link)]. In situ hybridization results are reported for embryos injected with 0.15 pmol and 0.05 pmol doses. All Xenopus experiments were performed in compliance with ethical regulations outlined by the NIH and institutional guidelines under Cincinnati Children's Hospital Medical Center Institutional Animal Care and Use Committee (IACUC) approved protocol IACUC2022-0026, approved 7/22/2022.
Experiments with <50% survival of control embryos were excluded. DAI scoring experiments were performed a minimum of 3 independent times while in situ hybridization staining was performed once. Data were analyzed by 1W-ANOVA or Chi-square test in Prism 9 (GraphPad). Graphs were created in Prism 9.