M205 stereomicroscope

A Leica M205 stereomicroscope was used for the examination of mating cultures on agar plates. Fluorescence microscopy was performed using a Leica DM6000 microscope. Cell wall staining was conducted using calcofluor white M2R (0.0004%) and emission was detected using a DAPI filter cube. Images were overlaid using ImageJ software.

Complete cDNAs of the river lamprey nogginA, nogginB, nogginC and nogginD genes were obtained by RT‒PCR using the primer pairs given in [17 (link)].
The resulting DNA was cloned and inserted into the pCS2 vector. Synthetic capped mRNA was synthesized by the mMessage Machine SP6 kit (Ambion, Naugatuck, CT, USA) for SP6 RNA polymerase.
The mRNAs of the clawed frog noggin1, noggin2, cerberus, chordin and wnt8 genes were synthesized based on cDNAs obtained in previous studies [14 (link),15 (link)].
A microinjection method was used to deliver synthetic mRNAs into embryos. Synthetic mRNA (10–100 ng of mRNA in 1 μL) mixed with the fluorescent dye FLD (fluorescein lysine dextran) was microinjected into developing clawed frog and river lamprey embryos using an Eppendorf injector (Hamburg, Germany). Injections into embryos were carried out at the stage of 4–8 blastomeres in a 4% Ficoll solution to minimize potential damage from the injection.
Assessment of Xl_Noggin1_Myc and Lf_BMPa_Flag protein expression by coimmunoprecipitation was carried out according to the methods of [14 (link)]. In one sample, 30 river lamprey embryos preinjected with synthetic mRNA were lysed.
Embryos were photographed using a Leica M205 stereomicroscope (Wetzlar, Germany).

All experiments with animals were performed according to the protocol of the Institutional Animal Care and Use Committee (IACUC) approved by the Bioethics Commission of the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences (IBCH RAS) and handled in accordance with the Animals (Scientific Procedures) Act 1986 and Helsinki Declaration. For microinjection experiments, the Xenopus laevis embryos were obtained, fertilized in vitro and dejellied in 2% cysteine in 0.1 × MMR (Marc’s Modified Ringer) solution. The microinjections were performed in 4% Ficoll in 0.1 × MMR. The incubation of embryos at alkaline pH was performed in 0.1 × MMR solution adjusted by NaOH to pH 8.4. Upon reaching stage 26–27, embryos were photographed on Leica M205 stereomicroscope.

Illustrations and measurements were produced using a Leica M205 stereomicroscope. Measurements followed Sissom (1990) and are given in mm. Trichobothrial notations followed Vachon (1974) , and the morphological terminology mostly followed Hjelle (1990) . The terminology of metasomal carination followed Vachon (1952) , and the terminology of pedipalp chelal carinae followed Soleglad and Sissom (2001) . Type series of the new species are deposited in the Museum of Hebei University, Baoding, China (MHBU).

Mice were killed and tissues were removed and rinsed in phosphate-buffered saline (PBS) (Gibco BRL, France). Stereomicroscopic pictures were taken using a M205 stereomicroscope (Leica) equipped with a color DFC450c camera (Leica). Histological studies using either periodic acid-Schiff (PAS) or alcian blue (AB)–PAS and immunofluorescence studies using specific primary polyclonal anti-Muc5b antibody10 (link) and anti-GFP antibody (Ab290, Abcam, France) were performed as described previously10 (link). For immunohistochemistry of the nose and the middle ear, excess soft tissue was removed. The nose and the middle ear were decalcified in 10% EDTA for 6 weeks and in 12% formic acid for 3 weeks, respectively. Histological and immunofluorescence analyses were performed on a Leica DM4000B. High-quality captures in bright field and fluorescence were performed and digitized for the cervix on a Carl Zeiss Axio Scan. Z1 scanner and processed with ZEN software.

Fluorescent images were captured using either a Leica M205 stereomicroscope with metal-halide lamp (two objectives—0.63× and 1.0×, motorized zoom with 7.8—160 × magnification range) with a DFC 7000 T camera and the software Leica Applications Suite X, v. 3.3 (Leica Microsystems, Germany), or a Leica SP8 confocal laser microscope (with HPL APO CS2 40× oil, numerical aperture of 1.30, and objective lens working distance = 0.24 mm). For image editing ImageJ 1.52a (National Institutes of Health, Maryland, USA. https://imagej.nih.gov/ij/) software was used. For all experiments three biological replicates were executed. Statistical analysis was performed using one-way ANOVA with a post-hoc Tukey test. For all endpoints, p value < 0.05 was considered statistically significant and the GraphPad Prism software, version 6.01 (California, USA) was used. Data were displayed as mean ± standard error.