Histosol

Adult Maritigrella crozieri were collected from the Florida Keys (Rawlinson, 2010 (link); Lapraz et al., 2013 (link)). They were fixed in a Petri-dish containing frozen 4% paraformaldehyde (diluted in phosphate buffered saline [PBS]) overnight at 4°C, rinsed in PBS (3 × 5 min, 5 × 1 hr washes) at room temperature and dehydrated in a step-wise ethanol series for histology and immunofluorescence, and in a methanol series for mRNA in situ hybridization. For histology, heads of adult worms (from the pharynx anteriorward) were dissected, cleared in histosol (National Diagnostics), and embedded in paraffin. Paraffin blocks were sectioned at 8–12 µm using a Leica (RM2125 RTF) microtome. Larval stages were fixed in 4% PFA in PBS for 20 min at room temperature, rinsed in PBS for five x 30 min washes and stored in 1% PBS-azide at 4°C for immunofluorescence, or dehydrated into 100% methanol and stored at −20°C for mRNA in situ hybridization.

Embryos were dissected in PBS and pieces including A1 were fixed in PEMFA (4 % formaldehyde in PEM buffer: 100 mM PIPES, 2.0 mM EGTA, 1.0 mM MgSO₄) at room temperature (RT) for 15–30 min, washed in PBT (PBS with 0.1 % Triton-X 100) and stored in ethanol at − 20 °C. Samples were cleared 3 × 10 min in Histosol (National Diagnostics) at RT, infiltrated with paraffin at 60 °C for 2–3 days and hardened in moulds at RT. Sections 6–8 μm thick were prepared on a Leica RM2125RTF microtome. Slides with sections were washed with Histosol, ethanol and re-hydrated to PBT. Slides were placed in a humidified chamber, blocked with 10 % sheep serum (Sigma-Aldrich) in PBT for 30 min at RT and incubated with Phospho-Histone H3 antibody (Invitrogen) diluted 1:130 at 4 °C overnight, Alexa Fluor 568 anti-rabbit secondary antibody (Invitrogen) diluted 1:300 at RT for 2 h and DAPI (Invitrogen) diluted 1:1000. Sections were imaged with a Leica TCS SP5 confocal microscope and photos processed using Fiji (https://fiji.sc).

Embryos were embedded in paraffin and sectioned at 7 μm as described in O’Neill et al., 2007 (link). Sectioned embryos were stained with Masson’s Trichrome as per Witten and Hall, 2003 (link) or Mayer’s Hematoxylin and Eosin by clearing in two rinses of Histosol (National Diagnostics), rehydration through serial dilutions of ethanol, staining for 15 min in Mayer’s Haematoxylin (Sigma), rinsing in running tap water for 20 min, rinsing briefly in 95% ethanol, and staining in 0.1% w/v Eosin Y (Sigma) in 95% ethanol for 2 min. Slides were then washed briefly in 95% ethanol, washed briefly with 100% ethanol, cleared with Histosol, and mounted with permount (Sigma).

The chicken Phox2b clone (Stanke et al., 1999 (link)) was a kind gift of Jean-François Brunet (Institut de Biologie de l'École Normale Supérieure, Paris, France). For in situ hybridization on paraffin wax sections, slides were de-waxed in Histosol (National Diagnostics) and rehydrated into diethylpyrocarbonate (DEPC)-treated (Sigma-Aldrich) PBS through a graded ethanol series. In situ hybridization was performed on sections as described (Miller et al., 2017 (link)).

Embryos were cleared with histosol (National Diagnostics) (20 min/wash, three times) at room temperature and transitioned into wax in a 1:1 molten paraffin:histosol solution (30 min/wash, twice) and placed in molten paraffin (RA Lamb Wax; Fisher Scientific) at 60°C overnight. Molten paraffin was then changed five times (each change lasting >1 h) before the tissue was transferred into a Peel‐A‐Way embedding mold (Sigma) for transverse sectioning. The embedded blocks were left to cool overnight and sectioned using a Leica RM2125 RTS microtome. Sections were mounted on Superfrost plus slides (VWR). The paraffin‐embedded sections were dewaxed in histosol (5 min/rinse, twice), 100% ethanol (5 min/rinse, twice) and grading into water through a series of descending ethanol concentrations (90%, 70%, and 50%, 5 min/rinse), followed by a final rinse in water (5 min). The slides were coverslipped with Fluoromount G containing DAPI (Southern Biotech) and cured overnight before imaging. The fluorescent micrographs were taken with Zeiss Axioscope A1 and combined into figure plates in Adobe Photoshop 2022.