Mmachine sp6 kit

The cd146-MO (5′-AGCAGTGCGGTGTAGGTCATTTCTC-3′) and vegfc-MO (5′-GAAAATCCAAATAAGTGCATTTTAG-3′) were used as previously reported^{25 (link)}. For mRNA synthesis, human vegfc full-length coding sequence cDNA (CDS) was cloned into the pCS2 + vector. Capped mRNA was synthesized using the mMACHINE SP6 Kit (Ambion). For overexpression of cytoplasmic domain-deleted CD146 under the hsp-70 promoter, the full-length CDS of zebrafish cytoplasmic domain-deleted cd146 was cloned into pDONR221 vector by BP reaction then was subcloned into a vector with hsp-70 promoter and GFP reporter by LR reaction (MultiSite Gateway Technology, Invitrogen). MOs, mRNAs and hsp70-cd146∆C-GFP plasmid were injected into one-cell stage zebrafish embryos at the yolk/blastomere boundary.

pCS2+ plasmids were linearized with NotI-HF^R(NEB), purified by gel extraction and then used as templates for in vitro transcription. In vitro transcription of mRNAs was performed using the mMachine SP6 kit (Ambion). Synthesized mRNA was purified by LiCl precipitation. Plasmid and mRNA probes were microinjected together or separately into four-cell stage embryos with a single injection into each ventral blastomere. mRNA constructs were injected in 10 nl single injections as follows: LifeAct-GFP, 6 ng/µl; H2B-RFP, 20 ng/µl; LSR-3xGFP, 10 ng/µl; SF9-3xGFP, 8 ng/µl; CA-MYPT, 100 ng/µl. For MCC-specific labeling of the actin cortex, pα-tubulin LifeAct-GFP and pα-tubulin LifeAct-RFP were injected. For MCC-specific expression of LSR, pα-tubulin angulin-1-GFP was injected. For goblet cell-specific labeling of the actin cortex, pNectin Utrophin-RFP. For goblet cell-specific expression of LSR, pNectin LSR-GFP and pNectin LSR-GFP were injected. All plasmids were injected at 7.5 ng/µl. Unless specified, LSR labeling is performed by expressing LSR-3xGFP mRNA. For LSR knockdown experiments, 23.8 ng of LSR MO#1 and 40 ng of LSR MO#2 were injected (Gene Tools). For LSR MO and CA-MYPT experiments, the LSR MOs and CA-MYPT were co-injected with the fluorescent nuclear marker H2B-RFP at the 16- and 32-cell stages to mosaically target integrating MCCs or the superficial epithelium.

The cDNAs of SlPIP2;1, SlPIP2;7 and SlPIP2;5 were subcloned into pCS107 vector using the restriction sites Xhol I and EcoR I. Capped cRNA transcripts were synthesized in vitro with mMACHINE SP6 Kit (Ambion) with Xba I linearized vector. Xenopus laevis oocytes of stages V and VI were isolated and defolliculated by digestion at room temperature for 1 h with 2 mg/ml collagenase A (Sigma) in ND96 solution (96 mM NaCl, 2 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂and 5 mM Hepes-NaOH, pH 7.4, 220 mosm/kg). A 50 nl volume of in vitro transcripts (50 ng) of the target gene, using the same volume of distilled water as negative control, was injected into the oocytes. Non-injected oocytes were also analyzed as a negative control. The oocytes were incubated at 18 °C for 48 h in ND96 solution supplemented with 10 μg/ml penicillin and 10 μg/ml streptomycin (Fig. S1). To measure the osmotic water permeability coefficient (Pf), a single oocyte was transferred to 5-fold diluted ND96 solution. Oocyte volume (V) was calculated from the measured area of each oocyte. The osmotic permeability coefficient (Pf) was calculated for the first 5 min using the formula Pf = V0[d(V0/V)/dt]/[S0 × VW(Osmin-Osmout)], with an initial volume (V0) of 9 × 10⁻⁴ cm³, an initial oocyte surface area (S0) of 0.045 cm², and a molar volume of water (V/V) of 18 cm³/mol39 (link).

To generate transgenic animals, wild‐type (WT) embryos were co‐injected with 50 ng·mL⁻¹ of Tol2(tg:mCherry) vector (donated by Opitz R) and 40 ng·mL⁻¹ of Tol2 transposase mRNA at the one‐cell stage. Capped mRNA encoding for Tol2 transposase was generated by in vitro transcription using an mMachine SP6 Kit (Ambion, San Diego, CA, USA) and NotI‐linearized pCS‐zT2TP plasmid [24] as a template. Injected embryos were examined with epifluorescence microscopy for mCherry expression in thyroid tissue using a confocal microscope (Nikon A1, Tokyo, Japan). Embryos displaying mosaic mCherry expression in the thyroid region were allowed to grow to adulthood. Individual F0 founders transmitting a robust and thyroid‐specific reporter signal to their progeny were identified by outcrossing WT animals.

Zebrafish embryos were injected using glass capillary needles (30-0020, Harvard Apparatus, MA, USA), which were pulled by a needle puller (P-97, Sutter Instrument) and attached to a microinjector system (PV820, World Precision Instruments). Microinjections of mRNAs and morpholinos (MOs) were performed at the one-cell stage. mRNAs were synthesized using mMACHINE SP6 kit (Ambion). The following mRNAs were injected: 70 pg membrane RFP (Iioka et al., 2004 (link)), 70 pg H2B GFP (Keller et al., 2008 (link)), 200 pg CAMypt1 (Schwayer et al., 2019 (link)), and 72 pg chk1 (Shamipour et al., 2019 (link)). The following MOs were injected: 4 ng e-cadherin MO, 4 ng control MO for e-cadherin (Babb and Marrs, 2004 (link)). To induce mesodermal progenitor cell fate, the following combination of mRNAs and MOs was injected: 100 pg ndr2l cyclops mRNA and 2 ng casanova MO (Krieg et al., 2008 (link)). To label the interstitial fluid, 1 nL of 0.6 mg/ml dextran Alexa Fluor 647 (10,000 MW; D22914, Invitrogen) was injected in the blastoderm of 1k-stage embryos (∼3 hpf).