E18 sprague dawley rat embryos

All animal procedures were approved by the University of Maryland Animal Use and Care committee. Dissociated hippocampal neuron cultures were prepared from both male and female E18 Sprague Dawley rat embryos (Charles River) as described previously (Dharmasri et al., 2023; Divakaruni et al., 2018 (link)). Neurons were plated on glass coverslips (18 mm #1.5, Warner Instruments) coated with poly-l-lysine at 30,000 cells per coverslip. Cells were grown in Neurobasal A + GlutaMax, gentamycin, and B27 supplement, with FUDR added at 1 week to suppress glial growth. Cells were fixed between 13–15 DIV for most experiments, between 6–8 or 20–22 DIV for developmental comparisons, and at 21 DIV for experiments with bassoon labeling.

Primary cortical neurons were established from embryonic day-18 (E18) Sprague–Dawley rat embryos (Charles River Labs, NY, USA). An eighteen day timed pregnant rat was euthanized using CO₂ and pups were removed, decapitated and the cortex was dissected in Hibernate-E media (Brain Bits LLC, IL, USA). Dissociated cortical neurons were obtained by incubating the cortex in EBSS containing 15 units/mL of papain (Worthington Biochemicals, NJ, USA) for 45 min at 37°C before triturating in neurobasal medium containing 20% fetal bovine serum (Hyclone, UT, USA), DNAse (0.2 mg/mL). Undissociated neurons were removed from the cell suspension by passing the cell suspension through a 40 µm cell strainer (Fisher Scientific, NY, USA). Neurons were centrifuged at 2000 g for 3 min at 20°C and the pellet was resuspended in neurobasal medium supplemented with B27, penicillin (100 U/mL), streptomycin (100 U/mL) and L-glutamine (0.5 mM, Invitrogen, NY, USA). Neurons were then plated at a density of 150 000 cells/mL on circular glass coverslips and 6-well tissue culture dishes, coated with poly-L-lysine (50 µg/mL, Sigma Chemicals, MO, USA), and incubated in a humidified atmosphere containing 5% CO₂: 95% O₂ at 37°C.

HEK293, HeLa, and SH‐SY5Y cells (ATCC) were cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma) supplemented with 10% FBS (Biosera) and 1 mM sodium pyruvate (Sigma) in a 5% CO₂ atmosphere at 37°C. Cells were transfected with plasmid DNA using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions or polyethylenimine (PEI) (stock 1 mM; 3 μl/μg plasmid). HeLa, HEK293, and SH‐SY5Y cells were siRNA transfected using Lipofectamine RNAiMax or Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Cells were used for experiments 4 days after siRNA transfection.
Cortical neurons were isolated from E18 Sprague Dawley rat embryos (Charles River) and cultured on glass coverslips coated with poly‐L‐lysine in 12‐ or 24‐well plates in neurobasal medium supplemented with B27 supplement (Invitrogen), 100 IU/ml penicillin, 100 mg/ml streptomycin, and 2 mM L‐glutamine. Cortical neurons (DIV5–6) were transfected using Lipofectamine LTX with PLUS reagent according to the manufacturer's instructions (Thermofisher). Briefly, neurons were placed in fresh culture medium with a DNA:PLUS:LTX ratio of 1:0.5:0.5 (0.5–1 μg DNA/100,000 cells/cm²). After 6 h, the transfection mix was replaced with conditioned medium. For lentiviral transduction, neurons were exposed to 4TU/cell in fresh culture medium overnight.

Wild type C57BL6/N (C57BL/6NTac, 8–10 weeks old) and Swiss-Webster (8–10 weeks) mice were purchased from Taconic. The UBC-GFP (C57BL/6-Tg(UBC-GFP)30Scha/J, 8–13 weeks old; JAX #004353)(Schaefer et al., 2001 (link)) and R26R–YFP (B6.Cg–Gt(ROSA)26Sor^{tm3(CAG−EYFP)Hze}/J; JAX #007903) (Madisen et al., 2010 (link)) mice were obtained from the Jackson Laboratory. The Nkx3-1^Cre allele has been previously described (Lin et al., 2007 (link); Thomsen et al., 2008 (link)). As hosts for renal grafts, R2G2 mice (B6;129- Rag2^tm1FwaIl2rg^tm1Rsky/DwIHsd, 8–15 weeks old) were purchased from Envigo, and NOD/SCID mice (NOD.Cg-Prkdc^scid/J, 8–14 weeks old; JAX #001303) were purchased from the Jackson Laboratory. To obtain urogenital mesenchyme for tissue recombination and renal grafting, we used E18 Sprague-Dawley rat embryos from timed matings (Charles River #400). Animal studies were approved by and conducted according to standards set by the Columbia University Irving Medical Center (CUIMC) Institutional Animal Care and Use Committee (IACUC).

The use of animals and all surgical procedures described in this article were carried out according to The guide to the Care and Use of Experimental Animals of the Canadian Council on Animal Care. The ethical approval was obtained from the Animal care and ethics committee of the Centre de Recherche du Centre Hospitalier de l’Université de Montréal (CRCHUM) (protocol number N14002 NLs). Primary cortical cultures were prepared from E18 Sprague Dawley rat embryos (Charles River, Montréal, Québec, Canada). To collect the embryos, the rat was euthanized in a CO₂ chamber. The cerebral cortices were treated with trypsin (0.025% at 37°C for 20 min). The reaction was stopped with trypsin inhibitor solution containing DNAse. Neurons were dissociated by several passages through a Pasteur pipette. The cells were then plated either on glass coverslips or on culture dishes coated with poly-D-lysine (Sigma, Oakville, ON, Canada). The neurons were maintained in neurobasal medium (Invitrogen, Burlington, ON, Canada) supplemented with glutamax (Invitrogen) and B27 (Invitrogen). HeLa cells (ATCC, Manassas, VA, USA) were cultured in EMEM supplemented with L-glutamine (ATCC, Manassas, VA, USA) and with 10% foetal bovine serum (Hyclone) (Thermo scientific) at 37°C in a humidified 5% CO₂ incubator.