GoKawiilExperimental model and subject details
Zebrafish husbandry
Zebrafish were reared at 28.5 °C in 14–10-h light–dark cycles (conductivity of 1,000 μS, adjusted via Instant Ocean Sea Salt (approximately 30 g l−1), pH 7.0, adjusted using sodium bicarbonate)69. Zebrafish from 5 to 14 days post-fertilization were fed rotifers and used for experiments. All experiments complied with protocols approved by the Institutional Animal Care and Use Committee of Janelia Research Campus. Zebrafish sex cannot be determined until approximately 4 weeks post-fertilization70, so the sex of the experimental animals was unknown. Where relevant, fish were randomized across conditions.
No blinding was used in either data collection or analysis. Blinding during data collection was not possible because the experimental condition determined the acquisition protocol and was therefore necessarily known to the experimenter at the microscope. Blinding during analysis was not applied because all reported quantities were extracted by automated pipelines using identical parameters across conditions.
Danionella cerebrum husbandry
Danionella cerebrum were reared at 26.5 °C in 14–10-h light–dark cycles (conductivity of 450 μS, pH 7.5). Feeding protocols were adjusted according to age: (1) at 5–28 days post-fertilization, rotifers were administered once daily, (2) at 16–28 days post-fertilization, in addition to rotifers, GEMMA 75 was provided twice daily, and (3) at 29 days post-fertilization and beyond, the diet was composed of GEMMA 75 twice daily and Artemia once daily. Adult Danionella cerebrum were maintained in group housing with stock density of approximately 45 fish in 3.5-l tanks (Tecniplast). Fish younger than 6 weeks of age are sexually immature and could not be sexed; the sex of fish older than 6 weeks of age is mentioned in the main text. For egg collection, 10-cm-long custom-made acrylic tubes were used. All experiments complied with protocols approved by the Institutional Animal Care and Use Committee of Janelia Research Campus.
Zebrafish transgenics and transgenesis
Transgenic zebrafish were maintained in the Casper or Nacre background71. All lines were generated using the Tol2 system72 and genes were codon optimized using CodonZ73. Codon-optimized GCaMP7f and jRGECO1b were synthesized (Twist) and used for subsequent cloning. For cloning, restriction digest cloning was used throughout and all genes (GCaMP7f, jRGECO1b and tTA) were cloned with a preceding Kozak sequence and followed by an SV40 poly(A) signal sequence. Plasmids (150 ng μl−1), along with Tol2 transposase mRNA (50 ng μl−1), were co-injected (0.5 nl total injected volume) into one-cell stage embryos. Embryos were screened at 7 days post-fertilization for expression, and positive embryos were reared to maturity. At maturity, these adults were individually screened for dense expression in progeny, and the best founders were retained. We note that due to the non-deterministic landing site of the transgene, founders have variation in expression and need to be carefully screened for dense expression (Extended Data Fig. 12).
The ubi:tTA and TRE elements were obtained from multiple plasmids, a gift from D. Feliciano and I. Espinosa-Medina. These included a ubiquitous promoter containing vector or p5E-ubi7 (Addgene 27320), a vector containing the tTA advanced Tet-off transcriptional activator from pTet-Off Advanced Vector (631070, Takara) inserted into the multiple-cloning site of the pME entry vector74, and a vector containing the tetracycline-responsive element promoter p5E-TRE75. To generate the Tg(ubi:tTA;TRE:jRGECO1b) animals, the ubi:tTA;TRE elements were cloned and a codon-optimized jRGECO1b sequence placed downstream. To generate the Tg(ubi:tTA);Tg(TRE:GCaMP7f) animals, plasmids containing ubi:tTA and TRE:GCaMP7f were independently cloned and co-injected at equimolarity. To generate the Tg(foxj1a:GCaMP7f) animals, the foxj1a promoter was cloned (Addgene plasmid 163829)36 and a codon-optimized GCaMP7f sequence placed downstream using restriction digest cloning. To generate the Tg(elavl3:gtACR2-eYFP) transgenics, the promoter was cloned using a known elavl3 promoter sequence76 and gtACR2-eYFP sequence placed downstream77. To generate the Tg(β-actin2:mCherry-CAAX; myl7:GFP) transgenic line the bactin2 promoter was cloned (Addgene plasmid 82583)78 and the mCherry-CAAX sequence placed downstream. For optogenetic activation of motor vagal neurons, the transgenic lines Tg(VAChTa:Gal4)79 and Tg(UAS:CoChR-eGFP)jf44 (ref. 13) were utilized. To record activity of the sympathetic ganglia, the transgenic lines Tg(th:Gal4)42 and Tg(UAS:GCaMP6f)jf46 (ref. 13) were crossed and imaged. To quantify blood vessel diameter and track blood flow, the transgenic lines Tg(flk1:dsRED-CAAX)80 and Tg(gata1:dsRED)81 were imaged. To check the colocalization of neurons and astrocytes with the identified ‘brain-border’ population, and its location relative to the brain’s basement membrane, the transgenic lines Tg(elavl3:H2B-jRGECO1b)82, Tg(gfap:jRGECO1b)13 and TgBAC(lamC1:lamC1-sfGFP)83 were respectively used. Additional lines used for expansion microscopy were: Tg(isl1CREST-hsp70l:mRFP)84, Tg(phox2bb:eGFP)85 and Tg(foxj1a:eGFP)86.
Danionella cerebrum transgenics and transgenesis
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