csf1r CRISPants phenocopy existing csf1r microglia mutants. (A) Neutral Red (NR⁺) images and quantification of wild-type (WT), csf1ra^−/− and csf1ra CRISPant zebrafish larvae at 3 dpf. (B) Indel spectrum of a pool of csf1ra CRISPants calculated by TIDE. (C) NR images and quantification of WT and spi1b CRISPant zebrafish larvae at 3 dpf. (D) Indel spectrum of a representative individual spi1b CRISPant calculated by TIDE. The R² value represents reliability of the indel spectrum. ***P<0.001. One-way ANOVA and Student's t-test. Each dot represents one larva. Error bars represent s.d.

SpotNGlia semi-automatically counts microglia numbers. (A) Examples of z-stack images of NR-stained larvae and a schematic representation of the SpotNGlia analysis pipeline. (B) SpotNGlia output of test dataset with both manual (blue) and automated (red) brain segmentation and NR⁺ microglia annotation. (C) Box plots showing Jaccard and Dice indices for accuracy of brain segmentation and F1, precision and recall scores for the accuracy of NR⁺microglia annotation. This is a Tukey boxplot: it uses the median and interquartile range (IQR) for the box. The whiskers extend to the most extreme data within 1.5×IQR. Data outside 1.5×IQR are considered outliers. (D) Correlation between manually and automated microglia quantification after manual correction for segmented brain area. Error bars represent s.d.

Reverse genetic screen reveals zebrafish il34 as a regulator of microglia development. (A) Accumulated data from all gRNA injections showing the number of NR⁺microglia as quantified with SpotNGlia. Magenta bars represent genes showing a significant reduction in microglia numbers upon CRISPR/Cas9-based targeting (black bar, control; green bars, genes with non-significant reduction in microglia numbers). (B) NR⁺ microglia numbers in 3 dpf zebrafish larvae injected with gRNA-Cas9 RNPs targeting il34. Controls in A and B are non-injected WT larvae. (C) A −5 bp deletion in exon 1 of il34 directly introduces a stop codon. (D) NR⁺microglia numbers in il34 mutants with a premature stop codon in exon 5 and their heterozygous and WT siblings at 3 dpf. (E) GFP⁺ microglia in the optic tecti (dashed lines) of 3 dpf il34 mutants and controls, and quantification of their numbers and the fraction of microglia containing more than one protrusion (ramified microglia). Controls in D and E are WT (il34^+/+) larvae. *P<0.05, **P<0.01, ***P<0.001. One-way ANOVA and Student's t-test. Bonferroni correction for multiple testing. Scale bars: 100 µm. Each dot represents one larva. Error bars represent s.d.

Il34 does not affect proliferation but does affect the distribution of YSMs to target organs. (A) NR⁺ microglia numbers in il34 mutants and their heterozygous and WT siblings at 5 dpf. (B) EdU/L-plastin staining of microglia in the optic tecti (dashed lines) of 4 dpf il34 mutants and WT controls, and quantification of microglia numbers, EdU⁺ microglia numbers and the fraction of EdU⁺ microglia among total numbers. (C) In vivo imaging of GFP⁺macrophages located on the yolk sac (dashed lines) in il34 mutants and WT controls, transgenic for mpeg1-GFP, and quantification at 48 hpf. YSMs with more than one protrusion were counted as branched YSMs. (D) In vivo imaging of mpeg1-GFP⁺ macrophages located in the head region (dashed lines) in il34 mutants and WT controls, and quantification at 48 hpf. (E) In vivo imaging of GFP⁺ macrophages located in the head region (dashed lines) in il34 mutants and WT controls, and quantification at 3 dpf. a, outline of the head region. (F) In vivo imaging of mpeg1-GFP⁺macrophages located in the tail (dashed lines) in il34 mutants and WT controls, and quantification. b, outline of the embryonic region/trunk region. Scale bars: 100 µm. *P<0.05, **P<0.01, ***P<0.001. One-way ANOVA and Student's t-test. Each dot represents one larva. Error bars represent s.d.