Fig. 6

Stable foxp1b zebrafish mutants have hypertrophic adipose but undergo severely reduced hypertrophic remodelling in response to a high-fat diet (HFD). (a) Phylogenetic tree showing relatedness of zebrafish Foxp1a and Foxp1b amino acid sequences to human, mouse, opossum, and coelacanth Foxp1. Scale bar indicates substitutions per site. (b) Overview of human FOXP1 domain structure showing polyglutamine (polyQ), coiled-coil, and forkhead domains. Zoomed view of the DNA-binding forkhead domain showing structural features, including helices (helices 1–5) and beta-strands (s1–s3). Amino acids involved in DNA binding are highlighted in blue; residues at the FOXP domain-swapped dimer interface are highlighted in orange. Wild-type zebrafish Foxp1a and Foxp1b sequences are aligned to human FOXP1, along with the ed116 (foxp1a) and ed125 (foxp1b) mutant alleles. Grey boxes indicate the addition of nonsense peptide sequence followed by a premature stop codon. (c) Western blot showing reduction of Foxp1 protein in foxp1a;foxp1b double mutants compared to wild-type. β-Actin serves as a loading control. Asterisk indicates the Foxp1 band. (d) Nile Red fluorescence images showing adipose lipid distribution (black signal) in wild-type, foxp1aed116, foxp1bed125, and double foxp1aed116;foxp1bed125 zebrafish mutants. e, eye. Asterisk indicates lipid accumulation within the liver in double mutants. Scale bar is 1 mm. (e) Violin plots of fish size (standard length, mm) in foxp1aed116, foxp1bed125, and double foxp1aed116;foxp1bed125 mutants compared to wild-type siblings. (f) Violin plots of normalised adipose area in the same genotypes. (g) Violin plots of average lipid droplet (LD) diameter in the same genotypes. (h) Schematic of the HFD feeding experiment. Zebrafish were Nile Red-imaged at 35 days post fertilisation (dpf) to establish baseline adipose measurements, then subjected to a 14-day HFD (2 hr daily immersion in 5% chicken egg yolk) or control diet (2 hr daily immersion in system water), in addition to normal feeding. Post-diet Nile Red imaging was performed at 49 dpf. Subcutaneous adipose tissue was divided into anterior-posterior strata for spatial analysis. 200 μm strata are numbered anterior (1) to posterior. (i) Segmented subcutaneous adipose LDs from representative wild-type, foxp1a, and foxp1b fish on control diet (left) and after HFD (right), colour-coded by LD diameter. Strata boundaries (200 μm) are indicated by dashed lines. (j) Average LD diameter per stratum for wild-type and foxp1b fish on control diet (grey or salmon) and HFD (black or pink). Thin lines represent individual fish; thick lines represent group means. (k) HFD effect on LD diameter per stratum (HFD minus control diet) for wild-type (black) and foxp1b (pink). Filled circles indicate strata where the HFD effect is significant (BH-adjusted p<0.05). (l) As in (J), comparing wild-type and foxp1a. (m) As in (k), comparing wild-type (black) and foxp1a (teal). Statistical tests in (e–g) were one-way ANOVA followed by Tukey’s HSD post hoc test. **p<0.01, ***p<0.001, ns = not significant.