NAD de novo synthesis is performed by the yolk sac during organogenesis and later by the embryonic liver.

(A) Schematic overview of the NAD synthesis pathways. The NAD de novo Synthesis Pathway (blue box) converts the essential amino acid L-tryptophan to NAD and is predominantly performed by the liver (Liu et al., 2018). The Preiss-Handler Pathway (purple box) converts nicotinic acid to NAD in the kidney. The Salvage Pathway (orange box), performed in all cells, salvages the product of NAD consuming enzymes, nicotinamide (NAM), back to NAD. When NAD levels are in excess, NAM is converted to 1-methylnicotinamide (1MNA), N-methyl-2-pyridone-5-carboxamide (2PY) and N-methyl-4-pyridone-5-carboxamide (4PY) and excreted. Enzymes are in red, metabolites in black. (B) Re-analysis of published scRNA-seq data (Mu et al., 2020) via DotPlot showing average log normalised expression (heatmap colour) and proportion of cells (circle size) of requisite NAD de novo Synthesis Pathway Genes in the hepatocyte lineage between embryonic day (E) 9.5 and E15.5. (C) HAAO enzyme activity in embryonic liver from E12.5 to E17.5. (D-H) Concentration of core NAD de novo Synthesis Pathway metabolites in the embryonic liver (E14.5) and yolk sac (E10.5-14.5). Metabolite concentrations are normalised to wet weight measured at dissection. (I,J) HAAO enzyme activity in placenta (I) and visceral yolk sac (J) samples collected between E11.5 to E17.5. Haao+/+ (black circle), Haao+/- (blue square) and Haao-/- (red triangle). Bars indicate the mean ± standard deviation. See Table S1 for HAAO activity numerical values and Tables S2 and S3 for metabolite concentration values, including those of additional NAD-related metabolites. 3HK = 3-hydroxykynurenine, 3HAA = 3-hydroxyanthranilic acid, NAMN = nicotinic acid mononucleotide, KA = kynurenic acid, AA = anthranilic acid, XA = xanthurenic acid, QA = quinolinic acid, NaMN = nicotinic acid mononucleotide, NaAD = nicotinic acid adenine dinucleotide, NADP = nicotinamide adenine dinucleotide phosphate, NMN = nicotinamide mononucleotide, <LOD = below the limit of detection.

The yolk sac performs NAD de novo synthesis from E10.5 onwards. (A-B) Heatmap (A) and bar graphs (B) showing changes in core NAD de novo NAD Synthesis Pathway gene expression in Haao+/- visceral yolk sacs between E10.5 and E17.5 quantified by RT-qPCR (n = 4). Expression is calculated as log2 fold-changes relative to gene expression at E10.5 and normalised to two housekeeping genes, Ubc and Ywhaz. Bars indicate the mean ± standard deviation. Statistical significance was determined by one-way ANOVA. (C-D) NAD-related metabolite concentrations in visceral yolk sacs at embryonic day (E) 10.5, E12.5 and E14.5 were compared to metabolite concentrations in E14.5 embryonic livers. (C) Principal component analysis (PCA) of the concentrations of NAD and 11 related metabolites in the three tissues at each embryonic stage. Embryonic liver (green) and yolk sac (light blue, indigo, purple). The primary determinants of variance in the NAD metabolome were gestational age (PC 1) and Haao genotype (PC 2). (D) Heatmap showing wet weight normalised, log10 transformed, and Pareto scaled concentration of each quantified metabolite across all samples, tissues and timepoints. Consistent with the PCA, the metabolite profile of E10.5 yolk sacs clustered to that of E14.5 embryonic liver. Hierarchical clustering was performed using Euclidean as distance measure and Ward as a clustering method. See Tables S2 and S3 for numerical metabolite concentration values.

Congenital NAD Deficiency Disorder (CNDD) phenotypes and NAD deficiency can be induced in Haao-/- embryos by restricting maternal NAD precursor supply for three days. (A) Schematic overview of the dietary treatment to induce NAD deficiency and embryo malformations. Pregnant mice were provided Breeder diet, rich in L-tryptophan and vitamin B3, throughout gestation except for a three-day window between embryonic day (E) 7.5 and E10.5 during which feed lacking any NAD-precursor and drinking water supplemented with 1 g/L L-tryptophan and either 15 mg/L (NW15) or 3.5 mg/L (NW3.5) nicotinic acid was given. Red arrows indicate tissue collection timepoints. (B) Weights of Haao+/- (square) or Haao-/- (triangle) embryos at embryonic day (E) 18.5 collected from mothers on NW3.5 diet. Weights represent wet weight measured at dissection. Statistical significance was determined by Student’s t-test with**** = p<0.0001. Bars indicate the mean ± standard deviation. (C) Summary of the phenotypic outcomes of E18.5 embryos generated from the NW3.5 diet condition. Each bar represents a litter, with the graph on the left summarising data of entire litters and the other two separating the Haao+/- and Haao-/- embryos of each litter. Colours indicate the number of different congenital malformations observed in each embryo. Dead embryos (black) represented early resorptions and could not be genotyped.

The yolk sac NAD metabolome is affected by maternal dietary limitation of NAD precursors that causes embryo malformations. (A-G) Concentration of NAD-related metabolites in E10.5 yolk sacs normalised to total protein content. (A) L-tryptophan (TRP), (B) 3-hydroxyanthranilic acid (3HAA), (C) quinolinic acid (QA), (D) nicotinic acid mononucleotide (NAMN), (E) nicotinic acid adenine dinucleotide (NAAD), (F) NAD+, (G) nicotinamide (NAM). (H) Wet weight of E10.5 embryos from the NW15 (purple) and NW3.5 (orange) diet conditions, measured at dissection. Bars indicate the mean ± standard deviation. The Haao genotype and diet are indicated below each graph. Statistical significance in (A-G) was calculated by one-way ANOVA with Tukey’s multiple comparisons test with ns = not significant, * = p<0.05, ** = p<0.01, and **** = p<0.0001. Statistical significance in (H) was calculated by one-way ANOVA with the p value indicated above graph. <LOD = below the limit of detection. For numerical values, see Table S5.

Perturbation of yolk sac NAD metabolome at E10.5 correlates with metabolic alterations in the embryo. (A-H) Concentration of NAD-related metabolites in E10.5 embryos normalised to total protein content. (A) 3-hydroxyanthranilic acid (3HAA), (B) quinolinic acid (QA), (C) nicotinic acid mononucleotide (NAMN), (D) nicotinic acid adenine dinucleotide (NAAD), (E) NAD+, (F) nicotinamide (NAM), (G) N-methyl-4-pyridone-5-carboxamide (4PY), (H) N-methyl-2-pyridone-5-carboxamide (2PY). Bars indicate the mean ± standard deviation. Statistical significance in (A-H) was calculated by one-way ANOVA with Tukey’s multiple comparisons test with ns = not significant, * = p<0.05, ** = p<0.01, *** = p<0.001, and **** = p<0.0001. <LOD = below the limit of detection. For numerical values, see Table S6. (I) Heatmap showing significant Pearson’s correlation (p < 0.01; −0.45 < r > 0.45) between normalised metabolite levels in E10.5 yolk sacs and embryos. Correlations that did not reach significance are denoted as light blue coloured squares. (J, K) Correlation of 3HAA (J) and NAM (K) concentration in corresponding E10.5 yolk sac and embryo samples. The Pearson correlation coefficient (r) and coefficient of determination (r2) are indicated in each graph. Each datapoint represents an E10.5 embryo and corresponding pooled yolk sac sample.

The extraembryonic endoderm and subsequent yolk sac endoderm express NAD de novo Synthesis Pathway genes before expression commences in the embryonic liver, in both mouse and human. (A) t-SNE projection of re-analysed single-cell RNA-seq data from pooled E9.5 and E10.5 visceral yolk sacs previously generated by Zhao and Choi (Zhao and Choi, 2019). (B) DotPlot showing average expression (heatmap colour) and proportion of cells (circle size) of essential NAD de novo Synthesis Pathway genes in E9.5/E10.5 yolk sac populations. (C) DotPlot for essential NAD de novo Synthesis Pathway genes in the extraembryonic endoderm (ExE) population of the mouse gastrulation atlas (Pijuan-Sala et al., 2019) from E6.5 to E8.5. (D) Schematic timeline comparing organogenesis length and yolk sac duration during mouse and human development. Stages and equivalences are approximations based on figures from https://hdbratlas.org/comparison-HvM.html and published staging criteria (Kaufman, 1992, O’Rahilly and Müller, 2010) (E) Heatmap showing z-normalised expression of NAD de novo Synthesis Pathway genes in re-analysed bulk RNA-seq samples for individual human yolk sacs, published in Cindrova-Davies et al. (Cindrova-Davies et al., 2017). (F) DotPlot for requisite NAD de novo Synthesis Pathway gene expression in human yolk sac populations between 3-8 post-conceptional weeks (PCW). scRNA-seq data acquired from Goh et al. (Goh et al., 2023) and re-analysed. (G) DotPlot of pathway gene expression in human yolk sac endoderm population between 3-8 PCW. (H) DotPlot of human hepatocyte lineage previously published in Wesley et al. (Wesley et al., 2022). ExE endoderm = extraembryonic endoderm, CS = Carnegie Stage, HB = hepatoblast, FH = foetal hepatocytes, AH = adult hepatocytes.

RidgePlots show that the time of onset, expression level, and proportion of hepatocyte-lineage cells expressing NAD de novo synthesis genes varies, including for Kmo (A), Kynu (B), Haao (C) and Qprt (D) from E9.5 to E15.5. Data previously generated in Mu et al. (Mu et al., 2020).

NAD de novo Synthesis Pathway activity is absent from the mouse placenta. DotPlot showing proportion of cells (circle size) expressing requisite pathway genes in different placental cell populations at E9.5 (black), E10.5 (yellow), E12.5 (blue) and E14.5 (orange) using scRNA-seq generated by Marsh and Blelloch (Marsh and Blelloch, 2020). Placental populations were clustered by lineage: junctional zone trophoblast (yellow), labyrinth layer trophoblast (orange), endothelium (pink), mesenchyme (purple), immune/blood cells (indigo), and maternal decidual stroma (brown).

NAD de novo Synthesis Pathway gene expression is negligible in the E10.5 embryo. DotPlot shows the proportion of cells (circle size) expressing requisite pathway genes in E10.5 embryo cell populations. scRNA-seq generated as part of the ‘mouse organogenesis cell atlas’ (MOCA) by Cao et al. (Cao et al., 2019) and analysed using ShinyCell (Ouyang et al., 2021). Gene expression data is not scaled.

HAAO enzyme activity in embryonic liver, yolk sac, and placenta at different stages in gestation.

NAD metabolite concentrations in embryonic liver at E14.5 by Haao genotype, as measured by UHPLC-MS/MS.

NAD metabolite concentrations in the visceral yolk sac at E10.5, E12.5 and E14.5 by Haao genotype, as measured by UHPLC-MS/MS.

Summary of congenital malformation types and incidence in embryos at E18.5 arising from maternal NW3.5 diet provision between E7.5 and E10.5.

NAD metabolite concentrations in the E10.5 embryo and yolk sac with maternal NW15 or NW3.5 diet provision, normalised by protein.

NAD metabolite concentrations in the E10.5 embryo and yolk sac with maternal NW15 or NW3.5 diet provision, normalised by wet weight.

Primers for RT-qPCR

Summary of published RNA-seq datasets used and assessed in this study