One crisis, diverse impacts—Tissue-specificity of folate deficiency-induced circulation defects in zebrafish larvae

<div><p>Folate (vitamin B9) is an essential nutrient required for cell survival, proliferation, differentiation and therefore embryogenesis. Folate deficiency has been associated with many diseases, including congenital heart diseases and megaloblastic anemia, yet the mechanisms underlying these remains elusive. Here, we examine the impact of folate deficiency on the development of the circulation system using a zebrafish transgenic line which displays inducible folate deficiency. Impaired hematopoiesis includes decreased hemoglobin levels, decreased erythrocyte number, increased erythrocyte size and aberrant <i>c-myb</i> expression pattern were observed in folate deficient embryos. Cardiac defects, including smaller chamber size, aberrant cardiac function and <i>cmlc2</i> expression pattern, were also apparent in folate deficient embryos. Characterization of intracellular folate content in folate deficiency revealed a differential fluctuation among the different folate derivatives that carry a single carbon group at different oxidation levels. Rescue attempts by folic acid and nucleotides resulted in differential responses among affected tissues, suggesting that different pathomechanisms are involved in folate deficiency-induced anomalies in a tissue-specific manner. The results of the current study provide an explanation for the inconsistent outcome observed clinically in patients suffering from folate deficiency and/or receiving folate supplementation. This study also supports the use of this model for further research on the defective cardiogenesis and hematopoiesis caused by folate deficiency.</p></div

Prospective causal links involved in FD-induced developmental defects of the zebrafish circulation system.

<p>FD increases embryonic oxidative stress, leading to activated Erk (I) and impeded cell migration (II), which contributed to impeded hematopoiesis and cardiogenesis, respectively. FD also disturbed one-carbon metabolism, which interferes with nucleotide synthesis (III) and cell proliferation/hematopoiesis. The impeded one-carbon metabolism may also disturb intracellular methylation potential, which hampers primordial cell migration (II), leading to cardiogenic defects. This scheme is depicted based on the results reported in the current study (solid lines) and those in the literature (dashed line).</p

Zebrafish larval hematopoiesis and response to rescuing agents.

<p>(A, B) Hemoglobin of larvae in control and FD groups, with/without folate supplementation, were stained with o-dianisidine at 3 dpf. Hemoglobin signals were distributed most abundantly in the heart (dashed rectangles) and common cardinal veins (arrowheads) of control larvae (normal). Ectopic accumulation of hemoglobin in caudal veins (arrows) was often observed in FD larvae (mild and severe). The severity of anomalies was categorized and quantified based on the level and distribution of hemoglobin signals in larval heart and common cardinal veins. The images shown were the lateral (the upper panel) and ventral (the lower panel) views of larvae. Average of at least six independent experiments with the total sample number of 51–139 for each group are reported. (C, D) The relative number and size of embryonic erythrocytes were analyzed with flow cytometry for both control and FD embryos of 2-dpf generated by crossing Tg (hsp:EGFP-γGH) and Tg (gata1:dsRed). The numbers of erythrocytes were presented as the percentage of red fluorescent cells to total cell number. The size of erythrocytes was normalized with those of control larvae. Presented are data collected from at least three independent experiments with a total embryo number of approximately 30–40 for each group. (E) Hematopoiesis in both control and FD embryos was characterized by whole mount <i>in situ</i> hybridization with a riboprobe specific to <i>c-myb</i>, a hematopoietic stem cells marker. Reduced signals (arrowheads) with spatially and temporally altered distribution (arrows) were observed in embryos with severe folate deficiency. The larval responses to rescuing agents were quantified based on the distribution patterns of the <i>c-myb</i> signal at 32 hpf larvae (F) as shown in (E), and on the hemoglobin level (G) as shown in (A). There were approximately 10 to 40 larvae included for each group. (H) The 1-dpf wild-type larvae exposed to folic acid or 5-CHO-THF for 1 hour were examined for oxidative stress with H2DCFDA staining. C or CTL, heat-shocked non-fluorescent transgenic control; M or MFD, mild folate deficiency; S or SFD, severe folate deficiency; 5-CHO, 5-formyltetrahydrofolate; NAC, N-acetyl-L-cysteine; FA, folic acid. *, p<0.05; **, p<0.01; ***, p<0.001.</p

Folate and folate-mediated one-carbon metabolism.

<p>(A) Folate is comprised of a pteridine ring, p-aminobenzoic acid and glutamyl moieties with 5 to 8 glutamate residues attached in γ-linkage. The one-carbon units are attached to the N5- and/or N10-position of the pteridine ring at the oxidation levels of formate, formaldehyde and methanol. (B) The one-carbon units carried by reduced folate are involved in cytosolic, mitochondrial and nuclear folate pools and used for the biosynthesis of purines, thymidylate, amino acids and S-adenosylmethionine (SAM). Over-expressed γ-glutamylhydrolase (γGH) facilitates intracellular folate exportation by converting polyglutamylfolates (folate-Glu_n) to monoglutamylfolates (folate-Glu₁), leading to intracellular folate deficiency (thickened circle and arrows in shadowed box). Enzyme abbreviations: DHFR, dihydrofolate reductase; MTHFD, methylenetetrahydrofolate dehydrogenase; FDH, 10-formyltetrahydrofolate dehydrogenase; GART, glycinamide ribonucleotide transformylase; AICART, aminoimidazolecarboxamide ribonucleotide transformylase; MTHFS, 5,10-methenyltetrahydrofolate synthetase; SHMT, Serine hydroxymethyltransferase; MTHFR, methylenetetrahydrofolate reductase; TS, thymidylate synthase; MTR, 5-methyltetrahydrofolate-homocysteine methyltransferase; MAT, methionine adenosyl transferase; MT, methyltransferase; SAHH, S-adenosylhomocysteine hydrolase.</p

Folate deficiency impeded embryonic cells migration.

<p>The cultured A375 human melanoma cells (A) and A549 alveolar basal epithelial adenocarcinomic cells (B) were transfected with plasmids expressing either EGFP or EGFP-γGH fusion protein and subjected to the wound-healing assay. The wound was scratched and recorded for the scratched area immediately (0 hr) and again one day later (24 hr). The migration of A375 (C) and A549 (D) cells was evaluated by the “healed area”, which was calculated as described in Materials and Methods. Presented are the data collected from approximately 20 different wound areas from at least 3 to 6 independent repeats. (E) Wild-type zebrafish embryos at 64-cell stage were injected with the plasmids expressing either EGFP or EGFP-γGH into one single cell and continuously recorded for the migration of injected cells following the green fluorescence. Each colored line represents a single cell migratory track in one-hour recording period. The migratory parameters of recorded cells, including average speed (F), total distance (G), maximum distance (H) and maximum speed (I), were calculated with the on-line software CellTracker (v1.0, F. Piccinini et al., 2015) on MATLAB R2015a system. Zebrafish larvae of 31 hpf were subjected to WISH with the riboprobe specific to <i>sox10</i> to track the migration of neural crest cells. (J, K) The extent of neural crest cells (arrows) migration in FD embryos with/without NAC exposure was graded and quantified based on the following criteria: completely evacuated from the neural crest (0), still visible and below (+1) or above (+2, delayed) the trunk mid-line (dotted line). CTL, heat-shocked non-fluorescent transgenic control; FD, folate deficiency; NAC, N-acetyl-L-cysteine; 5-CHO, 5-formyltetrahydrfolate. *, p<0.05; **, p<0.01; ***, p<0.001.</p