Aminolevulinate synthase 2 mediates erythrocyte differentiation by regulating larval globin expression during Xenopus primary hematopoiesis

AbstractHemoglobin synthesis by erythrocytes continues throughout a vertebrate’s lifetime. The mechanism of mammalian heme synthesis has been studied for many years; aminolevulinate synthase 2 (ALAS2), a heme synthetase, is associated with X-linked dominant protoporphyria in humans. Amphibian and mammalian blood cells differ, but little is known about amphibian embryonic hemoglobin synthesis. We investigated the function of the Xenopus alas2 gene (Xalas2) in primitive amphibian erythrocytes and found that it is first expressed in primitive erythroid cells before hemoglobin alpha 3 subunit (hba3) during primary hematopoiesis and in the posterior ventral blood islands at the tailbud stage. Xalas2 is not expressed during secondary hematopoiesis in the dorsal lateral plate. Hemoglobin was barely detectable by o-dianisidine staining and hba3 transcript levels decreased in Xalas2-knockdown embryos. These results suggest that Xalas2 might be able to synthesize hemoglobin during hematopoiesis and mediate erythrocyte differentiation by regulating hba3 expression in Xenopus laevis

Different Human Copper-Zinc Superoxide Dismutase Mutants, SOD1G93A and SOD1H46R, Exert Distinct Harmful Effects on Gross Phenotype in Mice

Amyotrophic lateral sclerosis (ALS) is a heterogeneous group of fatal neurodegenerative diseases characterized by a selective loss of motor neurons in the brain and spinal cord. Creation of transgenic mice expressing mutant Cu/Zn superoxide dismutase (SOD1), as ALS models, has made an enormous impact on progress of the ALS studies. Recently, it has been recognized that genetic background and gender affect many physiological and pathological phenotypes. However, no systematic studies focusing on such effects using ALS models other than SOD1G93A mice have been conducted. To clarify the effects of genetic background and gender on gross phenotypes among different ALS models, we here conducted a comparative analysis of growth curves and lifespans using congenic lines of SOD1G93A and SOD1H46R mice on two different genetic backgrounds; C57BL/6N (B6) and FVB/N (FVB). Copy number of the transgene and their expression between SOD1G93A and SOD1H46R lines were comparable. B6 congenic mutant SOD1 transgenic lines irrespective of their mutation and gender differences lived longer than corresponding FVB lines. Notably, the G93A mutation caused severer disease phenotypes than did the H46R mutation, where SOD1G93A mice, particularly on a FVB background, showed more extensive body weight loss and earlier death. Gender effect on survival also solely emerged in FVB congenic SOD1G93A mice. Conversely, consistent with our previous study using B6 lines, lack of Als2, a murine homolog for the recessive juvenile ALS causative gene, in FVB congenic SOD1H46R, but not SOD1G93A, mice resulted in an earlier death, implying a genetic background-independent but mutation-dependent phenotypic modification. These results indicate that SOD1G93A- and SOD1H46R-mediated toxicity and their associated pathogenic pathways are not identical. Further, distinctive injurious effects resulted from different SOD1 mutations, which are associated with genetic background and/or gender, suggests the presence of several genetic modifiers of disease expression in the mouse genome

Comparisons of the mutant SOD1 protein levels in different mutant <i>SOD1</i> transgenic mouse lines.

<p>(A) Representative data for western blot analysis of the human mutant SOD1 proteins (hSOD1). The 1% Triton X100-soluble fractions of the spinal cord from mice at 12 weeks of age with different genders (F; female, M; male), genotypes (G93A; <i>SOD1^G93A</i>, H46R; <i>SOD1^H46R</i>), and genetic backgrounds (B6; C57BL/6, FVB; FVB/N) are analyzed. β-actin serves as control. (B) Quantitative analyses of western blotting for mutant SOD1 in the spinal cord from 12 week-old mice. Densitometric data for immunoreactive signals are normalized by the levels of β-actin. There are no significant differences in the mean values between groups with different genders, genotypes, and genetic backgrounds. Values are mean ± SEM (n = 4) in an arbitrary unit. Statistical significance is evaluated by ANOVA with Bonferroni's <i>post hoc</i> test.</p

Effect of genetic background on survival in different mutant <i>SOD1</i> transgenic mouse lines.

<p>(A) Survival curves for C57BL/6 (B6) congenic <i>SOD1^G93A</i> transgenic female mice (B6_G93A F) (closed circle: n = 19) and FVB/N (FVB) congenic <i>SOD1^G93A</i> transgenic female mice (FVB_G93A F) (open square: n = 20). (B) Survival curves for B6 congenic <i>SOD1^G93A</i> transgenic male mice (B6_G93A M) (closed circle: n = 15) and FVB congenic <i>SOD1^G93A</i> transgenic male mice (FVB_G93A M) (open square: n = 17). (C) Survival curves for B6 congenic <i>SOD1^H46R</i> transgenic female mice (B6_H46R F) (closed circle: n = 31) and FVB congenic <i>SOD1^H46R</i> transgenic female mice (FVB_H46R F) (open square: n = 20). (D) Survival curves for B6 congenic <i>SOD1^H46R</i> transgenic male mice (B6_H46R M) (closed circle: n = 63) and FVB congenic <i>SOD1^H46R</i> transgenic male mice (FVB_H46R M) (open square: n = 27). Kaplan-Meier analysis with Log-rank (Mantel-Cox) test identifies significant differences in survival for mutant SOD1 transgenic lines between B6 and FVB backgrounds (<i>p</i><0.0001).</p

Growth curves for ALS2 deficient <i>SOD1</i> transgenic mice on a FVB background.

<p>(A) Growth curves for FVB/N (FVB) congenic female mice [wild-type (FVB_WT F; open triangle, n = 9–16), <i>SOD1^G93A</i> (FVB_G93A F; closed circle, n = 9–27), <i>SOD1^G93A</i>;<i>Als2</i>^−/− (FVB_G93A_Als2−/− F; open square, n = 5–20)]. (B) Growth curves for FVB congenic male mice [wild-type (FVB_WT M; open triangle, n = 15–19), <i>SOD1^G93A</i> (FVB_G93A M; closed circle, n = 6–17), <i>SOD1^G93A</i>;<i>Als2</i>^−/− (FVB_G93A_Als2−/− M; open square, n = 4–10)]. (C) Growth curves for FVB congenic female mice [wild-type (FVB_WT F; open triangle, n = 9–16), <i>SOD1^H46R</i> (FVB_H46R F; closed circle, n = 14–20), <i>SOD1^H46R</i>;<i>Als2</i>^−/− (FVB_H46R_Als2−/− F; open square, n = 8–15)]. (D) Growth curves for FVB congenic male mice [wild-type (FVB_WT M; open triangle, n = 15–19), <i>SOD1^H46R</i> (FVB_H46R M; closed circle, n = 19–28), <i>SOD1^H46R</i>;<i>Als2</i>^−/− (FVB_H46R_Als2−/− M; open square, n = 4–10)]. (A–D) Data are omitted from analysis when the numbers of live animals of the particular genotype at a particular age are <4. Values are mean ± SD. Statistical significances are evaluated by ANOVA with Bonferroni's <i>post hoc</i> test. Degree of body weight loss associated with disease progression in male FVB <i>SOD1^H46R</i> mice is greater than that in male FVB <i>SOD1^H46R</i>;<i>Als2</i>^−/−SOD1^H46R mice (***<i>p</i><0.001). The values for all mutant SOD1 transgenic mice with or without ALS2 are significantly lower than those for age-matched WT animals (levels of significance are not shown).</p

Effect of different types of the <i>SOD1</i> mutation on survival.

<p>(A) Survival curves for C57BL/6 (B6) congenic <i>SOD1^G93A</i> transgenic female mice (B6_G93A F) (closed circle: n = 19) and B6 congenic <i>SOD1^H46R</i> transgenic female mice (B6_H46R F) (open square: n = 31). (B) Survival curves for B6 congenic <i>SOD1^G93A</i> transgenic male mice (B6_G93A M) (closed circle: n = 15) and B6 congenic <i>SOD1^H46R</i> transgenic male mice (B6_H46R M) (open square: n = 63). (C) Survival curves for FVB/N (FVB) congenic <i>SOD1^G93A</i> transgenic female mice (FVB_G93A F) (closed circle: n = 20) and FVB congenic <i>SOD1^H46R</i> transgenic female mice (FVB_H46R F) (open square: n = 20). (D) Survival curves for FVB congenic <i>SOD1^G93A</i> transgenic male mice (FVB_G93A M) (closed circle: n = 17) and FVB congenic <i>SOD1^H46R</i> transgenic male mice (FVB_H46R M) (open square: n = 27). Kaplan-Meier analysis with Log-rank (Mantel-Cox) test identifies significant differences in survival between B6_G93A F and B6_H46R F (<i>p</i> = 0.0210), B6_G93A M and B6_H46R M (<i>p</i> = 0.0004), FVB_G93A F and FVB_H46R F (<i>p</i><0.0001), and FVB_G93A M and FVB_H46R M (<i>p</i><0.0001).</p

Effect of gender on survival in different mutant <i>SOD1</i> transgenic mouse lines.

<p>(A) Survival curves for C57BL/6 (B6) congenic <i>SOD1^G93A</i> transgenic mice (B6_G93A) [female (F); closed circle: n = 19, male (M); open square: n = 15]. (B) Survival curves for B6 congenic <i>SOD1^H46R</i> transgenic mice (B6_H46R) (F; closed circle: n = 31, M; open square: n = 63). (C) Survival curves for FVB/N (FVB) congenic <i>SOD1^G93A</i> transgenic mice (FVB_G93A) (F; closed circle: n = 20, M; open square: n = 17). (D) Survival curves for FVB congenic <i>SOD1^H46R</i> transgenic mice (FVB_H46R) (F; closed circle: n = 20, M; open square: n = 27). Kaplan-Meier analysis with Log-rank (Mantel-Cox) test reveals a significant gender difference in FVB_G93A (<i>p</i> = 0.003), but not in B6_G93A (<i>p</i> = 0.7344), B6_H46R (<i>p</i> = 0.3723), and FVB_H46R (<i>p</i> = 0.4439).</p

Summary of the quantitative analysis of the transgenes and their expression.

<p>Values are mean ± SD.</p>*a<p>ΔCt = (Ct for human SOD1)−(Ct for mouse Sod1).</p>*b<p>ΔCt = (Ct for human SOD1 mRNA)−(Ct for β-actin mRNA).</p

Lifespan of the mutant <i>SOD1</i> transgenic mouse lines used in this study.

*<p>Values are mean ± SD.</p

Copy numbers of the transgene and the levels of its transcript.

<p>(A) Comparison of the difference in threshold cycle (ΔCT) between the human <i>SOD1</i> transgene (SOD1) and a reference mouse <i>Sod1</i> gene (Sod1) in <i>SOD1^G93A</i> and <i>SOD1^H46R</i> transgenic mice. There are no significant differences in the mean values between groups with different genders (F; female, M; male), genotypes (G93A; <i>SOD1^G93A</i>, H46R; <i>SOD1^H46R</i>), and genetic backgrounds (B6; C57BL/6, FVB; FVB/N). (B) Comparison of the ΔCT between the human <i>SOD1</i> and the mouse <i>Actb</i> transcripts in <i>SOD1^G93A</i> and <i>SOD1^H46R</i> transgenic mice. There are no significant differences in the mean values between groups with different genders, genotypes, and genetic backgrounds. All values are mean ± SD (n = 4). Statistical significance is evaluated by ANOVA with Bonferroni's <i>post hoc</i> test.</p