QDPR homologues in Danio rerio regulate melanin synthesis, early gliogenesis, and glutamine homeostasis

Abstract

Dihydropteridine reductase (QDPR) catalyzes the recycling of tetrahydrobiopterin (BH4), a cofactor in dopamine, serotonin, and phenylalanine metabolism. QDPR-deficient patients develop neurological symptoms including hypokinesia, truncal hypotonia, intellectual disability and seizures. The underlying pathomechanisms are poorly understood. We established a zebrafish model for QDPR deficiency and analyzed the expression as well as function of all zebrafish QDPR homologues during embryonic development. The homologues qdpra is essential for pigmentation and phenylalanine metabolism. Qdprb1 is expressed in the proliferative zones of the optic tectum and eye. Knockdown of qdprb1 leads to up-regulation of pro-proliferative genes and increased number of phospho-histone3 positive mitotic cells. Expression of neuronal and astroglial marker genes is concomitantly decreased. Qdprb1 hypomorphic embryos develop microcephaly and reduced eye size indicating a role for qdprb1 in the transition from cell proliferation to differentiation. Glutamine accumulation biochemically accompanies the developmental changes. Our findings provide novel insights into the neuropathogenesis of QDPR deficiency.

Fig 1. Characterization of Qdpra.

(A) WISH of qdpra at 24 hpf (dorsal view, anterior to the left) shows staining in retinal pigment epithelium (red arrow) and neural crest cells/melanophore precursor (black arrow). At 48 hpf qdpra transcripts are found in the retinal pigment epithelium, choroid fissure (red arrow) and neural crest cells (black arrow). At 72 hpf and more pronounced at 120 hpf (lateral views, anterior to the left), staining is present in the liver (blue arrow). (B) Lateral views with anterior to the left and (C) dorsal views with anterior to the top of embryos at stages indicated. Knockdown of qdpra results in reduced pigments in the eye (asterisk) at 26 hpf (B) and overall diminished pigmentation at 72 hpf (C). (D) At 72 hpf melanin content is reduced by 20% (of wildtype) in Qdpra hypomorphic zebrafish. (E) Amino acid analysis shows hyperphenylalaninemia and normal tyrosine upon qdpra knockdown.

Fig 2. Characterization of Qdprb1.

(A) WISH of qdprb1 at 24 hpf (dorsal view, anterior to the left) shows staining in eye (red arrow) and mid-hindbrain boundary (black arrow), at 48 hpf (lateral view, anterior to the left) in the optic tectum (blue arrow) and the mid-hindbrain boundary (black arrow) and CMZ (inset; red arrow, dorsal view, anterior to the left), and at 72 hpf (dorsal view, anterior to the left) in proliferative regions of the optic tectum (black arrow, left picture), CMZ (red arrow, right picture, dorsal view of the eye), as well as inner retinal layer (black arrow). (B) Lateral views, anterior to the left. Comparison of wildtype embryos at 18 hpf, 24 hpf and 72 hpf and qdprb1 hypomorphic embryos exhibit abnormal midbrain (red arrow) and anterior hindbrain (blue arrow) morphology and microcephaly.

Fig 3. Biochemical analysis of Qdprb1 hypomorphic embryos.

Amino acid analysis at 72 hpf shows reduced phenylalanine and unchanged tyrosine levels upon (A) as well as a strong increase of glutamine content (B) upon qdprb1 knockdown. (C) Ratio of glutamine over glutamate in Qdprb1 hypomorphic embryos remains unchanged during gastrulation and early segmentation, but it starts increasing at the onset of the observed morphological phenotype (18 hpf) coinciding with the start of neuro- and gliogenesis. This pattern is not found in wildtype zebrafish.

Fig 4. Reduced brain size but increased number of pH3-positive cells upon qdprb1 knockdown.

(A) Dorsal views, anterior to the left. At 3 dpf tg(HuC/D:GFP) transgenic zebrafish show a decreased size of the optic tectum and eye upon qdprb1 knockdown, which can be rescued by co-injecting qdprb1 mRNA. The insets show dorsal views of the left eye, which is reduced in size but still layered upon qdprb1 suppression. This phenotype is rescued upon co-injection of qdprb1 mRNA. The overall GFP signal remains unchanged. (B) The brain area of Qdprb1 hypormorphic embryos is reduced by about 15% compared to wildtypes and rescued by co-injection of qdprb1 mRNA. (C) Z-stack overlays of DAPI (pink) and pH3 (green) staining of dorsally imaged retinas reveal an increased number of pH3-positive retinal cells in Qdprb1 hypormorphic embryos (3 dpf), which are not restricted to the CMZ like found in wildtype retinas. Number and distribution of pH3-positive cells is restored by co-injection of qdprb1 mRNA. (D) Statistical analysis shows a significant increase in pH3 positive cells upon qdprb1 knockdown (n = 6) in comparison to wildtype zebrafish (n = 7), which could be rescued by addition of qdprb1 mRNA (n = 3). (E) Z-confocal image of DAPI staining (pink) of the retina at 3 dpf shows retinal layers although decreased overall size and broadened CMZ in qdprb1 hypomorphic embryos.

Fig 5. Expression of astroglial markers at 72hpf.

(A) Lateral views with anterior to the left. WISH of the BH₄ de novo synthesis pathway initiator and dopaminergic neuron marker, gch1, shows unchanged staining upon qdprb1 knockdown. (B) RT-qPCR analysis reveals strongly reduced expression of glula in qdprb1 hypomorphic embryos, which is rescued by qdprb1 mRNA co-injection. (C) Lateral views with anterior to the left. This finding is corroborated by WISH experiments highlighting that glula expression is lost in the eye (asterisk in inset) and reduced in the mid/hindbrain (asterisk). (D) Also gfap expression is reduced by qdprb1 knockdown and can be rescued by qdprb1 mRNA co-injection. (E) RT-qPCR analysis of astrocytic glutamate transporters show an almost complete loss of slc1a2a, which is confirmed by WISH (F)–dorsal views focused on the eye. Asterisk indicates the effect on slc1a2a expression. Slc1a2a expression is normalized in qdprb1 mRNA co-injected embryos (E). (G) Lateral views with anterior to the left. WISH of slc1a2b reveals mildly reduced staining in midbrain and eye (inset), confirming that both SLC1A2 homologues are affected in Qdprb1 hypomorphic embryos.

Fig 6. Glutamine exposure partially mimics qdprb1 knockdown.

(A) Lateral views with anterior to the left. Zebrafish exposed to 20 mM glutamine from sphere stage till 72 hpf display smaller heads and eyes. (B) RT-qPCR analysis zebrafish early exposed to glutamine (20 mM) shows reduced expression of qdprb1, gfap, and slc1a2a. (C) Late exposure to glutamine starting at 48 hpf does not affect the expression of these genes.