We present an ATS zebrafish model, generated by MO-based knockdown of the slc2a10
gene, which encodes the glut10 protein. Two MOs, targeting the slc2a10
start codon and the exon 2–intron 2 donor splice site, respectively, produced identical phenotypes underscoring the specificity of the MOs for slc2a10
as the likelihood of both MOs mistargeting the same gene is very small (23
). The most prominent features of the morphants were a bowed/wavy appearance of the notochord and tail region and cardiovascular insufficiency. Cardiovascular abnormalities included incomplete and irregular patterning especially of the venous plexus and the intersegmental vessels. The heart rate was significantly reduced and blood pooling frequently observed in the heart and tail regions. These circulatory abnormalities may represent developmental precursors to lesions observed in human ATS: tortuosity and aneurysms of the large blood vessels.
In morphant fish, the notochord appeared bowed, kinked and shortened. The notochord consists of large mesodermal cells, packed within a sheath of connective tissue. It represents a primitive form of cartilage that defines the primitive longitudinal skeletal axis of the embryo that guides the formation of the vertebral column. It also provides key signals to the development of other mesodermal derivatives, including the vasculature (24
). Therefore, it is unclear whether the notochord abnormalities contribute to the vascular patterning defects that we observed following slc2a10
knockdown. It has been shown that early curvature of the notochord in zebrafish embryos can result in a scoliotic adult phenotype (25
). Similar to our zebrafish model, vertebral column abnormalities, including scoliosis, have been observed in human ATS (5
Previously, it has been shown that TGFβ signaling is upregulated in vascular smooth muscle cells of ATS patients (6
). This might, at least in part, be responsible for the phenotypic abnormalities encountered in ATS patients, especially because a link between elevated TGFβ signaling and connective tissue defects has been shown in related syndromes, including the Marfan, Loeys–Dietz and some cutis laxa syndromes (8
). Surprisingly, we found downregulation, rather than upregulation, of total-body TGFβ signaling in slc2a10
knockdown zebrafish embryos based on five lines of evidence. First, treatment of wild-type embryos with a tgfbr1 inhibitor resulted in a phenotype similar to the ATS zebrafish model, with a bowed notochord/tail region and comparable cardiovascular abnormalities. Secondly, treatment of scl2a10
morphants with tgfbr1 inhibitor aggravated the phenotype. Thirdly, transcriptional profiling showed a significant correlation between the mRNA profile of slc2a10
knockdown and tgfbr1 inhibition. The genes affected by both treatments are related to the development of the cardiovascular system, the eye, neurogenesis and cartilage formation. Involvement of TGFβ signaling in these functions has been demonstrated before (2
). Fourthly, the TGFβ reporter construct 3TP-lux showed reduced promoter activity in response to slc2a10
knockdown. Fifthly, slc2a10
knockdown partially rescued the deleterious effects of reduced levels of smad7, but not vice versa
. This suggests that glut10 exerts its effect on TGFβ signaling downstream of smads.
It is possible that TGFβ downregulation during early embryogenesis causes a compensatory upregulation later in development. Such a mechanism has been shown in a Tgfbr1
) knockout mouse model. A compensatory upregulation of the ALK5 downstream pathway was noted in these mice to be mediated by activin/ALK4 signaling (30
). Also, reduced TGFβ signaling caused by initially elevated sequestration may be followed by an excessive TGFβ release from a defective ECM later in life. Impaired elastic fiber formation, an important feature of ATS patients (31
), results in a higher amount of ‘bare’ microfibrils that can sequester TGFβ in the ECM. As TGFβ activation is dependent on mechanical forces (32
), TGFβ release may increase severely once sufficient intravascular pressure exists, a physiological variable that increases through development.
Our expression study also provides new insights into the specific molecular mechanisms involved in the ATS phenotype, as some pathways are altered by slc2a10 knockdown but not by tgfbr1 inhibition. A key finding is the downregulation of major players in cellular respiration, a process that converts glucose to the high-energy compound ATP through sequential steps of glycolysis in the cytoplasm, the Szent–Györgyi–Krebs cycle, and the oxidative phosphorylation in the mitochondria. In addition, specifically affected genes in the slc2a10 knockdown model involve the reactive oxygen species production pathway, heme biosynthesis and Ca2+ homeostasis, all important mitochondrial functions. Thus, the differential expression pattern overall points to a contribution of mitochondrial dysfunction in the phenotype caused by the loss of glut10 function in the zebrafish embryo.
Mitochondrial dysfunction in slc2a10
knockdown embryos was confirmed by our extracellular flux measurements. In spite of relatively preserved mitochondrial morphology, the loss of glut10 caused reduced overall respiration and reduced maximal flux of the electron transport chain in response to uncoupler administration. Reduced electron transport chain activity in slc2a10
knockdown embryos is consistent with reduced gene expression of electron transport chain components NADH-ubiquinone oxidoreductase 1 alpha/beta subcomplex (ndufab1) and cytochrome C1 (cyc1) (Supplementary Material, Table S2
Mitochondrial dysfunction observed through altered transcriptional profiles in our study is consistent with the recent finding that GLUT10 is required for dehydroascorbic acid (DHA) transport into the mitochondria (33
). DHA is converted to the antioxidant ascorbic acid that reduces reactive oxygen species generated as a result of oxidative phosphorylation. Consequently, defective recycling of DHA in the absence of GLUT10 results in increased sensitivity of cells to oxidative damage (33
), which is expected to lead to alterations in the expression of genes required for mitochondrial function as observed in our study.
GLUT10 deficiency results in severe cardiovascular and connective tissue manifestations in both humans (5
) and zebrafish (this study). In contrast, inactivating mutations in mouse Glut10 result in a mild, subclinical phenotype (13
). Differences in vitamin C metabolism among species may explain these observations. Some vertebrates, including humans and teleost fish but not mice, lack gulonolactone oxidase, a key enzyme in the biosynthetic pathway of vitamin C (34
). These organisms depend on dietary vitamin C and efficient intracellular recycling of this antioxidant and thus may be more susceptible to the loss of GLUT10, a DHA transporter.
Because a primary mitochondrial abnormality in our study led to decreased expression of TGFβ target genes, we conclude that at least a part of the TGFβ signaling pathway is dependent on mitochondrial function. Consistent with this notion, several studies highlighted connections between mitochondria, oxidative stress and TGFβ signaling (35
). This may occur through the coupling of intracellular oxidative pathways and TGFβ signaling by the renin–angiotensin pathway. Indeed, the angiotensinogen transcript is downregulated in the slc2a10
knockdown model and this molecule is known to enhance TGFβ signaling and ECM metabolism (39
Mitochondrial dysfunction in relation to oxidative stress has recently been shown to be involved in the pathogenesis of other connective tissue disorders related to ATS. Mutations in the PYCR1
gene encoding Δ-1-pyrroline-5-carboxylate reductase 1, an mitochondrial enzyme involved in proline metabolism, cause autosomal-recessive cutis laxa type IIB, wrinkly skin syndrome and geroderma osteodysplasticum (41
). Together with our findings, this illustrates that proper mitochondrial function is essential for the development and maintenance of connective tissues, in part through interactions with the TGFβ signaling pathway.