Mtmr8 is essential for vasculature development in zebrafish embryos
© Mei et al; licensee BioMed Central Ltd. 2010
Received: 15 January 2010
Accepted: 5 September 2010
Published: 5 September 2010
Embryonic morphogenesis of vascular and muscular systems is tightly coordinated, and a functional cooperation of Mtmr8 with PI3K in actin filament modeling and muscle development has been revealed in zebrafish. Here, we attempt to explore the function of Mtmr8 in vasculature development parallel to its function in muscle development.
During early stage of somitogenesis, mtmr8 expression was detected in both somitic mesodem and ventral mesoderm. Knockdown of mtmr8 by morpholino impairs arterial endothelial marker expression, and results in endothelial cell reduction and vasculogenesis defects, such as retardation in intersegmental vessel development and interruption of trunk dorsal aorta. Moreover, mtmr8 morphants show loss of arterial endothelial cell identity in dorsal aorta, which is effectively rescued by low concentration of PI3K inhibitor, and by over-expression of dnPKA mRNA or vegf mRNA. Interestingly, mtmr8 expression is up-regulated when zebrafish embryos are treated with specific inhibitor of Hedgehog pathway that abolishes arterial marker expression.
These data indicate that Mtmr8 is essential for vasculature development in zebrafish embryos, and may play a role in arterial specification through repressing PI3K activity. It is suggested that Mtmr8 should represent a novel element of the Hedgehog/PI3K/VEGF signaling cascade that controls arterial specification.
MTM (myotubularin myopathy) family factors are members of the growing class of dual-specificity phosphatases (DSPs) including PTEN, which can dephosphorylate the products of phosphoinositide 3-kinases (PI3K), and are negative regulators of the PI3K/Akt signaling pathways. A potential function for PI3K and PTEN has been suggested in both angiogenic signaling[2, 3] and various models of muscle defects. And, dual-specific phosphatase-5 (Dusp-5) has been identified to play a functional role in vascular development through counteracting the function of Snrk-1, a serine threonine kinase in angioblast development. However, functions of MTM family members in these processes are not clear.
In vertebrates, vascular and muscular systems are tightly connected. And, the cells in dorsal aorta and myotomal muscle are both derived from mesoderm. Endothelial cells (ECs) forming intersegmental vessel (SE) migrate following a path initially along the somite boundary and later between notochord and somitic tissue. These data suggested that a dynamic connection between somitogenesis and vasculature development might exist, and the connection might be influenced by some signaling molecules. For example, semaphorin-plexin-signaling was proved to play significant roles in both somitogenesis and vasculature formation in zebrafish. Furthermore, zebrafish perlecan was demonstrated to play a central function in skeletal muscle and cardiovascular development.
Several signaling pathways, such as vascular endothelial growth factor (VEGF) signaling, and PI3K/Akt and Hedgehog (Hh) signaling which genetically interacts with each other[8–10], had been shown to be involved in vascular formation. In vitro studies suggested a role for AKT/PKB as a downstream effector of VEGF signaling[11, 12]. Moreover, over-expression of the downstream effector AKT/PKB rescued VEGF receptor blockade. In addition, activation of protein kinase A (PKA), a negative regulator of hedgehog pathway, effectively inhibited Akt, which demonstrated a direct role for AKT in regulating Hedgehog signaling[13, 14]. Myocardial Hh activation triggered by FGF signaling, is essential for Vegf expression. However, these signaling interactions form complicated network and need further explanation.
Specific genes controlling artery/vein specification have been identified in different vertebrate species. In zebrafish embryo, sonic hedgehog (Shh) and Vegf, expressed in the notochord and somite respectively, are required for arterial specification[9, 17]. Conversely, activation of PI3K/AKT signaling in angioblasts promotes venous specification. However, the precise mechanism of signaling interactions in vascular development remains to be elucidated. Interestingly, our recent study has shown a functional cooperation of the MTM family member Mtmr8 with PI3K in muscle development in zebrafish, and revealed a possible participation of Mtmr8 in the Hedgehog (Hh) pathway. To explore the function of Mtmr8 in vasculature development, here, we further observed the vascular development defects of Mtmr8 loss-of-function. Subsequently, we found that the Mtmr8, as a negative regulator of PI3K, affected the Hedgehog and Vegf pathway in the blood vessel development in vivo. The data indicate that Mtmr8 is essential for vasculature development in zebrafish.
Developmental defects of hemato-vascular progenitors in mtmr8 morphants
Determination of abnormal vascular phenotypes in mtmr8 morphant embryos
Expression defects of molecular marker genes for artery/vein and vascular endothelium in mtmr8 morphants
Moreover, the effects of mtmr8 knockdown on vasculogenesis were judged by endogenous alkaline phosphatase activity. In Cont-MO embryos (Figure 4K, L), the endogenous alkaline phosphatase activity labeled the major cerebral vessels and the subintestinal vessels (SIVs). However, all Mtmr8 morphants displayed an obvious reduction in the head signal and a total absence of SIV labeling (Figure 4M, N). The obvious effects of mtmr8 knockdown on endothelial cell alkaline phosphatase activity further suggest that the vascular changes should be specific to endothelial cells, and the endothelial cell marker gene fli1 should be primary target for the mtmr8 functions.
Mtmr8 mediates PI3K/Akt signaling for artery specification
In mtmr8 morphants, the expression of artery marker gene ephrinB2a was severely reduced (Figure 4D) in the trunk region, but the reduced levels were variable, and might be classified into three kinds: normal, similar to consistent expression in wild-type (Figure 5J), mild defect, patchy expression in some cells along the DA (Figure 5K), severe defect, almost no any ephrinB2a expression in the trunk DA (Figure 5L). In previous studies, when treated with 10 μM LY294002, about half of the mtmr8 morphants could be further deteriorated in muscle development, and the others were mildly or not obviously affected (Data not shown). However, 4 μM LY294002 had no obvious effect on the muscle phenotype of the morphants. Interestingly, when 4 μM LY294002 was used to treat the mtmr8 morphants, a significantly increasing proportion of normal embryos was found in the morphants (Figure 5M). Subsequently, we checked phosphorylation status of another early arterial progenitor marker ERK (p42/44 MAP kinase) in the mtmr8 morphants by Western blot analysis, and found that loss of Mtmr8 function significantly reduces ERK phosphorylation (Figure 5N). The data indicate that Mtmr8 negatively mediates PI3K/Akt signaling for artery specification.
Mtmr8 is essential for normal vasculature development through regulating Hedgehog and Vegf signaling pathways
Vascular function is tightly regulated by Vegf signaling pathway. To assess whether Mtmr8 influenced the developing vasculature through Vegf pathway, we treated 10 hpf embryos with a Vegf receptor tyrosine kinase inhibitor, SU5416. In comparison with control embryos (Figure 6G), the embryos incubated with 5 μM of SU5416 failed to show vascular expression of mtmr8 in the trunk at 30 hpf (Figure 6H), and the reduced transcript levels were detected under different doses of SU5416 by Q-RT-PCR (Figure 6K), suggesting that Mtmr8 might act downstream of Vegf to determine arterial cell fate. Moreover, we checked the expression of vegf165 in Mtmr8 morphants and control embryos, and found a significant down-regulation of vegf165 expression within the somites at 26 hpf (Figure 6I, J), and the quantitative down-regulation change was confirmed by Q-RT-PCR (Figure 6L).
Previous studies have showed that mutations in most myotubularin family genes are causative for human neuromuscular disorders. And, knockdown of zebrafish myotubularin and mtmr8 also impairs the embryo muscle development[19, 32]. Moreover, Mtmr8 has been shown to negatively regulate the PI3K/AKT pathway. In the current study, we have further found that mtmr8 knockdown leads to activation of PI3K/Akt signaling, which impairs arterial endothelial marker gene expression, and results in endothelial cell impairment and vasculogenesis defects. Significantly, mtmr8 expression has been detected in somitic and lateral mesoderm, where the angioblasts are committed to endothelial lineage differentiation and restricted to arterial or venous fate[19, 33]. And, significant expression reduction of endothelial progenitor marker genes in mtmr8 morphants indicates that the impaired arterial differentiation should be the consequence of defects in endothelial cell formation. These results suggest that mtmr8 should be essential for the endothelial cell differentiation and vasculature development through repressing the activity of PI3K in zebrafish.
In conclusion, our findings indicate that Mtmr8 functions as a key regulator of somitic muscle development and vasculature development in zebrafish embryos. The signaling pathways that function during vascular development are highly conserved. Further analysis for Mtmr8 function will be essential to understand the complex signaling hierarchy for the differentiation of endothelial cells and the development of vasculature in vertebrates.
Danio rerio wild-type (WT) AB strain, and Tg(kdrl:GFP) la116 transgenic zebrafish (previous Tg(flk1:GFP) la116 )  were used. Zebrafish were maintained and staged as described. When necessary, embryos were anesthetized with 0.003% tricaine (Sigma). All of the experimental researches on zebrafish were performed with the approval of the animal ethics committee in the Institute of Hydrobiology, Chinese Academy of Sciences.
Antisense morpholino and mRNA microinjection
The sequences and doses of the injected morpholinos were described previously. mtmr8, dnPKA, scl, vegf121 and vegf165 cDNAs were subcloned into PCS2+ plasmid for in vitro synthesis of capped mRNA, using SP6 RNA polymerase (Ambion), following the manufacturer's instructions. A mis-matched mtmr8 mRNA (mis-mtmr8) was constructed by using mis-match forward prime 5'-ATGGAGCACATCATAACGCCCAAAGTCG-3' (underlines indicate the changed nucleotide). All mRNA were re-suspended in water and co-injected at a concentration of 50-100 ng/μL.
Whole-mount in situ hybridization, endogenous alkaline phosphatase-based vessel staining and histological analysis
Embryos at different stages were collected and fixed as described. Purified plasmids were linearized by selected restriction enzymes and used as templates for in vitro transcription using T7 or Sp6 RNA polymerase to generate DIG-labeled (Roche) sense and anti-sense probes. Whole mount in situ hybridization was performed as described previously[36, 37].
At 72 hpf, embryos were fixed in 4% paraformaldehyde and dehydrated in methanol. Later, they were rehydrated to PBST and then used for staining, as described by Serbedzija et al . Histological analysis was performed according to methods described by Lawson et al .
Real-time quantitative PCR (Q-RT-PCR) and Western blot detection
Real-time quantitative PCR was performed by DNA Engine Chromo 4 Real-Time System (MJ Research) with SYBR Green I Dye according to a previous report. The ratio of these genes to β-actin in control embryos was set to 1 (100%), and the expression of all morpholino-injected or drug-treated embryos were normalized relative to this value. All samples were analyzed in triplicates and the results were expressed relative to the expression of β-actin using the 2 (-delta delta C(T)) method . Data are presented as mean ± SD of three separate experiments.
Western blot detection for the ratio between phospho-p44/42 MAP kinase (Thr202/Tyr204) and p44/42 MAP kinase in developing zebrafish larvae was performed essentially as described. The membranes were washed and incubated with Alkaline Phosphatase (AP)-conjugated secondary antibodies. Images of blots were captured with a scanner, and quantitative densitometric analysis was performed using Scion Image.
PI3K inhibitor, LY294002 (Promega) was dissolved in DMSO at stock concentration of 50 mM. For experiments, embryos were incubated in embryo media containing 0-50 μM of the drug from 10 hpf to 24 hpf. Control embryos were treated with the equivalent amount of DMSO solution (1%≤).
Cyclopamine and SU5416 were obtained from Calbiochem and were used at a concentration of 0-50 μm and 0-5 μM respectively. Zebrafish embryos were soaked in 6-well plates and were treated with the drugs from 10 hpf, until they were processed for in situ experiments. Control embryos were treated in embryo medium containing equivalent amount of DMSO solution (1% < ).
All the experiments were performed in triplicates (about 30 embryos in each experiment group). Data were presented as mean ± SD. The data in morphant embryos compared to the control (set to 1) were assessed by one-way analysis of variance (ANOVA), followed by the Tukey's post hoc test for multiple comparisons. A probability (*P) of <0.05 was considered statistically significant.
The authors wish to thank Shuo Lin (University of California, Los Angeles) for the Tg(kdrl:GFP) la116 transgenic zebrafish.
This work was supported by the National Major Basic Research Program (grant No. 2010CB126300), the Innovation Project of Chinese Academy of Sciences (grant No. KSCX2-YW-N-020), the Autonomous Research Project of State Key Laboratory of Freshwater Ecology and Biotechnology (2008FBZ15), and the Innovation Projects of IHB, CAS (grant Nos.075A01-1-301, 085A02-1-301).
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