Neural tube development depends on notochord-derived Sonic hedgehog released into the sclerotome

Sonic hedgehog (Shh), produced in notochord and floor plate, is necessary both for neural and mesodermal development. To reach the myotome, Shh has to traverse the sclerotome. By loss and gain of Shh function, and floor plate deletions, we report that sclerotomal Shh is also necessary for neural tube development. Reducing the amount of Shh in sclerotome by membrane-tethered hedgehog-interacting protein or by Patched1, but not by dominant active Patched, decreased motoneuron numbers while also compromising myotome differentiation. These effects were a specific and direct consequence of reducing Shh. In addition, grafting notochords in a basal, but not apical location vis-a-vis the tube, profoundly affected motoneuron development, suggesting that initial ligand presentation occurs at the basal side of epithelia corresponding to the sclerotome-neural tube interface.Collectively, our results reveal that the sclerotome is a potential site of a Shh gradient that coordinates development of mesodermal and neural progenitors.Summary statementShh that transits through the sclerotome is presented to the neuroepithelium from its basal aspect to affect motoneuron development.

Mesodermal Development From Reprogrammed Fanconi Anemia Cells Is Affected by ALDH2 Enzymatic Activity

Abstract 648 Introduction Fanconi anemia (FA) is a genome instability disorder with clinical characteristics including progressive bone marrow failure (BMF), developmental abnormalities, and increased occurrence of leukemia and cancer. To date 15 genes have been implicated in FA, and their products form a common DNA repair network often referred to as “FA pathway”. Following DNA damage or replication stress, the FA pathway is activated, leading to the monoubiquitination of FANCD2 and FANCI proteins (the ID complex). The monoubiquitinated ID complex is loaded on damaged chromatin with subnuclear foci formation, and mediates homologous recombination. Since cells derived from FA patients are hypersensitive to treatments that induce DNA interstrand cross-links (ICLs), the FA pathway has been considered to function in ICL repair. However, it still remains unclear what type of endogenous DNA damage is repaired through the FA pathway and is the cause of phenotypes in FA patients. Recent studies have suggested that cells deficient in the FA pathway are also sensitive to formaldehyde and acetaldehyde. Aldehydes may create DNA adducts including ICLs or protein DNA crosslinking. These results raise a possibility that the FA pathway prevents BMF by mitigating genotoxicity due to endogenous aldehydes. It has been known that ALDH2 deficiency resulting from Glu487Lys substitution (A allele) is prevalent in East Asian populations. While the Glu487 form (G allele) is proficient in aldehyde catabolism, even the GA heterozygote displayed strongly reduced catalysis because ALDH2 is a tetrameric enzyme and the variant form can suppress the activity in a dominant negative manner. Therefore some Japanese FA patients are expected to be deficient in ALDH2, providing an opportunity to test role of ALDH2 and aldehyde metabolism in human FA patients. Results and discussion In FA fetus, p53/p21 axis has already activated in fetal liver (Ceccaldi, Cell stem cell, 2012), indicating the possibility that hematopoietic defects in FA patients originates from an earlier developmental stage. Since human hematopoietic system originates from embryonic mesoderm, we set out to estimate the role of ALDH2 and FANCA pathway during early embryogenesis. For this, we reprogrammed somatic cells from a patient with ALDH2 GA genotype and observed their in vitro mesodermal differentiation. We first introduced reprogramming factors into fibroblasts by episomal vectors, and obtained colonies which are morphologically compatible with human induced pluripotent stem cells (iPSCs). These iPSC-like cells (designated as FA-iPLCs) showed close similarity to conventional ES/iPSCs regarding marker gene expressions and differentiation ability into three germ layers. We obtained gene-complemented FA-iPLCs (designated as cFA-iPLCs) for control study. To evaluate the impact of ALDH2 activity on iPSC- or iPLC-derived mesodermal differentiation, we next adapted the previously reported serum-free monolayer culture system. Both FA- and cFA-iPLCs showed similar differentiation manners with conventional embryonic stem cells and iPSCs, and percentages of KDR+ mesodermal progenitors including KDR+CD34+ common hemoangiogenic progenitors were comparable. Notably, ALDH2 agonist Alda1 did increase only FA-iPLC-derived mesodermal progenitors but not cFA-iPLCs. These data supported the hypothesis that mesodermal development towards hematopoietic cells in human can be affected by ALDH2 activity in the absence of FA pathway. To confirm the hypothesis, next we set out to assess whether the variation in ALDH2 affects symptoms in Japanese FA patients. Strikingly, we found that progression of BMF was strongly accelerated in heterozygous carrier of the variant A allele compared to homozygous GG patients. Furthermore we looked at occurrence of leukemia and/or myelodysplasia and the somatic developments. Interestingly, these were not significantly difference between patients with each variation of ALDH2, indicating the possibility that aldehydes affect only in early hematopoietic development, not other mesodermal tissues. Overall, our results from FA-iPLCs and clinical study indicate that the variation in ALDH2 affects the occurrence of bone marrow failure in FA patients, and that hematopoietic defect in FA patients is caused by aldehydes in early mesodermal developmental stage. Disclosures: No relevant conflicts of interest to declare.

Canonical NF-κ B Pathway Is Required for Early Mesodermal Differentiation from the Murine Embryonic Stem Cells.

NF-κB family of transcription factors play important roles in cell growth and survival as well as stress and immune responses through the target gene expression. We previously reported that NF-κB family proteins participate in both primitive and definitive hematopoiesis by preventing reactive oxygen species (ROS)-mediated apoptosis. However, their functions in mesodermal development, which is the earlier process to produce hematopoietic cells, has not been elucidated. So, in the present study, we inducibly expressed IκBSR with a Tet-off system in murine embryonic stem (ES) cells and evaluated its effects on mesodermal differentiation. In this system, IκBSR can inhibit the function of canonical NF-κB pathway as a dominant negative mutant in response to tetracycline removal from the culture medium. After 4.0–4.5-day cultures on the OP9 cell layer with differentiation medium deprived of leukemia inhibitory factor (LIF), about 30% of cultured cells developed into the vascular endothelial growth factor receptor 2 (VEGFR2)+ lateral mesodermal cells, which have the potential to differentiate into endothelial cells, hematopoietic cells, and cardiomyocytes. When Tet was deprived from the culture medium, the induced IκBSR inhibited the development of VEGFR2+ cells by inducing apoptosis via the ROS accumulation. However, even if this apoptosis was prevented by the anti-oxidants, the cultured cells did not express VEGFR2. As for this mechanism, we found that although the VEGFR2 promoter was activated by p65, c-Rel, and p50 in response to LIF deprivation in ES cells, IκBSR suppressed this induction almost completely using luciferase assays and ChIP assays. These results suggest that IκBSR blocks the development of lateral mesodermal cells from undifferentiated ES cells through the down-regulated expression of VEGFR2 in concurrence with the induction of apoptosis. To further analyze the effects of IκBSR on mesodermal development, we performed cDNA microarray analysis using differentiated ES cells deprived of Tet for 2 days. Among 4,277 genes, 123 genes were significantly down-regulated and 105 were up-regulated by induction of IκBSR. Among known essential genes for mesodermal development, the expression of platelet derived growth factor receptor α (PDGFRα), which is induced in paraxial mesoderm, was also suppressed by IκBSR significantly. Furthermore, in the prolonged cultures, we found that, although the development of E-cadherin (ECD)+/PDGFRα+ bi-potent mesendodermal cells at day 3.0–3.5 was not affected by IκBSR, it inhibited the subsequent development of ECD−/PDGFRα+ paraxial mesodermal cells at day 5.0–6.0. In accord with this result, we found that IκBSR sustained the expression of Mixl1, one of the intrinsic mesendodermal markers in differentiated ES cells, by semi-quantitive RT-PCR analysis. Together, these results suggest that the canonical NF-κB pathway is required for early mesodermal differentiation from mesendoderm stage through the expression of various essential molecules, thereby regulating their morphogenetic behaviors and cell-fate decisions. We are now evaluating the in vivo effects of IκBSR on the mesodermal development using the tetraploid chimeric mice model, and would like to further disclose the role of NF-κB pathway in our presentation.