A phenotypic rescue approach identifies lineage regionalization defects in a mouse model of DiGeorge syndrome

ABSTRACTTBX1 is a key regulator of pharyngeal apparatus (PhAp) development. Vitamin B12 treatment partially rescues aortic arch patterning defects of Tbx1+/- embryos. Here we show that it also improves cardiac outflow tract septation and branchiomeric muscle anomalies of Tbx1 hypomorphic mutants. At molecular level, the in vivo vB12 treatment let us to identify genes that were dysregulated by Tbx1 haploinsufficiency and rescued by treatment. We found that SLUG, encoded by the rescued gene Snai2, identified a population of mesodermal cells that was partially overlapping with but distinct from ISL1+ and TBX1+ populations. In addition, SLUG+ cells were mislocalized and had a greater tendency to aggregate in Tbx1+/- and Tbx1-/- embryos and vB12 treatment restore cellular distribution. Adjacent neural crest-derived mesenchymal cells, which do not express TBX1, were also affected, showing enhanced segregation from cardiopharyngeal mesodermal cells. We propose that TBX1 regulates cell distribution in core mesoderm and the arrangement of multiple lineages within the PhAp.

Pharmacological Rescue of the Brain Cortex Phenotype of Tbx1 Mouse Mutants: Significance for 22q11.2 Deletion Syndrome

ObjectivesTbx1 mutant mice are a widely used model of 22q11.2 deletion syndrome (22q11.2DS) because they manifest a broad spectrum of physical and behavioral abnormalities that is similar to that found in 22q11.2DS patients. In Tbx1 mutants, brain abnormalities include changes in cortical cytoarchitecture, hypothesized to be caused by the precocious differentiation of cortical progenitors. The objectives of this research are to identify drugs that have efficacy against the brain phenotype, and through a phenotypic rescue approach, gain insights into the pathogenetic mechanisms underlying Tbx1 haploinsufficiency.Experimental ApproachDisease model: Tbx1 heterozygous and homozygous embryos. We tested the ability of two FDA-approved drugs, the LSD1 inhibitor Tranylcypromine and Vitamin B12, to rescue the Tbx1 mutant cortical phenotype. Both drugs have proven efficacy against the cardiovascular phenotype, albeit at a much reduced level compared to the rescue achieved in the brain.MethodsIn situ hybridization and immunostaining of histological brain sections using a subset of molecular markers that label specific cortical regions or cell types. Appropriate quantification and statistical analysis of gene and protein expression were applied to identify cortical abnormalities and to determine the level of phenotypic rescue achieved.ResultsCortical abnormalities observed in Tbx1 mutant embryos were fully rescued by both drugs. Intriguingly, rescue was obtained with both drugs in Tbx1 homozygous mutants, indicating that they function through mechanisms that do not depend upon Tbx1 function. This was particularly surprising for Vitamin B12, which was identified through its ability to increase Tbx1 gene expression.ConclusionTo our knowledge, this is only the second example of drugs to be identified that ameliorate phenotypes caused by the mutation of a single gene from the 22q11.2 homologous region of the mouse genome. This one drug-one gene approach might be important because there is evidence that the brain phenotype in 22q11.2DS patients is multigenic in origin, unlike the physical phenotypes, which are overwhelmingly attributable to Tbx1 haploinsufficiency. Therefore, effective treatments will likely involve the use of multiple drugs that are targeted to the function of specific genes within the deleted region.

Restoration of FVIII Function and Phenotypic Rescue in Hemophilia A Mice by Transplantation of MSCs Derived From F8-Modified iPSCs

Hemophilia A (HA), an X-linked recessive congenital bleeding disorder, affects 80%–85% of patients with hemophilia. Nearly half of severe cases of hemophilia are caused by a 0.6-Mb genomic inversion (Inv22) that disrupts F8. Although viral-based gene therapy has shown therapeutic effects for hemophilia B (HB), this promising approach is not applicable for HA at the present stage; this limitation is mainly due to the large size of F8 cDNA, which far exceeds the adeno-associated virus (AAV) packaging capacity. We previously reported an in situ genetic correction of Inv22 in HA patient-specific induced pluripotent stem cells (HA-iPSCs) by using TALENs. We also investigated an alternative strategy for targeted gene addition, in which cDNA of the B-domain deleted F8 (BDDF8) was targeted at the rDNA locus of HA-iPSCs using TALENickases to restore FVIII function. Mesenchymal stem cells (MSCs) have low immunogenicity and can secrete FVIII under physiological conditions; in this study, MSCs were differentiated from F8-corrected iPSCs, BDDF8-iPSCs, and HA-iPSCs. Differentiated MSCs were characterized, and FVIII expression efficacy in MSCs was verified in vitro. The three types of MSCs were introduced into HA mice via intravenous injection. Long-term engraftment with restoration of FVIII function and phenotypic rescue was observed in HA mice transplanted with F8-corrected iMSCs and BDDF8-iMSCs. Our findings suggest that ex vivo gene therapy using iMSCs derived from F8-modified iPSCs can be feasible, effective, and promising for the clinical translation of therapeutic gene editing of HA and other genetic birth defects, particularly those that involve large sequence variants.

Pharmacological rescue of the brain cortex phenotype of Tbx1 mouse mutants: significance for 22q11.2 deletion syndrome

ABSTRACTObjectivesTbx1 mutant mice are a widely used model of 22q11.2 deletion syndrome (22q11.2DS) because they manifest a broad spectrum of physical and behavioral abnormalities that is similar to that found in 22q11.2DS patients. In Tbx1 mutants, brain abnormalities include changes in cortical cytoarchitecture, hypothesized to be caused by the precocious differentiation of cortical progenitors. The objectives of this research are to identify drugs that have efficacy against the brain phenotype, and through a phenotypic rescue approach, gain insights into the pathogenetic mechanisms underlying Tbx1 haploinsufficiency.Experimental approachDisease modelTbx1 heterozygous and homozygous embryos. We tested the ability of two FDA-approved drugs, the LSD1 inhibitor Tranylcypromine and Vitamin B12, to rescue the Tbx1 mutant cortical phenotype. Both drugs have proven efficacy against the cardiovascular phenotype, albeit at a much reduced level compared to the rescue achieved in the brain.Methodsin situ hybridization and immunostaining of histological brain sections using a subset of molecular markers that label specific cortical regions or cell types. Appropriate quantification and statistical analysis of gene and protein expression were applied to identify cortical abnormalities and to determine the level of phenotypic rescue achieved.ResultsCortical abnormalities observed in Tbx1 mutant embryos were fully rescued by both drugs. Intriguingly, rescue was obtained with both drugs in Tbx1 homozygous mutants, indicating that they function through mechanisms that do not depend upon Tbx1 function. This was particularly surprising for Vitamin B12, which was identified through its ability to increase Tbx1 gene expression.ConclusionsTo our knowledge, this is only the second example of drugs to be identified that ameliorate phenotypes caused by the mutation of a single gene from the 22q11.2 homologous region of the mouse genome. This one drug-one gene approach might be important because there is evidence that the brain phenotype in 22q11.2DS patients is multigenic in origin, unlike the physical phenotypes, which are overwhelmingly attributable to Tbx1 haploinsufficiency. Therefore, effective treatments will likely involve the use of multiple drugs that are targeted to the function of specific genes within the deleted region.

Kinetic modeling of stem cell transcriptome dynamics to identify regulatory modules of normal and disturbed neuroectodermal differentiation

Thousands of transcriptome data sets are available, but approaches for their use in dynamic cell response modelling are few, especially for processes affected simultaneously by two orthogonal influencing variables. We approached this problem for neuroepithelial development of human pluripotent stem cells (differentiation variable), in the presence or absence of valproic acid (signaling variable). Using few basic assumptions (sequential differentiation states of cells; discrete on/off states for individual genes in these states), and time-resolved transcriptome data, a comprehensive model of spontaneous and perturbed gene expression dynamics was developed. The model made reliable predictions (average correlation of 0.85 between predicted and subsequently tested expression values). Even regulations predicted to be non-monotonic were successfully validated by PCR in new sets of experiments. Transient patterns of gene regulation were identified from model predictions. They pointed towards activation of Wnt signaling as a candidate pathway leading to a redirection of differentiation away from neuroepithelial cells towards neural crest. Intervention experiments, using a Wnt/beta-catenin antagonist, led to a phenotypic rescue of this disturbed differentiation. Thus, our broadly applicable model allows the analysis of transcriptome changes in complex time/perturbation matrices.