Thymosin β4 is Not Required for Embryonic Viability or Vascular Development

Abstract

Rationale

Rossdeutsch et al. describe a requirement for Thymosin β4 (Tβ4) in vascular development. Impaired mural cell migration, differentiation, partial embryonic lethality, and hemorrhaging were observed following analysis of two lines of mice, one of which was germline null for Tβ4, and another in which Tβ4 was knocked down by endothelial specific expression of Tβ4 shRNA. These data are in direct contrast to our published global and cardiac specific Tβ4 knockout lines. Thus the role of Tβ4 needs to be clarified to understand its importance in cardiovascular development.

Objective

To investigate and clarify the role of Tβ4 in vascular smooth muscle cell development and vessel stability.

Methods and Results

Examination of Tβ4 global knockouts did not demonstrate embryonic hemorrhaging, altered mural cell development or lethality. Endothelial specific knockouts also did not exhibit any embryonic lethality and were viable to adulthood.

Conclusions

Analysis of our Tβ4 global and cardiac- and endothelial-specific knockout models demonstrated that Tβ4 is dispensable for embryonic viability and vascular development.

Introduction

Understanding signaling factors that regulate formation of the vasculature can lend significant insight into both development and disease. Thymosin β4 (Tβ4) is a 43 amino acid factor initially found to interact with G-actin and regulate F-actin formation¹. Recent studies have indicated that administration of Tβ4 to either post-ischemia reperfusion or myocardial infarction models can improve cardiovascular function and abrogate scar formation, primarily through de novo vasculature formation^2-4. Early studies using an shRNA knockdown approach suggested that Tβ4 acted as a key regulator of blood vessel formation (angiogenesis, vasculogenesis and arteriogenesis)⁵. However, our studies using both global and cardiac specific knockout approaches found Tβ4 to be dispensable for embryonic viability and vessel development⁶.

In a recent paper, Rossdeutsch et al. describe a requirement for Tβ4 in vascular development⁷. Impaired mural cell migration and differentiation was observed following analysis of two lines of mice, one of which was germline null for Tβ4, and another in which Tβ4 was knocked down by endothelial specific expression of Tβ4 shRNA. Rossdeutsch et al. report that global knockout of Tβ4 resulted in partial lethality at the start of the study that was decreased in subsequent generations later during the study, an incompletely penetrant hemorrhagic phenotype and decreased mural cell overage of the aorta⁷. Tβ4 knockdown in endothelium also resulted in partial lethality and decreased mural cell coverage⁷. These data are in direct contrast to our published Tβ4-knockout model⁶. Thus, to clarify the role of Tβ4 in vascular formation we have further analyzed our global knockouts of Tβ4 and an endothelial-specific Tβ4 knockout not previously reported. Consistent with our previous results, we did not observe an evident phenotype, alteration in vascular development or embryonic lethality. We conclude from these data that Tβ4 is not required for embryonic viability or vascular development.

Materials and Methods

Animal Care

All animal procedures were performed and approved by the UCSD Animal Care and Use Committee.

Generation of tβ4 Floxed, Knockout and Endothelial-Specific Knockout mice

tβ4-targeted mice were generated and used as previously described to a C57/B6J background⁶. Endothelial-specific knockout mice were generated by crossing tβ4^f/f female mice to Tie2-Cre male mice.

Immunofluorescence Analyses

Frozen sections from E14.5, E13.5 and E12.5 embryos were isolated and stained as previously described⁶.

Results

Mural Cell Coverage and Development Are Not Altered in Global Null tβ4 Mutants

To clarify the role of Tβ4 in large vessel development, we examined our global tβ4- knockout mice from E12.5 to E14.5 (Figures 1-2). Initial observation in these stages revealed no gross abnormalities or hemorrhage between wild type and knockout samples. To examine large vessel defects we first examined aortas in E14.5 embryos (Figure 1). At this stage, no differences were observed between examined wild type and knockout embryos. To rule out developmental and mural cell recruitment defects, E12.5 and E13.5 aortas were stained with antibodies to αSMA and PDGFRβ, two markers of vascular smooth muscle cells (Figures 2&3) ^6-8. No differences were observed between control and Tβ4 null embryos.

Figure 1. tβ4-loss does not alter developing vasculature at E14.5.

A&B∷ E14.5 Wildtype and Tβ4 knockout aorta. A:Blue Dapi, Green CD31 Red αSMA or B: Blue Dapi, Green CD31, Red PDGFRβ. 40X C: 13.5 Wildtype and Tβ4 knockout left ventricle. Blue Dapi, Red Tβ4. 20X

Figure 2. tβ4-loss does not alter developing vasculature at E12.5 or E13.5.

A&B: E12.5 Wildtype and Tβ4 knockout aortae stained in A:Blue Dapi, Green CD31 Red αSMA or B: Blue Dapi, Green CD31, Red PDGFRβ. C&D: E13.5 Wild type and Tβ4 knockout aorta stained in C:Blue Dapi, Green CD31 Red αSMA or D: Blue Dapi, Green CD31, Red PDGFRβ. 40X

Endothelial-Specific Deletion of tβ4 Does Not Result in Embryonic Lethality

Rossdeutsch et al. observed 12% lethality in embryos with endothelial specific shRNA mediated knockdown of Tβ4. As stated in previous publications, shRNA knockdown can have off-target effects^6,9. To further investigate this possibility, we used the same Tie2-Cre employed by Rossduetsch et al.^7,10. Our tβ4^f/f females⁶ were bred to Tie2-Cre male mice¹¹. Male Tie2-Cre mice were also utilized by Rossdeutsch et al. in their knockdown studies. Out of 34 postnatal male mice, 18 were tβ4^f/y and 16 were tβ4^f/y;Tie2-Cre⁺, demonstrating lack of embryonic lethality in endothelial specific knockouts of Tβ4. These data are similar to our global and cardiac specific knockouts of Tβ4⁶; where we did not observe any postnatal lethality or vascular phenotype.

Discussion

Rossdeutsch and co-workers report that global tβ4-knockout mice present with hemorrhage and defective mural cell recruitment, resulting in partial lethality between E10.5 and E14.5⁷. These data are in direct contrast to our published global tβ4-knockout model⁶. In the Discussion section of their manuscript, Rossdeutsch and co-workers refer to our paper⁷, and state that, from our data,“Tβ4 knockout aortas had an apparent reduction in α-SMA+ cell coverage within the vessel wall relative to wild-type controls at E14.5”⁷. We believe this to be a misinterpretation of our data. In our publication⁶ we did not report, nor observe, differences in mural cell coverage in any of our samples at E14.5. Data showing αSMA staining of aortic vascular smooth muscle were meant to illustrate maintenance of vascular smooth muscle within the aorta, and were not meant to be a quantitative assessment. However, given the observations by Rossdeutsch et al, we have specifically examined aortas in multiple sections from E12.5 to E14.5 Tβ4 null and control embryos and found no differences between control and Tβ4 null embryos (Figures 1 and 2). We also did not observe any hemorrhaging in any of the embryos in our studies. Thus, our data do not support an essential role for Tβ4 in vessel development.

Rossdeutsch et al. also suggest that differences in observed phenotypes between our Tβ4 null mutants and theirs can be attributed to either strain background dependent effects or distinct gene targeting strategies⁷. Initially, Rossdeutsch et al. observed 40% lethality of their global null Tβ4 mice in a mixed C57/B6J and 129Sv background, with reduction to 20% lethality when bred into a pure C57/B6J background. However, our global null mice were bred into a C57/B6J background and yet did not display any phenotype⁶. It is possible that differences in observed phenotypes between the two Tβ4 null mutant lines may result from variables such as environment, diet, and infectious state which may differ between the two mouse facilities.

An alternative explanation to strain or facility-dependent phenotype is that the targeted ES cells used to establish the Rossdeutsch mutant mouse line contained a Tβ4- independent lethal mutation(s) that may have been bred out over successive generations. Moreover, the possibility raised by Rossdeutsch et al. that differences in targeting strategies, in particular the retention of the Neo cassette in the Rossdeutsch et al. targeted allele, could also account for discrepant phenotypes is a possibility. However, if this is the case, it is unlikely to reflect differences in targeting Tβ4 function, as exon 2, encompassing 75% of the coding sequence, is deleted in both knockouts and similarly Tβ4 protein is absent, as demonstrated by protein analysis, in both knockouts. Therefore, if the different phenotypes are a result of retention of the Neo gene in the Rossdeutsch Tβ4 null allele, observed phenotypes may result from non-Tβ4 related functions.

Rossdeutsch et al. argue that there are no “off-target” effects with their shRNA gene silencing approach and state “the differences between our global knockout and knockdown model are probably attributed to the fact that RNAi targeting in vivo, when sufficiently optimal to abrogate expression of the target gene, can result in a more severe phenotype than a corresponding global-null. Genetic ablation via homologous recombination through the germline, leading to complete loss of function from the outset in development, may be partially compensated for by functional orthologues, whereas RNAi-mediated efficient knockdown, occurring rapidly and at a defined developmental stage, may not be permissive for compensation.”⁷ Our previously published cardiac specific (Nkx2.5-Cre, αMHC-Cre), ⁶ and endothelial cell specific (Tie2-Cre) (reported here) ablations of our Tβ4 floxed alleles, have demonstrated that targeted deletion during development does not result in an evident phenotype, thus calling into question this argument by Rossdeutsch et al. to address discrepancies between their global Tβ4 null and their conditional shRNA-Tβ4 knockdown phenotypes.

In summary, our data with global and conditional Tβ4 knockout mice suggest that the shRNA knockdown approach used by Rossdeutsch et al may have ‘off-target’ effects which result in observed phenotypes. The phenotype observed in their global Tβ4 knockout mice may result from perturbation of Tβ4-independent events. Together, these data do not support an essential role for Tβ4 in vessel development or embryonic viability.

Non-standard Abbreviations and Acronyms

αSMA

α-smooth muscle actin

CD31

Platelet endothelial cell adhesion molecule

ES

Embryonic Stem (Cells)

PDGFRβ

Platelet-derived growth factor subunit b

ShRNA

short hairpin RNA

Tβ4

thymosin beta 4