Mesodermal Patterning Activity of the Transcription Factor SCL.

The transcription factor SCL is critically required for the establishment of the hematopoietic lineage as well as for proper endothelial development, however it is not required for maintenance of HSCs or endothelial cells in the adult. Conflicting data exists regarding the developmental function of SCL, namely whether it acts as a master regulator, actively patterning mesoderm toward hematopoietic/endothelial development at the expense of other lineages, or is merely necessary to maintain the earliest committed hematopoietic and endothelial precursors. To answer this question in a mammalian model, we have engineered murine ES cells with a conditional doxycycline-inducible SCL transgene, and evaluated the effects of pulses of SCL expression at defined time points during in vitro development. During embryoid body differentiation, a pulse of SCL expression during mesodermal patterning results in enhanced hematopoiesis at later time points, as assayed by CFC activity and FACS analysis, as well significantly increasing the frequency of bipotent hematopoietic/endothelial hemangioblast CFCs. Concomitantly, SCL represses cardiac development, assayed by beating cardiomyocyte frequency, and molecular markers of the cardiac lineage. SCL also represses paraxial mesodermal developmental potential, severely reducing both the earliest PDGFαR⁺/Flk-1^neg paraxial progenitors, as well as repressing levels of later markers including Pax3. Through chimeric embryoid bodies in which SCL cells are mixed with GFP-labelled ES cells, we show formally that this effect is cell-autonomous. Finally, we tested whether expression of SCL would enable hematopoiesis during ES cell monolayer differentiation. Hematopoiesis requires inter-germ layer inductive signaling which occurs in 3 dimensions but not in monolayer culture, thus ES cells do not spontaneously differentiate into blood in this 2 dimensional culture. We show that when mesoderm is produced during monolayer culture of wild-type ES cells, or control cells not expressing SCL, it is biased towards paraxial or cardiac differentiation. However when given a pulse of SCL expression, these pathways are repressed, PDGFαR^neg/Flk-1⁺ presumptive lateral plate progenitors are produced, and abundant hematopoietic differentiation occurs. This data in the mammalian system thus strongly supports the former hypothesis, of an active role for SCL in mesodermal patterning.