# Oct4 Contributes to Mesodermal Differentiation by Sustaining the Proliferative Capacity of Early Mesodermal Progenitors

## TL;DR

Oct4 helps mesodermal cells stay proliferative during early development, which is important for proper differentiation into mesodermal lineages.

## Contribution

Oct4 sustains the proliferative capacity of early mesodermal progenitors, supporting mesodermal differentiation.

## Key findings

- Oct4 and Brachyury are transiently co-expressed in early mesodermal progenitors.
- Loss of Oct4 reduces EB size and causes cell cycle arrest in G0/G1 phase.
- Oct4 loss diminishes cardiomyocyte formation and cannot be rescued by increasing cell numbers.

## Abstract

Oct4 is well established as a core regulator of pluripotency, yet emerging evidence points to an additional role in lineage specification during the exit from the pluripotent state. Although Oct4 expression has been observed in early mesodermal progenitors, its precise function in this developmental context remains unclear. To investigate this, we employed embryoid bodies (EBs) as a model of spontaneous differentiation that recapitulates key aspects of early embryonic development in vitro. In accordance with previous studies, reporter assay revealed a distinct temporal pattern characterized by the strong, transient co-expression of Oct4 and the early mesoderm-specifying marker gene Brachyury within a narrow developmental window, consistent with the Oct4 role in early mesodermal progenitors. We further examined the consequences of the Oct4 loss at early stages of this differentiation. Conditional knockout of the Oct4 gene resulted in a significant reduction in EB size and accumulation of cells in the G0/G1 phase, indicating a critical requirement for Oct4 in maintaining cell proliferation. Despite this defect, cells retained the ability to initiate multilineage differentiation, albeit with reduced expression of Brachyury and elevated expression of endodermal markers FoxA2 and Sox17. Interestingly, the formation of beating cardiomyocyte-like structures was also diminished following Oct4 loss and could not be rescued by simply increasing cell numbers. Taken together, these findings highlight an important Oct4 function in mesodermal differentiation, mediated through the maintenance of proliferative capacity of early mesodermal progenitors.