The CCAAT/Enhancer Binding Protein Beta (cebpb) is essential for the development of enveloping layer (EVL) in zebrafish

Epiboly, the coordinated spreading of the enveloping layer (EVL), the yolk syncytial layer (YSL) and the deep cells (DCs) toward the vegetal pole, is required for gastrulation. Interference with the EVL leads to early developmental failure. However, the genetic factors underlying EVL differentiation are so far unknown. By in-depth analyzing of single-cell sequencing data from 4 h post fertilization zebrafish embryos, we identified 105 genes (FDR<0.05) which showed highly specific EVL expression, among which the transcription factor cebpb and genes (krt18, cldne, cldnb, krt4, krt5) involved in cell-cell junction formation were exclusively expressed in EVL. To explore the role of cebpb in EVL differentiation, we knocked down its expression by morpholino-labeled antisense RNA. The morphant embryos showed disrupted EVL and developmental termination prior to epiboly. In accordance, expression of the EVL specific cell-adhesion molecule genes were significantly reduced. Thus, cebpb is the earliest EVL marker and essential for EVL differentiation in zebrafish. The elucidation of the specific EVL gene set provides us new insights into the molecular mechanisms underlying EVL differentiation.