BMAL1 and CLOCK control polarization and lumen formation during mammary alveologenesis in 3D culture


  • Jacob Larsen Purdue University
  • Aridany Suarez-Trujillo Berry University
  • Kelsey Teeple Purdue University
  • Jenna Schoonmaker Purdue University
  • Karen Plaut Purdue University
  • Theresa Casey Purdue University



Mammary gland, Acini, 3D cultures, BMAL1, CLOCK, Circadian timing system


Circadian clock disruption decreased mammary development and impaired lactation in late gestation cows and mice. Transcriptional targets of the circadian clock genes BMAL1 and CLOCK include the cell-cell and cell-ECM proteins e-cadherin (CDH1) and zonula occludens-1 (ZO-1). The polarization and lumen formation of milk-producing acini is dependent on these junctional proteins. We hypothesized that if BMAL1 and CLOCK were disrupted, mammary epithelial cells (HC11) will have a reduced ability to form acini. Our objective was to measure the effect of BMAL1 gene deletion (BMAL1-KO) and CLOCK protein reduction (shCLOCK) in HC11 cells on the formation of acini and expression of CDH1 and ZO-1 in three-dimensional (3D) cultures. Cells were plated on Matrigel, embedded, and cultured to create 3D structures. ImageJ software was used to quantify the acini and found that the BMAL1-KO and shCLOCK lines formed fewer and smaller acini (p < 0.05). Confocal microscopy revealed that CDH1 was located along lateral membranes and ZO-1 was apically located in all three lines. Levels of CDH1 and ZO-1 were greater (p < 0.05) per unit volume of cell in the shCLOCK cell line. Live/dead staining of cells found little to no cell death across three lines. Transmission electron microscopy (TEM) of acini indicated less polarization of cells within BMAL1-KO and shCLOCK acini. Polarization of cells is required for formation of 3D structures and requires coordination of temporal-spatial cues. Disruption of circadian clocks results disturbs temporal organization of cellular processes and causes a reduced ability of cells to polarize and form acini.


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How to Cite

Larsen, J., Suarez-Trujillo, A., Teeple, K., Schoonmaker, J., Plaut, K., & Casey, T. (2023). BMAL1 and CLOCK control polarization and lumen formation during mammary alveologenesis in 3D culture. Journal of Student Research, 11(3).



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