Spindle Orientation Coordinates Cell Fate Decisions During Epidermal Morphogenesis

Asymmetric cell divisions (ACDs) drive cell fate specification and the formation of complex tissue architecture. This often requires orienting the mitotic spindle to position daughter cells and/or segregate cell fate determinants. However, the mechanisms that properly position the mitotic spindle and link spindle orientation to cell fate specification are not fully understood. During mouse embryogenesis, oriented divisions drive epidermal morphogenesis. Disrupting oriented divisions results in differentiation defects, loss of barrier function, and lethality. ACDs have also been observed during hair follicle morphogenesis and have been suggested to produce specific populations of hair follicle cells, though this has not been directly tested. In the developing epidermis, spindle orientation requires a conserved cortical protein complex of LGN/NuMA/dynein-dynactin. This complex is thought to function by generating pulling forces on astral microtubules. However, the factors that regulate astral microtubule dynamics and their association with this cortical complex have not been well studied. In this work, I explored the role of the microtubule catastrophe factor KIF18B in regulating microtubule dynamics to promote spindle orientation in keratinocytes. Utilizing two individually isolated control and KIF18B knockout (KIF18BKO) cell lines, I demonstrated that KIF18B is required for spindle alignment in cultured keratinocytes. Mitotic spindles in KIF18BKO cells exhibited an increase in both the length and number of astral microtubules. Microtubules more frequently touched the cortex in KO cells than in control cells. Furthermore, live-imaging revealed that loss of KIF18B dramatically altered microtubule dynamics; astral microtubules of KIF18BKO cells showed an increase in growth lifetime and growth displacement compared to controls. KIF18B’s regulation of microtubules was restricted to the astral microtubules, which is of note, as these are the microtubules that interact with the cell cortex as well as the spindle orientation machinery. I propose that KIF18B promotes astral microtubule catastrophe, which is essential to maintain proper microtubule dynamics and orient the mitotic spindle. Unexpectedly, I discovered that KIF18B accumulates at the cell cortex during mitosis, colocalizing with the conserved spindle orientation machinery. This cortical localization has not been previously reported. KIF18B cortical localization mimicked that of NuMA. During metaphase, KIF18B polarized to one side of the cell cortex. However, during anaphase, KIF18B became bipolar and expanded along the cell cortex. This localization pattern was NuMA-dependent in both metaphase and anaphase and did not require microtubules. In vivo I found that KIF18B was required for oriented cell divisions within the hair placode, the first stage of hair follicle morphogenesis, but was not essential in the interfollicular epidermis. Disrupting spindle orientation in the placode, using mutations in either KIF18B or NuMA, resulted in aberrant expression of Sox9, which serves as a cell fate marker of the inner region of adult hair follicle cells. Additionally, I showed that treatment of mice with Wnt inhibitors phenocopied the abnormal Sox9 expression seen in ACD mutants. These data lead me to hypothesize that asymmetric segregation of Wnt regulators plays a role in asymmetric cell division of basal placode cells. My data functionally link spindle orientation to cell fate decisions during hair follicle morphogenesis for the first time. Taken together, my data demonstrate a role for regulated microtubule dynamics in spindle orientation in epidermal cells. My work also highlights the importance of spindle orientation during asymmetric cell division to dictate cell fate specification.