The cellular and molecular mechanisms underlying ventral midline patterning and morphogenesis in the amniote midbrain

Abstract

The floorplate (FP) is located at the ventral midline of the developing neural tube, and is involved in patterning and specification of ventral and dorsal cell fates. The FP has long been known to pattern ventral cell fates via secretion of Sonic Hedgehog (SHH). However, the mechanism by which the FP is specified is controversial due to species variations where SHH is differentially required for FP specification in fish and mouse. In Chapter 3, we show that, similar to the fish, the amniote anterior neural plate can be divided into medial (MFP) and lateral (LFP) subdivisions which differentially require SHH and FOXA2 for their specification, and that FOXA2, but not SHH, is sufficient to induce the entire midbrain FP pattern. In addition, we show that all three midbrain signaling centers are physically continuous and interconvertible, with their specification depending on SHH. Prior to the expression of SHH protein, the ventral midline undergoes a morphogenetic event called median hinge point (MHP) formation which buckles the flat neural plate and lifts the neural folds which ultimately fuse into a cylindrical neural tube. Previous studies in the lab have shown that Bone Morphogenetic Proteins (BMP) modulate HP formation. HP formation involves dynamic cell-shape changes, which result in HP cells becoming wedge-shaped. Multiple mechanisms (constriction of the adherens belt via cytoskeletal and junctional remodeling, and polarized endocytosis) have been proposed to explain this shape change. However, they do not explain how reduction in apical area can be achieved in the amniote neural plate where cells are bipolar and only slender processes contact the apical surface in non-mitotic cells. In Chapter 4, we develop an early electroporation technique which is used in Chapter 5 to visualize HP formation in real time as part of a novel 3D explant system. Our results suggest that BMP attenuation regulates cell cycle progression by increasing the duration of G1 and S phases, and causes a subset of cells to prematurely exit the cell cycle and undergo sub-apical G2-M transition, similar to what is seen in the MHP where there is reduced mitotic index and cells undergo mitosis sub-apically.