Non-muscle myosin IIA and IIB differentially regulate cell shape changes during zebrafish brain morphogenesis

Abstract

During brain morphogenesis, the neuroepithelium must fold in specific regions to delineate functional units, and give rise to conserved embryonic brain shape. Individual cell shape changes are the basis for the morphogenetic events that occur during whole tissue shaping. We used the zebrafish to study the molecular mechanisms that regulate the first fold in the vertebrate brain, the highly conserved midbrain-hindbrain boundary (MHB). Since the contractile state of the neuroepithelium is tightly regulated by non-muscle myosin II (NMII) activity, we tested the role of NMIIA and NMIIB in regulating cell shape changes that occur during MHB morphogenesis. Using morpholino knockdown, we show that NMIIA and NMIIB are both required for normal MHB tissue angle. Quantification of cell shapes revealed that NMIIA is required for the shortening of cells specifically at the MHB constriction (MHBC), while NMIIB is required for the proper width of cells throughout the MHB region. NMIIA and NMIIB knockdown also correlated with abnormal distribution of actin within the cells of the MHBC. Thus, NMIIA and NMIIB perform distinct functions in regulating cell shape during MHB morphogenesis.

Calcium signals drive cell shape changes during zebrafish midbrain–hindbrain boundary formation

“…We hypothesized that decreasing Ca 2+ with 2-APB would specifically rescue short cells induced by NMII overactivation (Figure 4A). The mypt1 morphants revealed cells that were both shorter and wider than controls, as we previously reported (Figure 4B; Gutzman and Sive, 2010; Gutzman et al. , 2015).…”

“…All mGFP live confocal imaging was conducted as previously described (Graeden and Sive, 2009; Gutzman et al. , 2015) using a Nikon CS2 scanning confocal microscope.…”

“…MO details are as follows: mypt1 MO (5′-ATTTTTTGTGACTTACTCAGCGATG-3′; Gutzman et al. , 2008, 2015), calmodulin 1a (ENSDART00000034580), and calm1a MO (5′-CCACAAACAGACTGCCTTACCTGCA-3′). Zebra­fish p53 MO (5′-GCGCCATTGCTTTTGCAAGAATTG-3′) was used in conjunction with the calm1a MO at equal concentration.…”

“…Using the zebrafish MHB as a model to understand brain morphogenesis, we characterized the basic cell shape changes that are required to fold the neuroepithelium in this region (Gutzman et al. , 2008, 2015). Zebrafish MHB formation is initiated at the 18-somite stage (ss) after the neural tube has formed.…”

A Genetically Encoded Biosensor Strategy for Quantifying Non-muscle Myosin II Phosphorylation Dynamics in Living Cells and Organisms

“…NMMII activity has been broadly implicated in shaping organisms and tissues (Mammoto and Ingber, 2010; Siedlik and Nelson, 2015). Polarized contraction, either in select groups of cells or compartmentalized within individual cells, is thought to help shape organisms (Aldaz et al, 2013), organs (Gutzman et al, 2015) and even finer structures (Ewald et al, 2008; Kim et al, 2013). Because both filament orientation and the duration they persist are thought to be vitally important for polarized contraction (Kasza et al, 2014), an optical approach for measuring myosin RLC phosphorylation would be useful for understanding how NMMII activity is controlled and sustained throughout development.…”