Spatiotemporal Control of Fly Morphogenesis

The process of morphogenesis, by which an organism develops its shape, is one of the least well understood pillars of developmental biology (1). Research on model organisms has emphasized the importance of particular genes and molecular pathways. However, current models of how these directly relate to growth on the macroscopic scale, particularly in humans, remain simplistic at best (2). For their part, Karen Kasza and other members of the Zallen lab of Sloan Kettering Institute have discovered how the coordinated actions of the proteins actin, myosin II, and rho-kinase contribute to the early macroscopic development in the model organism Drosophila melanogaster.

Drosophila

A Fly embryo just after lengthening, before the onset of cell division. Copyright: Keller Lab/Janelia Farm Research Campus/HHMI

Drosophila embryos consist of a layer of epithelial cells surrounding a yolk center. During the first two hours of development, these embryos double in length along the anterior-posterior (AP) axis (3). Through green fluorescent protein (GFP) microscopy, members of the Zallen lab identified actin-myosin contraction has as the primary agent. Actin, the multi-unit filamental protein also responsible for muscle contraction, was specifically found to accumulate at the edges of the cell membrane perpendicular to the AP axis. This localized buildup causes asymmetrical tension, resulting in epithelial cells lengthening along the AP axis (3).

One of the key characteristics of the actin-myosin contraction is that its activity is controlled through the addition or subtraction of a phosphate group to myosin itself. To examine what effect the activity on myosin had on the timing or degree of lengthening, the Zallen lab genetically modified wild-type flies to express either a more active (EE) or less active (AA) myosin phosphovariant. In both cases, replacement of the wild type (WT) with either the EE or AA phosphovariant caused a decrease in the degree of anterior-posterior lengthening (3).

From this information, Dr. Kasza and her colleagues came up with a number of theories. Intuitively, the degree of lengthening should depend primarily two characteristics. It should depend on the magnitude of the tension applied, whereby not applying enough tension should lower the degree of lengthening. Additionally, it should depend on the orientation of lengthening; actin oriented in the wrong direction (i.e. parallel to the AP axis) should also decrease the amount of lengthening. Since changes to actin’s activity had a detrimental effect on lengthening, the Zallen lab next examined the protein responsible for phosphorylating myosin, rho-kinase (3).

Flies genetically engineered to not express rho-kinase (knockouts) showed none of the polarity effects of their wild-type counterparts. Rather, actin marked through GFP localized everywhere along the cell membrane. Rho-kinase is necessary for specific actin localization to the cell membrane perpendicular to the AP axis, as changing the activity level of the protein through phosphovariants negatively affects the degree of lengthening.

Future research by the Dr. Kasza and her colleagues at the Zallen lab hopes to take advantage of optogenetics to further examine the mechanisms of rho-kinase and related proteins(RhoGEF2, RhoA), particularly for their potential function in humans.

Sources:

1. R, T. (2005, February 30). Can morphogenesis be understood in terms of physical rules? Journal of Biosciences, 87-92.

2. Davies, J. (2013). Mechanisms of Morphogenesis. Academic Press.

3. Kasza, K., Farrell, D., & Zallen, J. (2014). Spatiotemporal control of epithelial remodeling by regulated myosin phosphorylation. Proceedings of the National Academy of Sciences, 11732-11737.

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