Seeing is believing: a novel way to look at mammalian development

In-vivo imaging of the dynamic behaviors of tissues and cells during organ formation in mammals has been particularly challenging, due to their in utero development. This article describes a novel, prolonged and robust live imaging system for visualizing the formation of a variety of embryonic tissues in the mid-gestation mouse embryo.

HFSP Long-Term Fellow R’ada Massarwa and colleagues
authored on Fri, 04 January 2013

Live imaging of the mouse embryo is very challenging as the embryo develops in-utero, unlike other model organisms such as Drosophila, Xenopus, zebrafish and chick embryos. A particularly challenging stage of mouse embryogenesis is between E8.5 to E9.5, when the heart begins to beat and the embryo is rapidly expanding in size and is undergoing major changes in morphology, including the formation of the early brain, spinal cord and face.

We have overcome these challenges by developing a robust live imaging system in which the formation of these tissues can be visualized in a whole living embryo in real time. Using this novel imaging system we followed the dynamics of neural tube closure in the mouse embryo. Neural tube closure is one of the most complex morphological processes during embryogenesis in vertebrates, including humans. We visualized neural tube closure from its initiation, when the neural tissue is a flat sheet, through the bending and meeting of the neural folds to complete the formation of the neural tube, the embryonic precursor of the brain and spinal cord. Our study contributed new insights into the dynamics of neural tube closure including evidence supporting the existence of two different and independent morphological processes, zipping and inflection of the neural folds. We found that these two processes are coordinated both spatially and temporally to promote neural tube closure. In addition, we used the Cre/LoxP genetic tool to fluorescently label the non-neural ectoderm cells that border the neural tissue. This showed that the non-neural ectoderm is highly active during neural tube closure. These cells undergo a variety of shape changes including rosette formation and they extend cellular processes such as filopodia, lamellipodia and membrane ruffles at different regions along the neural tube.

We then expanded upon the information gained through the study of normal embryos to determine how neural tube closure goes awry in a mutant mouse with a cranial neural tube defect. An advantage of our system is the ability to image more than one embryo simultaneously, a crucial parameter in studies of mutant embryos that cannot be distinguished at the start of the experiment. By quantitating the dynamics of zipping and inflection at different times and regions along the neural tube we determined the onset of the defect and visualized how the compounding disruption to these processes leads to severely altered cranial morphogenesis.

Figure: The dissected embryo inside the yolk sac is added to a drop of freshly prepared culture media on the surface of a filter paper. The yolk sac is opened starting at the top furthest away from the embryo and peeled down around the embryo, maintaining its connection with the ventral side of the embryo. The yolk sac is then adhered to the surface of the filter to anchor the embryo to the filter. Different embryos at different developmental stages can be stretched and mounted at different angles, to expose a variety of tissues at different regions of the embryo. In order to expose the neural folds to image neural tube closure, the embryo is extended by gently pushing both the head and the tail down onto the filter in the direction of the yellow arrows. The tissue will adhere to the surface of the filter allowing the neural tissue to be visualized. A view from the top with the four embryos mounted on the filter paper for imaging neural tube closure.

Neural tube defects are the second most common birth defect in humans. Although the genetic mutations underlying human neural tube defects are essentially unknown, the mouse is an excellent model for mammalian neural tube closure and there are over 200 gene mutations in mice that cause neural tube defects. Our imaging system provides a robust means to elucidate how the cell behaviors of neural tube closure are disrupted in these mutants. This novel dynamic and live imaging method provides an unprecedented view of mammalian development that should be applicable to the study of many embryonic tissues in the mid-gestation embryo. 

 

Reference

In toto live imaging of mouse morphogenesis and new insights into neural tube closure. Massarwa R. & Niswander L. J. Development 140, 226-236 (2013).

Pubmed link