Characterization and Classification of Zebrafish Brain Morphology Mutants

Abstract

The mechanisms by which the vertebrate brain achieves its threedimensional structure are clearly complex, requiring the functions of many genes. Using the zebrafish as a model, we have begun to define genes required for brain morphogenesis, including brain ventricle formation, by studying 16 mutants previously identified as having embryonic brain morphology defects. We report the phenotypic characterization of these mutants at several timepoints, using brain ventricle dye injection, imaging, and immunohistochemistry with neuronal markers. Most of these mutants display early phenotypes, affecting initial brain shaping, whereas others show later phenotypes, affecting brain ventricle expansion. In the early phenotype group, we further define four phenotypic classes and corresponding functions required for brain morphogenesis. Although we did not use known genotypes for this classification, basing it solely on phenotypes, many mutants with defects in functionally related genes clustered in a single class. In particular, Class 1 mutants show midline separation defects, corresponding to epithelial junction defects; Class 2 mutants show reduced brain ventricle size; Class 3 mutants show midbrain-hindbrain abnormalities, corresponding to basement membrane defects; and Class 4 mutants show absence of ventricle lumen inflation, corresponding to defective ion pumping. Later brain ventricle expansion requires the extracellular matrix, cardiovascular circulation, and transcription/splicing-dependent events. We suggest that these mutants define processes likely to be used during brain morphogenesis throughout the vertebrates. Anat Rec, 292:94-106, 2009. 2008

Tubulogenesis

“…At least two distinct mechanisms can provide the force needed to separate cell surfaces and generate a lumen between closely apposed cell surfaces. In zebrafish brain ventricle development, the Na + /K + -ATPase containing the Snakehead alpha subunit (Atp1a1) is required to 'inflate' the ventricle, presumably through ion transport generating hydrostatic pressure to separate cell surfaces (Lowery et al, 2009). During development of the mouse aorta, by contrast, electrostatic repulsion of heavily sialylated proteins is required to separate the lumenal faces of the aortic cells (Strilić et al, 2010).…”

Manual Drainage of the Zebrafish Embryonic Brain Ventricles

“…However, there appear to be hundreds of uncharacterized proteins in the eCSF, which was obtained after choroid plexus formation, later than the time-points demonstrated here. Additionally, our lab and others have identified zebrafish mutants with abnormal brain ventricle size or brain defects [29][30][31] , and may have abnormalities in eCSF composition and function. This method readily allows for isolating and testing putative abnormal eCSF from mutants or under different environmental conditions.…”

Comparative analysis of neurulation: First impressions do not count

“…Continued cavitation (lumen formation) is thought to involve apical membrane biogenesis (Munson et al, 2008), to establish an epithelial seam that divides the left and right halves of the neural rod, lumen inflation, and localized cell proliferation (Lowery and Sive, 2005). While hinge points are not observed during neurulation in the zebrafish, they are involved in shaping the lumen after the neural rod stage (Gutzman et al, 2008;Nyholm et al, 2009) and may be controlled by the same molecular mechanisms that regulate hinge point formation in amniotes (Lowery et al, 2009;Nyholm et al, 2009). …”