The characterisation o f gimpy categorised as one of the 'dwarf class of mutants (Stemple et a l, 1996) and given

the name gimpy (gip).

5.2.2 Embryonic morphology

5.2.2.1 Live examination o f embryos reveals a number o f defects

The gimpy phenotype is clearly discernible from 20-22hpf, even at a gross level (compare wild-type embryos in fig. 5.1a with mutants in fig. 5.1b). The embryos are clearly shorter than their sibs, with a reduced yolk plug extension and somites which are narrowed in their anterior-posterior aspect (compare fig. 5.1c and fig. 5.Id). A more detailed examination reveals the detail of the neural and notochordal defects. The midbrain-hindbrain boundary is shifted anteriorly in gip (fig. 5.2b), although this may be a function of the overall shortening of the body, and the vesicles of the hindbrain appear to be swollen and more conspicuous (arrowheads, fig. 5.2b). The developing lens protrudes from the presumptive retina (fig. 5.2d) and the shape of the eye is irregular compared to wild-type sibs. Finally, using Nomarski optics, the notochord phenotype is obvious at 24hpf. The notochord precursor cells fail to vacuolate (seen as the 'pits' in the notochord cells, fig. 5.2e) and no discrete boundary forms between the notochord and the dorsal and ventral tissue flanking it (compare figs. 5.2e and 5.2f). At this stage, gip embryos are capable of movement by means of a mild shuddering when touched, but they fail to display the stereotyped writhing of their wild-type sibs.

By 48hpf, the mutant embryos are beginning to degenerate. The circulatory system fails to form correctly, leading to embryos with varying degrees of heart and tissue oedema. The embryos lose their spontaneous movements and become paralysed and the pectoral fin buds fail in their outgrowth, remaining as stumps. However, pigmentation forms as for the wild-type, suggesting that some migration of the neural crest is occurring normally. Death occurs between three and six days after fertilisation, with the oedema gradually worsening and embryos becoming necrotic - those surviving past five days would never be viable as their jaw defects, coupled with their paralysis, would mean that they could not feed.

a) W ild-type sibs

h) Gimpy mutants

c), d) higher pow er magnification live photos of embryos in a), b)

The gimpy m utant has a dw arf phenotype, such that em bryos are sm aller than their wild-type sibs (compare lengths of yolk plug extension, c, d). The somites, s, are also narrower with a less well-defined shape.

s somites y yolk plug extension

a wt

a) wild-type sib, lateral view b) gimpy, lateral view

c) wild-type sib, antero-dorsal view d) gimpy, antero-dorsal view e) wild-type sib, lateral trunk f) gimpy, lateral tmnk

Arrowheads in b show swollen hindbrain vesicles in the mutant. The distance between the eye, e, and the midbrain-hindbrain boundary, mhb, is reduced in gimpy

com pared to sibs. Additionally, the lens, 1, appears to protm de in the m utant and the notochord, nc, fails to vacuolate.

e eye 1 lens mhb m idbrain-hindbrain boundary nc notochord

V

dtS

i

5.2.2.2 The ultrastructure o f the notochord is severely disrupted

To examine further the notochordal phenotype, transmission electron microscopy was carried out on ultrathin, transverse sections cut from comparable regions of the trunk in both wild-type and gip embryos. Even at a lower power (figs. 5.3a, b) the disruption in the mutant is clear. In the wild-type, there is a large, open region in the notochord (fig. 5.3a) traversed by the membranes of the vacuolated cells. Looking at a higher magnification, there is an outer layer of cells with numerous mitochondria and endoplasmic reticulum surrounding the vacuolar cells (data not shown). The whole structure is encompassed by the notochordal sheath, composed of densely packed collagen II fibres (Eikenberry et al., 1984) wrapped in an elastic sheath. However in gip, the morphology described for the wild-type has all but disappeared. There is a remnant of a vacuole which is meagre compared to the wild-type (fig. 5.3b) and the clearly defined subcellular and supporting structures are lacking, having been replaced by amorphous, dense debris - even at a high magnification (data not shown), no distinct structure is apparent and only an insignificant attempt at making the notochord sheath is visible (fig. 5.3b).

5.2.3 Patterning o f the central nervous system

5.2.3.1 Immunostaining for anti-acetylated tubulin reveals severe defects in the axon scaffold and the embryonic brain

As might be expected from the morphology of the brain, there is considerable mispatteming of the axon scaffold at 48hpf, as shown by the expression of the acetylated tubulin epitope (Wilson et al., 1990). The telencephalon is reduced and shifted ventrally in gip and, strikingly, the tectum appears to be absent aside from a few stray projections. The trigeminal ganglion, however, appears to be correctly positioned, taking into account the smaller size of the embryo (figs. 5.4a, b). The major tracts crossing the midline are affected though - their positioning appears to be normal in gip, but there is defasciculation, such that the anterior commissure and the tract of the post-optic commissure are less-tightly bundled (figs. 5.4c-f). The lateral line projection is also missing.

The swollen, over-vesiculated hindbrain has a surprising neural phenotype when examined using this technique. It could be proposed that, since the hindbrain appears to be more segmentally organised than usual, this would be reflected in strictly arranged arrays of hindbrain commissural intemeurons. However, this is