All panels show embryos stained with acetylated tubulin and opsin antibodies, at 60 hours post fertilization.
Panels A, B and C are lateral views. In panel A, staining in the trunk labels the spinal motor axons and the Rohon-Beard neurons.
Panels B and C show embryos that have the eyes removed. Panel B is focused on the midline of the embryonic brain. The tracts of the posterior commissure and the habenular commissure run either side of the epiphysis. The telencephalon shows staining, and the tracts of the anterior commissure, the postoptic commissure and the supraoptic tract all remain in a similar orientation to each other as seen at 32 hours post fertilization.
Panel C is focused on the surface of the embryo. The trigeminal ganglion can be seen, along with a projection from it that would normally run ventral to the eye (removed).
Panel D is a ventral view of the forebrain. The anterior and post-optic commissures can be seen to cross the midline. The bilateral olfactory placodes can be seen at the anterior extreme of the embryo. The lens and retina of the eyes are labelled and the optic nerve can be seen projecting from the eye and crossing the midline.
Panel E is a high magnification dorsal view of the epiphysis. The midline photoreceptors are labelled with opsin antibody. The projection neurons either side of the midline are labelled with tubulin antibody. The posterior commissure and habenular commissure (only partially in the plane of focus) both cross the midline at this point.
Panels F and G are dorsal views of the brain. Panel F shows the hindbrain commissural intemeurons, which project across the hindbrain at regular intervals. Panel G is focused on glial cells, which are arranged in bilateral rows at similar intervals to the hindbrain commissural intemeurons.
Abbreviations:
AC = anterior commissure cb = cerebellum
DVDT = dorsoventral diencephalic tract HC = habenular commissure
PC = posterior commissure POC = postoptic commissure SOT = supraoptic tract
TAC = tract of the anterior commissure THC = tract of the habenular commissure TPC = tract of the posterior commissure TPOC = tract of the postoptic commissure trig = trigeminal ganglion
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observed in wild type embryos (Shanmugalingam et a l, 2000). This is suggested to result from mis-speciflcation of a population of midline forebrain cells (due to loss of FgfS activity), which subsequently mis-express axon guidance cues (possibly twhh, sixS and/or pax2.1).
A screen was carried out to specifically isolate mutants displaying defects in axon path finding within the retinotectal system (Karlstrom et a l, 1996; Karlstrom et a l,
1997), which resulted in the identification of 30 alleles including no isthmus {noi), detour {dtr) and you-too (yot) involved in this process. Further analysis o f these mutants enables a greater understanding of brain development at a molecular level. The noi line was previously shown to encode the Pax2 protein (Krauss et a l, 1991). Pax2 has been shown to be directly involved in guiding axons o f both the forebrain commissures (AC and POC) and the retinotectal axons across the midline (Macdonald et a l, 1997).
Detailed analysis of the dtr mutant suggests is functions as a downstream component in the Hedgehog pathway, (Chandrasekhar et a l, 1999). The yot mutant similarly shows disrupted Hedgehog signalling. The yo t locus encodes Gli-2, a protein with both activation and repressive function acting in response to Hedgehog signalling (Karlstrom et a l, 1999). The Hedgehog pathway is involved in both general forebrain patterning (through its midline activity) and directly in the regulation o f growth cone extension o f the axonal projections (Trousse et a l, 2001). The identification of additional mutants will act to further our understanding o f neural patterning and axon guidance.
5.1.5.4 The pineal complex.
The pineal complex (or epiphysis) is a located in the dorsal diencephalon o f the zebrafish brain. It is part o f the neuro-endocrine system, involved in circadian regulation o f the organism by means of sensitivity to light. The zebrafish epiphysis is
responsive to light has been shown to produce melatonin, (Cahill, 1996), secretion being greatest during the hours of darkness (Kazimi & Cahill, 1999). Two distinct photoreceptive structures lie within the pineal complex - the pineal gland and the parapineal. The parapineal lies asymmetrically in the brain. This is a phenomenon observed throughout species that possess such a structure (Concha & Wilson, 2001), and a consistent placing of the organ to the left side o f the embryo is dependent upon the Nodal signalling pathway in zebrafish (Concha et al., 2000; Liang et a l, 2000). The photoreceptors labelled in our screening protocol, (without permeablising the embryo skin), are the photoreceptors of the pineal gland, which are located on the midline. The parapineal gland sits deeper within the dorsal brain tissue and so is not exposed to the antibody.
As part of our forebrain screens both at UCL and in Tuebingen, we wished to find mutants displaying defects in pineal development. Two distinct cell types were labelled by our protocol within the pineal organ: firstly projection neurons, which are located in two bilateral rows within the pineal complex and stained by acetylated Tubulin antibody, and second, a population o f photoreceptive neurons that lie between the projection neurons, stained by anti-Opsin antibody.
Opsin is a phototransducing molecule, thus it is activated by light and relays this information to other molecules within the cell. Opsins are found in both the pineal organ and the retina, and different Opsins are capable o f responding to different wavelengths of light. In zebrafish, as in other teleosts investigated (halibut, herring and cod). Opsin immuoreactivity is observed in the pineal organ prior to its expression in the retinal photoreceptors (Forsell et al., 2001). At both embryonic stages at which we screened, anti-Opsin antibody labelling was observed exclusively in the pineal gland (panels 5.2F and 5.3E).
5.1.6 Data from two large-scale genetic screens in zebrafish.
The work presented here is part of the data generated from two large scale genetic screens, each spanning two years and involving the contribution o f a very large
number of people. Both screens used ENU mutagenesis and diploid embryos were generated for morphological observation and antibody labelling.
The number and types of potential mutants (identified in the first round of screening) are presented here, with a number of phenotypic examples given. Many o f the potential mutants have yet to be re-screened and their status as mutants confirmed, while research projects focused on other lines such as ul48 and u320, characterised and presented within this thesis (chapters 6 and 7), are at a stage where publication is, hopefully imminent.
5.2
Results.
5.2.1 The UCL genetic screen.
The UCL genetic screen ran for two years between 1998 and 2000. The screen, co ordinated by Corinne Houart, Steve Wilson and Nigel Holder, was conducted by all members o f the UCL fish group that were present during that period. The initial stage involved screening progeny from around 400 families descended from N-ethyl-N- nitrosourea (ENU) treated males. Later stages involved re-screening and mutant characterisation, which are on going within the UCL fish group.
5.2.2 Mutants identified from the UCL genetic screen.
More than 20 novel mutant zebrafish lines have been isolated from an original pool of nearly 100 lines that were identified as potentially interesting mutants from the first round of screening. Lines from the original pool were discarded if the observed phenotype was not repeated in subsequent matings or, more commonly, further analysis of the line deemed the phenotype to be not o f significant interest.
The lines identified in the UCL screen were named with a ‘w’ prefix followed by the family number. The novel allele carried within each line also takes this name. A brief description of four novel mutant lines I identified follows:
5.2.3 Novel mutant line shows defects in laterality.
This mutant line was identified by morphological observation at 24 hours post fertilisation by Anukampa Barth (AB) and myself. A reversal of heart positioning within the thorax was observed during screening and thus line u28 has been named heart reversed (htr“^^). A preliminary characterisation o f laterality in htr^^^ mutants has been conducted in our lab by A.B., and behavioural studies are underway in collaboration with Richard Andrew’s research group at the University of Sussex.
Asymmetry across the left-right axis is a highly conserved phenomenon among vertebrates (Goldstein et a l, 1998), with a number o f organs including the heart, spleen, liver, elements of the brain and most external structures showing a differential development either side of the midline (Concha & Wilson, 2001; Morelli et al., 2001). A number of proteins have been identified with a role in asymmetric patterning, including Nodal, Lefty2 and Pitx2, (reviewed by Hamada et al., 2002). Defects in patterning o f the left-right axis broadly fall into two main classes - an inversion {situs inversus), where mutant embryos show a reversal o f the asymmetric patterning observed in wild type, or more commonly, a randomisation o f left-right patterning resulting in only half the mutant embryos exhibiting a reversed phenotype. The randomised-type mutants can be further split into two groups - those that exhibit consistent laterality throughout different organ systems and those that show a variety of lateralities within an individual embryo (heterotaxia). A more detailed description of laterality mutants suggests five classes of phenotypes (Bisgrove & Yost, 2001).
We have observed incorrect heart looping in a variable percentage o f embryos from line htr“^^, which results in the heart being placed on the right side of the torso (see figure 5.4A & B). Due to the variable percentage o f embryos displaying such a phenotype in any given batch from this line, we are currently unable to reach a