Strategy for Marker Based Screening

1. Collect and sort 100 fertile embryos from each pair mating. Separate into two dishes of 50 embryos. Individual pairs or F1 females must be labeled and kept separately. Space constraints will determine the number of pairs or females that can be screened at any given time, since the fish must be kept separate until marker screening is done and it may take a number of weeks until enough individual crosses or squeezes are generated to perform an en masse marker-based screen.

2. Allow the embryos to develop to the stages to be screened morphologically (see Subheading 4. for specific details on morphological screening). One dish of 2550 embryos is fixed for use in a marker-based screen. At least half of the embryos are kept for morphological screening and structural stains at later stages. We fix 25 embryos at bud and early pharyngula stages for use in an in situ-based screen. Embryos of different stages from the same parent(s) are combined in small baskets and incubated through the in situ protocol in 24-well tissue-culture plates. A similar approach can be used for screening with antibodies to specific antigens. We routinely screen embryos from 30-50 individual broods at a time. It is possible to scale up the in situ protocol to use larger well number microtiter plates, such as 96-well plates, moved through common solution baths. However, it is difficult to keep large numbers of pairs or individual females separate for any length of time in a small facility. Thus, the number of broods screened at any given time will be dictated by this space constraint.

3. Allow to develop to 3 d, and screen for defects in organogenesis if a diploid screen is being employed. Collect embryos for structural stains at appropriate stages. If no phenotype has been detected at any stages, pairs or females should be returned to original tanks in diploid screens, or to a separate tank in haploid-or gynogenetic-based screens.

Choosing markers: A large number of cloned genes and cell-type-specific antibodies have been generated that allow visualization of specific regions or molecular processes within the zebrafish embryo. These markers can often reveal subtle changes in the molecular phenotype of a given mutation that may not be detectable morphologically. They may also implicate a mutation that has an otherwise uninteresting phenotype in a specific molecular process. Some examples are given in Table 1.

Table 1

In Situ and Antibodies Availiable for Marker Based Screening of Early Segmentation and Pharyngula Stages in Zebrafisha

Germ layer

Marker

Region expressed

Ref.

Neuroectoderm zash 1a and 1b tel. and di., r1-6 18

pax 2 optic stalk, mes/rhomb, and dorsal 21 spinal neurons pax 6 retina, lens, and dorsal spinal neurons 22

shh fp, ventral di. 24

wnt 1 mes/rhomb 26

krx 20 r3 and 5 28

rtk 1 r2 and 4 29

pou 2 r2 and 4 30

axial fp, ventral di 31

collagen type 2 fp 32

ZN1* 1° motor neurons, most differentiating 33 neurons

ZN12* reticulospinal and Rohon-Beard neurons 33

axial noto., pcp 31

znot noto 34

gsc pcp 35

shh noto., pcp 24

Brachyury noto 36

snail 1 paraxial 37

twist noto., somite 38

aThis is by no means an exhaustive list and is meant to provide the reader with a starting point for designing marker screens. Abbreviations: di—diencephalon, fp—floorplate, mes—mesencephalon, noto—notochord, pcp—prechordal plate mesoderm, r—rhombomere, rhomb— rhombencephalon, tel—telencephalon.

Mesoderm

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