Worm Breeder's Gazette 10(3): 114

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Localization of mab-5 RNA

Michael Weir, Michael Costa and Cynthia Kenyon

The mab-5 gene functions in a position-specific fashion to allow 
ectodermal and mesodermal precursor cells located in the posterior 
body region to generate cell types and body structures that give that 
region its characteristic identity.  Mutations in mab-5 cause a number 
of cells in the posterior part of the nematode to be transformed such 
that they follow developmental pathways similar to their anterior 
homologues.  mab-5 is cell autonomous, indicating that the function of 
the mab-5 gene is normally required in these posterior cells.  In 
principle, one way in which mab-5 activity could be targeted to 
posterior cells would be by localization of the mab-5 transcript, 
either by regulation of transcription initiation or transcript 
stability.  We have used in situ hybridization to tissue sections to 
show that the mab-5 RNA is in fact localized to the posterior part of 
the larva.
How is expression of mab-5 localized?  One possibility is that 
localization depends upon interactions with other patterning genes 
which themselves might be localized in their expression.  Now that we 
have a molecular probe to measure mab-5 function, we can test for 
changes in mab-5 expression in animals mutant for putative interacting 
genes.  Currently, we are looking at mab-5 RNA in lin-22 mutants.  We 
think that lin-22 may interact with mab-5 mutants, anterior V cells 
adopt posterior-specific fates in a mab-5-dependent fashion.
A striking feature of our localization result in wildtype larvae is 
that there is a correlation of mab-5's expression domain with the 
domain affected by mab-5 mutations.  This kind of correlation has been 
observed previously in Drosophila, where the growing consensus is that 
the expression of genes in overlapping patterns may be important for 
the combinatorial specification of developmental pathways.  It will be 
interesting to see how other nematode patterning genes are localized 
in their expression (in space, sublineages, sexes and time).  Below, 
we describe our in situ protocol (see Costa et al., 1988).
In Situ Hybridization.
Our in situ method is similar to that used for Drosophila embryos (
see Hafen et al., 1983; Kornberg et al., 1985; Edgar et al., 1986).  
Nematodes were grown on NGM plates and harvested in M9.  Eggs were 
isolated by hypochlorite treatments and incubated to larval stages on 
NGM plates (Sulston and Hodgkin, 1988).  Larvae were washed with M9 
and then treated with 4% paraformaldehyde in PBS, 0.03% triton-X for 
15 min, washed in two changes of PBS, centrifuged briefly, and the 
supernatant discarded.  The pelleted larvae were transferred onto 
parafilm, and mixed with OCT embedding compound (Miles Scientific) 
prior to freezing on powdered dry ice.  At this stage, specimens could 
be stored in air-tight containers at -70 C for extended periods (weeks)
.  Eight micron frozen sections were cut at -12 C and placed on 
polylysine treated slides (Sigma P1399) which were immediately placed 
on a slide warmer for 2 min.  Within 2 hr after cutting, sections were 
fixed for 20 min in 4% paraformaldehyde in PBS followed by treatment 
with 3X PBS (5 min), 1X PBS (2 X 5 min), and 2 min in each of 30%, 60%,
80%, 95% and 100% ethanol, and air dried.  Dehydrated sections could 
be stored at room temperature.
Treatment of sections prior to hybridization was as follows: 0.2N 
HCl (20 min, room temp.); H2O (5 min); 2X SSC (30 min, preheated to 70 
C); H2O (2 min, room temp.); pronase in 50mM Tris-HCl pH 7.5, 5mM EDTA 
for 10 min [10-50 units/ml (Calbiochem), predigested at 37 C for 4 hr];
1X PBS with 2 mg/ml glycine (30 sec); 1X PBS (2 X 30 sec); 
immediately transferred to 4% paraformaldehyde in PBS (20 min); 3X PBS 
(5 min); 1X PBS (5 min); 0.1M triethanolamine (Triethanolamine pH8 
with HCl) with 0.5% acetic anhydride (stirred vigorously for 5 min); 
1X PBS (2 X 5 min); 2 min each with 30%, 60%, 80%, 95%, and 100% 
DNA probes were prepared according to Kornberg et al., 1985.  
Plasmid DNA (40  g/ml) was digested with DNase I (Worthington: DPFF) 
at several test concentrations between 0.4 and 0.8  g/ml in 50 mM Tris-
HCl, pH 8, 10mM MgCl2.  Digestion was for 1 hr (25 C) followed by 
inactivation at 65 C for 15 min and storage at -70 C.  Aliquots of 0.4 
g digested DNA at 20 g/ml were radioactively labelled with 50 Ci of 
[35S]dATP (700 Ci/mmol) and 50 M dCTP, dGTP, and TTP, and 8 units of 
DNA polymerase I (Boehringer-Mannheim, nuclease free).  Labelling was 
for 2 hr at 16 C.  The reaction was terminated with the addition of 25.
0 l of 10mg/ml denatured salmon sperm DNA with 50mM EDTA and treatment 
at 65 C for 10 min.  Unincorporated nucleotides were removed by spin 
dialysis through Sephadex G-50.  Samples of DNA from the different 
DNase treatments were fractionated on a 7M urea-5% acrylamide gel and 
autoradiographed.  DNase-treated DNA with fragments between 35 and 135 
bp was used for in situ hybridization.
Before applying to slides, labelled DNA was heated to 100 C for 3 
min and diluted 10-fold in hybridization solution (10 mM Tris-HCl, pH 
7.5, 0.6M NaCl, 1mM EDTA, 0.25 mg/ml yeast tRNA, 1X Denhardt's 
solution, 10% dextran sulfate (Pharmacia), 50% Dowex AG deionized 
formamide).  18 l of this hybridization mix was applied to each slide 
and 22mm square coverslips were applied and sealed with rubber cement (
Carter's).  Hybridization was for 20-24 hr at 37 C, followed by six 
changes of wash (50% formamide (practical grade), 0.6 M NaCl, 10 mM 
Tris-HCl, pH 7.5, 1 mM EDTA) over 18 hr at 37 C, and dehydration 
through 70% and 95% ethanol containing 0.3 M ammonium acetate (2 X 5 
min each).
After drying, slides were dipped in 50% NTB-2 emulsion (Kodak) in 0.
3 M ammonium acetate.  Autoradiography was for 1 month at 4 C.  Slides 
were developed in Kodak D19 (2.5 min, 16 C) and fixed in Kodak rapid 
fix for 3 min (16 C).  Slides were treated with DAPI (0.1  g/ml) for 
10 min and coverslips mounted with fluoromount-G (Southern