Norwegian Arabidopsis Research Center

Please contact:
Reidunn B. Aalen
or
Ellen Andersen Department of Molecular Biosciences
Pb 1041, N-0316 Oslo, Norway.

In situ hybridization is a useful technique for determining spatial patterns of gene expression by hybridizing the complimentary strand of a nucleotide probe to the sequence of interest. The method involves several steps and takes a considerable amount of time from start to finish, it takes a week just to fix and embed tissue.
When planning an in situ experiment there are several things you should think through before starting. We have tried to outline some of the critical steps below, such as choice of tissue, probes and sectioning planes. These parameters will vary and has to be determined for each experiment.
For other steps like the fixation of tissue, synthesis of probes and the hybridization process we use standard protocols.

• seedlings - shoot apical meristem
• flowers - from buds to flowers containing mature ovules, including inflorescence meristem
• embryo - from globular stage to mature embryos

Probes are complementary sequences of nucleotide bases (RNA or DNA) to the specific mRNA sequence of interest. These probes can be as small as 20-40 bp or be up to 1000 bp. We prefer probes of about 500 bp.
We use RNA probes and Roche DIG labeling system.

• STM (shootmeristemless)
  Long J, Barton MK.
  Initiation of axillary and floral meristems in Arabidopsis. Dev Biol. 2000 Feb 15;218(2):341-53.
• WUS (wuschel)
  Schoof H, Lenhard M, Haecker A, Mayer KF, Jurgens G, Laux T. The stem cell population of Arabidopsis shoot meristems in maintained by a regulatory loop between the CLAVATA and WUSCHEL genes. Cell. 2000 Mar 17;100(6):635-44.
• YABBY
  Siegfried KR, Eshed Y, Baum SF, Otsuga D, Drews GN, Bowman JL. Members of the YABBY gene family specify abaxial cell fate in Arabidopsis. Development. 1999 Sep;126(18):4117-28.
• HAESA
  Jinn TL, Stone JM, Walker JC. HAESA, an Arabidopsis leucine-rich repeat receptor kinase, controls floral organ abscission. Genes Dev. 2000 Jan 1;14(1):108-17.
• SCR (scarecrow)
  Wysocka-Diller JW, Helariutta Y, Fukaki H, Malamy JE, Benfey PN. Molecular analysis of SCARECROW function reveals a radial patterning mechanism common to root and shoot. Development. 2000 Feb;127(3):595-603.
• ML1 (meristem layer 1)
  Sessions A, Weigel D, Yanofsky MF. Related Articles, Links The Arabidopsis thaliana MERISTEM LAYER 1 promoter specifies epidermal expression in meristems and young primordia. Plant J. 1999 Oct;20(2):259-63.
• REV (revoluta)
  Otsuga D, DeGuzman B, Prigge MJ, Drews GN, Clark SE. Related Articles, Links REVOLUTA regulates meristem initiation at lateral positions. Plant J. 2001 Jan;25(2):223-36.

STM in early torpedo stage embryo.	WUS in flower bud.	YABBY in heart stage embryo.