Mishima Marek Jindra

LABORATORY OF MOLECULAR GENETICS

CREW MEMBERS – and their models

·       Marek JINDRA, Ph.D. (Head) – Drosophila, Bombyx mori, Manduca sexta

·       Masako ASAHINA, Ph.D. – C. elegans, Bombyx mori, Manduca sexta

·       Ivana GAZIOVA, M.S. – Drosophila

·       Mirka UHLIROVA, M.S. – Bombyx mori, Manduca sexta

·       Maruska SILHANKOVA, M.S. – C. elegans

 

MISSION

We study gene functions in vivo.  We apply molecular and reverse genetic methods on invertebrate models.  In focus of our projects are transcription factors and coactivators, and their roles in important biological processes:

·         Steroid signaling

·         Molting and Metamorphosis

·         Stress response

 

 

MODELS                METHODS                  PROJECTS                  PROTOCOLS                 PUBLICATIONS

 

EXPERIMENTAL MODELS

In some projects, we take advantage of classic models allowing superb genetic manipulations:

·         the fruit fly Drosophila melanogaster

·         the nematode Caenorhabditis elegans

Besides that, we strive to develop methods that will enable causal genetic tests also in non-drosophilid insects, moths (Lepidoptera):

·         the silkmoth Bombyx mori

·         the tobacco hornworm Manduca sexta

Why work on moths?

·         moths represent typical insect development more faithfully than the highly advanced Drosophila

·         large size facilitates developmental studies and work on isolated organs

·         many relevant genes have been characterized in Bombyx and Manduca


 

KEY METHODS (see Protocols):

·         Transposon (P-element) mutagenesis: Drosophila

·         Trimethylpsoralene-UV mutagenesis: C. elegans

·         RNA interference (RNAi): C. elegans, insects

·         Germline transformation: Drosophila, C. elegans, Bombyx mori

·         Organ culture of epidermis: Manduca sexta

 

PROJECTS

1.     Biological roles of a new type transcriptional coactivator MBF1 in Drosophila and C. elegans

CREW:               Marek JINDRA, Ivana GAZIOVA, Mirka UHLIROVA, Masako ASAHINA

ALLIES:             Susumu HIROSE, Yash HIROMI (NIG, Japan)

AIMS:                 Find out which biological processes require MBF1. Uncover molecular and genetic interactions between MBF1 and its partner transcription factors.

STRATEGY:      Determine the loss-of-function phenotypes under normal and biased conditions using mbf1- deletion mutants (Drosophila) and RNAi (C. elegans). Using transgenic and molecular methods, screen for MBF1 interacting proteins.

PROGRESS:      Physically and genetically interacting MBF1 partners have been found among Drosophila bZIP transcription factors. MBF1 is involved in morphogenesis (Liu et al., in preparation) and stress response (Jindra et al., in preparation).

 

 

2.     Roles of nuclear receptors of the steroid signaling pathway in non-drosophilid insects: A reverse genetic approach

CREW:               Mirka UHLIROVA, Masako ASAHINA, Marek JINDRA

ALLIES:             Lynn M. RIDDIFORD (University of Washington, Seattle, USA), Ernst A. WIMMER (University of Bayreuth, Germany)

AIMS:                 Develop methods that would allow causal testing of gene functions in moths (Lepidoptera). Through inactivation of genes encoding nuclear receptors, reveal their roles in the ecdysteroid control of insect molting and metamorphosis.

STRATEGY:      Employ germline transformation and inducible ectopic expression to interfere with gene functions in vivo using RNAi or overexpression of dominant negative proteins.

PROGRESS:      Successful germline transformation of the silkmoth Bombyx mori with a transposon vector piggyBac and heat-inducible expression of a transgenic mRNA and protein in vivo (Uhlirova et al., 2002) were prerequisites for our current efforts to produce conditional gene knock-downs using RNAi.

 

 

3.     Molecular characterization and function of the ecdysoneless gene of Drosophila

CREW:               Ivana GAZIOVA, Marek JINDRA

ALLIES:             Vincent HENRICH (University of North Carolina, Greensboro, USA)

AIMS:                 Identify the protein encoded by ecdysoneless (ecd), determine its regulation and role in the steroid signaling and development.

STRATEGY:      Clone ecd via transformation rescue of lethal ecd mutants with genomic DNA fragments from the region where ecd maps genetically. Once ecd is idetified, explore its function using Drosophila genetics and molecular biology.

PROGRESS:      Genetic rescue of ecd mutants was achieved using P-element mediated transformation. Mutations within the ecd gene were identified for several ecd alleles and expression of the gene has been examined at the level of mRNA (Gaziova et al., in preparation).

 

 

4.     Role of the nuclear receptor NHR-25 (Ftz-F1) in C. elegans development, molting and steroid signaling

CREW:               Masako ASAHINA, Maruska SILHANKOVA, Marek JINDRA

ALLIES:             Marta KOSTROUCHOVA (Charles University, Prague, Czech Rep.)

AIMS:                 Define the roles of this conserved nuclear receptor in gonadal differentiation and molting, identify its target genes and show their relationships to steroid regulation.

STRATEGY:      Identify genes dependent on NHR-25 by testing expression of transgenic GFP reporters, driven by regulatory regions of selected candidate genes, in nhr-25 mutant and RNA interference background.

PROGRESS:      Isolation of an nhr-25 deletion mutant and RNAi revealed that NHR-25 is necessary for the completion of embryogenesis. RNAi showed additional requirements for NHR-25 in molting and the differentiation of the gonad and vulva, all consistent with nhr-25 expression patterns (Asahina et al., 2000).