NKX transcription factors form a large subfamily of homeo-domain containing proteins related to Drosophila NK genes. Based on their expression patterns in vertebrates and some functional studies it is believed that NKX genes play a role in morphogenesis and differentiation of specific cell types. We have identified several NKX genes in mouse and chicken that are expressed either in neuroectoderm or in mesoderm and mesendoderm or derivatives derived thereof (1).To investigate the functions of NKX transcription factors in mouse targeted gene disruption in ES cells will be applied to generate mouse mutants and analyse their phenotypes.

The NKX2.3 gene. NKX2.3 is expressed in epithelium of branchial arches and tongue, and in gut mesoderm during mouse development and postnatally (2). The NKX2.3 -/- mutant shows postnatal lethality and retarded development of intestinal epithelium and defects in secondary lymphoid organs. In spleen and mucosal lymph nodes the precise anatomical organisation of lymphocytes within these tissues is disturbed, probably due to the lack of the cell adhesion molecule MAdCAM-1 that is not expressed in the NKX2.3 mutant (3). The role of MAdCAM-1 for lymphocyte homing and immune response will be further investigated. We also want to know the reasons for the gut defect and lethality.

The NKX2.9 gene. NKX2.9 is related in structure and its expression to NKX2.2. Both genes are expressed exclusively in ventral domains of the CNS in a spatio-temporal overlapping patterns (4). The NKX2.2 gene has been knocked out by others and shows dorsalizing transformation of neuronal cell types. We are inactivating the NKX2.9 genes to determine its role in conjunction with NKX2.2.

The NKX3.1 gene. NKX3.1 is highly expressed in somites, prostate, and pituitary. We have mutated the gene in mice by the insertion of the LacZ gene. The mutant shows normal somite development most likely because NKX3.2 is also expressed in somites and may be redundant. However, the NKX3.1 mutant has defects in prostate development and in some salivary glands. The phenotype will be investigated further. We also plan to generate the double mtant for NKX3.1 and NKX3.2 in order to learn more about their roles in somitogenesis.

The NKX 3.2 gene. We have isolated the NKX3.2 genes from mouse and chicken and found them expressed asymmetrically in early lateral plate mesoderm in both organisms. Interestingly, in chicken NKX3.2 transcripts appear on the left side, while in mouse the gene is expressed on the right side (5). The unilateral expression is reversed in inv/inv mice which show situs inversus. Thus NKX 3.2 seems to be part of the left-right asymmetry system. We will investigate signals affecting the one-sided expression of NKX3.2 in mouse and chicken and analyse its role in establishing asymmetry in visceral organs.

The projects decribed above will be supported by attempts to identify important signalling pathways for the expression of these genes in Drosophila. For this purpose, transgenes in Drosophila will be tested in a wide variety of mutants. We also plan to perform functional studies for our genes in C elegans by inhibition of homologues with RNAi. Finally, we like to identify interaction partners for NKX factors by established methods like yeast to hybrid screens.

References.

1. Pabst, O., Herbrand, H., Takuma N., Arnold, H.H. (1999) NKX2 gene expression in neuroectoderm but not in mesendodermally derived structures depends on sonic hedgehog in mouse embryos. Dev. Genes Evol. in press

2. Pabst,O., Schneider,A., Brand,T. and Arnold, H.H. (1997) The mouse Nkx 2-3 homeodomain gene is expressed in gut mesenchyme during pre- and postnatal development Dev. Dyn. 209, 29-35

3. Pabst, O., Zweigerdt, R. and Arnold, H.H. (1999) Targeted disruption of the homeobox transcription factor Nkx2-3 in mice results in postnatal lethality and abnormal development of small intestine and spleen. Development, 126, 2215-2225

4. Pabst, O., Herbrand, H., and Arnold, H.H. (1998) Nkx-2.9 is a novel homeobox transcription factor which demarcates ventral domains in the developing mouse CNS. Mech. Dev. 73, 85-93

5. Schneider, A., Mijalski, T., Schlange, T., Dai, W., Overbeek, P., Arnold, H.H., Brand,T. (1999) The homeobox gene NKX3.2 is a target of L-R signalling and is expressed on opposite sides in chicken and mous e embryos.Curr. Biol. 9, 911-914