Ldb1, a ubiquitously expressed LIM domain binding protein, is essential in a number of tissues during development. It interacts with Gata1, Tal1, E2A and Lmo2 to form a transcription factor complex regulating late erythroid genes. We identify a number of novel Ldb1 interacting proteins in erythroleukaemic cells, in particular the repressor protein Eto-2 (and its family member Mtgr1), the cyclin-dependent kinase Cdk9, and the bridging factor Lmo4. MO-mediated knockdowns in zebrafish show these factors to be essential for definitive haematopoiesis. In accordance with the zebrafish results these factors are coexpressed in prehaematopoietic cells of the early mouse embryo, although we originally identified the complex in late erythroid cells. Based on the change in subcellullar localisation of Eto-2 we postulate that it plays a central role in the transition from the migration and expansion phase of the prehaematopoietic cells to the establishment of definitive haematopoietic stem cells.

SUMMARYThe Lhx2 transcription factor plays essential roles in morphogenesis and patterning of ectodermal derivatives as well as in controlling stem cell activity. Here, we show that during murine skin morphogenesis, Lhx2 is expressed in the hair follicle (HF) buds, whereas in postnatal telogen HFs Lhx2 + cells reside in the stem cell-enriched epithelial compartments (bulge, secondary hair germ) and co-express selected stem cell markers (Sox9, Tcf4 and Lgr5). Remarkably, Lhx2+ cells represent the vast majority of cells in the bulge and secondary hair germ that proliferate in response to skin injury. This is functionally important, as wound reepithelization is significantly retarded in heterozygous Lhx2 knockout (+/-) mice, whereas anagen onset in the HFs located closely to the wound is accelerated compared with wild-type mice. Cell proliferation in the bulge and the number of Sox9 + and Tcf4 + cells in the HFs closely adjacent to the wound in Lhx2 +/-mice are decreased in comparison with wild-type controls, whereas expression of Lgr5 and cell proliferation in the secondary hair germ are increased. Furthermore, acceleration of wound-induced anagen development in Lhx2 +/-mice is inhibited by administration of Lgr5 siRNA. Finally, Chip-on-chip/ChIP-qPCR and reporter assay analyses identified Sox9, Tcf4 and Lgr5 as direct Lhx2 targets in keratinocytes. These data strongly suggest that Lhx2 positively regulates Sox9 and Tcf4 in the bulge cells, and promotes wound re-epithelization, whereas it simultaneously negatively regulates Lgr5 in the secondary hair germ and inhibits HF cycling. Thus, Lhx2 operates as an important regulator of epithelial stem cell activity in the skin response to injury.

The molecular basis of the characteristic hair growth disorder in nude mice that carry a defective Whn transcription factor gene is unknown. A comparison of mRNA populations from wild-type and nude mice back skin by representational difference analysis revealed the absence of acidic hair keratin gene 3 (mHa3) mRNA in mutant mice. Whn and acidic hair keratin genes are co-expressed in hair follicles, nail forming regions and filiform papillae of the tongue: expression of the mHa3 gene is generally detectable about 1 day after Whn mRNA and rapidly ceases in its absence. Whn is strongly expressed during the anagen (growth) phase of the hair cycle in matrix, cortex and outer root sheath; its expression rapidly declines during catagen and is undetectable in telogen phases. In nude mice, low levels of mHa3 expression are maintained in nails and whisker follicles, whereas expression is completely absent in pelage hair follicles and filiform papillae. Thus, the nude phenotype represents the first example of an inherited skin disorder that is associated with the loss of expression rather than structural mutation of keratin genes. The distinct molecular difference between pelage and whisker follicles correlates with the improved mechanical stability of vibrissae in nude mice, implicating mHa3 as an important structural component of the hair shaft.