Hindbrain patterning: Krox20 couples segmentation and specification of regional identity.

We have previously demonstrated that inactivation of the Krox20 gene led to the disappearance of its segmental expression territories in the hindbrain, the rhombomeres (r) 3 and 5. We now performed a detailed analysis of the fate of prospective r3 and r5 cells in Krox20 mutant embryos. Genetic fate mapping indicates that at least some of these cells persist in the absence of a functional Krox20 protein and uncovers the requirement for autoregulatory mechanisms in the expansion and maintenance of Krox20-expressing territories. Analysis of even-numbered rhombomere molecular markers demonstrates that in Krox20-null embryos, r3 cells acquire r2 or r4 identity, and r5 cells acquire r6 identity. Finally, study of embryonic chimaeras between Krox20 homozygous mutant and wild-type cells shows that the mingling properties of r3/r5 mutant cells are changed towards those of even-numbered rhombomere cells. Together, these data demonstrate that Krox20 is essential to the generation of alternating odd- and even-numbered territories in the hindbrain and that it acts by coupling the processes of segment formation, cell segregation and specification of regional identity.

Krox-20 patterns the hindbrain through both cell-autonomous and non cell-autonomous mechanisms.

The Krox-20 gene encodes a zinc finger transcription factor, which has been shown previously, by targeted inactivation in the mouse, to be required for the development of rhombomeres (r) 3 and 5 in the segmented embryonic hindbrain. In the present work, Krox-20 was expressed ectopically in the developing chick hindbrain by use of electroporation. We demonstrate that Krox-20 expression is sufficient to confer odd-numbered rhombomere characteristics to r2, r4, and r6 cells, presumably in a cell-autonomous manner. Therefore, Krox-20, appears as the major determinant of odd-numbered identity within the hindbrain. In addition, we provide evidence for the existence of a non cell-autonomous autoactivation mechanism allowing recruitment of Krox-20-positive cells from even-numbered territories by neighboring Krox-20-expressing cells. On the basis of these observations, we propose that Krox-20 regulates multiple, intertwined steps in segmental patterning: Initial activation of Krox-20 in a few cells leads to the segregation, homogenization, and possibly expansion of territories to which Krox-20 in addition confers an odd-numbered identity.

A novel mechanism for P element homing in Drosophila.

P element insertion is essentially random at the scale of the genome. However, P elements containing regulatory sequences from Drosophila engrailed and polyhomeotic genes and from the Bithorax and Antennapedia complexes show some insertional specificity by frequently inserting near the parent gene (homing) and/or near genes containing Polycomb group response elements (preferential insertion). This phenomenon is thought to be mediated by Polycomb group proteins. In this report, we describe a case of homing of P elements containing regulatory sequences of the linotte gene. This homing occurs with high frequency (up to 20% of the lines) and high precision (inserted into a region of <1 kilobase). We present evidence showing that it is not mediated by Polycomb group proteins but by a new, as yet unknown, mechanism. We also suggest that P element homing could be a more frequent phenomenon than generally assumed and that it could become a powerful tool of Drosophila reverse genetics, for which there is no other described gene targeting technique.

The receptor tyrosine kinase gene linotte is required for neuronal pathway selection in the Drosophila mushroom bodies.

The linotte (lio) mutant was first isolated as a memory mutant. The lio gene encodes a putative receptor tyrosine kinase (RTK), homologous to the human protein RYK. This gene has been independently identified in a screen for embryonic nervous system axonal guidance defects and called derailed (drl). Here, we report that linotte mutants present structural brain defects in the adult central complex (CX) and mushroom bodies (MB). linotte and derailed are allelic for this phenotype, which can be rescued by a drl+ transgene. The Lio RTK is expressed preferentially in the adult CX and MB. Our results suggest that, analogous to its role within the embryonic nervous system, the Lio RTK is involved in neuronal pathway selection during adult brain development.

The Drosophila learning and memory gene linotte encodes a putative receptor tyrosine kinase homologous to the human RYK gene product.

The linotte mutant was isolated on the basis of its learning and memory deficit. Interestingly, linotte individuals carrying a null mutation are viable, indicating that the linotte gene is not required for vital functions. We show here that the linotte gene encodes a putative receptor tyrosine kinase, homologous to the human protein RYK. These products are unique among receptor tyrosine kinases, since they possess a short extra cellular domain, and a modified intracellular catalytic domain. In particular, the subdomains directly involved in ATP binding and phosphotransfer reaction display remarkable variations. These results suggest that linotte is part of a novel signal transduction cascade involved in learning and memory.