Acal, a new vessel that negatively regulates JNK signaling. Luis Daniel Ríos-Barrera, Juan Rafael Riesgo-Escovar. Developmental Neurobiology Dept., Neurobiology Institute, Universidad Nacional Autónoma de México, Queretaro, Mexico.

   The Jun N-terminal kinase (JNK) is part of a conserved signaling pathway that controls dorsal closure in the Drosophila embryo. Gain and loss of function conditions for the JNK pathway result in defects in dorsal closure, visible as dorsal holes in cuticle preparations. Here, we characterize a new dorsal open gene named acal in JNK signaling. The acal transcription unit is conserved among arthropods; however its molecular function is unclear as it has no conserved open reading frames. By cellular fractionation and RT-PCR, it is present in the nucleus, and by Northern blot, the primary transcript is processed to fragments smaller than 100 pb. These results suggest a non-coding RNA. Mutations in acal are lethal and result in cuticular dorsal holes (hence its name, meaning boat in the Nahuatl language). Mutant phenotype analysis by means of puc-lacZ, a reporter of JNK activity, revealed ectopic activation of the pathway. Similarly, heterozygosity for basket, the JNK gene, partially restored the acal homozygous phenotype, showing that acal inhibits JNK signaling during dorsal closure. acal is expressed in the epidermis during dorsal closure stages. Targeting acal expression to the ectoderm or to the lateral epidermis using the UAS-Gal4 system rescues the embryonic mutant phenotype. The expression pattern of raw, a negative regulator of JNK signaling during dorsal closure, is very similar to acal. Using in situ hybridization we found that epidermal acal expression disappears in raw mutants, suggesting raw acts upstream of acal during dorsal closure. We then turned our attention to thorax closure to study acal and raw. Thorax closure is a process analogous to dorsal closure during metamorphosis, also controlled by JNK signaling. Over-expression of acal or raw in the thorax using the UAS-Gal4 system results in a mild thoracic cleft phenotype. However, over-expression of both genes at the same time results in a significantly stronger phenotype. Taken together, our results show that acal is a novel negative regulator of JNK signaling downstream of Raw.