Whereas the mechanisms of early Drosophila mesoderm formation have been studied in much detail, the subsequent processes determining regional identities within the mesoderm remain largely unknown. Here, we describe two homeo box genes, tinman (tin) and bagpipe (hap), which spatially subdivide the mesoderm and

On the basis of homeo box cross‐homology we have isolated the pair‐rule gene even‐skipped (eve) of Drosophila. The eve transcription unit appears to be less than 1.5 kb in length, and encodes a single mRNA of approximately 1.4 kb. The nucleotide sequence of genomic and cDNA clones indicates that the eve protein is composed of 376 amino acid residues, and that its homeo domain shares only approximately 50% amino acid identity with the homeo domains of previously characterized genes. Using antibodies raised against a beta‐galactosidase fusion protein we show that the eve protein is distributed in a series of seven transverse stripes at the cellular blastoderm stage, and is localized primarily within the nuclear regions of those embryonic cells that express the gene. After gastrulation, seven weakly stained stripes of eve expression appear, resulting in a transient pattern that consists of a total of 14 evenly spaced stripes. Both the original and new stripes gradually disappear during germ band elongation. A second expression pattern emerges during neurogenesis, whereby eve protein is detected in discrete subsets of neurons in each of the ventral ganglia.

After gastrulation, progenitor cells of the cardiac, visceral and body wall musculature arise at defined positions within the mesodermal layer of the Drosophila embryo. The regulatory mechanisms underlying this process of pattern formation are largely unknown, although ablation experiments carried out in other insects indicate that inductive influences from ectodermal cells have major roles in embryonic mesoderm differentiation. An early and important event in the regional subdivision of the mesoderm is the restriction of tinman expression to dorsal mesodermal cells. Genetic analysis has shown that this homeobox gene controls the formation of the visceral musculature and the heart from dorsal portions of the mesoderm. We now show that an inductive signal from dorsal ectodermal cells is required for activation of tinman in the underlying mesoderm and present evidence that Decapentaplegic (Dpp), a member of the transforming growth factor-beta superfamily, serves as a signalling molecule in this process. This demonstrates that the spatial expression of dpp in the ectoderm determines which cells of the mesoderm become competent to develop into visceral mesoderm and the heart.

The secreted protein Jelly belly (Jeb) is required for an essential signalling event in Drosophila muscle development. In the absence of functional Jeb, visceral muscle precursors are normally specified but fail to migrate and differentiate. The structure and distribution of Jeb protein implies that Jeb functions as a signal to organize the development of visceral muscles. Here we show that the Jeb receptor is the Drosophila homologue of anaplastic lymphoma kinase (Alk), a receptor tyrosine kinase of the insulin receptor superfamily. Human ALK was originally identified as a proto-oncogene, but its normal function in mammals is not known. In Drosophila, localized Jeb activates Alk and the downstream Ras/mitogen-activated protein kinase cascade to specify a select group of visceral muscle precursors as muscle-patterning pioneers. Jeb/Alk signalling induces the myoblast fusion gene dumbfounded (duf; also known as kirre) as well as org-1, a Drosophila homologue of mammalian TBX1, in these cells.

We report the cloning and primary structure of the Drosophila insulin receptor gene (inr), functional expression of the predicted polypeptide, and the isolation of mutations in the inr locus. Our data indicate that the structure and processing of the Drosophila insulin proreceptor are somewhat different from those of the mammalian insulin and IGF 1 receptor precursors. The INR proreceptor (M(r) 280 kDa) is processed proteolytically to generate an insulin‐binding alpha subunit (M(r) 120 kDa) and a beta subunit (M(r) 170 kDa) with protein tyrosine kinase domain. The INR beta 170 subunit contains a novel domain at the carboxyterminal side of the tyrosine kinase, in the form of a 60 kDa extension which contains multiple potential tyrosine autophosphorylation sites. This 60 kDa C‐terminal domain undergoes cell‐specific proteolytic cleavage which leads to the generation of a total of four polypeptides (alpha 120, beta 170, beta 90 and a free 60 kDa C‐terminus) from the inr gene. These subunits assemble into mature INR receptors with the structures alpha 2(beta 170)2 or alpha 2(beta 90)2. Mammalian insulin stimulates tyrosine phosphorylation of both types of beta subunits, which in turn allows the beta 170, but not the beta 90 subunit, to bind directly to p85 SH2 domains of PI‐3 kinase. It is likely that the two different isoforms of INR have different signaling potentials. Finally, we show that loss of function mutations in the inr gene, induced by either a P‐element insertion occurring within the predicted ORF, or by ethylmethane sulfonate treatment, render pleiotropic recessive phenotypes that lead to embryonic lethality. The activity of inr appears to be required in the embryonic epidermis and nervous system among others, since development of the cuticle, as well as the peripheral and central nervous systems are affected by inr mutations.

Genetic screens are powerful tools to identify the genes required for a given biological process. However, for technical reasons, comprehensive screens have been restricted to very few model organisms. Therefore, although deep sequencing is revealing the genes of ever more insect species, the functional studies predominantly focus on candidate genes previously identified in Drosophila, which is biasing research towards conserved gene functions. RNAi screens in other organisms promise to reduce this bias. Here we present the results of the iBeetle screen, a large-scale, unbiased RNAi screen in the red flour beetle, Tribolium castaneum, which identifies gene functions in embryonic and postembryonic development, physiology and cell biology. The utility of Tribolium as a screening platform is demonstrated by the identification of genes involved in insect epithelial adhesion. This work transcends the restrictions of the candidate gene approach and opens fields of research not accessible in Drosophila.

The Heartless (Htl) FGF receptor is required for the differentiation of a variety of mesodermal tissues in the Drosophila embryo, yet its ligand is not known. Here we identify two new FGF genes, thisbe (ths) and pyramus (pyr), which probably encode the elusive ligands for this receptor. The two genes exhibit dynamic patterns of expression in epithelial tissues adjacent to Htl-expressing mesoderm derivatives, including the neurogenic ectoderm, stomadeum, and hindgut. Embryos that lack ths + and pyr + exhibit defects related to those seen in htl mutants, including delayed mesodermal migration during gastrulation and a loss of cardiac tissues and hindgut musculature. The misexpression of Ths in wild-type and mutant embryos suggests that FGF signaling is required for both cell migration and the transcriptional induction of cardiac gene expression. The characterization of htl and ths regulatory DNAs indicates that high levels of the maternal Dorsal gradient directly activate htl expression, whereas low levels activate ths. It is therefore possible to describe FGF signaling and other aspects of gastrulation as a direct manifestation of discrete threshold readouts of the Dorsal gradient.

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