Cell growth, division, and death are important determinants of tissue and animal size [1
], and disruption in their balance can lead to physiological disorders including cancer [2
]. While the mechanisms that integrate cell division checkpoints with cell death are relatively well studied [3
], less is known about the relationship between cell growth and death.
Apoptosis and autophagic cell death are the two most prominent morphological forms of cell death that occur during animal development [4
]. The mechanisms that regulate apoptosis have been extensively studied, but far less is known about autophagic cell death. Drosophila
larval salivary glands are an excellent system for investigating autophagic cell death during development. A rise in the steroid hormone 20-hydroxyecdysone (ecdysone) 12 hours after puparium formation triggers future adult head eversion, salivary gland cell death, and the synchronized degradation of salivary gland cells is completed by 16 hours after puparium formation [6
]. Both caspases and autophagy are induced following the rise in ecdysone that triggers cell death [7
]. Caspases and autophagy function in an additive manner in dying salivary glands, as evidenced by the finding that the combined inhibition of both caspases and autophagy results in a stronger salivary gland persistence phenotype than inhibition of either caspases or autophagy alone [9
]. Cell growth stops prior to salivary gland cell death, and maintenance of growth is sufficient to suppress both autophagy and degradation of this tissue [9
], but the mechanism that regulates this growth arrest is not clear.
The insulin-triggered class I phosphoinositide 3-kinase (PI3K) pathway is highly conserved and regulates cell and tissue growth [10
]. Binding of insulin to the insulin receptor leads to the phosphorylation of the receptor and the insulin receptor substrate protein Chico [11
]. This phosphorylation cascade activates the catalytic subunit Dp110 of the class I PI3K pathway [10
]. Activated Dp110 converts phosphatidylinositol-4, 5-P (2) to the second messenger phosphatidylinositol-3, 4, 5-P (3) (PIP3). The pleckstrin homology (PH) domain of Akt interacts with PIP3 on the cell membrane and activates the downstream effector target-of-rapamycin (TOR), an evolutionarily conserved kinase [13
]. TOR influences a wide range of cellular processes such as protein translation, cell cycle progression, growth and autophagy. Although activation of the class I PI3K pathway by expression of either activated Ras, Dp110, or Akt is sufficient to inhibit autophagy and degradation of salivary glands [9
], the mechanism that is responsible for the regulation of PI3K-dependent growth arrest in this tissue is not fully understood.
Recent studies in Drosophila
have identified the evolutionarily conserved Warts (Wts) signaling pathway as an important regulator of tissue growth. Wts, Fat, Merlin (Mer), Expanded (Ex), Hippo (Hpo), Salvador (Sav), and Mats are members of a kinase cascade that negatively regulates tissue growth [14
]. Mutations in any of these recessive genes causes increased cell division, and several of these mutants exhibit decreased cell death. These Wts pathway defects in cell division and cell death are caused by altered levels of the cell cycle regulator Cyclin E and the inhibitor of apoptosis DIAP1 [20
]. Wts, also known as the large tumor suppressor Lats, encodes a NDR family kinase that phosphorylates the transcriptional coactivator Yorkie (Yki) [28
], and inactivates Yki by exclusion from the nucleus [29
]. Yki, the orthologue of mammalian Yes associated protein Yap, is a positive regulator of growth, and over-expression of Yki results in overgrowth phenotypes in tissues that resemble loss-of-function mutations in members of the Wts pathway [28
]. Yki functions with the TEAD/TEF DNA binding family member protein Scalloped (Sd) to regulate transcription of the inhibitor of apoptosis diap1
], and presumably other Wts signaling targets, including the microRNA bantam
and the cell cycle regulator cyclin E
]. While mutations in Wts pathway genes, and over-expression of Yki, cause tissue overgrowth, it is not clear how this pathway influences cell growth, and if the PI3K pathway is influenced by this tumor suppressor pathway.
Here we show that wts mutant salivary glands fail to arrest growth, exhibit decreased caspase activity, have attenuated autophagy, and are not degraded. Although previous studies indicate that expression of Yki phenocopies wts mutants, this was not the case in salivary glands. By contrast, expression of the Yki target bantam was sufficient to induce cell growth and inhibit salivary gland cell death. However, bantam loss-of-function mutations failed to suppress the wts mutant salivary gland persistence phenotype. These data suggest that Wts is capable of regulating growth and autophagy independent of Yki. wts mutants had altered PI3K markers and required the function of TOR and chico to inhibit salivary gland degradation. These data suggest that Wts influences the PI3K signaling pathway, growth, and autophagy in a manner that is distinct from its regulation of Yki.