Questions currently under investigation

1. How intercellular signalling leads to tubule cell allocation and the patterned eversion of tubule primordia.                 

The Malpighian tubule cells arise from the future hindgut, becoming distinct as they bud out to form small protuberances. The primordial cells are marked out before they evert from the hindgut by the expression of two transcription factors (Krüppel and Cut) and a cell adhesion molecule (Fasciclin II).

The cascade of gene interactions which leads to this tubule cell specific expression is incompletely understood. We have shown that the juxtaposition of two groups of cells, posterior gut ectoderm with endoderm, creates a boundary from which the tubule cells arise. Signalling across this boundary is required for tubule cell specification and activates the Wnt pathway to regulate the expression of Krüppel and cut (Ainsworth et al, 2000, Hatton-Ellis et al, 2006 submitted).  All aspects of tubules development fail in embryos mutant for both Krüppel and cut , but not for each alone.

The four Malpighian tubules develop as 2 pairs; the anterior pair is longer and projects forwards through the body cavity, while the posterior, shorter pair projects backwards (see figure). Interestingly the eversion and later morphogenesis of each pair are dictated by threshold levels of TGF-beta signalling. High levels of pathway activation (through both Dpp and Screw) promote the expression of targets, including the Dorsocross family of transcription factors, leading to anterior tubule eversion. Low pathway activity, ensured by the expression of the pathway antagonist Brinker in the posterior tubule primordia, promotes the development of this pair of tubules. Uniform high or low levels of pathway activation in the tubule primordia result in the failure of one pair of tubules to evert, so that a subset of Kr/cut-expressing cells remain in the hindgut.

These results have intriguing parallels with vertebrate kidney development in which Wnt and TGF-beta signalling play important roles in regulating branching of the ureteric bud (analogous to tubule eversion) as well as growth of the nephron. We now plan to discover how activation of Wnt signalling leads to the expression of early tubule genes and to identify downstream genes that are regulated by Kr and cut.  We are also investigating the targets of Dorsocross and Brinker so that we can work out how the combined activity of this network of transcriptional regulators drives the changes in cell adhesion and shape that result in the first morphogenetic movements of the tubules as they bud out from the gut tube.

References

2. How establishing cell fate in tubule primordia leads to patterned cell proliferation.

Cell division in the tubules depends on the specification of two cell populations:
i) the tip cell lineage – mitogenic signalling cells which secrete the EGF ligand, Spitz
ii) the competence domain – cells that can respond to EGF signalling

Both cell types are specified by competitive interactions within a cluster of cells that express the proneural genes. These interactions are mediated by the neurogenic genes including Delta, the signal, and Notch, the receptor. The tip cell progenitor (the tip mother cell)  continues to express proneural genes and divides once, asymmetrically, to produce the tip cell and its sibling, both of which express rhomboid and therefore secrete EGF (Spitz) the mitogenic signal.

The same process of competitive signalling produces a restricted domain of cells that are competent to respond to EGF. These cells have expressed proneural genes but are repressed by Notch activation. As a result they are primed to respond to EGF by the expression of targets, pointed and seven-up. EGF signalling then activates Pointed by phosphorylation and further up-regulates seven-up expression to levels where it activates the cell cycle regulators Cyclin E and String.

The important consequence of specifying these two cell fates is that cell division in the tubules is tightly regulated, so that hyperactivation of mitogenic signalling does not lead to over-proliferation of tubule cells (and the development of tumours).

We now plan to analyse how cell division is terminated in the developing tubules ( and also how the patterning of cell differentiation (e.g. secretory vs. reabsorbtive cell types) relates to this early specification of mitotically active vs. quiescent cell types.

References

• Hoch, M., Broadie, K., Jäckle, H. and Skaer, H. (1994) Sequential fates in a single cell are established by the neurogenic cascade in the Malpighian tubules of Drosophila.  Development 120: 3439 - 3450.
• Kerber, B., Fellert, S. and Hoch, M. (1998) seven-up, the Drosophila homolog of the COUP-TF orphan receptors, controls cell proliferation in the insect kidney. Genes Dev. 12: 1781-1786.
• Wan, S., Cato, A-M. and Skaer, H. (2000) Multiple signalling pathways establish cell fate and number in Drosophila Malpighian tubules. Dev. Biol. 217: 153-165.
• Sudarsan, V., Pasalodos-Sanchez, S., Wan, S., Gampel, A. and Skaer, H. (2002) A genetic hierarchy establishes mitogenic signalling and mitotic competence in the renal tubules of Drosophila. Development. 129: 935-944.

 3. How cells outside the tubules are recruited, becoming polarised as they integrate into the epithelium.

Organs are made up of cells from separate origins, whose development and differentiation must be integrated to produce a physiologically coherent structure. For example during the development of the kidney, a series of interactions between the epithelial, mesonephric duct and the surrounding metanephric mesenchyme leads to the formation of renal tubules. Cells of the metanephric mesenchyme first induce branching of the mesonephric duct, to form the ureteric buds, and then respond to signals derived from them. As a result, mesenchymal cells are recruited to the buds, undergoing a mesenchymal-to-epithelial transition as they condense to form nephrons.

In contrast the simple renal tubules of invertebrates, such as insect Malpighian tubules, have always been thought to arise from single tissue primordia, epithelial buds which grow by cell division and enlargement, and from which a range of specialised subtypes differentiate.

We have uncovered unexpected parallels between the development of Drosophila Malpighian tubules and vertebrate nephrogenesis. We have shown that the Malpighian tubules also derive from two cell populations: ectodermal epithelial buds and surrounding mesenchymal mesoderm. The mesenchymal cells are recruited to the growing tubules, undergoing a mesenchymal-to-epithelial transition as they integrate with them, and they subsequently differentiate as a physiologically distinctive subset of tubule cells, the stellate cells.

Strikingly the normal incorporation of stellate cells and the later physiological activity of the mature tubules depend on the activity of hibris, an orthologue of mammalian NEPHRIN.

We now plan to analyse the molecular pathways that direct stellate cell specification, the recognition signals between tubule and stellate cells, the pathways that recruits stellate cells and bring about the transition from a migratory cell type to a polarised epithelial cell. A particular focus will be the role of Nephrin and its partners in these cell activities.

References

• The  Kidney: From Normal Development to Congenital Abnormalities. ed: P., Vize, A., Woolf  & J., Bard. (2003)  Academic Press.
• Denholm, B., Sudarsan, V., Pasalodos Sanchez, S., Artero, R., Lawrence, P, Maddrell, S., Baylies, M. and Skaer, H. (2003) Dual origin of the renal tubules in Drosophila: mesodermal cells integrate and polarise to establish secretory function.  Curr. Biol. 13: 1052-1057

 

4. How changes in cell shape and arrangement lead to tubule morphogenesis and how tissue interactions result in stereotypic pathfinding in the two pairs of tubules.

In a new project we are using the highly reproducible morphogenesis of the Malpighian tubules as a model to investigate the relative contributions of tissue autonomous changes (cell shape, adhesion and juxtaposition) and non-autonomous interactions (with guide-post and target tissues) in the control of tubule shape and 3-dimensional arrangement in the body cavity.

Our aim is to identify the genes that regulate the cellular changes involved in the convergent-extension movements that result in tubule elongation and to characterise the signalling pathways and their targets that underlie the tissue interactions that guide the anterior versus posterior movements of the different tubule pairs.

References

• Jung, A., Denholm, B., Skaer, H. & Affolter, M. (2005) Renal tubule development in Drosophila: a closer look at the cellular level. J.American Soc.Nephrology 16: 322–328