Organogenesis MCB141 part III Spring 2012 Lecture 9 Organogenesis of the endoderm and branching morphogenesis of the kidney 1. While we have concentrated mostly on the nervous system and the derivatives of the paraxial mesoderm, this lecture illustrates the development of the endoderm and of the intermediate mesoderm. The endoderm is discussed in Gilbert6, G9 471-9. 2. Along its length the endoderm develops different digestive organs, but also various structures are induced by interactions with the surrounding mesoderm, these include the thyroid and parathyroid glands (endocrine organs) from the pharynx, the lungs, the liver and pancreas. 3. As with the nervous system and paraxial mesoderm, the anterior to posterior domains of the endoderm are defined by different gene expression domains. Notably Sox2 is expressed in the anterior and Cdx2 in the posterior domain. The foregut, region that forms the liver, and pancreas is defined by expression of Pdx1. The ability of these regions to respond to signals is set up (competence) in part by pioneer transcription factors which access the closed chromatin and prepare it for binding of other transcription factors. The pioneer transcription factors, expressed along the length of the endoderm include foxa1 and foxa2. Note that these transcription factors are not all gut-specific, e.g. Sox2 is expressed in the nervous system, and foxa2 is expressed in the node, notochord, and the floor plate. 4. As an example of the interactions, the pdx1 expressing domain can be induced to form Liver by signaling from adjacent tissues. Both FGF and BMP signaling are needed, and the source can vary in different species-in the mouse, FGF is made by the adjacent heart, and BMP by the adjacent mesenchyme (the septum transversum mesenchyme). This is another example of the general observation that as development proceeds, the different tissues become differently competent to respond to signals, and adjacent new signaling centers also arise through the development of new organs or tissues that express the signals. 5. Many of the derivatives of the endoderm, such as the endocrine and exocrine glands, and lungs, exploit branching morphogenesis to elaborate a large surface area. Lung development is an example, and an elaborate series of interactions between mesoderm and endoderm using almost every signal you can remember mediate the outgrowth and branching morphogenesis. 6. Much of what we understand about branching comes from the simpler insect tracheal system, where breathless (FGF) from the tissues interact with branchless (FGFreceptor) and Sprouty (an intracellular signaling antagonist) in the tracheole affect the amount of branching. This suggests a mechanism for how branching is promoted, by local growth of the tip, followed by induction of the antagonist and local inhibition of signaling; new high points of signaling next to the tip would then promote new growth points. This is still a model, and the detailed mechanism of branching morphogenesis is still not well understood. 7. Kidney development llustrates the principles of epithelial mesenchymal interactions and subsequent branching morphogenesis. G8 460-469; G7 pp 477-485, Gilbert6. The kidney has evolved from a simple structure (the pronephros) to a complex branched organ (metanephros). During mammalian development, vestiges of the most primitive kidney can be seen, but are never functional. However, in the larval stages of amphibians and fishes, the pronephros, with its simple single tube, is the functional unit. The mesonephric kidney is the adult form in amphibians, but in mammals this too is replaced by the metanephric kidey. Gilbert6 8. The Pronephros of the amphibian illustrates early development of a simple kidney. Pictures that illustrate the different kidneys and their development can be seen in this review (fig. 3, 1 and 2). Cells aggregate into a glomus (cf. Glomerulus) which filters blood into the coelomic cavity. Just as in the mammalian kidney, filtrate is collected into tubules, which recover materials, and waste passes into pronephric duct. Organogenesis MCB141 part III Spring 2012 Lecture 9

Metanephric kidney development. 9. The kidney forms in a region of overlapping transcription factor expression, and is initiated in the caudal trunk by hox gene expression. Loss of hox11 deletes the metanephric kidney and ectopic expression in more anterior regions can induce transformation of that region into metanephros, illustrating that the A/P nature of the mesoderm determines the formation of adjacent organs. 10. Many other genes involved in kidney development have been found to be mutated in human patients. For example, loss of Wt1 causes Wilm’s tumor, and loss of Pax2 causes hypoplasia of kidneys. 11.The Wolffian duct forms along the entire length of the pro-, meso, and metanephric kiney regions. After the pro- and meso-nephros degenerate, the Wolffian duct takes on a new role in males where it forms epididymis and vas deferens. This anterior portion of the duct degenerates in females. 12.At the level of the metanephros, the Ureteric bud interacts with the surrounding mesenchyme. Mesenchyme promotes the growth and branching of the bud. The kidney rudiments can be explanted and observed in culture. This was a classic experiment to show the interactions between tissues, and the mesenchymal to epithelial changes that occur in the nephrogenic mesenchyme. The ureteric bud and the nephrogenic mesenchyme reciprocally interact to promote branching and differentiation of the kidney tubules. 13.In the kidney, several Wnts and GDNF (Glial derived neurotrophic factor) are involved in growth and branching. GDNF interacts with the ret receptor, which is preferentially expressed at the tip of the growing bud to induce proliferation. Recombination experiments show that GDNF is needed in the nephrogenic mesenchyme, while Ret is needed in the ureteric bud. 14.Is GDNF an instructive factor in kidney development? If so, it would be expected to be expressed around the tips of the buds, but instead it is expressed uniformly in the metanephric mesenchyme. An experiment that shows the permissive nature of the GDNF signal is to replace its expression in the mesenchyme, with expression in the duct, using a transgene. the hoxb7 promoter has been used to drive GDNF (and GFP) expression in the epithelium. Surprisingly this gives good rescue of kidney development, showing that it doesn’t matter much where the GDNF is expressed. Instead, the GDNF and Ret combination is permissive for proliferation of the epithelial/duct cells. Thus GDNF is a permissive signal for proliferation of the duct cells, which then permits branching, rather than being an instructive signal for branching. 15.This point is confirmed by making chimeras between Ret+ and Ret- cells. Only the Ret+ cells can occupy the positions at the tips, where proliferation is active, while Ret- cells get left behind in the main ducts. 16. The tips of the buds induce the mesenchyme to condense and form the proximal tubule and gllomerular portion of the nephron. The reciprocal signaling between mesenchyme and tubule uses Wnt signaling. The growing bud induces aggregation of mesenchyme. Different Wnts act to maintain GDNF (Wnt11) and initiate condensation of mesenchyme (by activating Wnt4). Wnt4 is required in the mesenchyme for it to become epithelial and contribute to the tubules. These findings illustrate the general finding that different members of a family can have different effects, and that Wnts are not simply a generic signal. There must be some mechanism for specific Wnts activating specific pathways, though it is less clear why each functions in a different way. The most likely explanation is that they have different affinities for different frizzled receptors, which in turn are expressed in a cell-type specific way. Thus a similar signal can act cell type-specifically to transduce different downstream signals.