Proceedings: consideration of genetics in the design of induced pluripotent stem cell-based models of complex disease.

The goal of exploiting induced pluripotent stem cell (iPSC) technology for the discovery of new mechanisms and treatments of disease is being pursued by many laboratories, and analyses of rare monogenic diseases have already provided ample evidence that this approach has merit. Considering the enormous medical burden imposed by common chronic diseases, successful implementation of iPSC-based models has the potential for major impact on these diseases as well. Since common diseases represent complex traits with varying genetic and environmental contributions to disease manifestation, the use of iPSC technology poses unique challenges. In this perspective, we will consider how the genetics of complex disease and mechanisms underlying phenotypic variation affect experimental design.

Chimeric primates: embryonic stem cells need not apply.

In this issue, Tachibana et al. report the generation of the first chimeras from a nonhuman primate, the rhesus monkey. Unlike mice, rhesus chimeras fail to form when embryonic stem cells are injected into blastocysts. Instead, chimera formation is achieved by aggregation of several four-cell embryos.

Kidney development in the absence of Gdnf and Spry1 requires Fgf10.

GDNF signaling through the Ret receptor tyrosine kinase (RTK) is required for ureteric bud (UB) branching morphogenesis during kidney development in mice and humans. Furthermore, many other mutant genes that cause renal agenesis exert their effects via the GDNF/RET pathway. Therefore, RET signaling is believed to play a central role in renal organogenesis. Here, we re-examine the extent to which the functions of Gdnf and Ret are unique, by seeking conditions in which a kidney can develop in their absence. We find that in the absence of the negative regulator Spry1, Gdnf, and Ret are no longer required for extensive kidney development. Gdnf-/-;Spry1-/- or Ret-/-;Spry1-/- double mutants develop large kidneys with normal ureters, highly branched collecting ducts, extensive nephrogenesis, and normal histoarchitecture. However, despite extensive branching, the UB displays alterations in branch spacing, angle, and frequency. UB branching in the absence of Gdnf and Spry1 requires Fgf10 (which normally plays a minor role), as removal of even one copy of Fgf10 in Gdnf-/-;Spry1-/- mutants causes a complete failure of ureter and kidney development. In contrast to Gdnf or Ret mutations, renal agenesis caused by concomitant lack of the transcription factors ETV4 and ETV5 is not rescued by removing Spry1, consistent with their role downstream of both RET and FGFRs. This shows that, for many aspects of renal development, the balance between positive signaling by RTKs and negative regulation of this signaling by SPRY1 is more critical than the specific role of GDNF. Other signals, including FGF10, can perform many of the functions of GDNF, when SPRY1 is absent. But GDNF/RET signaling has an apparently unique function in determining normal branching pattern. In contrast to GDNF or FGF10, Etv4 and Etv5 represent a critical node in the RTK signaling network that cannot by bypassed by reducing the negative regulation of upstream signals.

A Cre transgene active in developing endodermal organs, heart, limb, and extra-ocular muscle.

Cre-mediated recombination, a method widely used in mice for tissue-specific inactivation of endogenous genes or activation of transgenes, is critically dependent on the availability of mouse lines in which Cre recombinase functions in the tissue of interest or its progenitors. Here we describe a transgenic mouse line, Osr1-cre, in which Cre is active from embryonic day (E)11.5 in a few specific tissues. These include the endoderm of the posterior foregut, midgut, hindgut, and developing urogenital system, the heart left atrium, extra-ocular muscle progenitors, and mesenchyme in particular regions of the limb. Furthermore, starting at E12.5, Cre functions in limb interdigital mesenchyme. Within the urogenital system, recombination appears to be virtually complete in the epithelium of the bladder and urethra just posterior to it by E14.5. In males, some of these urethral cells form the prostate. The spatiotemporal pattern of Cre activity in Osr1-cre makes it a unique resource among the lines available for Cre-mediated recombination experiments.

FGF8 is required for cell survival at distinct stages of nephrogenesis and for regulation of gene expression in nascent nephrons.

During kidney morphogenesis, the formation of nephrons begins when mesenchymal nephron progenitor cells aggregate and transform into epithelial vesicles that elongate and assume an S-shape. Cells in different regions of the S-shaped body subsequently differentiate into the morphologically and functionally distinct segments of the mature nephron. Here, we have used an allelic series of mutations to determine the role of the secreted signaling molecule FGF8 in nephrogenesis. In the absence of FGF8 signaling, nephron formation is initiated, but the nascent nephrons do not express Wnt4 or Lim1, and nephrogenesis does not progress to the S-shaped body stage. Furthermore, the nephron progenitor cells that reside in the peripheral zone, the outermost region of the developing kidney, are progressively lost. When FGF8 signaling is severely reduced rather than eliminated, mesenchymal cells differentiate into S-shaped bodies. However, the cells within these structures that normally differentiate into the tubular segments of the mature nephron undergo apoptosis, resulting in the formation of kidneys with severely truncated nephrons consisting of renal corpuscles connected to collecting ducts by an abnormally short tubular segment. Thus, unlike other FGF family members, which regulate growth and branching morphogenesis of the collecting duct system, Fgf8 encodes a factor essential for gene regulation and cell survival at distinct steps in nephrogenesis.

SLIT2-mediated ROBO2 signaling restricts kidney induction to a single site.

Kidney development occurs in a stereotypic position along the body axis. It begins when a single ureteric bud emerges from the nephric duct in response to GDNF secreted by the adjacent nephrogenic mesenchyme. Posterior restriction of Gdnf expression is considered critical for correct positioning of ureteric bud development. Here we show that mouse mutants lacking either SLIT2 or its receptor ROBO2, molecules known primarily for their function in axon guidance and cell migration, develop supernumerary ureteric buds that remain inappropriately connected to the nephric duct, and that the SLIT2/ROBO2 signal is transduced in the nephrogenic mesenchyme. Furthermore, we show that Gdnf expression is inappropriately maintained in anterior nephrogenic mesenchyme in these mutants. Thus our data identify an intercellular signaling system that restricts, directly or indirectly, the extent of the Gdnf expression domain, thereby precisely positioning the site of kidney induction.