Sall4 controls differentiation of pluripotent cells independently of the Nucleosome Remodelling and Deacetylation (NuRD) complex.

Sall4 is an essential transcription factor for early mammalian development and is frequently overexpressed in cancer. Although it is reported to play an important role in embryonic stem cell (ESC) self-renewal, whether it is an essential pluripotency factor has been disputed. Here, we show that Sall4 is dispensable for mouse ESC pluripotency. Sall4 is an enhancer-binding protein that prevents precocious activation of the neural gene expression programme in ESCs but is not required for maintenance of the pluripotency gene regulatory network. Although a proportion of Sall4 protein physically associates with the Nucleosome Remodelling and Deacetylase (NuRD) complex, Sall4 neither recruits NuRD to chromatin nor influences transcription via NuRD; rather, free Sall4 protein regulates transcription independently of NuRD. We propose a model whereby enhancer binding by Sall4 and other pluripotency-associated transcription factors is responsible for maintaining the balance between transcriptional programmes in pluripotent cells.Wellcome Trust (PhD Studentship; Senior Fellowship in the Basic Biomedical Sciences [098021/Z/11/Z]), Wellcome Trust and UK Medical Research Council core funding to the Cambridge Stem Cell Institute [079249/Z/06/I], European Union Seventh Framework Programme (FP7) Project ‘4DCellFate’This is the final version of the article. It first appeared from The Company of Biologists via http://dx.doi.org/10.1242/dev.13911

Phosphorylation of Kif26b Promotes Its Polyubiquitination and Subsequent Proteasomal Degradation during Kidney Development

Kif26b, a member of the kinesin superfamily proteins (KIFs), is essential for kidney development. Kif26b expression is restricted to the metanephric mesenchyme, and its transcription is regulated by a zinc finger transcriptional regulator Sall1. However, the mechanism(s) by which Kif26b protein is regulated remain unknown. Here, we demonstrate phosphorylation and subsequent polyubiquitination of Kif26b in the developing kidney. We find that Kif26b interacts with an E3 ubiquitin ligase, neural precursor cell expressed developmentally down-regulated protein 4 (Nedd4) in developing kidney. Phosphorylation of Kif26b at Thr-1859 and Ser-1962 by the cyclin-dependent kinases (CDKs) enhances the interaction of Kif26b with Nedd4. Nedd4 polyubiquitinates Kif26b and thereby promotes degradation of Kif26b via the ubiquitin-proteasome pathway. Furthermore, Kif26b lacks ATPase activity but does associate with microtubules. Nocodazole treatment not only disrupts the localization of Kif26b to microtubules but also promotes phosphorylation and polyubiquitination of Kif26b. These results suggest that the function of Kif26b is microtubule-based and that Kif26b degradation in the metanephric mesenchyme via the ubiquitin-proteasome pathway may be important for proper kidney development

Monkeys mutant for PKD1 recapitulate human autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) caused by PKD1 mutations is one of the most common hereditary disorders. However, the key pathological processes underlying cyst development and exacerbation in pre-symptomatic stages remain unknown, because rodent models do not recapitulate critical disease phenotypes, including disease onset in heterozygotes. Here, using CRISPR/Cas9, we generate ADPKD models with PKD1 mutations in cynomolgus monkeys. As in humans and mice, near-complete PKD1 depletion induces severe cyst formation mainly in collecting ducts. Importantly, unlike in mice, PKD1 heterozygote monkeys exhibit cyst formation perinatally in distal tubules, possibly reflecting the initial pathology in humans. Many monkeys in these models survive after cyst formation, and cysts progress with age. Furthermore, we succeed in generating selective heterozygous mutations using allele-specific targeting. We propose that our models elucidate the onset and progression of ADPKD, which will serve as a critical basis for establishing new therapeutic strategies, including drug treatments

Essential roles of Sall1 in kidney development

SALL1 is a mammalian homologue of the Drosophila region-specific homeotic gene spalt (sal) and heterozygous mutations in SALL1 in humans lead to Townes-Brocks syndrome. We isolated a mouse homologue of SALL1 (Sall1) and found that mice deficient in Sall1 die in the perinatal period with kidney agenesis. Sall1 is expressed in the metanephric mesenchyme surrounding ureteric bud and homozygous deletion of Sall1 results in an incomplete ureteric bud outgrowth. Therefore, Sall1 is essential for ureteric bud invasion, the initial key step for K development. We also generated mice in which a green fluorescent protein (GFP) gene was inserted into the Sall1 locus and we isolated the GFP-positive population from embryonic kidneys of these mice by fluorescence-activated cell sorting (FACS). We then compared gene expression profiles in the GFP-positive and -negative population using microarray analysis, followed by in situ hybridization. We detected many genes known to be important for metanephros development, and genes expressed abundantly in the metanephric mesenchyme. We also found groups of genes which are not known to be expressed in the metanephric mesenchyme. Thus a combination of microarray technology and Sall1-GFP mice is useful for systematic identification of genes expressed in the developing kidney

Induction of nephron progenitors and glomeruli from human pluripotent stem cells

Studies of kidney regeneration using stem cells have progressed rapidly in recent years. Our group has developed a protocol to induce nephron progenitors from both mouse and human pluripotent stem cells which is based on a revised model of early stage kidney specification. The induced progenitors readily reconstitute three-dimensional nephron structures, including glomeruli and renal tubules, in vitro. We can further generate human induced pluripotent stem cells (iPSCs), in which nephrin-expressing glomerular podocytes are tagged with green fluorescent protein (GFP). The sorted GFP-positive cells retain the podocyte-specific molecular and structural features. Upon transplantation, mouse endothelial cells of the host animals are integrated into the human iPSC-derived glomeruli, and the podocytes show further maturation. Other laboratories have reported different protocols to induce nephron structures from human iPSCs in vitro. These findings will accelerate our understanding of kidney development and diseases in humans

Notch2 Activation in the Embryonic Kidney Depletes Nephron Progenitors

Successive activation of Wnt4 and Notch2 generates nephrons from the metanephric mesenchyme. Mesenchymal-to-epithelial transition requires Wnt4, and normal development of the proximal nephron (epithelia of glomeruli and proximal tubules) requires Notch2. It is unknown, however, whether Notch2 dictates the fate of the proximal nephron directly. Here, we generated a mutant strain of mice with activated Notch2 in Six2-containing nephron progenitor cells of the metanephric mesenchyme. Notch2 activation did not skew the cell fate toward the proximal nephron but resulted in severe kidney dysgenesis and depletion of Six2-positive progenitors. We observed ectopic expression of Wnt4 and premature tubule formation, similar to the phenotype of Six2-deficient mice. Activation of Notch2 in the progenitor cells suppressed Pax2, an upstream regulator of Six2, possibly through Hesr genes. Taken together, these data suggest that a positive feedback loop exists between Notch2 and Wnt4, and that Notch2 stabilizes, rather than dictates, nephron fate by shutting down the maintenance of undifferentiated progenitor cells, thereby depleting this population

Sall4 Is Transiently Expressed in the Caudal Wolffian Duct and the Ureteric Bud, but Dispensable for Kidney Development.

The kidney, the metanephros, is formed by reciprocal interactions between the metanephric mesenchyme and the ureteric bud, the latter of which is derived from the Wolffian duct that elongates in the rostral-to-caudal direction. Sall1 expressed in the metanephric mesenchyme is essential for ureteric bud attraction in kidney development. Sall4, another member of the Sall gene family, is required for maintenance of embryonic stem cells and establishment of induced pluripotent stem cells, and is thus considered to be one of the stemness genes. Sall4 is also a causative gene for Okihiro syndrome and is essential for the formation of many organs in both humans and mice. However, its expression and role in kidney development remain unknown, despite the essential role of Sall1 in the metanephric mesenchyme. Here, we report that mouse Sall4 is expressed transiently in the Wolffian duct-derived lineage, and is nearly complementary to Sall1 expression. While Sall4 expression is excluded from the Wolffian duct at embryonic (E) day 9.5, Sall4 is expressed in the Wolffian duct weakly in the mesonephric region at E10.5 and more abundantly in the caudal metanephric region where ureteric budding occurs. Sall4 expression is highest at E11.5 in the Wolffian duct and ureteric bud, but disappears by E13.5. We further demonstrate that Sall4 deletion in the Wolffian duct and ureteric bud does not cause any apparent kidney phenotypes. Therefore, Sall4 is expressed transiently in the caudal Wolffian duct and the ureteric bud, but is dispensable for kidney development in mice

Selective In Vitro Propagation of Nephron Progenitors Derived from Embryos and Pluripotent Stem Cells

Nephron progenitors in the embryonic kidney propagate while generating differentiated nephrons. However, in mice, the progenitors terminally differentiate shortly after birth. Here, we report a method for selectively expanding nephron progenitors in vitro in an undifferentiated state. Combinatorial and concentration-dependent stimulation with LIF, FGF2/9, BMP7, and a WNT agonist is critical for expansion. The purified progenitors proliferated beyond the physiological limits observed in vivo, both for cell numbers and lifespan. Neonatal progenitors were maintained for a week, while progenitors from embryonic day 11.5 expanded 1,800-fold for nearly 20 days and still reconstituted 3D nephrons containing glomeruli and renal tubules. Furthermore, progenitors generated from mouse embryonic stem cells and human induced pluripotent cells could be expanded with retained nephron-forming potential. Thus, we have established in vitro conditions for promoting the propagation of nephron progenitors, which will be essential for dissecting the mechanisms of kidney organogenesis and for regenerative medicine

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熊本大