The basic unit of kidney function is the nephron. In the mouse, around 14,000 nephrons form in a 10-day period extending into early neonatal life, while the human fetus forms the adult complement of ...nephrons in a 32-week period completed prior to birth. This review discusses our current understanding of mammalian nephrogenesis: the contributing cell types and the regulatory processes at play. A conceptual developmental framework has emerged for the mouse kidney. This framework is now guiding studies of human kidney development enabled in part by in vitro systems of pluripotent stem cell-seeded nephrogenesis. A near future goal will be to translate our developmental knowledge-base to the productive engineering of new kidney structures for regenerative medicine.
Cellular interactions among nephron, interstitial, and collecting duct progenitors drive mammalian kidney development. In mice, Six2
nephron progenitor cells (NPCs) and Foxd1
interstitial progenitor ...cells (IPCs) form largely distinct lineage compartments at the onset of metanephric kidney development. Here, we used the method for analyzing RNA following intracellular sorting (MARIS) approach, single-cell transcriptional profiling,
hybridization, and immunolabeling to characterize the presumptive NPC and IPC compartments of the developing human kidney. As in mice, each progenitor population adopts a stereotypical arrangement in the human nephron-forming niche: NPCs capped outgrowing ureteric branch tips, whereas IPCs were sandwiched between the NPCs and the renal capsule. Unlike mouse NPCs, human NPCs displayed a transcriptional profile that overlapped substantially with the IPC transcriptional profile, and key IPC determinants, including
, were readily detected within SIX2
NPCs. Comparative gene expression profiling in human and mouse Six2/SIX2
NPCs showed broad agreement between the species but also identified species-biased expression of some genes. Notably, some human NPC-enriched genes, including
and
, are linked to human renal disease. We further explored the cellular diversity of mesenchymal cell types in the human nephrogenic niche through single-cell transcriptional profiling. Data analysis stratified NPCs into two main subpopulations and identified a third group of differentiating cells. These findings were confirmed by section
hybridization with novel human NPC markers predicted through the single-cell studies. This study provides a benchmark for the mesenchymal progenitors in the human nephrogenic niche and highlights species-variability in kidney developmental programs.
Sox9 encodes an essential transcriptional regulator of chondrocyte specification and differentiation. When Sox9 nuclear activity was compared with markers of chromatin organization and ...transcriptional activity in primary chondrocytes, we identified two distinct categories of target association. Class I sites cluster around the transcriptional start sites of highly expressed genes with no chondrocyte-specific signature. Here, Sox9 association reflects protein-protein association with basal transcriptional components. Class II sites highlight evolutionarily conserved active enhancers that direct chondrocyte-related gene activity through the direct binding of Sox9 dimer complexes to DNA. Sox9 binds through sites with sub-optimal binding affinity; the number and grouping of enhancers into super-enhancer clusters likely determines the levels of target gene expression. Interestingly, comparison of Sox9 action in distinct chondrocyte lineages points to similar regulatory strategies. In addition to providing insights into Sox family action, our comprehensive identification of the chondrocyte regulatory genome will facilitate the study of skeletal development and human disease.
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•Sox9 has two distinct modes of action on the chondrocyte genome•Sox9 associates indirectly with the TSS domain to engage basal cell activities•Sub-optimal Sox9-dimer binding to multiple enhancers regulates chondrocyte genes•The Sox9 program is similar in mesoderm- and neural-crest-derived chondrocytes
Ohba et al. provide an unbiased genome-wide view of the Sox9 regulatory landscape in the developing mammalian chondrocyte, identifying and functionally validating distinct regulatory modes of Sox9 action at engaged target genes.
A balance between Six2-dependent self-renewal and canonical Wnt signaling-directed commitment regulates mammalian nephrogenesis. Intersectional studies using chromatin immunoprecipitation and ...transcriptional profiling identified direct target genes shared by each pathway within nephron progenitors. Wnt4 and Fgf8 are essential for progenitor commitment; cis-regulatory modules flanking each gene are cobound by Six2 and β-catenin and are dependent on conserved Lef/Tcf binding sites for activity. In vitro and in vivo analyses suggest that Six2 and Lef/Tcf factors form a regulatory complex that promotes progenitor maintenance while entry of β-catenin into this complex promotes nephrogenesis. Alternative transcriptional responses associated with Six2 and β-catenin cobinding events occur through non-Lef/Tcf DNA binding mechanisms, highlighting the regulatory complexity downstream of Wnt signaling in the developing mammalian kidney.
► Identification of gene regulatory networks in nephron progenitors ► Maintenance and commitment pathways integrated at cis-regulatory modules ► Multiple roles for β-catenin complexes in progenitor programs
Nephron progenitors must balance Six2-dependent self-renewal with Wnt/β-catenin-directed differentiation. Park et al. performed genome-wide analyses of target genes regulated by Six2 and β-catenin in these cells. Their work identifies key cis-regulatory nodes and defines distinct ways in which these transcriptional regulators interact and coordinate the control of kidney gene expression.
Chronic kidney disease affects 10% of the population with notable differences in ethnic and sex-related susceptibility to kidney injury and disease. Kidney dysfunction leads to significant morbidity ...and mortality and chronic disease in other organ systems. A mouse-organ-centered understanding underlies rapid progress in human disease modeling and cellular approaches to repair damaged systems. To enhance an understanding of the mammalian kidney, we combined anatomy-guided single-cell RNA sequencing of the adult male and female mouse kidney with in situ expression studies and cell lineage tracing. These studies reveal cell diversity and marked sex differences, distinct organization and cell composition of nephrons dependent on the time of nephron specification, and lineage convergence, in which contiguous functionally related cell types are specified from nephron and collecting system progenitor populations. A searchable database, Kidney Cell Explorer (https://cello.shinyapps.io/kidneycellexplorer/), enables gene-cell relationships to be viewed in the anatomical framework of the kidney.
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•Proximal nephron segments show distinct expression profiles between the sexes•The time of nephron formation determines position and segmental cell diversity•Lineage convergence is observed at nephron-collecting system junctions•Data can be queried and viewed within an annotated anatomical database
Ransick et al. combined in-depth single-cell profiling of male and female adult kidneys with cluster registration to kidney structures to generate an anatomical atlas of the mammalian nephron and collecting system. Kidney Cell Explorer (https://cello.shinyapps.io/kidneycellexplorer/) enables gene-cell relationships to be viewed in the anatomical framework of the mammalian kidney.
The Hedgehog (HH) signaling pathway was discovered originally as a key pathway in embryonic patterning and development. Since its discovery, it has become increasingly clear that the HH pathway also ...plays important roles in a multitude of cancers. Therefore, HH signaling has emerged as a therapeutic target of interest for cancer therapy. In this review, we provide a brief overview of HH signaling and the key molecular players involved and offer an up-to-date summary of our current knowledge of endogenous and exogenous small molecules that modulate HH signaling. We discuss experiences and lessons learned from the decades-long efforts toward the development of cancer therapies targeting the HH pathway. Challenges to develop next-generation cancer therapies are highlighted.
The Hedgehog (HH) signaling pathway was discovered originally as a key pathway in embryonic patterning and development. Since its discovery, it has become increasingly clear that the HH pathway also plays important roles in a multitude of cancers. Therefore, HH signaling has emerged as a therapeutic target of interest for cancer therapy. In this review, we provide a brief overview of HH signaling and the key molecular players involved and offer an up-to-date summary of our current knowledge of endogenous and exogenous small molecules that modulate HH signaling. We discuss experiences and lessons learned from the decades-long efforts toward the development of cancer therapies targeting the HH pathway. Challenges to develop next-generation cancer therapies are highlighted.
The nephron is the functional unit of the kidney, but the mechanism of nephron formation during human development is unclear. We conducted a detailed analysis of nephron development in humans and ...mice by immunolabeling, and we compared human and mouse nephron patterning to describe conserved and divergent features. We created protein localization maps that highlight the emerging patterns along the proximal-distal axis of the developing nephron and benchmark expectations for localization of functionally important transcription factors, which revealed unanticipated cellular diversity. Moreover, we identified a novel nephron subdomain marked by
expression that we fate-mapped to the proximal mature nephron. Significant conservation was observed between human and mouse patterning. We also determined the time at which markers for mature nephron cell types first emerge-critical data for the renal organoid field. These findings have conceptual implications for the evolutionary processes driving the diversity of mammalian organ systems. Furthermore, these findings provide practical insights beyond those gained with mouse and rat models that will guide
efforts to harness the developmental programs necessary to build human kidney structures.
Human kidney function is underpinned by approximately 1,000,000 nephrons, although the number varies substantially, and low nephron number is linked to disease. Human kidney development initiates ...around 4 weeks of gestation and ends around 34-37 weeks of gestation. Over this period, a reiterative inductive process establishes the nephron complement. Studies have provided insightful anatomic descriptions of human kidney development, but the limited histologic views are not readily accessible to a broad audience. In this first paper in a series providing comprehensive insight into human kidney formation, we examined human kidney development in 135 anonymously donated human kidney specimens. We documented kidney development at a macroscopic and cellular level through histologic analysis, RNA
hybridization, immunofluorescence studies, and transcriptional profiling, contrasting human development (4-23 weeks) with mouse development at selected stages (embryonic day 15.5 and postnatal day 2). The high-resolution histologic interactive atlas of human kidney organogenesis generated can be viewed at the GUDMAP database (www.gudmap.org) together with three-dimensional reconstructions of key components of the data herein. At the anatomic level, human and mouse kidney development differ in timing, scale, and global features such as lobe formation and progenitor niche organization. The data also highlight differences in molecular and cellular features, including the expression and cellular distribution of anchor gene markers used to identify key cell types in mouse kidney studies. These data will facilitate and inform
efforts to generate human kidney structures and comparative functional analyses across mammalian species.
The renal corpuscle of the kidney comprises a glomerular vasculature embraced by podocytes and supported by mesangial myofibroblasts, which ensure plasma filtration at the podocyte-generated slit ...diaphragm. With a spectrum of podocyte-expressed gene mutations causing chronic disease, an enhanced understanding of podocyte development and function to create relevant in vitro podocyte models is a clinical imperative. To characterize podocyte development, scRNA-seq was performed on human fetal kidneys, identifying distinct transcriptional signatures accompanying the differentiation of functional podocytes from progenitors. Interestingly, organoid-generated podocytes exhibited highly similar, progressive transcriptional profiles despite an absence of the vasculature, although abnormal gene expression was pinpointed in late podocytes. On transplantation into mice, organoid-derived podocytes recruited the host vasculature and partially corrected transcriptional profiles. Thus, human podocyte development is mostly intrinsically regulated and vascular interactions refine maturation. These studies support the application of organoid-derived podocytes to model disease and to restore or replace normal kidney functions.
•scRNA-seq identifies distinct transcriptional profiles of human podocyte development•Comparative analyses show similarities and differences of in vitro-derived podocytes•Implantation study suggests vascular interactions improve in vitro podocyte maturation
Tran et al. performed single-cell RNA sequencing to provide an understanding of human podocyte development. Insights from the in vivo analysis was applied to extensively evaluate the formation of podocytes in vitro, highlighting autonomous programs of development and those requiring an interplay with adjacent cell types.
Neuronal subtype specification in the vertebrate neural tube is one of the best-studied examples of embryonic pattern formation. Distinct neuronal subtypes are generated in a precise spatial order ...from progenitor cells according to their location along the anterior-posterior and dorsal-ventral axes. Underpinning this organization is a complex network of multiple extrinsic and intrinsic factors. This review focuses on the molecular mechanisms and general strategies at play in ventral regions of the forming spinal cord, where sonic hedgehog-based morphogen signaling is a key determinant. We discuss recent advances in our understanding of these events and highlight unresolved questions.
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