Spatial transcriptional mapping of the human nephrogenic program.

Congenital abnormalities of the kidney and urinary tract are among the most common birth defects, affecting 3% of newborns. The human kidney forms around a million nephrons from a pool of nephron progenitors over a 30-week period of development. To establish a framework for human nephrogenesis, we spatially resolved a stereotypical process by which equipotent nephron progenitors generate a nephron anlage, then applied data-driven approaches to construct three-dimensional protein maps on anatomical models of the nephrogenic program. Single-cell RNA sequencing identified progenitor states, which were spatially mapped to the nephron anatomy, enabling the generation of functional gene networks predicting interactions within and between nephron cell types. Network mining identified known developmental disease genes and predicted targets of interest. The spatially resolved nephrogenic program made available through the Human Nephrogenesis Atlas (https://sckidney.flatironinstitute.org/) will facilitate an understanding of kidney development and disease and enhance efforts to generate new kidney structures.

A ß-catenin-driven switch in TCF/LEF transcription factor binding to DNA target sites promotes commitment of mammalian nephron progenitor cells.

The canonical Wnt pathway transcriptional co-activator ß-catenin regulates self-renewal and differentiation of mammalian nephron progenitor cells (NPCs). We modulated ß-catenin levels in NPC cultures using the GSK3 inhibitor CHIR99021 (CHIR) to examine opposing developmental actions of ß-catenin. Low CHIR-mediated maintenance and expansion of NPCs are independent of direct engagement of TCF/LEF/ß-catenin transcriptional complexes at low CHIR-dependent cell-cycle targets. In contrast, in high CHIR, TCF7/LEF1/ß-catenin complexes replaced TCF7L1/TCF7L2 binding on enhancers of differentiation-promoting target genes. Chromosome confirmation studies showed pre-established promoter-enhancer connections to these target genes in NPCs. High CHIR-associated de novo looping was observed in positive transcriptional feedback regulation to the canonical Wnt pathway. Thus, ß-catenin's direct transcriptional role is restricted to the induction of NPCs, where rising ß-catenin levels switch inhibitory TCF7L1/TCF7L2 complexes to activating LEF1/TCF7 complexes at primed gene targets poised for rapid initiation of a nephrogenic program.

Single-Cell Profiling Reveals Sex, Lineage, and Regional Diversity in the Mouse Kidney.

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.

In Vivo Developmental Trajectories of Human Podocyte Inform In Vitro Differentiation of Pluripotent Stem Cell-Derived Podocytes.

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.

Progressive Recruitment of Mesenchymal Progenitors Reveals a Time-Dependent Process of Cell Fate Acquisition in Mouse and Human Nephrogenesis.

Mammalian nephrons arise from a limited nephron progenitor pool through a reiterative inductive process extending over days (mouse) or weeks (human) of kidney development. Here, we present evidence that human nephron patterning reflects a time-dependent process of recruitment of mesenchymal progenitors into an epithelial nephron precursor. Progressive recruitment predicted from high-resolution image analysis and three-dimensional reconstruction of human nephrogenesis was confirmed through direct visualization and cell fate analysis of mouse kidney organ cultures. Single-cell RNA sequencing of the human nephrogenic niche provided molecular insights into these early patterning processes and predicted developmental trajectories adopted by nephron progenitor cells in forming segment-specific domains of the human nephron. The temporal-recruitment model for nephron polarity and patterning suggested by direct analysis of human kidney development provides a framework for integrating signaling pathways driving mammalian nephrogenesis.

Conserved and Divergent Features of Mesenchymal Progenitor Cell Types within the Cortical Nephrogenic Niche of the Human and Mouse Kidney.

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, in situ 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 FOXD1, 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 DAPL1 and COL9A2, 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 in situ 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.

Diversification of oral and aboral mesodermal regulatory states in pregastrular sea urchin embryos.

Specification of the non-skeletogenic mesoderm (NSM) in sea urchin embryos depends on Delta signaling. Signal reception leads to expression of regulatory genes that later contribute to the aboral NSM regulatory state. In oral NSM, this is replaced by a distinct oral regulatory state in consequence of Nodal signaling. Through regulome wide analysis we identify the homeobox gene not as an immediate Nodal target. not expression in NSM causes extinction of the aboral regulatory state in the oral NSM, and expression of a new suite of regulatory genes. All NSM specific regulatory genes are henceforth expressed exclusively, in oral or aboral domains, presaging the mesodermal cell types that will emerge. We have analyzed the regulatory linkages within the aboral NSM gene regulatory network. A linchpin of this network is gataE which as we show is a direct Gcm target and part of a feedback loop locking down the aboral regulatory state.

Cis-regulatory logic driving glial cells missing: self-sustaining circuitry in later embryogenesis.

The glial cells missing (gcm) regulatory gene of the sea urchin Strongylocentrotus purpuratus is first expressed in veg2 daughter cells as the genomic target of late cleavage stage Delta-Notch signaling from the skeletogenic mesoderm precursors. Gcm is required in veg2 progeny during late cleavages for the early phase of pigment cell precursor specification. Here we report on a later acting cis-regulatory module that assumes control of gcm expression by the early mesenchyme blastula stage and maintains it through pigment cell differentiation and dispersal. Cis-perturbation analyses reveal that the two critical elements within this late module are consensus matches to Gcm and Six1 binding sites. Significantly, six1 mRNA localizes to gcm+cells from the mesenchyme blastula stage onwards. Trans-perturbations with anti-sense morpholinos reveal a co-dependency between six1 and gcm. Six1 mRNA levels fall sharply after Gcm is depleted, while depleting Six1 leads to significant reductions in output of endogenous gcm or modular-reporters. These results support the conclusion gcm and six1 comprise a positive intergenic feedback loop in the mesodermal GRN. This often employed cross regulatory GRN feature here ensures self-sustaining gcm output in a cohort of fully specified pigment cell precursors at a relatively early developmental stage.

cis-regulatory processing of Notch signaling input to the sea urchin glial cells missing gene during mesoderm specification.

The glial cells missing regulatory gene of Strongylocentrotus purpuratus (spgcm) was proposed earlier to be the genomic target of Delta/Notch (D/N) signaling required for specification of the mesodermal precursors of pigment cells. Here, we show that microinjection of a spgcm antisense morpholino oligonucleotide results in larvae without pigment cells. Microinjection of an mRNA encoding a dominant negative form of Suppressor of Hairless (dn-Su(H)) results in reduced levels of spgcm mRNA, disruption of mesodermal founder cell specification and failure to produce pigment cells. These results confirm that this gene is required for pigment cell specification. Three cis-regulatory modules of the spgcm gene were identified, which when incorporated in a GFP expression construct recapitulate the early expression pattern of this gene. Spatial expression of this GFP expression construct is severely disrupted by co-expression of dn-Su(H) mRNA, confirming that spgcm is a direct target of canonical N signaling mediated through Su(H) inputs. cis-perturbation analysis by mutation of consensus Su(H) sites identified a conserved motif paired-site and a lone site in the middle module that function both to drive expression in SMC precursors which receive the Delta signal and to repress expression in ectopic locations which lack this signal. While these Su(H) target sites provide the cis-regulatory architecture with the core of an N signaling transcriptional response switch, both the on and off outputs from this module require additional inputs.

CBFbeta is a facultative Runx partner in the sea urchin embryo.

BACKGROUND: Runx proteins are developmentally important metazoan transcription factors that form a heterodimeric complex with the non-homologous protein Core Binding Factor beta (CBFbeta). CBFbeta allosterically enhances Runx DNA binding but does not bind DNA itself. We report the initial characterization of SpCBFbeta, the heterodimeric partner of SpRunt-1 from the sea urchin Stronylocentrotus purpuratus. RESULTS: SpCBFbeta is remarkably similar to its mammalian homologues, and like them it enhances the DNA binding of the Runt domain. SpCBFbeta is entirely of zygotic provenance and its expression is similar that of SpRunt-1, accumulating globally at late blastula stage then later localizing to endoderm and oral ectoderm. Unlike SpRunt-1, however, SpCBFbeta is enriched in the endodermal mid- and hindgut of the pluteus larva, and is not highly expressed in the foregut and ciliated band. We showed previously that morpholino antisense-mediated knockdown of SpRunt-1 leads to differentiation defects, as well as to extensive post-blastula stage apoptosis caused by under-expression of the Runx target gene SpPKC1. In contrast, we show here that knockdown of SpCBFbeta does not negatively impact cell survival or SpPKC1 expression, although it does lead to differentiation defects similar to those associated with SpRunt-1 deficiency. Moreover, SpRunt-1 containing a single amino acid substitution that abolishes its ability to interact with SpCBFbeta retains the ability to rescue cell survival in SpRunt-1 morphant embryos. Chromatin immunoprecipitation shows that while the CyIIIa promoter engages both proteins, the SpPKC1 promoter only engages SpRunt-1. CONCLUSION: SpCBFbeta is a facultative Runx partner that appears to be required specifically for cell differentiation.

An evolutionary constraint: strongly disfavored class of change in DNA sequence during divergence of cis-regulatory modules.

The DNA of functional cis-regulatory modules displays extensive sequence conservation in comparisons of genomes from modestly distant species. Patches of sequence that are several hundred base pairs in length within these modules are often seen to be 80-95% identical, although the flanking sequence cannot even be aligned. However, it is unlikely that base pairs located between the transcription factor target sites of cis-regulatory modules have sequence-dependent function, and the mechanism that constrains evolutionary change within cis-regulatory modules is incompletely understood. We chose five functionally characterized cis-regulatory modules from the Strongylocentrotus purpuratus (sea urchin) genome and obtained orthologous regulatory and flanking sequences from a bacterial artificial chromosome genome library of a congener, Strongylocentrotus franciscanus. As expected, single-nucleotide substitutions and small indels occur freely at many positions within the regulatory modules of these two species, as they do outside the regulatory modules. However, large indels (>20 bp) are statistically almost absent within the regulatory modules, although they are common in flanking intergenic or intronic sequence. The result helps to explain the patterns of evolutionary sequence divergence characteristic of cis-regulatory DNA.

Spdeadringer, a sea urchin embryo gene required separately in skeletogenic and oral ectoderm gene regulatory networks.

The Spdeadringer (Spdri) gene encodes an ARID-class transcription factor not previously known in sea urchin embryos. We show that Spdri is a key player in two separate developmental gene regulatory networks (GRNs). Spdri is expressed in a biphasic manner, first, after 12 h and until ingression in the skeletogenic descendants of the large micromeres; second, after about 20 h in the oral ectoderm, where its transcripts remain present at 30-50 mRNA molecules/cell far into development. In both territories, the periods of Spdri expression follow prior territorial specification events. The functional significance of each phase of expression was assessed by determining the effect of an alphaSpdri morpholino antisense oligonucleotide (MASO) on expression of 17 different mesodermal genes, 8 different oral ectoderm genes, and 18 other genes expressed specifically during endomesoderm specification. These effects were measured by quantitative PCR, supplemented by whole-mount in situ hybridization and morphological observations. Spdri is shown to act in the micromere descendants in the pathways that result in the expression of batteries of terminal skeletogenic genes. But, in the oral ectoderm, the same gene participates in the central GRN controlling oral ectoderm identity. Spdri is linked in the oral ectoderm GRN with several other genes encoding transcriptional regulators that are expressed specifically in various regions of the oral ectoderm. If its expression is blocked by treatment with alphaSpdri MASO, oral-specific features disappear and expression of the aboral ectoderm marker spec1 encompasses the whole of the ectoderm. In addition to disappearance of the oral ectoderm, morphological consequences of alphaSpdri MASO treatment include failure of spiculogenesis and of correct primary mesenchyme cell (pmc) patterning in the postgastrular embryo, and also failure of gastrulation. To further analyze these phenotypes, chimeric embryos were constructed consisting of two labeled micromeres combined with micromereless 4th cleavage host embryos; either the micromeres or the hosts contained alphaSpdri MASO. These experiments showed that, while Spdri expression is required autonomously for expression of skeletogenic genes prior to ingression, complete skeletogenesis also requires the expression of oral ectoderm patterning information. Presentation of this information on the oral side of the blastocoel in turn depends on Spdri expression in the oral ectoderm. Failure of gastrulation is not due to indirect interference with endomesodermal specification per se, since all endomesodermal genes tested function normally in alphaSpdri MASO embryos. Part of its cause is interference by alphaSpdri MASO with a late signaling function on the part of the micromere descendants that is needed to complete clearance of the Soxb1 repressor of gastrulation from the prospective endoderm, but in addition there is a nonautonomous oral ectoderm effect.

New early zygotic regulators expressed in endomesoderm of sea urchin embryos discovered by differential array hybridization.

Genes that are upregulated by LiCl treatment of sea urchin embryos and/or downregulated by injection into the egg of mRNA encoding an internal fragment of cadherin (Cad) were detected in a differential macroarray screen. The method was that recently described by J. P. Rast et al. (2000, Dev. Biol. 228, 270-296). Almost 10(5) clones from a 12-h cDNA library were screened. Measurements on internal standards showed that the screening procedure was sufficiently sensitive to afford detection of differentially expressed mRNAs of the most rare class, those present in only a few copies per average cell. The injection of Cad mRNA, which specifically blocks nuclearization of beta-catenin, resulted in many-fold decreases in the levels of transcripts of a suite of marker genes expressed zygotically during endomesoderm specification. These measurements substantiated the use of Cad mRNA as the basis for a differential screen for discovery of new endomesodermal genes. By use of the newly developed BioArray software for analysis of macroarray screens, 1106 clones representing differentially expressed genes and yielding useful sequence were recovered. The 367 clones that gave significant BLASTX matches to known cellular proteins fell into 264 nonredundant sequence classes. Those of particular interest for this work were clones encoding DNA-binding transcription factors, signal transduction pathway components, proteases, kinases, and phosphatases. Quantitative PCR analysis of 66 such selected clones revealed that the large majority of these clones had been selected because they are upregulated by LiCl treatment, which affects the expression of a much greater diversity and number of genes than are involved in endomesoderm specification. Seven transcript species were identified that responded sharply to injection of Cad mRNA, and that are not represented in maternal mRNA. Six of those encode transcription factors. We focused on three transcription factor genes of this set that were previously unknown in sea urchin embryos. By whole-mount in situ hybridization, these genes are expressed in specific domains of the endomesodermal territory. They are: (1) Speve, an evenskipped orthologue expressed very early in all vegetal blastomeres and then gradually shifting to veg(1) derivatives by the mesenchyme blastula stage; (2) Spgcm, an orthologue of the fruit fly gene glial cells missing, which is first expressed specifically and exclusively in part of the prospective secondary mesenchyme (mesodermal) domain at late-cleavage blastula stage; and (3) Spfoxc, which is first expressed in the early blastula only in the four small micromeres, and later only expressed in that coelomic pouch which gives rise to the mesoderm of the ventral surface of the adult rudiment.

A provisional regulatory gene network for specification of endomesoderm in the sea urchin embryo.

We present the current form of a provisional DNA sequence-based regulatory gene network that explains in outline how endomesodermal specification in the sea urchin embryo is controlled. The model of the network is in a continuous process of revision and growth as new genes are added and new experimental results become available; see http://www.its.caltech.edu/~mirsky/endomeso.htm (End-mes Gene Network Update) for the latest version. The network contains over 40 genes at present, many newly uncovered in the course of this work, and most encoding DNA-binding transcriptional regulatory factors. The architecture of the network was approached initially by construction of a logic model that integrated the extensive experimental evidence now available on endomesoderm specification. The internal linkages between genes in the network have been determined functionally, by measurement of the effects of regulatory perturbations on the expression of all relevant genes in the network. Five kinds of perturbation have been applied: (1) use of morpholino antisense oligonucleotides targeted to many of the key regulatory genes in the network; (2) transformation of other regulatory factors into dominant repressors by construction of Engrailed repressor domain fusions; (3) ectopic expression of given regulatory factors, from genetic expression constructs and from injected mRNAs; (4) blockade of the beta-catenin/Tcf pathway by introduction of mRNA encoding the intracellular domain of cadherin; and (5) blockade of the Notch signaling pathway by introduction of mRNA encoding the extracellular domain of the Notch receptor. The network model predicts the cis-regulatory inputs that link each gene into the network. Therefore, its architecture is testable by cis-regulatory analysis. Strongylocentrotus purpuratus and Lytechinus variegatus genomic BAC recombinants that include a large number of the genes in the network have been sequenced and annotated. Tests of the cis-regulatory predictions of the model are greatly facilitated by interspecific computational sequence comparison, which affords a rapid identification of likely cis-regulatory elements in advance of experimental analysis. The network specifies genomically encoded regulatory processes between early cleavage and gastrula stages. These control the specification of the micromere lineage and of the initial veg(2) endomesodermal domain; the blastula-stage separation of the central veg(2) mesodermal domain (i.e., the secondary mesenchyme progenitor field) from the peripheral veg(2) endodermal domain; the stabilization of specification state within these domains; and activation of some downstream differentiation genes. Each of the temporal-spatial phases of specification is represented in a subelement of the network model, that treats regulatory events within the relevant embryonic nuclei at particular stages.

A genomic regulatory network for development.

Development of the body plan is controlled by large networks of regulatory genes. A gene regulatory network that controls the specification of endoderm and mesoderm in the sea urchin embryo is summarized here. The network was derived from large-scale perturbation analyses, in combination with computational methodologies, genomic data, cis-regulatory analysis, and molecular embryology. The network contains over 40 genes at present, and each node can be directly verified at the DNA sequence level by cis-regulatory analysis. Its architecture reveals specific and general aspects of development, such as how given cells generate their ordained fates in the embryo and why the process moves inexorably forward in developmental time.