Netrin 1 directs vascular patterning and maturity in the developing kidney.

The intricate vascular system of the kidneys supports body fluid and organ homeostasis. However, little is known about how vascular architecture is established during kidney development. More specifically, how signals from the kidney influence vessel maturity and patterning remains poorly understood. Netrin 1 (Ntn1) is a secreted ligand that is crucial for vessel and neuronal guidance. Here, we demonstrate that Ntn1 is expressed by Foxd1+ stromal progenitors in the developing mouse kidney and conditional deletion (Foxd1GC/+;Ntn1fl/fl) results in hypoplastic kidneys with extended nephrogenesis. Wholemount 3D analyses additionally revealed the loss of a predictable vascular pattern in Foxd1GC/+;Ntn1fl/fl kidneys. As vascular patterning has been linked to vessel maturity, we investigated arterialization. Quantification of the CD31+ endothelium at E15.5 revealed no differences in metrics such as the number of branches or branch points, whereas the arterial vascular smooth muscle metrics were significantly reduced at both E15.5 and P0. In support of our observed phenotypes, whole kidney RNA-seq revealed disruptions to genes and programs associated with stromal cells, vasculature and differentiating nephrons. Together, our findings highlight the significance of Ntn1 to proper vascularization and kidney development.

Nephron progenitor cell commitment: Striking the right balance.

The filtering component of the kidney, the nephron, arises from a single progenitor population. These nephron progenitor cells (NPCs) both self-renew and differentiate throughout the course of kidney development ensuring sufficient nephron endowment. An appropriate balance of these processes must be struck as deficiencies in nephron numbers are associated with hypertension and kidney disease. This review will discuss the mechanisms and molecules supporting NPC maintenance and differentiation. A focus on recent work will highlight new molecular insights into NPC regulation and their dynamic behavior in both space and time.

Transcriptional regulatory control of mammalian nephron progenitors revealed by multi-factor cistromic analysis and genetic studies.

Nephron progenitor number determines nephron endowment; a reduced nephron count is linked to the onset of kidney disease. Several transcriptional regulators including Six2, Wt1, Osr1, Sall1, Eya1, Pax2, and Hox11 paralogues are required for specification and/or maintenance of nephron progenitors. However, little is known about the regulatory intersection of these players. Here, we have mapped nephron progenitor-specific transcriptional networks of Six2, Hoxd11, Osr1, and Wt1. We identified 373 multi-factor associated 'regulatory hotspots' around genes closely associated with progenitor programs. To examine their functional significance, we deleted 'hotspot' enhancer elements for Six2 and Wnt4. Removal of the distal enhancer for Six2 leads to a ~40% reduction in Six2 expression. When combined with a Six2 null allele, progeny display a premature depletion of nephron progenitors. Loss of the Wnt4 enhancer led to a significant reduction of Wnt4 expression in renal vesicles and a mildly hypoplastic kidney, a phenotype also enhanced in combination with a Wnt4 null mutation. To explore the regulatory landscape that supports proper target gene expression, we performed CTCF ChIP-seq to identify insulator-boundary regions. One such putative boundary lies between the Six2 and Six3 loci. Evidence for the functional significance of this boundary was obtained by deep sequencing of the radiation-induced Brachyrrhine (Br) mutant allele. We identified an inversion of the Six2/Six3 locus around the CTCF-bound boundary, removing Six2 from its distal enhancer regulation, but placed next to Six3 enhancer elements which support ectopic Six2 expression in the lens where Six3 is normally expressed. Six3 is now predicted to fall under control of the Six2 distal enhancer. Consistent with this view, we observed ectopic Six3 in nephron progenitors. 4C-seq supports the model for Six2 distal enhancer interactions in wild-type and Br/+ mouse kidneys. Together, these data expand our view of the regulatory genome and regulatory landscape underpinning mammalian nephrogenesis.

Differential regulation of mouse and human nephron progenitors by the Six family of transcriptional regulators.

Nephron endowment is determined by the self-renewal and induction of a nephron progenitor pool established at the onset of kidney development. In the mouse, the related transcriptional regulators Six1 and Six2 play non-overlapping roles in nephron progenitors. Transient Six1 activity prefigures, and is essential for, active nephrogenesis. By contrast, Six2 maintains later progenitor self-renewal from the onset of nephrogenesis. We compared the regulatory actions of Six2 in mouse and human nephron progenitors by chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq). Surprisingly, SIX1 was identified as a SIX2 target unique to the human nephron progenitors. Furthermore, RNA-seq and immunostaining revealed overlapping SIX1 and SIX2 activity in 16 week human fetal nephron progenitors. Comparative bioinformatic analysis of human SIX1 and SIX2 ChIP-seq showed each factor targeted a similar set of cis-regulatory modules binding an identical target recognition motif. In contrast to the mouse where Six2 binds its own enhancers but does not interact with DNA around Six1, both human SIX1 and SIX2 bind homologous SIX2 enhancers and putative enhancers positioned around SIX1. Transgenic analysis of a putative human SIX1 enhancer in the mouse revealed a transient, mouse-like, pre-nephrogenic, Six1 regulatory pattern. Together, these data demonstrate a divergence in SIX-factor regulation between mouse and human nephron progenitors. In the human, an auto/cross-regulatory loop drives continued SIX1 and SIX2 expression during active nephrogenesis. By contrast, the mouse establishes only an auto-regulatory Six2 loop. These data suggest differential SIX-factor regulation might have contributed to species differences in nephron progenitor programs such as the duration of nephrogenesis and the final nephron count.

Induction and patterning of the metanephric nephron.

The functional unit of the mammalian metanephric kidney is the nephron: a complex tubular structure dedicated to blood filtration and maintenance of several important physiological functions. Nephrons are assembled from a nephron-restricted pool of mesenchymal progenitors over an extensive developmental period that is completed prior to (human), or shortly after (mouse), birth. An appropriate balance in the expansion and commitment of nephron progenitors to nephron formation is essential for normal kidney function. Too few nephrons increase risk of kidney disease later in life while the failure of normal progenitor differentiation in Wilm's tumor patients leads to massive growth of a nephroblast population often necessitating surgical removal of the kidney. An inductive process within the metanephric mesenchyme leads to the formation of a pretubular aggregate which transitions into an epithelial renal vesicle: the precursor for nephron assembly. Growth, morphogenesis and patterning transform this simple cyst-like structure into a highly elongated mature nephron with distinct cell types positioned along a proximal (glomerular) to distal (connecting segment) axis of functional organization. This review discusses our current understanding of the specification, maintenance and commitment of nephron progenitors, and the regulatory processes that transform the renal vesicle into a nephron.

Comprehensive mapping of sensory and sympathetic innervation of the developing kidney.

The kidney functions as a finely tuned sensor to balance body fluid composition and filter out waste through complex coordinated mechanisms. This versatility requires tight neural control, with innervating efferent nerves playing a crucial role in regulating blood flow, glomerular filtration rate, water and sodium reabsorption, and renin release. In turn sensory afferents provide feedback to the central nervous system for the modulation of cardiovascular function. However, the cells targeted by sensory afferents and the physiological sensing mechanisms remain poorly characterized. Moreover, how the kidney is innervated during development to establish these functions remains elusive. Here, we utilized a combination of light-sheet and confocal microscopy to generate anatomical maps of kidney sensory and sympathetic nerves throughout development and resolve the establishment of functional crosstalk. Our analyses revealed that kidney innervation initiates at embryonic day (E)13.5 as the nerves associate with vascular smooth muscle cells and follow arterial differentiation. By E17.5 axonal projections associate with kidney structures such as glomeruli and tubules and the network continues to expand postnatally. These nerves are synapsin I-positive, highlighting ongoing axonogenesis and the potential for functional crosstalk. We show that sensory and sympathetic nerves innervate the kidney concomitantly and classify the sensory fibers as calcitonin gene related peptide (CGRP)+, substance P+, TRPV1+, and PIEZO2+, establishing the presence of PIEZO2 mechanosensory fibers in the kidney. Using retrograde tracing, we identified the primary dorsal root ganglia, T10-L2, from which PIEZO2+ sensory afferents project to the kidney. Taken together our findings elucidate the temporality of kidney innervation and resolve the identity of kidney sympathetic and sensory nerves.

Progenitor programming in mammalian nephrogenesis.

Nephrogenesis is dependent on the input of several transcriptional regulatory networks. However, the details of how these networks operate and converge to facilitate nephron progenitor specific programmes are largely unknown. To this end, recent studies have focused on identifying the precise regulatory mechanisms that modulate progenitor maintenance and induction. Continued focus on this area of research will help identify nephrogenic programmes which could be manipulated for therapeutic intervention of kidney disease.

Mapping of the podocin proximity-dependent proteome reveals novel components of the kidney podocyte foot process.

Introduction: The unique architecture of glomerular podocytes is integral to kidney filtration. Interdigitating foot processes extend from the podocyte cell body, wrap around fenestrated capillaries, and form specialized junctional complexes termed slit diaphragms to create a molecular sieve. However, the full complement of proteins which maintain foot process integrity, and how this localized proteome changes with disease, remain to be elucidated. Methods: Proximity-dependent biotin identification (BioID) enables the identification of spatially localized proteomes. To this end, we developed a novel in vivo BioID knock-in mouse model. We utilized the slit diaphragm protein podocin (Nphs2) to create a podocin-BioID fusion. Podocin-BioID localizes to the slit diaphragm, and biotin injection leads to podocyte-specific protein biotinylation. We isolated the biotinylated proteins and performed mass spectrometry to identify proximal interactors. Results and Discussion: Gene ontology analysis of 54 proteins specifically enriched in our podocin-BioID sample revealed 'cell junctions,' 'actin binding,' and 'cytoskeleton organization' as top terms. Known foot process components were identified, and we further uncovered two novel proteins: the tricellular junctional protein Ildr2 and the CDC42 and N-WASP interactor Fnbp1l. We confirmed that Ildr2 and Fnbp1l are expressed by podocytes and partially colocalize with podocin. Finally, we investigated how this proteome changes with age and uncovered a significant increase in Ildr2. This was confirmed by immunofluorescence on human kidney samples and suggests altered junctional composition may preserve podocyte integrity. Together, these assays have led to new insights into podocyte biology and support the efficacy of utilizing BioID in vivo to interrogate spatially localized proteomes in health, aging, and disease.

Altered binding affinity of SIX1-Q177R correlates with enhanced WNT5A and WNT pathway effector expression in Wilms tumor.

Wilms tumors present as an amalgam of varying proportions of tissues located within the developing kidney, one being the nephrogenic blastema comprising multipotent nephron progenitor cells (NPCs). The recurring missense mutation Q177R in NPC transcription factors SIX1 and SIX2 is most correlated with tumors of blastemal histology and is significantly associated with relapse. Yet, the transcriptional regulatory consequences of SIX1/2-Q177R that might promote tumor progression and recurrence have not been investigated extensively. Utilizing multiple Wilms tumor transcriptomic datasets, we identified upregulation of the gene encoding non-canonical WNT ligand WNT5A in addition to other WNT pathway effectors in SIX1/2-Q177R mutant tumors. SIX1 ChIP-seq datasets from Wilms tumors revealed shared binding sites for SIX1/SIX1-Q177R within a promoter of WNT5A and at putative distal cis-regulatory elements (CREs). We demonstrate colocalization of SIX1 and WNT5A in Wilms tumor tissue and utilize in vitro assays that support SIX1 and SIX1-Q177R activation of expression from the WNT5A CREs, as well as enhanced binding affinity within the WNT5A promoter that may promote the differential expression of WNT5A and other WNT pathway effectors associated with SIX1-Q177R tumors.

Wt1a, Foxc1a, and the Notch mediator Rbpj physically interact and regulate the formation of podocytes in zebrafish.

Podocytes help form the glomerular blood filtration barrier in the kidney and their injury or loss leads to renal disease. The Wilms' tumor suppressor-1 (Wt1) and the FoxC1/2 transcription factors, as well as Notch signaling, have been implicated as important regulators of podocyte fate. It is not known whether these factors work in parallel or sequentially on different gene targets, or as higher-order transcriptional complexes on common genes. Here, we use the zebrafish to demonstrate that embryos treated with morpholinos against wt1a, foxc1a, or the Notch transcriptional mediator rbpj develop fewer podocytes, as determined by wt1b, hey1 and nephrin expression, while embryos deficient in any two of these factors completely lack podocytes. From GST-pull-downs and co-immunoprecipitation experiments we show that Wt1a, Foxc1a, and Rbpj can physically interact with each other, whereas only Rbpj binds to the Notch intracellular domain (NICD). In transactivation assays, combinations of Wt1, FoxC1/2, and NICD synergistically induce the Hey1 promoter, and have additive or repressive effects on the Podocalyxin promoter, depending on dosage. Taken together, these data suggest that Wt1, FoxC1/2, and Notch signaling converge on common target genes where they physically interact to regulate a podocyte-specific gene program. These findings further our understanding of the transcriptional circuitry responsible for podocyte formation and differentiation during kidney development.

Innervation in organogenesis.

Proper innervation of peripheral organs helps to maintain physiological homeostasis and elicit responses to external stimuli. Disruptions to normal function can result in pathophysiological consequences. The establishment of connections and communication between the central nervous system and the peripheral organs is accomplished through the peripheral nervous system. Neuronal connections with target tissues arise from ganglia partitioned throughout the body. Organ innervation is initiated during development with stimuli being conducted through several types of neurons including sympathetic, parasympathetic, and sensory. While the physiological modulation of mature organs by these nerves is largely understood, their role in mammalian development is only beginning to be uncovered. Interactions with cells in target tissues can affect the development and eventual function of several organs, highlighting their significance. This chapter will cover the origin of peripheral neurons, factors mediating organ innervation, and the composition and function of organ-specific nerves during development. This emerging field aims to identify the functional contribution of innervation to development which will inform future investigations of normal and abnormal mammalian organogenesis, as well as contribute to regenerative and organ replacement efforts where nerve-derived signals may have significant implications for the advancement of such studies.

Sex differences in resilience to ferroptosis underlie sexual dimorphism in kidney injury and repair.

In both humans and mice, repair of acute kidney injury is worse in males than in females. Here, we provide evidence that this sexual dimorphism results from sex differences in ferroptosis, an iron-dependent, lipid-peroxidation-driven regulated cell death. Using genetic and single-cell transcriptomic approaches in mice, we report that female sex confers striking protection against ferroptosis, which was experimentally induced in proximal tubular (PT) cells by deleting glutathione peroxidase 4 (Gpx4). Single-cell transcriptomic analyses further identify the NFE2-related factor 2 (NRF2) antioxidant protective pathway as a female resilience mechanism against ferroptosis. Genetic inhibition and pharmacological activation studies show that NRF2 controls PT cell fate and plasticity by regulating ferroptosis. Importantly, pharmacological NRF2 activation protects male PT cells from ferroptosis and improves cellular plasticity as in females. Our data highlight NRF2 as a potential therapeutic target to prevent failed renal repair after acute kidney injury in both sexes by modulating cellular plasticity.

What's so Bor(a)ing about Aurora-A activation?

Aurora-A kinases are highly conserved mitotic kinases required for cell division. The regulation of Aurora-A activity is less highly conserved and currently poorly understood. Work by Knoblich and coworkers in this issue of Developmental Cell identifies the conserved protein, Aurora Borealis (Bora), as a key regulator of Aurora-A activity during mitosis.

TPX2 is required for postmitotic nuclear assembly in cell-free Xenopus laevis egg extracts.

Cell division in many metazoa is accompanied by the disassembly of the nuclear envelope and the assembly of the mitotic spindle. These dramatic structural rearrangements are reversed after mitosis, when the mitotic spindle is dismantled and the nuclear envelope reassembles. The targeting protein for XKlp2 (TPX2) plays important roles in mitotic spindle assembly. We report that TPX2 depletion from nuclear assembly extracts prepared from Xenopus laevis eggs results in the formation of nuclei that are only about one fifth the size of control nuclei. TPX2-depleted nuclei assemble nuclear envelopes, nuclear pore complexes, and a lamina, and they perform nuclear-specific functions, including DNA replication. We show that TPX2 interacts with lamina-associated polypeptide 2 (LAP2), a protein known to be required for nuclear assembly in interphase extracts and in vitro. LAP2 localization is disrupted in TPX2-depleted nuclei, suggesting that the interaction between TPX2 and LAP2 is required for postmitotic nuclear reformation.

Injection of Evans blue dye to fluorescently label and image intact vasculature.

Blood vessels perform critical functions in both health and disease. Understanding how vessels form, pattern and respond to damage is essential. However, labeling and imaging the vasculature to ascertain these properties can be difficult and time-consuming. Here, the authors present a novel methodology for rapidly and efficiently labeling whole vascular networks in vivo by exploiting the fluorescent properties of Evans blue. By combining the labeling with fluorescence microscopy, this method enables visualization of whole tissue vasculature for a fraction of the time and cost compared with traditional methods.

Ferroptotic stress promotes the accumulation of pro-inflammatory proximal tubular cells in maladaptive renal repair.

Overwhelming lipid peroxidation induces ferroptotic stress and ferroptosis, a non-apoptotic form of regulated cell death that has been implicated in maladaptive renal repair in mice and humans. Using single-cell transcriptomic and mouse genetic approaches, we show that proximal tubular (PT) cells develop a molecularly distinct, pro-inflammatory state following injury. While these inflammatory PT cells transiently appear after mild injury and return to their original state without inducing fibrosis, after severe injury they accumulate and contribute to persistent inflammation. This transient inflammatory PT state significantly downregulates glutathione metabolism genes, making the cells vulnerable to ferroptotic stress. Genetic induction of high ferroptotic stress in these cells after mild injury leads to the accumulation of the inflammatory PT cells, enhancing inflammation and fibrosis. Our study broadens the roles of ferroptotic stress from being a trigger of regulated cell death to include the promotion and accumulation of proinflammatory cells that underlie maladaptive repair.

The Xenopus TACC homologue, maskin, functions in mitotic spindle assembly.

Maskin is the Xenopus homolog of the transforming acidic coiled coil (TACC)-family of microtubule and centrosome-interacting proteins. Members of this family share a approximately 200 amino acid coiled coil motif at their C-termini, but have only limited homology outside of this domain. In all species examined thus far, perturbations of TACC proteins lead to disruptions of cell cycle progression and/or embryonic lethality. In Drosophila, Caenorhabditis elegans, and humans, these disruptions have been attributed to mitotic spindle assembly defects, and the TACC proteins in these organisms are thought to function as structural components of the spindle. In contrast, cell division failure in early Xenopus embryo blastomeres has been attributed to a role of maskin in regulating the translation of, among others, cyclin B1 mRNA. In this study, we show that maskin, like other TACC proteins, plays a direct role in mitotic spindle assembly in Xenopus egg extracts and that this role is independent of cyclin B. Maskin immunodepletion and add-back experiments demonstrate that maskin, or a maskin-associated activity, is required for two distinct steps during spindle assembly in Xenopus egg extracts that can be distinguished by their response to "rescue" experiments. Defects in the "early" step, manifested by greatly reduced aster size during early time points in maskin-depleted extracts, can be rescued by readdition of purified full-length maskin. Moreover, defects in this step can also be rescued by addition of only the TACC-domain of maskin. In contrast, defects in the "late" step during spindle assembly, manifested by abnormal spindles at later time points, cannot be rescued by readdition of maskin. We show that maskin interacts with a number of proteins in egg extracts, including XMAP215, a known modulator of microtubule dynamics, and CPEB, a protein that is involved in translational regulation of important cell cycle regulators. Maskin depletion from egg extracts results in compromised microtubule asters and spindles and the mislocalization of XMAP215, but CPEB localization is unaffected. Together, these data suggest that in addition to its previously reported role as a translational regulator, maskin is also important for mitotic spindle assembly.

Wnt11 directs nephron progenitor polarity and motile behavior ultimately determining nephron endowment.

A normal endowment of nephrons in the mammalian kidney requires a balance of nephron progenitor self-renewal and differentiation throughout development. Here, we provide evidence for a novel action of ureteric branch tip-derived Wnt11 in progenitor cell organization and interactions within the nephrogenic niche, ultimately determining nephron endowment. In Wnt11 mutants, nephron progenitors dispersed from their restricted niche, intermixing with interstitial progenitors. Nephron progenitor differentiation was accelerated, kidneys were significantly smaller, and the nephron progenitor pool was prematurely exhausted, halving the final nephron count. Interestingly, RNA-seq revealed no significant differences in gene expression. Live imaging of nephron progenitors showed that in the absence of Wnt11 they lose stable attachments to the ureteric branch tips, continuously detaching and reattaching. Further, the polarized distribution of several markers within nephron progenitors is disrupted. Together these data highlight the importance of Wnt11 signaling in directing nephron progenitor behavior which determines a normal nephrogenic program.

Six2 and Wnt regulate self-renewal and commitment of nephron progenitors through shared gene regulatory networks.

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 ß-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 ß-catenin into this complex promotes nephrogenesis. Alternative transcriptional responses associated with Six2 and ß-catenin cobinding events occur through non-Lef/Tcf DNA binding mechanisms, highlighting the regulatory complexity downstream of Wnt signaling in the developing mammalian kidney.

Myosin-10 and actin filaments are essential for mitotic spindle function.

Mitotic spindles are microtubule-based structures responsible for chromosome partitioning during cell division. Although the roles of microtubules and microtubule-based motors in mitotic spindles are well established, whether or not actin filaments (F-actin) and F-actin-based motors (myosins) are required components of mitotic spindles has long been controversial. Based on the demonstration that myosin-10 (Myo10) is important for assembly of meiotic spindles, we assessed the role of this unconventional myosin, as well as F-actin, in mitotic spindles. We find that Myo10 localizes to mitotic spindle poles and is essential for proper spindle anchoring, normal spindle length, spindle pole integrity, and progression through metaphase. Furthermore, we show that F-actin localizes to mitotic spindles in dynamic cables that surround the spindle and extend between the spindle and the cortex. Remarkably, although proper anchoring depends on both F-actin and Myo10, the requirement for Myo10 in spindle pole integrity is F-actin independent, whereas F-actin and Myo10 actually play antagonistic roles in maintenance of spindle length.