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Background: Low nephron number has a direct impact on the development of hypertension and chronic kidney disease later in life. While intrauterine growth restriction caused by maternal low protein diet (LPD) is thought to be a significant cause of reduced nephron endowment in impoverished communities, its influence on the cellular and molecular processes which drive nephron formation are poorly understood., Methods: We conducted a comprehensive characterization of the impact of LPD on kidney development using tomographic and confocal imaging to quantify changes in branching morphogenesis and the cellular and morphological features of nephrogenic niches across development. These analyses were paired with single-cell RNA sequencing to dissect the transcriptional changes that LPD imposes during renal development to affect nephron number., Results: Single cell analysis at E14.5 and P0 revealed differences in the expression of genes and pathways involved in metabolism, cell cycle, epigenetic regulators and reciprocal inductive signals in most cell types analyzed, yielding imbalances and shifts in cellular energy production and cellular trajectories. In the nephron progenitor cells, LPD impeded cellular commitment and differentiation towards pre-tubular and renal vesicle structures. Confocal microscopy revealed a reduction in the number of pre-tubular aggregates and proliferation in nephron progenitor cells. We also found changes in branching morphogenesis, with a reduction in cell proliferation in the ureteric tips as well as reduced tip and tip parent lengths by optical projection tomography which causes patterning defects., Conclusions: This unique profiling demonstrates how a fetal programming defect leads to low nephron endowment which is intricately linked to changes in both branching morphogenesis and the commitment of nephron progenitor cells. The commitment of progenitor cells is pivotal for nephron formation and is significantly influenced by nutritional factors, with a low protein diet driving alterations in this program which directly results in a reduced nephron endowment., Significance Statement: While a mother's diet can negatively impact the number of nephrons in the kidneys of her offspring, the root cellular and molecular drivers of these deficits have not been rigorously explored. In this study we use advanced imaging and gene expression analysis in mouse models to define how a maternal low protein diet, analogous to that of impoverished communities, results in reduced nephron endowment. We find that low protein diet has pleiotropic effects on metabolism and the normal developmental programs of gene expression. These profoundly impact the process of branching morphogenesis necessary to establish niches for nephron generation and change cell behaviors which regulate how and when nephron progenitor cells commit to differentiation.

A mechanistic connection between aging and development is largely unexplored. Through profiling age-related chromatin and transcriptional changes across 22 murine cell types, analyzed alongside previous mouse and human organismal maturation datasets, we uncovered a transcription factor binding site (TFBS) signature common to both processes. Early-life candidate cis-regulatory elements (cCREs), progressively losing accessibility during maturation and aging, are enriched for cell-type identity TFBSs. Conversely, cCREs gaining accessibility throughout life have a lower abundance of cell identity TFBSs but elevated activator protein 1 (AP-1) levels. We implicate TF redistribution toward these AP-1 TFBS-rich cCREs, in synergy with mild downregulation of cell identity TFs, as driving early-life cCRE accessibility loss and altering developmental and metabolic gene expression. Such remodeling can be triggered by elevating AP-1 or depleting repressive H3K27me3. We propose that AP-1-linked chromatin opening drives organismal maturation by disrupting cell identity TFBS-rich cCREs, thereby reprogramming transcriptome and cell function, a mechanism hijacked in aging through ongoing chromatin opening., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a leading cause of kidney failure and is associated with substantial morbidity and mortality. Interstitial inflammation is attributed to the action of infiltrating macrophages and is a feature thought to aggravate disease progression. Here, we investigated the therapeutic potential of the anti-inflammatory IL37b cytokine as a treatment for ADPKD using genetic mouse models, demonstrating that transgenic expression of human IL37b reduced collecting duct cyst burden in both early and adult-onset ADPKD rodent models. Moreover, injection of recombinant human IL37b could also reduce cyst burden in early onset ADPKD mice, an observation not associated with increased macrophage number at early stages of cyst formation. Interestingly, transgenic IL37b expression also did not alter macrophage numbers in advanced disease. Whole kidney RNA-seq highlighted an IL37b-mediated upregulation of the interferon signaling pathway and single-cell RNA-seq established that these changes originate at least partly from kidney resident macrophages. We further found that blocking type I interferon signaling in mice expressing IL37b resulted in increased cyst number, confirming this as an important pathway by which IL37b exerts its beneficial effects. Thus, our studies show that IL37b promotes interferon signaling in kidney resident macrophages which suppresses cyst initiation, identifying this protein as a potential therapy for ADPKD., (Copyright © 2024 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Aurora Kinase A (AURKA) promotes cell proliferation and is overexpressed in different types of polycystic kidney disease (PKD). To understand AURKA's role in regulating renal cyst development we conditionally deleted the gene in mouse models of Autosomal Dominant PKD (ADPKD) and Joubert Syndrome, caused by Polycystin 1 (Pkd1) and Inositol polyphosphate-5-phosphatase E (Inpp5e) mutations respectively. We show that while Aurka is dispensable for collecting duct development and homeostasis, its deletion prevents cyst formation in both disease models. Cross-comparison of transcriptional changes implicated AKT signaling in cyst prevention and we show that (i) AURKA and AKT physically interact, (ii) AURKA regulates AKT activity in a kinase-independent manner and (iii) inhibition of AKT can reduce disease severity. AKT activation also regulates Aurka expression, creating a feed-forward loop driving renal cystogenesis. We find that the AURKA kinase inhibitor Alisertib stabilises the AURKA protein, agonizing its cystogenic functions. These studies identify AURKA as a master regulator of renal cyst development in different types of PKD, functioning in-part via AKT., (© 2024. The Author(s).)

Crosstalk between cardiac cells is critical for heart performance. Here we show that vascular cells within human cardiac organoids (hCOs) enhance their maturation, force of contraction, and utility in disease modeling. Herein we optimize our protocol to generate vascular populations in addition to epicardial, fibroblast, and cardiomyocyte cells that self-organize into in-vivo-like structures in hCOs. We identify mechanisms of communication between endothelial cells, pericytes, fibroblasts, and cardiomyocytes that ultimately contribute to cardiac organoid maturation. In particular, (1) endothelial-derived LAMA5 regulates expression of mature sarcomeric proteins and contractility, and (2) paracrine platelet-derived growth factor receptor β (PDGFRβ) signaling from vascular cells upregulates matrix deposition to augment hCO contractile force. Finally, we demonstrate that vascular cells determine the magnitude of diastolic dysfunction caused by inflammatory factors and identify a paracrine role of endothelin driving dysfunction. Together this study highlights the importance and role of vascular cells in organoid models., Competing Interests: Declaration of interests R.J.M., J.E.H., G.A.Q.-R., and E.R.P. are listed as co-inventors on pending patents that relate to cardiac organoid maturation and cardiac regeneration therapeutics. E.R.P. and J.E.H. are listed as co-inventors on pending patents that relate to endothelial cells in 3D cardiac products. J.E.H. is a co-inventor on licensed patents relating to engineered heart muscle. R.J.M., E.R.P., and J.E.H. are co-founders, scientific advisors, and stockholders in Dynomics., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The kidney plays an integral role in filtering the blood-removing metabolic by-products from the body and regulating blood pressure. This requires the establishment of large numbers of efficient and specialized blood filtering units (nephrons) that incorporate a system for vascular exchange and nutrient reabsorption as well as a collecting duct system to remove waste (urine) from the body. Kidney development is a dynamic process which generates these structures through a delicately balanced program of self-renewal and commitment of nephron progenitor cells that inhabit a constantly evolving cellular niche at the tips of a branching ureteric "tree." The former cells build the nephrons and the latter the collecting duct system. Maintaining these processes across fetal development is critical for establishing the normal "endowment" of nephrons in the kidney and perturbations to this process are associated both with mutations in integral genes and with alterations to the fetal environment., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Studies of gene-targeted mice identified the roles of the different pro-survival BCL-2 proteins during embryogenesis. However, little is known about the role(s) of these proteins in adults in response to cytotoxic stresses, such as treatment with anti-cancer agents. We investigated the role of BCL-XL in adult mice using a strategy where prior bone marrow transplantation allowed for loss of BCL-XL exclusively in non-hematopoietic tissues to prevent anemia caused by BCL-XL deficiency in erythroid cells. Unexpectedly, the combination of total body γ-irradiation (TBI) and genetic loss of Bcl-x caused secondary anemia resulting from chronic renal failure due to apoptosis of renal tubular epithelium with secondary obstructive nephropathy. These findings identify a critical protective role of BCL-XL in the adult kidney and inform on the use of BCL-XL inhibitors in combination with DNA damage-inducing drugs for cancer therapy. Encouragingly, the combination of DNA damage-inducing anti-cancer therapy plus a BCL-XL inhibitor could be tolerated in mice, at least when applied sequentially., (© 2020 The Authors.)

Mutations in the lipid transport protein ABCA12 cause the life-threatening skin condition harlequin ichthyosis (HI), which is characterized by the loss of skin barrier function, inflammation, and dehydration. Inflammatory responses in HI increase disease severity by impairing keratinocyte differentiation, suggesting amelioration of this phenotype as a possible therapy for the condition. Existing treatments for HI are based around the use of retinoids, but their value in treating patients during the neonatal period has been questioned relative to other improved management regimens, and their long-term use is associated with side effects. We have developed a conditional mouse model to demonstrate that topical application of the aminosalicylic acid derivatives 5ASA or 4ASA considerably improves HI keratinocyte differentiation without the undesirable side effects of the retinoid acitretin and salicylic acid (aspirin). Analysis of changes in gene expression shows that 4ASA in particular elicits compensatory upregulation of a large family of barrier function-related genes, many of which are associated with other ichthyoses, identifying this compound as a lead candidate for developing topical treatments for HI., Competing Interests: D.L.C., G.M.A.U., and I.M.S. are inventors on patent applications WO 2016/145488 A1, PCT/AU2016/050185, US 15/557405, India 201727036282, Europe 16764064.8, Australia 2016232987, Canada 2984949, Japan 2017-549008, and China 201680028353.X, related to ASA treatment of ichthyosis skin diseases and received funding from Factor Therapeutics under a research collaboration and option agreement., (© 2020 The Authors.)

Branching morphogenesis is an integral developmental mechanism central to the formation of a range of organs including the kidney, lung, pancreas and mammary gland. The ramified networks of epithelial tubules it establishes are critical for the processes of secretion, excretion and exchange mediated by these tissues. In the kidney, branching serves to establish the collecting duct system that transports urine from the nephrons into the renal pelvis, ureter and finally the bladder. Generally speaking, the formation of these networks in different organs begins with the specification and differentiation of simple bud-like organ anlage, which then undergo a process of elaboration, typically by bifurcation. This process is often governed by the interaction of progenitor cells at the tips of the epithelia with neighboring mesenchymal cell populations which direct the branching process and which often themselves differentiate to form part of the adult organ. In the kidney, the tips of ureteric bud elaborate through a dynamic cell signaling relationship with overlying nephron progenitor cell populations. These cells sequentially commit to differentiation and the resulting nephrons reintegrate with the ureteric epithelium as development progresses. This review will describe recent advances in understanding the how the elaboration of the ureteric bud is patterned and consider the extent to which this process is shared with other organs., (© 2020 American Association for Anatomy.)

Laminin alpha 5 (LAMA5) is a member of a large family of proteins that trimerise and then polymerise to form a central component of all basement membranes. Consequently, the protein plays an instrumental role in shaping the normal development of the kidney, skin, neural tube, lung and limb, and many other organs and tissues. Pathogenic mutations in some laminins have been shown to cause a range of largely syndromic conditions affecting the competency of the basement membranes to which they contribute. We report the identification of a mutation in the polymerisation domain of LAMA5 in a patient with a complex syndromic disease characterised by defects in kidney, craniofacial and limb development, and by a range of other congenital defects. Using CRISPR-generated mouse models and biochemical assays, we demonstrate the pathogenicity of this variant, showing that the change results in a failure of the polymerisation of α/β/γ laminin trimers. Comparing these in vivo phenotypes with those apparent upon gene deletion in mice provides insights into the specific functional importance of laminin polymerisation during development and tissue homeostasis., Competing Interests: Competing interestsA.J.M. has received research grant funding from Sanofi-Genzyme and has membership of an Advisory Board for Otsuka, neither of which are related to this project or work., (© 2020. Published by The Company of Biologists Ltd.)

Dysregulation of lipid homeostasis is intimately associated with defects in insulin secretion, a key feature of type 2 diabetes. Here, we explore the role of the putative lipid transporter ABCA12 in regulating insulin secretion from β-cells. Mice with β-cell-specific deletion of Abca12 display impaired glucose-stimulated insulin secretion and eventual islet inflammation and β-cell death. ABCA12's action in the pancreas is independent of changes in the abundance of two other cholesterol transporters, ABCA1 and ABCG1, or of changes in cellular cholesterol or ceramide content. Instead, loss of ABCA12 results in defects in the genesis and fusion of insulin secretory granules and increases in the abundance of lipid rafts at the cell membrane. These changes are associated with dysregulation of the small GTPase CDC42 and with decreased actin polymerisation. Our findings establish a new, pleiotropic role for ABCA12 in regulating pancreatic lipid homeostasis and insulin secretion., (© 2020 The Authors.)

Polycystic kidney disease (PKD) results in the formation of renal cysts that can impair function leading to renal failure. DNA damage accumulates in renal epithelial cells in PKD, but the molecular mechanisms are unclear and are investigated here. Phosphoinositide 3-kinase (PI3K)/AKT signaling activates mammalian target of rapamycin complex 1 (mTORC1) and hyperactivation of mTORC1 is a common event in PKD; however, mTORC1 inhibitors have yielded disappointing results in clinical trials. Here, we demonstrate AKT and mTORC1 hyperactivation in two representative murine PKD models (renal epithelial-specific Inpp5e knockout and collecting duct-specific Pkd1 deletion) and identify a downstream signaling network that contributes to DNA damage accumulation. Inpp5e- and Pkd1-null renal epithelial cells showed DNA damage including double-stranded DNA breaks associated with increased replication fork numbers, multinucleation and centrosome amplification. mTORC1 activated CAD, which promotes de novo pyrimidine synthesis, to sustain cell proliferation. AKT, but not mTORC1, inhibited the DNA repair/replication fork origin firing regulator TOPBP1, which impacts on DNA damage and cell proliferation. Notably, Inpp5e- and Pkd1-null renal epithelial cell spheroid formation defects were rescued by AKT inhibition. These data reveal that AKT hyperactivation contributes to DNA damage accumulation in multiple forms of PKD and cooperates with mTORC1 to promote cell proliferation. Hyperactivation of AKT may play a causal role in PKD by regulating DNA damage and cell proliferation, independent of mTORC1, and AKT inhibition may be a novel therapeutic approach for PKD., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

Ciliated bronchial epithelium 1 (CBE1) is a microtubule-associated protein localized to the manchette and developing flagellum during spermiogenesis and is associated with sperm maturation arrest in humans. It was hypothesized that CBE1 functions in microtubule-mediated transport mechanisms and sperm tail formation. To test this hypothesis, we analyzed Cbe1 expression and localization during spermiogenesis, and in mouse inner medullary collecting duct-3 (IMCD3) cells as a model of ciliogenesis. Furthermore, we generated and analyzed the fertility of a Cbe1 mutant mouse line. Mice containing a homozygous deletion in the long forms of Cbe1 were born at a lower frequency than predicted by Mendelian inheritance; however, adult male mice were fertile. An in-depth analysis of the Cbe1 gene revealed alternative transcript variants, which were not affected by the exon 2 mutation. To assess whether short variants compensate for the loss of long variants, exons 2 and 4 (which affect all variants) were individually mutated in IMCD3 cells and the effects on cell proliferation and ciliogenesis were analyzed. In wild-type IMCD3 cells, both variants were upregulated during cilia assembly. CBE1 protein was not a structural component of cilia; rather, CBE1 localized to the mitochondria and the contractile ring of dividing IMCD3 cells. Although IMCD3 cells carrying the mutation in long variants showed no phenotypic alterations, the mutation in exon 4 resulted in a significantly decreased proliferation rate. This study reveals that long isoforms of CBE1 are not essential for male fertility. Data, however, suggest that CBE1 is associated with intramanchette transport and midpiece formation of the sperm tail., (Copyright © 2019 Endocrine Society.)

Adamts18 encodes a secreted metalloprotease restricted to branch-tip progenitor pools directing the morphogenesis of multiple mammalian organs. Adamts18 was targeted to explore a potential role in branching morphogenesis. In the kidney, an arborized collecting system develops through extensive branching morphogenesis of an initial epithelial outgrowth of the mesonephric duct, the ureteric bud. Adamts18 mutants displayed a weakly penetrant phenotype: duplicated ureteric outgrowths forming enlarged, bi-lobed kidneys with an increased nephron endowment. In contrast, Adamts18 mutants showed a fully penetrant lung phenotype: epithelial growth was markedly reduced and early secondary branching scaled to the reduced length of the primary airways. Furthermore, there was a pronounced delay in the appearance of differentiated cell types in both proximal and distally positions of the developing airways. Adamts18 is closely related to Adamts16. In the kidney but not the lung, broad epithelial Adamts16 expression overlaps Adamts18 in branch tips. However, compound Adamts16/18 mutants displayed a comparable low penetrance duplicated ureteric phenotype, ruling out a possible role for Adamts16 as a functional modifier of the Adamts18 kidney phenotype. Given the predicted action of secreted Adamts18 metalloprotease, and broad expression of Adamts18 in branching organ systems, these findings suggest distinct requirements for matrix modelling in the morphogenesis of epithelial networks., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Mutations in subunits of the cilia-specific cytoplasmic dynein-2 (CD2) complex cause short-rib thoracic dystrophy syndromes (SRTDs), characterized by impaired bone growth and life-threatening perinatal respiratory complications. Different SRTD mutations result in varying disease severities. It remains unresolved whether this reflects the extent of retained hypomorphic protein functions or relative importance of the affected subunits for the activity of the CD2 holoenzyme. To define the contribution of the LC8-type dynein light chain subunit to the CD2 complex, we have generated Dynll1-deficient mouse strains, including the first-ever conditional knockout (KO) mutant for any CD2 subunit. Germline Dynll1 KO mice exhibit a severe ciliopathy-like phenotype similar to mice lacking another CD2 subunit, Dync2li1. Limb mesoderm-specific loss of Dynll1 results in severe bone shortening similar to human SRTD patients. Mechanistically, loss of Dynll1 leads to a partial depletion of other SRTD-related CD2 subunits, severely impaired retrograde intra-flagellar transport, significant thickening of primary cilia and cilia signaling defects. Interestingly, phenotypes of Dynll1-deficient mice are very similar to entirely cilia-deficient Kif3a/Ift88-null mice, except that they never present with polydactyly and retain relatively higher signaling outputs in parts of the hedgehog pathway. Compared to complete loss of Dynll1, maintaining very low DYNLL1 levels in mice lacking the Dynll1-transcription factor ASCIZ (ATMIN) results in significantly attenuated phenotypes and improved CD2 protein levels. The results suggest that primary cilia can maintain some functionality in the absence of intact CD2 complexes and provide a viable animal model for the analysis of the underlying bone development defects of SRTDs., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Kidneys are bilateral organs required to maintain homeostasis in the body through the regulation of fluid composition and the excretion of metabolic waste products. The initial steps in organ development are characterized by cellular interactions which regulate both the position and number of kidneys formed. Once established, further development is driven by orchestrated interactions between progenitor cell populations which serve to establish both nephrons-the functional unit of the organ which filters the blood-and the complex ramified collecting duct system which transports urine to the bladder. The delicate balance involved in these processes is reflected in the emerging family of genetic or environmental factors which, when perturbed, give rise to defects in organ development or function later in life. This article is categorized under: Vertebrate Organogenesis > From a Tubular Primordium: Branched Birth Defects > Organ Anomalies., (© 2018 Wiley Periodicals, Inc.)

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., Competing Interests: LO, AC, KS, NL, PW, LC, AJ, AA, OM, JB, IS, ML, AM No competing interests declared, (© 2018, O'Brien et al.)

Background: Duodenal atresia (DA) is a congenital obstruction of the duodenum, which affects 1 in 7000 pregnancies and requires major surgery in the 1st days of life. Three morphological DA types are described. In humans, the association between DA and Down syndrome suggests an underlying, albeit elusive, genetic etiology. In mice, interruption of fibroblast growth factor 10 ( Fgf10 ) gene signaling results in DA in 30-50% of embryos, supporting a genetic etiology. This study aims to validate the spectrum of DA in two novel strains of Fgf10 knock-out mice, in preparation for future and translational research. Methods: Two novel CRISPR Fgf10 knock-out mouse strains were derived and embryos generated by heterozygous plug-mating. E15.5-E19.5 embryos were genotyped with respect to Fgf10 and micro-dissected to determine the presence and type of DA. Results: One twenty seven embryos (32 wild-type, 34 heterozygous, 61 null) were analyzed. No wild-type or heterozygous embryos had DA. However, 74% of Fgf10 null embryos had DA (49% type 1, 18% type 2, and 33% type 3). Conclusion: Our CRISPR-derived strains showed higher penetrance of DA due to single-gene deletion of Fgf10 in mice than previously reported. Further, the DA type distribution in these mice more closely reiterated that observed in humans. Future experiments will document RNA and protein expression of FGF10 and its key downstream signaling targets in normal and atretic duodenum. This includes exploitation of modern, high-fidelity developmental tools, e.g., Fgf10 flox/+ -tomato flox/flox mice.

Identifying soluble factors that influence epidermal integrity is critical for the development of preventative and therapeutic strategies for disorders such as ichthyosis, psoriasis, dermatitis and epidermal cancers. The transcription factor Grainyhead-like 3 (GRHL3) is essential for maintaining barrier integrity and preventing development of cutaneous squamous cell carcinoma (SCC); however, how loss of this factor, which in the skin is expressed exclusively within suprabasal epidermal layers triggers proliferation of basal keratinocytes, had thus far remained elusive. Our present study identifies thymus and activation-regulated chemokine (TARC) as a novel soluble chemokine mediator of keratinocyte proliferation following loss of GRHL3. Knockdown of GRHL3 in human keratinocytes showed that of 42 cytokines examined, TARC was the only significantly upregulated chemokine. Mouse skin lacking Grhl3 presented an inflammatory response with hallmarks of TARC activation, including heightened induction of blood clotting, increased infiltration of mast cells and pro-inflammatory T cells, increased expression of the pro-proliferative/pro-inflammatory markers CD3 and pSTAT3, and significantly elevated basal keratinocyte proliferation. Treatment of skin cultures lacking Grhl3 with the broad spectrum anti-inflammatory 5-aminosalicylic acid (5ASA) partially restored epidermal differentiation, indicating that abnormal keratinocyte proliferation/differentiation balance is a key driver of barrier dysfunction following loss of Grhl3, and providing a promising therapeutic avenue in the treatment of GRHL3-mediated epidermal disorders.

Apoptotic cell death removes unwanted cells and is regulated by interactions between pro-survival and pro-apoptotic members of the BCL-2 protein family. The regulation of apoptosis is thought to be crucial for normal embryonic development. Accordingly, complete loss of pro-survival MCL-1 or BCL-XL (BCL2L1) causes embryonic lethality. However, it is not known whether minor reductions in pro-survival proteins could cause developmental abnormalities. We explored the rate-limiting roles of MCL-1 and BCL-XL in development and show that combined loss of single alleles of Mcl-1 and Bcl-x causes neonatal lethality. Mcl-1 +/- ;Bcl-x +/- mice display craniofacial anomalies, but additional loss of a single allele of pro-apoptotic Bim (Bcl2l11) restores normal development. These findings demonstrate that the control of cell survival during embryogenesis is finely balanced and suggest that some human craniofacial defects, for which causes are currently unknown, may be due to subtle imbalances between pro-survival and pro-apoptotic BCL-2 family members., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Branching morphogenesis of the ureteric bud is integral to kidney development; establishing the collecting ducts of the adult organ and driving organ expansion via peripheral interactions with nephron progenitor cells. A recent study suggested that termination of tip branching within the developing kidney involved stochastic exhaustion in response to nephron formation, with such a termination event representing a unifying developmental process evident in many organs. To examine this possibility, we have profiled the impact of nephron formation and maturation on elaboration of the ureteric bud during mouse kidney development. We find a distinct absence of random branch termination events within the kidney or evidence that nephrogenesis impacts the branching program or cell proliferation in either tip or progenitor cell niches. Instead, organogenesis proceeds in a manner indifferent to the development of these structures. Hence, stochastic cessation of branching is not a unifying developmental feature in all branching organs., Competing Interests: KS, AC, VL, JL, LJ, NH, IS No competing interests declared, ML has consulted for and received research funding from Organovo Inc., (© 2018, Short et al.)

The regulation of final nephron number in the kidney is poorly understood. Cessation of nephron formation occurs when the self-renewing nephron progenitor population commits to differentiation. Transcription factors within this progenitor population, such as SIX2, are assumed to control expression of genes promoting self-renewal such that homozygous Six2 deletion results in premature commitment and an early halt to kidney development. In contrast, Six2 heterozygotes were assumed to be unaffected. Using quantitative morphometry, we found a paradoxical 18% increase in ureteric branching and final nephron number in Six2 heterozygotes, despite evidence for reduced levels of SIX2 protein and transcript. This was accompanied by a clear shift in nephron progenitor identity with a distinct subset of downregulated progenitor genes such as Cited1 and Meox1 while other genes were unaffected. The net result was an increase in nephron progenitor proliferation, as assessed by elevated EdU (5-ethynyl-2'-deoxyuridine) labeling, an increase in MYC protein, and transcriptional upregulation of MYC target genes. Heterozygosity for Six2 on an Fgf20-/- background resulted in premature differentiation of the progenitor population, confirming that progenitor regulation is compromised in Six2 heterozygotes. Overall, our studies reveal a unique dose response of nephron progenitors to the level of SIX2 protein in which the role of SIX2 in progenitor proliferation versus self-renewal is separable., (Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Metanephric kidney development is orchestrated by the iterative branching morphogenesis of the ureteric bud. We describe an underlying patterning associated with the ramification of this structure and show that this pattern is conserved between developing kidneys, in different parts of the organ and across developmental time. This regularity is associated with a highly reproducible branching asymmetry that is consistent with locally operative growth mechanisms. We then develop a class of tip state models to represent elaboration of the ureteric tree and describe rules for 'half-delay' branching morphogenesis that describe almost perfectly the patterning of this structure. Spatial analysis suggests that the observed asymmetry may arise from mutual suppression of bifurcation, but not extension, between the growing ureteric tips, and demonstrates that disruption of patterning occurs in mouse mutants in which the distribution of tips on the surface of the kidney is altered. These findings demonstrate that kidney development occurs by way of a highly conserved reiterative pattern of asymmetric bifurcation that is governed by intrinsic and locally operative mechanisms., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

In laboratory animals and in human, centrally penetrant ghrelin receptor agonists, given systemically or orally, cause defecation. Animal studies show that the effect is due to activation of ghrelin receptors in the spinal lumbosacral defecation centers. However, it is not known whether there is a physiological role of ghrelin or the ghrelin receptor in the control of defecation. Using immunohistochemistry and immunoassay, we detected and measured ghrelin in the stomach, but were unable to detect ghrelin by either method in the lumbosacral spinal cord, or other regions of the CNS In rats in which the thoracic spinal cord was transected 5 weeks before, the effects of a ghrelin agonist on colorectal propulsion were significantly enhanced, but defecation caused by water avoidance stress (WAS) was reduced. In knockout rats that expressed no ghrelin and in wild-type rats, WAS-induced defecation was reduced by a ghrelin receptor antagonist, to similar extents. We conclude that the ghrelin receptors of the lumbosacral defecation centers have a physiological role in the control of defecation, but that their role is not dependent on ghrelin. This implies that a transmitter other than ghrelin engages the ghrelin receptor or a ghrelin receptor complex., (© 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

The kidney develops as an outgrowth of the epithelial nephric duct known as the ureteric bud, in a position specified by a range of rostral and caudal factors which serve to ensure two kidneys form in the appropriate positions in the body. At its simplest level, kidney development can be viewed as the process by which this single bud then undergoes a process of arborisation to form a complex connected network of ducts which will serve to drain urine from the nephrons in the adult organ. The process of bud elaboration is dictated by factors expressed by both the bud itself and by surrounding cells of the metanephric mesenchyme which control cell division and bifurcation. These cells play two critical roles. Firstly, they potentiate the ongoing elaboration of the ureteric tree: remove them and branching ceases. Secondly, they harbour progenitor cells which are fated to undergo their own process of tubulogenesis to form the nephrons of the adult organ. In this chapter, we will discuss how the ureteric bud arises in the developing embryo, how it undergoes branching, how we can measure and study this process and finally the likely relevance that this process has for our understanding of congenital and acquired kidney disease.

The mammalian kidney develops from a simple epithelial bud to an arborized network of tubules, which are fated to form the ureter, renal pelvis and collecting ducts. This process of ductal elaboration is achieved through an ancient developmental mechanism known as branching morphogenesis that is widely employed in glandular organs, the vasculature and lungs. It breaks up large solid tissues facilitating secretion, excretion and gas exchange, depending on the tissue. In the kidney, growth of the ureteric bud is driven by interactions between progenitor cells in the tips of the epithelial tree and their mesenchymal 'caps'. The cells of the cap mesenchyme give rise to nephrons; therefore, the interaction between these two cell populations is likely to be a critical driver of nephron number, which is determined during gestation. These cellular interactions are potentially affected by genetic mutations (congenital kidney diseases) and by changes in the fetal environment. Understanding the aetiology of congenital and acquired kidney diseases therefore requires a full appreciation of the processes involved in establishing the cellular architecture of the kidney and of the factors that affect the commitment of progenitor cells to form nephrons.

Polycystic kidney disease (PKD) is a common cause of renal failure with few effective treatments. INPP5E is an inositol polyphosphate 5-phosphatase that dephosphorylates phosphoinositide 3-kinase (PI3K)-generated PI(3,4,5)P 3 and is mutated in ciliopathy syndromes. Germline Inpp5e deletion is embryonically lethal, attributed to cilia stability defects, and is associated with polycystic kidneys. However, the molecular mechanisms responsible for PKD development upon Inpp5e loss remain unknown. Here, we show conditional inactivation of Inpp5e in mouse kidney epithelium results in severe PKD and renal failure, associated with a partial reduction in cilia number and hyperactivation of PI3K/Akt and downstream mammalian target of rapamycin complex 1 (mTORC1) signaling. Treatment with an mTORC1 inhibitor improved kidney morphology and function, but did not affect cilia number or length. Therefore, we identify Inpp5e as an essential inhibitor of the PI3K/Akt/mTORC1 signaling axis in renal epithelial cells, and demonstrate a critical role for Inpp5e-dependent mTORC1 regulation in PKD suppression., (© The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Sexual dimorphism is a prominent feature of renal physiology and as a consequence, it differentially affects predisposition to many adult kidney diseases. Furthermore the left and right kidneys differ in terms of their position, size and involvement in congenital malformations of the urogenital tract. We set out to determine whether differences in the program of branching morphogenesis that establishes the basic architecture of the kidney were apparent with respect to either sex or laterality in mouse embryonic kidneys. This was achieved using a combination of optical projection tomography imaging and computational analysis of many spatial metrics describing the branched ureteric tree. We undertook a comprehensive assessment of twelve aspects of ureteric morphology across developmental time and we found no consistent differences between kidneys of different sexes or laterality. These results suggest that dimorphism is established after birth or at a physiological or cellular level that is not reflected in the morphology of the ureteric tree.

Kidney development is initiated by the outgrowth of an epithelial ureteric bud into a population of mesenchymal cells. Reciprocal morphogenetic responses between these two populations generate a highly branched epithelial ureteric tree with the mesenchyme differentiating into nephrons, the functional units of the kidney. While we understand some of the mechanisms involved, current knowledge fails to explain the variability of organ sizes and nephron endowment in mice and humans. Here we present a spatially-averaged mathematical model of kidney morphogenesis in which the growth of the two key populations is described by a system of time-dependant ordinary differential equations. We assume that branching is symmetric and is invoked when the number of epithelial cells per tip reaches a threshold value. This process continues until the number of mesenchymal cells falls below a critical value that triggers cessation of branching. The mathematical model and its predictions are validated against experimentally quantified C57Bl6 mouse embryonic kidneys. Numerical simulations are performed to determine how the final number of branches changes as key system parameters are varied (such as the growth rate of tip cells, mesenchyme cells, or component cell population exit rate). Our results predict that the developing kidney responds differently to loss of cap and tip cells. They also indicate that the final number of kidney branches is less sensitive to changes in the growth rate of the ureteric tip cells than to changes in the growth rate of the mesenchymal cells. By inference, increasing the growth rate of mesenchymal cells should maximise branch number. Our model also provides a framework for predicting the branching outcome when ureteric tip or mesenchyme cells change behaviour in response to different genetic or environmental developmental stresses., (Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.)

Epigenetic mechanisms involved in the establishment of lung epithelial cell lineage identities during development are largely unknown. Here, we explored the role of the histone methyltransferase Ezh2 during lung lineage determination. Loss of Ezh2 in the lung epithelium leads to defective lung formation and perinatal mortality. We show that Ezh2 is crucial for airway lineage specification and alveolarization. Using optical projection tomography imaging, we found that branching morphogenesis is affected in Ezh2 conditional knockout mice and the remaining bronchioles are abnormal, lacking terminally differentiated secretory club cells. Remarkably, RNA-seq analysis revealed the upregulation of basal genes in Ezh2-deficient epithelium. Three-dimensional imaging for keratin 5 further showed the unexpected presence of a layer of basal cells from the proximal airways to the distal bronchioles in E16.5 embryos. ChIP-seq analysis indicated the presence of Ezh2-mediated repressive marks on the genomic loci of some but not all basal genes, suggesting an indirect mechanism of action of Ezh2. We found that loss of Ezh2 de-represses insulin-like growth factor 1 (Igf1) expression and that modulation of IGF1 signaling ex vivo in wild-type lungs could induce basal cell differentiation. Altogether, our work reveals an unexpected role for Ezh2 in controlling basal cell fate determination in the embryonic lung endoderm, mediated in part by repression of Igf1 expression., (© 2015. Published by The Company of Biologists Ltd.)

Mouse models play a key role in the understanding gene function, human development and disease. In 2007, the Australian Government provided funding to establish the Monash University embryonic stem cell-to-mouse (ES2M) facility. This was part of the broader Australian Phenomics Network, a national infrastructure initiative aimed at maximising access to global resources for understanding gene function in the mouse. The remit of the ES2M facility is to provide subsidised access for Australian biomedical researchers to the ES cell resources available from the International Knockout Mouse Consortium (IKMC). The stated aim of the IKMC is to generate a genetically modified mouse ES cell line for all of the ~23,000 genes in the mouse genome. The principal function of the Monash University ES2M service is to import genetically modified ES cells into Australia and to convert them into live mice with the potential to study human disease. Through advantages of economy of scale and established relationships with ES cell repositories worldwide, we have created over 110 germline mouse strains sourced from all of the major ES providers worldwide. We comment on our experience in generating these mouse lines; providing a snapshot of a "clients" perspective of using the IKMC resource and one which we hope will serve as a guide to other institutions or organisations contemplating establishing a similar centralised service.

Bifurcating developmental branching morphogenesis gives rise to complex organs such as the lung and the ureteric tree of the kidney. However, a few quantitative methods or tools exist to compare and distinguish, at a structural level, the critical features of these important biological systems. Here we develop novel graph alignment techniques to quantify the structural differences of rooted bifurcating trees and demonstrate their application in the analysis of developing kidneys from in normal and mutant mice. We have developed two graph based metrics: graph discordance, which measures how well the graphs representing the branching structures of distinct trees graphs can be aligned or overlayed; and graph inclusion, which measures the degree of containment of a tree graph within another. To demonstrate the application of these approaches we first benchmark the discordance metric on a data set of 32 normal and 28Tgfβ(+/-) mutant mouse ureteric trees. We find that the discordance metric better distinguishes control and mutant mouse kidneys than alternative metrics based on graph size and fingerprints - the distribution of tip depths. Using this metric we then show that the structure of the mutant trees follows the same pattern as the normal kidneys, but undergo a major delay in elaboration at later stages. Analysis of both controls and mutants using the inclusion metric gives strong support to the hypothesis that ureteric tree growth is stereotypic. Additionally, we present a new generalised multi-tree alignment algorithm that minimises the sum of pairwise graph discordance and which can be used to generate maximum consensus trees that represent the archetype for fixed developmental stages. These tools represent an advance in the analysis and quantification of branching patterns and will be invaluable in gaining a deeper understanding of the mechanisms that drive development. All code is being made available with documentation and example data with this publication., (Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.)

Harlequin ichthyosis (HI) is a severe skin disease which leads to neonatal death in ∼50% of cases. It is the result of mutations in ABCA12, a protein that transports lipids required to establish the protective skin barrier needed after birth. To better understand the life-threatening newborn HI phenotype, we analysed the developing epidermis for consequences of lipid dysregulation in mouse models. We observed a pro-inflammatory signature which was characterized by chemokine upregulation in embryonic skin which is distinct from that seen in other types of ichthyosis. Inflammation also persisted in grafted HI skin. To examine the contribution of inflammation to disease development, we overexpressed interleukin-37b to globally suppress fetal inflammation, observing considerable improvements in keratinocyte differentiation. These studies highlight inflammation as an unexpected contributor to HI disease development in utero, and suggest that inhibiting inflammation may reduce disease severity., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Mutations in inositol polyphosphate 5-phosphatase E (INPP5E) cause the ciliopathies known as Joubert and MORM syndromes; however, the role of INPP5E in ciliary biology is not well understood. Here, we describe an interaction between INPP5E and AURKA, a centrosomal kinase that regulates mitosis and ciliary disassembly, and we show that this interaction is important for the stability of primary cilia. Furthermore, AURKA phosphorylates INPP5E and thereby increases its 5-phosphatase activity, which in turn promotes transcriptional downregulation of AURKA, partly through an AKT-dependent mechanism. These findings establish the first direct link between AURKA and phosphoinositide signaling and suggest that the function of INPP5E in cilia is at least partly mediated by its interactions with AURKA., (© 2015. Published by The Company of Biologists Ltd.)

Developmental branching morphogenesis establishes organ architecture, and it is driven by iterative interactions between epithelial and mesenchymal progenitor cell populations. We describe an approach for analyzing this interaction and how it contributes to organ development. After initial in vivo cell labeling with the nucleoside analog 5-ethynyl-2'-deoxyuridine (EdU) and tissue-specific antibodies, optical projection tomography (OPT) and confocal microscopy are used to image the developing organ. These imaging data then inform a second analysis phase that quantifies (using Imaris and Tree Surveyor software), models and integrates these events at a cell and tissue level in 3D space and across developmental time. The protocol establishes a benchmark for assessing the impact of genetic change or fetal environment on organogenesis that does not rely on ex vivo organ culture or section-based reconstruction. By using this approach, examination of two developmental stages for an organ such as the kidney can be undertaken by a postdoctoral-level researcher in 6 weeks, with a full developmental analysis in mouse achievable in 5 months.

Keratins are cytoskeletal intermediate filament proteins that are increasingly being recognised for their diverse cellular functions. Here we report the consequences of germ line inactivation of Keratin 76 (Krt76) in mice. Homozygous disruption of this epidermally expressed gene causes neonatal skin flaking, hyperpigmentation, inflammation, impaired wound healing, and death prior to 12 weeks of age. We show that this phenotype is associated with functionally defective tight junctions that are characterised by mislocalization of the integral protein CLDN1. We further demonstrate that KRT76 interacts with CLDN1 and propose that this interaction is necessary to correctly position CLDN1 in tight junctions. The mislocalization of CLDN1 has been associated in various dermopathies, including the inflammatory disease, psoriasis. These observations establish a previously unknown connection between the intermediate filament cytoskeleton network and tight junctions and showcase Krt76 null mice as a possible model to study aberrant tight junction driven skin diseases.

The skin is a highly regenerative organ which plays critical roles in protecting the body and sensing its environment. Consequently, morbidity and mortality associated with skin defects represent a significant health issue. To identify genes important in skin development and homeostasis, we have applied a high throughput, multi-parameter phenotype screen to the conditional targeted mutant mice generated by the Wellcome Trust Sanger Institute's Mouse Genetics Project (Sanger-MGP). A total of 562 different mouse lines were subjected to a variety of tests assessing cutaneous expression, macroscopic clinical disease, histological change, hair follicle cycling, and aberrant marker expression. Cutaneous lesions were associated with mutations in 23 different genes. Many of these were not previously associated with skin disease in the organ (Mysm1, Vangl1, Trpc4ap, Nom1, Sparc, Farp2, and Prkab1), while others were ascribed new cutaneous functions on the basis of the screening approach (Krt76, Lrig1, Myo5a, Nsun2, and Nf1). The integration of these skin specific screening protocols into the Sanger-MGP primary phenotyping pipelines marks the largest reported reverse genetic screen undertaken in any organ and defines approaches to maximise the productivity of future projects of this nature, while flagging genes for further characterisation.