Deep learning is widely applicable to phenotyping embryonic development and disease.

Genome editing simplifies the generation of new animal models for congenital disorders. However, the detailed and unbiased phenotypic assessment of altered embryonic development remains a challenge. Here, we explore how deep learning (U-Net) can automate segmentation tasks in various imaging modalities, and we quantify phenotypes of altered renal, neural and craniofacial development in Xenopus embryos in comparison with normal variability. We demonstrate the utility of this approach in embryos with polycystic kidneys (pkd1 and pkd2) and craniofacial dysmorphia (six1). We highlight how in toto light-sheet microscopy facilitates accurate reconstruction of brain and craniofacial structures within X. tropicalis embryos upon dyrk1a and six1 loss of function or treatment with retinoic acid inhibitors. These tools increase the sensitivity and throughput of evaluating developmental malformations caused by chemical or genetic disruption. Furthermore, we provide a library of pre-trained networks and detailed instructions for applying deep learning to the reader's own datasets. We demonstrate the versatility, precision and scalability of deep neural network phenotyping on embryonic disease models. By combining light-sheet microscopy and deep learning, we provide a framework for higher-throughput characterization of embryonic model organisms. This article has an associated 'The people behind the papers' interview.

Characterization of early postzygotic somatic mutations through multi-organ analysis.

Mosaicisms caused by postzygotic mutational events are of increasing interest because of their potential association with various human diseases. Postzygotic somatic mutations have not been well characterized however in terms of their developmental lineage in humans. We conducted whole-genome sequencing (WGS) and targeted deep sequencing in 15 organs across three developmental lineages from a single male fetus with polycystic kidney disease (PKD) of 21 weeks gestational age. This fetus had no detectable neurological abnormalities at autopsy but germline mutations in the PKHD1 gene were identified that may have been associated with the PKD. Eight early embryonic mosaic variants with no alteration of protein function were detected. These variants were thought to have occurred at the two or four cell stages after fertilization with a mutational pattern involving frequent C>T and T>C transitions. In our current analyses, no tendency toward organ-specific mutation occurrences was found as the eight variants were detected in all 15 organs. However different allele fractions of these variants were found in different organs, suggesting a tissue-specific asymmetric growth of cells that reflected the developmental germ layer of each organ. This indicated that somatic mutation occurrences, even in early embryogenesis, can affect specific organ development or disease. Our current analyses demonstrate that multi-organ analysis is helpful for understanding genomic mosaicism. Our results also provide insights into the biological role of mosaicism in embryonic development and disease.

Cilia, ciliopathies and hedgehog-related forebrain developmental disorders.

Development of the forebrain critically depends on the Sonic Hedgehog (Shh) signaling pathway, as illustrated in humans by the frequent perturbation of this pathway in holoprosencephaly, a condition defined as a defect in the formation of midline structures of the forebrain and face. The Shh pathway requires functional primary cilia, microtubule-based organelles present on virtually every cell and acting as cellular antennae to receive and transduce diverse chemical, mechanical or light signals. The dysfunction of cilia in humans leads to inherited diseases called ciliopathies, which often affect many organs and show diverse manifestations including forebrain malformations for the most severe forms. The purpose of this review is to provide the reader with a framework to understand the developmental origin of the forebrain defects observed in severe ciliopathies with respect to perturbations of the Shh pathway. We propose that many of these defects can be interpreted as an imbalance in the ratio of activator to repressor forms of the Gli transcription factors, which are effectors of the Shh pathway. We also discuss the complexity of ciliopathies and their relationships with forebrain disorders such as holoprosencephaly or malformations of cortical development, and emphasize the need for a closer examination of forebrain defects in ciliopathies, not only through the lens of animal models but also taking advantage of the increasing potential of the research on human tissues and organoids.

Spatiotemporal dynamics and heterogeneity of renal lymphatics in mammalian development and cystic kidney disease.

Heterogeneity of lymphatic vessels during embryogenesis is critical for organ-specific lymphatic function. Little is known about lymphatics in the developing kidney, despite their established roles in pathology of the mature organ. We performed three-dimensional imaging to characterize lymphatic vessel formation in the mammalian embryonic kidney at single-cell resolution. In mouse, we visually and quantitatively assessed the development of kidney lymphatic vessels, remodeling from a ring-like anastomosis under the nascent renal pelvis; a site of VEGF-C expression, to form a patent vascular plexus. We identified a heterogenous population of lymphatic endothelial cell clusters in mouse and human embryonic kidneys. Exogenous VEGF-C expanded the lymphatic population in explanted mouse embryonic kidneys. Finally, we characterized complex kidney lymphatic abnormalities in a genetic mouse model of polycystic kidney disease. Our study provides novel insights into the development of kidney lymphatic vasculature; a system which likely has fundamental roles in renal development, physiology and disease.

Lkb1 deficiency confers glutamine dependency in polycystic kidney disease.

Polycystic kidney disease (PKD) is a common genetic disorder characterized by the growth of fluid-filled cysts in the kidneys. Several studies reported that the serine-threonine kinase Lkb1 is dysregulated in PKD. Here we show that genetic ablation of Lkb1 in the embryonic ureteric bud has no effects on tubule formation, maintenance, or growth. However, co-ablation of Lkb1 and Tsc1, an mTOR repressor, results in an early developing, aggressive form of PKD. We find that both loss of Lkb1 and loss of Pkd1 render cells dependent on glutamine for growth. Metabolomics analysis suggests that Lkb1 mutant kidneys require glutamine for non-essential amino acid and glutathione metabolism. Inhibition of glutamine metabolism in both Lkb1/Tsc1 and Pkd1 mutant mice significantly reduces cyst progression. Thus, we identify a role for Lkb1 in glutamine metabolism within the kidney epithelia and suggest that drugs targeting glutamine metabolism may help reduce cyst number and/or size in PKD.

Kidney: polycystic kidney disease.

Polycystic kidney disease (PKD) is a life-threatening genetic disorder characterized by the presence of fluid-filled cysts primarily in the kidneys. PKD can be inherited as autosomal recessive (ARPKD) or autosomal dominant (ADPKD) traits. Mutations in either the PKD1 or PKD2 genes, which encode polycystin 1 and polycystin 2, are the underlying cause of ADPKD. Progressive cyst formation and renal enlargement lead to renal insufficiency in these patients, which need to be managed by lifelong dialysis or renal transplantation. While characteristic features of PKD are abnormalities in epithelial cell proliferation, fluid secretion, extracellular matrix and differentiation, the molecular mechanisms underlying these events are not understood. Here we review the progress that has been made in defining the function of the polycystins, and how disruption of these functions may be involved in cystogenesis.

Fetal syringomyelia.

We explored the prevalence of syringomyelia in a series of 113 cases of fetal dysraphism and hindbrain crowding, of gestational age ranging from 17.5 to 34 weeks with the vast majority less than 26 weeks gestational age. We found syringomyelia in 13 cases of Chiari II malformations, 5 cases of Omphalocele/Exostrophy/Imperforate anus/Spinal abnormality (OEIS), 2 cases of Meckel Gruber syndrome and in a single pair of pyopagus conjoined twins. Secondary injury was not uncommon, with vernicomyelia in Chiari malformations, infarct like histology, or old hemorrhage in 8 cases of syringomyelia. Vernicomyelia did not occur in the absence of syrinx formation. The syringes extended from the sites of dysraphism, in ascending or descending patterns. The syringes were usually in a major proportion anatomically distinct from a dilated or denuded central canal and tended to be dorsal and paramedian or median. We suggest that fetal syringomyelia in Chiari II malformation and other dysraphic states is often established prior to midgestation, has contributions from the primary malformation as well as from secondary in utero injury and is anatomically and pathophysiologically distinct from post natal syringomyelia secondary to hindbrain crowding.

Expression of Nek1 during kidney development and cyst formation in multiple nephron segments in the Nek1-deficient kat2J mouse model of polycystic kidney disease.

BACKGROUND: Neks, mammalian orthologs of the fungal protein kinase never-in-mitosis A, have been implicated in the pathogenesis of polycystic kidney disease. Among them, Nek1 is the primary protein inactivated in kat2J mouse models of PKD. RESULT: We report the expression pattern of Nek1 and characterize the renal cysts that develop in kat2J mice. Nek1 is detectable in all murine tissues but its expression in wild type and kat2J heterozygous kidneys decrease as the kidneys mature, especially in tubular epithelial cells. In the embryonic kidney, Nek1 expression is most prominent in cells that will become podocytes and proximal tubules. Kidney development in kat2J homozygous mice is aberrant early, before the appearance of gross cysts: developing cortical zones are thin, populated by immature glomeruli, and characterized by excessive apoptosis of several cell types. Cysts in kat2J homozygous mice form postnatally in Bowman's space as well as different tubular subtypes. Late in life, kat2J heterozygous mice form renal cysts and the cells lining these cysts lack staining for Nek1. The primary cilia of cells lining cysts in kat2J homozygous mice are morphologically diverse: in some cells they are unusually long and in others there are multiple cilia of varying lengths. CONCLUSION: Our studies indicate that Nek1 deficiency leads to disordered kidney maturation, and cysts throughout the nephron.

Ex vivo modeling of chemical synergy in prenatal kidney cystogenesis.

Cyclic adenosine monophosphate (cAMP) drives genetic polycystic kidney disease (PKD) cystogenesis. Yet within certain PKD families, striking differences in disease severity exist between affected individuals, and genomic and/or environmental modifying factors have been evoked to explain these observations. We hypothesized that PKD cystogenesis is accentuated by an aberrant fetal milieu, specifically by glucocorticoids. The extent and nature of cystogenesis was assessed in explanted wild-type mouse embryonic metanephroi, using 8-Br-cAMP as a chemical to mimic genetic PKD and the glucocorticoid dexamethasone as the environmental modulator. Cysts and glomeruli were quantified by an observer blinded to culture conditions, and tubules were phenotyped using specific markers. Dexamethasone or 8-Br-cAMP applied on their own produced cysts predominantly arising in proximal tubules and descending limbs of loops of Henle. When applied together, however, dexamethasone over a wide concentration range synergized with 8-Br-cAMP to generate a more severe, glomerulocystic, phenotype; we note that prominent glomerular cysts have been reported in autosomal dominant PKD fetal kidneys. Our data support the idea that an adverse antenatal environment exacerbates renal cystogenesis.

The Meckel syndrome protein meckelin (TMEM67) is a key regulator of cilia function but is not required for tissue planar polarity.

Meckel syndrome (MKS) is a lethal disorder associated with renal cystic disease, encephalocele, ductal plate malformation and polydactyly. MKS is genetically heterogeneous and part of a growing list of syndromes called ciliopathies, disorders resulting from defective cilia. TMEM67 mutation (MKS3) is a major cause of MKS and the related ciliopathy Joubert syndrome, although the complete etiology of the disease is not well understood. To further investigate MKS3, we analyzed phenotypes in the Tmem67 null mouse (bpck) and in zebrafish tmem67 morphants. Phenotypes similar to those in human MKS and other ciliopathy models were observed, with additional eye, skeletal and inner ear abnormalities characterized in the bpck mouse. The observed disorganized stereociliary bundles in the bpck inner ear and the convergent extension defects in zebrafish morphants are similar to those found in planar cell polarity (PCP) mutants, a pathway suggested to be defective in ciliopathies. However, analysis of classical vertebrate PCP readouts in the bpck mouse and ciliary organization analysis in tmem67 morphants did not support a global loss of planar polarity. Canonical Wnt signaling was upregulated in cyst linings and isolated fibroblasts from the bpck mouse, but was unchanged in the retina and cochlea tissue, suggesting that increased Wnt signaling may only be linked to MKS3 phenotypes associated with elevated proliferation. Together, these data suggest that defective cilia loading, but not a global loss of ciliogenesis, basal body docking or PCP signaling leads to dysfunctional cilia in MKS3 tissues.

Calcium ameliorates renal cyst growth in metanephric organ culture: a morphological study.

Polycystic kidney disease (PKD) is associated with alterations in developmental processes that severely affect kidney integrity, often leading to fatal consequences. It has been suggested that dysfunctional calcium (Ca2+) regulation associated with the PKD phenotype is consequent to mutations affecting the pkd1 gene. Previously, it has been observed that blocking calcium along with cAMP allowed tubular epithelial cells to enter the proliferative phase that culminated in a cyst-like phenotype. In this regard, mouse metanephroi, (embryonic day 13.5, E13.5) were used to study morphological and ultrastructural effects of calcium replenishment on 8-bromocyclic 3'5'cyclic adenosine monophosphate (8-Br-cAMP)-induced cyst-like tubular dilations. Phase contrast microscopy of 8-Br-cAMP-treated metanephroi exhibited numerous dilated tubules that continued to increase in size for 4 days in culture. The effects of 8-Br-cAMP on renal tubular epithelia were assessed by histopathological and electron microscopic analyses. Transmission electron microscopy revealed changes such as increased vacuolation, swollen mitochondria, chromatin condensation, and disrupted cell membrane in tubular epithelia of 8-Br-cAMP-treated metanephroi. Concurrent treatments with calcium-channel agonists (calcium ionophore A23187 and phorbol-12-myristate-13-acetate) and 8-Br-cAMP abolished cAMP-induced morphometric and ultrastructural alterations. Calcium replenishment rescued tubular epithelial cells from mitogenic effects of cAMP and restored normal morphology at cellular and sub-cellular levels as verified by histopathological and ultrastructural examinations.

CaMK-II is a PKD2 target that promotes pronephric kidney development and stabilizes cilia.

Intracellular Ca²âº signals influence gastrulation, neurogenesis and organogenesis through pathways that are still being defined. One potential Ca²âº mediator of many of these morphogenic processes is CaMK-II, a conserved calmodulin-dependent protein kinase. Prolonged Ca²âº stimulation converts CaMK-II into an activated state that, in the zebrafish, is detected in the forebrain, ear and kidney. Autosomal dominant polycystic kidney disease has been linked to mutations in the Ca²âº-conducting TRP family member PKD2, the suppression of which in vertebrate model organisms results in kidney cysts. Both PKD2-deficient and CaMK-II-deficient zebrafish embryos fail to form pronephric ducts properly, and exhibit anterior cysts and destabilized cloacal cilia. PKD2 suppression inactivates CaMK-II in pronephric cells and cilia, whereas constitutively active CaMK-II restores pronephric duct formation in pkd2 morphants. PKD2 and CaMK-II deficiencies are synergistic, supporting their existence in the same genetic pathway. We conclude that CaMK-II is a crucial effector of PKD2 Ca²âº that both promotes morphogenesis of the pronephric kidney and stabilizes primary cloacal cilia.

[Prenatal diagnosis of hyperechogenic kidneys: A study of 17 cases]. / Diagnostic anténatal des reins hyperéchogènes : à propos de 17 cas.

OBJECTIVES: To evaluate the prenatal diagnosis and the prognostic value of ultrasound in case of fetal hyperechogenic kidneys. PATIENTS AND METHODS: Seventeen prenatally diagnosed cases of hyperechogenic kidneys were retrospectively reviewed at the University Hospital of Lille from 1997 to 2008. The clinical and ultrasound data were compared to the postnatal follow-up and the long-term prognosis. RESULTS: The aetiologies are nine recessive polycystic kidney diseases, three dominant, two Bardet-Biedl syndromes and three cases of transient renal hyperechogenicity. No renal ultrasonographic criterion is specific of aetiology. Five pregnancies were terminated. We observed one neonatal death and 11 survivors (median follow-up: 30months) including two infants with hypertension. All oligohydramnios (n=8) were associated with poor prenatal outcomes (terminations of pregnancy, neonatal death or hypertension) compared to the other nine with normal amniotic fluid volume (nine children symptom-free). Kidneys less or equal to +4 S.D. and a normal amniotic fluid volume were associated with a good prognosis (n=7, seven symptom-free). CONCLUSION: The fetal kidneys characteristics on prenatal ultrasound fail to provide an accurate etiological diagnosis. Only congenital defects and family history adjust the aetiology. Amniotic fluid volume and fetal kidney size are the best prenatal predictors of outcome.

Prenatal MRI findings of polycystic kidney disease associated with holoprosencephaly.

Holoprosencephaly (HPE) and polycystic kidney disease (PKD) are genetically heterogeneous anomalies which can make up part of various syndromes or chromosomal anomalies. Due to the rapid lethality prognosis, early and precise prenatal diagnosis would be of great value. This case report describes extensive PKD involvement, already present in utero, in a patient with HPE and subdural effusion visible by MR imaging. The detailed anatomic information obtained by the MR imaging can guide the surgical planning and can aid antenatal counseling.

Distinct roles and regulations for HoxD genes in metanephric kidney development.

Hox genes encode homeodomain-containing proteins that control embryonic development in multiple contexts. Up to 30 Hox genes, distributed among all four clusters, are expressed during mammalian kidney morphogenesis, but functional redundancy between them has made a detailed functional account difficult to achieve. We have investigated the role of the HoxD cluster through comparative molecular embryological analysis of a set of mouse strains carrying targeted genomic rearrangements such as deletions, duplications, and inversions. This analysis allowed us to uncover and genetically dissect the complex role of the HoxD cluster. Regulation of metanephric mesenchyme-ureteric bud interactions and maintenance of structural integrity of tubular epithelia are differentially controlled by some Hoxd genes during renal development, consistent with their specific expression profiles. We also provide evidence for a kidney-specific form of colinearity that underlies the differential expression of two distinct sets of genes located on both sides and overlapping at the Hoxd9 locus. These insights further our knowledge of the genetic control of kidney morphogenesis and may contribute to understanding certain congenital kidney malformations, including polycystic kidney disease and renal hypoplasia.

Kidney-specific inactivation of the Pkd1 gene induces rapid cyst formation in developing kidneys and a slow onset of disease in adult mice.

Autosomal dominant polycystic kidney disease, caused by mutations in the PKD1 gene, is characterized by progressive deterioration of kidney function due to the formation of thousands of cysts leading to kidney failure in mid-life or later. How cysts develop and grow is currently unknown, although extensive research revealed a plethora of cellular changes in cyst lining cells. We have constructed a tamoxifen-inducible, kidney epithelium-specific Pkd1-deletion mouse model. Upon administration of tamoxifen to these mice, a genomic fragment containing exons 2-11 of the Pkd1-gene is specifically deleted in the kidneys and cysts are formed. Interestingly, the timing of Pkd1-deletion has strong effects on the phenotype. At 1 month upon gene disruption, adult mice develop only a very mild cystic phenotype showing some small cysts and dilated tubules. Young mice, however, show massive cyst formation. In these mice, at the moment of gene disruption, cell proliferation takes place to elongate the nephron. Our data indicate that Pkd1 gene deficiency does not initiate sufficient autonomous cell proliferation leading to cyst formation and that additional stimuli are required. Furthermore, we show that one germ-line mutation of Pkd1 is already associated with increased proliferation.

Are therapeutic stem cells justified in bilateral multicystic kidney disease? A review of literature with insights into the embryology.

Aim was to describe the challenges faced in the management of bilateral multicystic kidney disease (MCKD). A case of antenatally detected bilateral polycystic disease was referred at 28 weeks of gestation. The patient was advised to continue pregnancy till term and be in regular follow-up. Postnatally, the male baby passed urine in normal stream and was diagnosed as bilateral multicystic kidney disease on ultrasonography. He developed symptoms of renal failure. The baby was operated with right pyeloplasty and left pyelostomy, as the left ureter was atretic. The histopathology was consistent with bilateral multicystic kidney disease. Postoperatively, the baby was stable with intermittent episodes of metabolic acidosis that were managed medically and with peritoneal dialysis. Autologous stem cells were injected at the age of 1 year into the aorta at the level of the renal arteries clamping the aorta below. Repeat biopsy at time of stem cell injection showed 5/10 glomeruli showing global sclerosis on right side and 5/15 glomeruli showing global sclerosis on left side. The only improvement seen was in decreased doses of medicines to keep the child metabolically stable. The baby kept struggling but succumbed at the age of 17 months and 15 days. Post mortem bilateral renal biopsies demonstrated presence of primitive renal tubules and blastemal cells that were not demonstrated earlier. Survival for few months in bilateral multicystic kidney disease is thus possible with adequate treatment, the novel use of stem cells in these cases may prove beneficial in future though it is too early to comment further.

Differential diagnosis of fetal hyperechogenic cystic kidneys unrelated to renal tract anomalies: A multicenter study.

OBJECTIVES: To identify important factors in the differential diagnosis of renal cysts associated with hyperechogenic kidneys. METHODS: This was a retrospective multicenter study. We identified 93 fetuses presenting between 1990 and 2002 with hyperechogenic kidneys and which had a diagnosis of nephropathy confirmed later. We analyzed retrospectively the prenatal ultrasound findings of those fetuses which were found sonographically to have renal cysts. RESULTS: Of the 93 fetuses presenting with hyperechogenic kidneys and with a later diagnosis of nephropathy, there were 28 with autosomal dominant polycystic kidney disease (ADPKD), 31 with autosomal recessive polycystic kidney disease (ARPKD), 11 with Bardet-Biedl syndrome, nine with Meckel-Gruber syndrome, six with Ivemark II syndrome, one with Jarcho-Levin syndrome, one with Beemer syndrome and one with Meckel-like syndrome. One third of the fetuses (30/93) had renal cysts. Cystic characteristics (size, location, number) were not very useful for diagnosis; more useful was diagnosis of an associated malformation. Three (11%) of the fetuses with ADPKD had cysts, as did nine (29%) of those with ARPKD, three (27%) of those with Bardet-Biedl syndrome, all (100%) of those with Meckel-Gruber syndrome, three (50%) of those with Ivemark II syndrome, and each of the three cases with other syndromes (Jarcho-Levin, Beemer and Meckel-like syndromes). None of the cases with trisomy 13 had cysts. There were no associated malformations in the 12 cases with renal cysts and polycystic kidney disease; the other 18 cases with renal cysts were associated with malformations that were often specific, such as polydactyly in Bardet-Biedl and Beemer syndromes, occipital defect and Dandy-Walker malformation in Meckel-Gruber or Meckel-Gruber-like syndromes, and thoracic and/or vertebral abnormalities in Jarcho-Levin and Beemer syndromes. CONCLUSION: Renal cysts associated with hyperechogenic kidneys are not rare. The clue to diagnosis is the demonstration of an associated malformation. If no malformation is found, the main diagnosis remains polycystic kidney disease, i.e. ARPKD or ADPKD.

Early embryonic renal tubules of wild-type and polycystic kidney disease kidneys respond to cAMP stimulation with cystic fibrosis transmembrane conductance regulator/Na(+),K(+),2Cl(-) Co-transporter-dependent cystic dilation.

Metanephric organ culture has been used to determine whether embryonic kidney tubules can be stimulated by cAMP to form cysts. Under basal culture conditions, wild-type kidneys from embryonic day 13.5 to 15.5 mice grow in size and continue ureteric bud branching and tubule formation over a 4- to 5-d period. Treatment of these kidneys with 8-Br-cAMP or the cAMP agonist forskolin induced the formation of dilated tubules within 1 h, which enlarged over several days and resulted in dramatically expanded cyst-like structures of proximal tubule and collecting duct origin. Tubule dilation was reversible upon withdrawal of 8-Br-cAMP and was inhibited by the cAMP-dependent protein kinase inhibitor H89 and the cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor CFTR(inh)172. For further testing of the role of CFTR, metanephric cultures were prepared from mice with a targeted mutation of the Cftr gene. In contrast to kidneys from wild-type mice, those from Cftr -/- mice showed no evidence of tubular dilation in response to 8-Br-cAMP, indicating that CFTR Cl(-) channels are functional in embryonic kidneys and are required for cAMP-driven tubule expansion. A requirement for transepithelial Cl(-) transport was demonstrated by inhibiting the basolateral Na(+),K(+),2Cl(-) co-transporter with bumetanide, which effectively blocked all cAMP-stimulated tubular dilation. For determination of whether cystic dilation occurs to a greater extent in PKD kidneys in response to cAMP, Pkd1(m1Bei) -/- embryonic kidneys were treated with 8-Br-cAMP and were found to form rapidly CFTR- and Na(+),K(+),2Cl(-) co-transporter-dependent cysts that were three- to six-fold larger than those of wild-type kidneys. These results suggest that cAMP can stimulate fluid secretion early in renal tubule development during the time when renal cysts first appear in PKD kidneys and that PKD-deficient renal tubules are predisposed to abnormally increased cyst expansion in response to elevated levels of cAMP.