Prenatal diagnosis of rhombencephalosynapsis: neuroimaging features and severity of vermian anomaly.

OBJECTIVES: To describe the prenatal neuroimaging spectrum of rhombencephalosynapsis (RES) and criteria for its classification according to the severity of vermian anomaly. METHODS: In this multicenter retrospective study of fetuses with RES between 2002 and 2020, the medical records and brain ultrasound and magnetic resonance images were evaluated comprehensively to determine the severity of the vermian anomaly and the presence of associated brain findings. RES was classified, according to the pattern of vermian agenesis and the extent of the fusion of the hemispheres, as complete RES (complete absence of the vermis) or partial RES (further classified according to the part of the vermis that was missing and, consequently, the region of hemispheric fusion, as anterior, posterior, severe or mixed RES). Findings were compared between cases with complete and those with partial RES. RESULTS: Included in the study were 62 fetuses with a gestational age ranging between 12 and 37 weeks. Most had complete absence of the vermis (complete RES, 77.4% of cases), a 'round-shaped' cerebellum on axial views (72.6%) and a transverse cerebellar diameter (TCD) < 3rd centile (87.1%). Among the 22.6% of cases with partial RES, 6.5% were classified as severe partial, 6.5% as partial anterior, 8.1% as partial mixed and 1.6% as partial posterior. Half of these cases presented with normal or nearly normal cerebellar morphology and 28.5% had a TCD within the normal limits. Infratentorially, the fourth ventricle was abnormal in 88.7% of cases overall, and anomalies of the midbrain and pons were frequent (93.5% and 77.4%, respectively). Ventriculomegaly was observed in 80.6% of all cases, being more severe in cases with complete RES than in those with partial RES, with high rates of parenchymal and septal disruption. CONCLUSIONS: This study provides prenatal neuroimaging criteria for the diagnosis and classification of RES, and identification of related features, using ultrasound and magnetic resonance imaging. According to our findings, a diagnosis of RES should be considered in fetuses with a small TCD (severe cerebellar hypoplasia) and/or a round-shaped cerebellum on axial views, during the second or third trimester, especially when associated with ventriculomegaly. Partial RES is more common than previously thought, but presents an extreme diagnostic challenge, especially in cases with normal or nearly-normal cerebellar morphobiometric features. © 2021 International Society of Ultrasound in Obstetrics and Gynecology.

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.

Loss of the neurodevelopmental Joubert syndrome causing protein, Ahi1, causes motor and muscle development delays independent of central nervous system involvement.

Joubert syndrome (JBTS) is a predominantly autosomal recessive neurodevelopmental disorder that presents with characteristic malformations of the cerebellar vermis, superior cerebellar peduncles and midbrain in humans. Accompanying these malformations are a heterogeneous set of clinical symptoms, which frequently include deficits in motor and muscle function, such as hypotonia (low muscle tone) and ataxia (clumsiness). These symptoms are attributed to improper development of the hindbrain, but no direct evidence has been reported linking these in JBTS. Here, we describe muscle developmental defects in a mouse with a targeted deletion of the Abelson helper integration site 1 gene, Ahi1, one of the genes known to cause JBTS in humans. While FVB/NJ Ahi1-/- mice display no gross malformations of the cerebellum, deficits are observed in several measures of motor function, strength, and body development. Specifically, Ahi1-/- mice show delayed physical development, delays in surface reflex righting as neonates, and reductions in grip strength and spontaneous locomotor activity as adults. Additionally, Ahi1-/- mice showed evidence of muscle-specific contributions to this phenotype, such as reductions in 1) myoblast differentiation potential in vitro, 2) muscle desmin expression, and 3) overall muscle mass, myonuclear domain, and muscle fiber cross-sectional area. Together, these data suggest that loss of Ahi1 may cause abnormalities in the differentiation of myoblasts to mature muscle cells. Moreover, Ahi1 loss impacts muscle development directly, outside of any indirect impact of cerebellar malformations, revealing a novel myogenic cause for hypotonia in JBTS.

Congenital basis of posterior fossa anomalies.

The classification of posterior fossa congenital anomalies has been a controversial topic. Advances in genetics and imaging have allowed a better understanding of the embryologic development of these abnormalities. A new classification schema correlates the embryologic, morphologic, and genetic bases of these anomalies in order to better distinguish and describe them. Although they provide a better understanding of the clinical aspects and genetics of these disorders, it is crucial for the radiologist to be able to diagnose the congenital posterior fossa anomalies based on their morphology, since neuroimaging is usually the initial step when these disorders are suspected. We divide the most common posterior fossa congenital anomalies into two groups: 1) hindbrain malformations, including diseases with cerebellar or vermian agenesis, aplasia or hypoplasia and cystic posterior fossa anomalies; and 2) cranial vault malformations. In addition, we will review the embryologic development of the posterior fossa and, from the perspective of embryonic development, will describe the imaging appearance of congenital posterior fossa anomalies. Knowledge of the developmental bases of these malformations facilitates detection of the morphological changes identified on imaging, allowing accurate differentiation and diagnosis of congenital posterior fossa anomalies.

Imaging and differential diagnosis of suprarenal masses in the fetus.

Prenatal sonography and magnetic resonance imaging of suprarenal fetal masses is presented, along with clinical information and follow-up. Imaging pearls and differential considerations for each diagnosis will be discussed. Fetal suprarenal mass diagnoses include neuroblastoma, extralobar pulmonary sequestration, congenital adrenal hyperplasia, partial multicystic dysplastic kidney, renal duplication, urinoma, gastric duplication cyst, and splenic cyst. Recognizing the range of malignant and benign suprarenal fetal masses that can present on prenatal imaging can help guide patient counseling and management.

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.

Vertebrate kidney tubules elongate using a planar cell polarity-dependent, rosette-based mechanism of convergent extension.

Cystic kidney diseases are a global public health burden, affecting over 12 million people. Although much is known about the genetics of kidney development and disease, the cellular mechanisms driving normal kidney tubule elongation remain unclear. Here, we used in vivo imaging to show for the first time that mediolaterally oriented cell intercalation is fundamental to vertebrate kidney morphogenesis. Unexpectedly, we found that kidney tubule elongation is driven in large part by a myosin-dependent, multicellular rosette-based mechanism, previously only described in Drosophila melanogaster. In contrast to findings in Drosophila, however, non-canonical Wnt and planar cell polarity (PCP) signaling is required to control rosette topology and orientation during vertebrate kidney tubule elongation. These data resolve long-standing questions concerning the role of PCP signaling in the developing kidney and, moreover, establish rosette-based intercalation as a deeply conserved cellular engine for epithelial morphogenesis.

Investigating embryonic expression patterns and evolution of AHI1 and CEP290 genes, implicated in Joubert syndrome.

Joubert syndrome and related diseases (JSRD) are developmental cerebello-oculo-renal syndromes with phenotypes including cerebellar hypoplasia, retinal dystrophy and nephronophthisis (a cystic kidney disease). We have utilised the MRC-Wellcome Trust Human Developmental Biology Resource (HDBR), to perform in-situ hybridisation studies on embryonic tissues, revealing an early onset neuronal, retinal and renal expression pattern for AHI1. An almost identical pattern of expression is seen with CEP290 in human embryonic and fetal tissue. A novel finding is that both AHI1 and CEP290 demonstrate strong expression within the developing choroid plexus, a ciliated structure important for central nervous system development. To test if AHI1 and CEP290 may have co-evolved, we carried out a genomic survey of a large group of organisms across eukaryotic evolution. We found that, in animals, ahi1 and cep290 are almost always found together; however in other organisms either one may be found independent of the other. Finally, we tested in murine epithelial cells if Ahi1 was required for recruitment of Cep290 to the centrosome. We found no obvious differences in Cep290 localisation in the presence or absence of Ahi1, suggesting that, while Ahi1 and Cep290 may function together in the whole organism, they are not interdependent for localisation within a single cell. Taken together these data support a role for AHI1 and CEP290 in multiple organs throughout development and we suggest that this accounts for the wide phenotypic spectrum of AHI1 and CEP290 mutations in man.

p120 catenin is required for normal renal tubulogenesis and glomerulogenesis.

Defects in the development or maintenance of tubule diameter correlate with polycystic kidney disease. Here, we report that absence of the cadherin regulator p120 catenin (p120ctn) from the renal mesenchyme prior to tubule formation leads to decreased cadherin levels with abnormal morphologies of early tubule structures and developing glomeruli. In addition, mutant mice develop cystic kidney disease, with markedly increased tubule diameter and cellular proliferation, and detached luminal cells only in proximal tubules. The p120ctn homolog Arvcf is specifically absent from embryonic proximal tubules, consistent with the specificity of the proximal tubular phenotype. p120ctn knockdown in renal epithelial cells in 3D culture results in a similar cystic phenotype with reduced levels of E-cadherin and active RhoA. We find that E-cadherin knockdown, but not RhoA inhibition, phenocopies p120ctn knockdown. Taken together, our data show that p120ctn is required for early tubule and glomerular morphogenesis, as well as control of luminal diameter, probably through regulation of cadherins.

Dicer regulates the development of nephrogenic and ureteric compartments in the mammalian kidney.

MicroRNAs (miRNAs) are a large and growing class of small, non-coding, regulatory RNAs that control gene expression predominantly at the post-transcriptional level. The production of most functional miRNAs depends on the enzymatic activity of Dicer, an RNase III class enzyme. To address the potential action of Dicer-dependent miRNAs in mammalian kidney development, we conditionally ablated Dicer function within cells of nephron lineage and the ureteric bud-derived collecting duct system. Six2Cre-mediated removal of Dicer activity from the progenitors of the nephron epithelium led to elevated apoptosis and premature termination of nephrogenesis. Thus, Dicer action is important for maintaining the viability of this critical self-renewing progenitor pool and, consequently, development of a normal nephron complement. HoxB7Cre-mediated removal of Dicer function from the ureteric bud epithelium led to the development of renal cysts. This was preceded by excessive cell proliferation and apoptosis, and accompanied by disrupted ciliogenesis within the ureteric bud epithelium. Dicer removal also disrupted branching morphogenesis with the phenotype correlating with downregulation of Wnt11 and c-Ret expression at ureteric tips. Thus Dicer, and by inference Dicer-dependent miRNA activity, have distinct regulatory roles within different components of the developing mouse kidney. Furthermore, an understanding of miRNA action may provide new insights into the etiology and pathogenesis of renal cyst-based kidney disease.

Pitchfork regulates primary cilia disassembly and left-right asymmetry.

A variety of developmental disorders have been associated with ciliary defects, yet the controls that govern cilia disassembly are largely unknown. Here we report a mouse embryonic node gene, which we named Pitchfork (Pifo). Pifo associates with ciliary targeting complexes and accumulates at the basal body during cilia disassembly. Haploinsufficiency causes a unique node cilia duplication phenotype, left-right asymmetry defects, and heart failure. This phenotype is likely relevant in humans, because we identified a heterozygous R80K PIFO mutation in a fetus with situs inversus and cystic liver and kidneys, and in patient with double-outflow right ventricle. We show that PIFO, but not R80K PIFO, is sufficient to activate Aurora A, a protooncogenic kinase that induces cilia retraction, and that Pifo/PIFO mutation causes cilia retraction, basal body liberation, and overreplication defects. Thus, the observation of a disassembly phenotype in vivo provides an entry point to understand and categorize ciliary disease. AUTHOR AUDIO:

Genetic and physical interaction between the NPHP5 and NPHP6 gene products.

Nephronophthisis (NPHP) is an autosomal recessive cystic kidney disease, caused by mutations of at least nine different genes. Several extrarenal manifestations characterize this disorder, including cerebellar defects, situs inversus and retinitis pigmentosa. While the clinical manifestations vary significantly in NPHP, mutations of NPHP5 and NPHP6 are always associated with progressive blindness. This clinical finding suggests that the gene products, nephrocystin-5 and nephrocystin-6, participate in overlapping signaling pathways to maintain photoreceptor homeostasis. To analyze the genetic interaction between these two proteins in more detail, we studied zebrafish embryos after depletion of NPHP5 and NPHP6. Knockdown of zebrafish zNPHP5 and zNPHP6 produced similar phenotypes, and synergistic effects were observed after the combined knockdown of zNPHP5 and zNPHP6. The N-terminal domain of nephrocystin-6-bound nephrocystin-5, and mapping studies delineated the interacting site from amino acid 696 to 896 of NPHP6. In Xenopus laevis, knockdown of NPHP5 caused substantial neural tube closure defects. This phenotype was copied by expression of the nephrocystin-5-binding fragment of nephrocystin-6, and rescued by co-expression of nephrocystin-5, supporting a physical interaction between both gene products in vivo. Since the N- and C-terminal fragments of nephrocystin-6 engage in the formation of homo- and heteromeric protein complexes, conformational changes seem to regulate the interaction of nephrocystin-6 with its binding partners.

Activated extracellular signal-regulated kinase correlates with cyst formation and transforming growth factor-beta expression in fetal obstructive uropathy.

Human renal dysplasia is frequently associated with urinary tract obstruction and the abnormal expression of mitogen-activated protein kinase (MAPK). Here, we determined the renal responses and MAPK expression in developing kidneys that were obstructed in fetal lambs. Kidneys were harvested at various times after obstruction (gestation day 60) through normal term (day 145). Dilation of Bowman's capsule and proximal tubules was seen 2 days after obstruction and involved the whole cortex 18 days later, with numerous cysts present throughout the kidney at term. The proliferation marker Ki-67 and transforming growth factor-beta (TGF-beta) were detected 2 days after obstruction and progressively increased in tubules, cysts, and the interstitium. In control kidneys, p38 was expressed in tubules only during the fetal stage, whereas phosphorylated extracellular signal-regulated kinase (P-ERK) was limited to ureteric buds and collecting ducts at all stages examined. However, Jun-N-terminal kinase (JNK) was absent in the fetal kidney but present in tubules at term. In obstructed kidneys, cyst epithelia were positive for p38 and P-ERK but negative for JNK throughout all stages. These studies show that P-ERK correlated spatially and temporally with Ki-67 and TGF-beta expression, which suggests that ERK may contribute to cyst formation and fibrosis in the obstructed fetal kidney.

Expression profiles of congenital renal dysplasia reveal new insights into renal development and disease.

Congenital renal dysplasia (RD) is a major cause of renal failure in the pediatric population. Although molecular and genetic aspects of RD have been studied in animal models, limited studies have been done in human RD primarily due to lack of available material. To identify novel genes that are associated with RD and normal kidney development, we performed microarray analysis on total RNA extracted from age-matched fetal kidneys of normal and RD patients. In midgestational RD kidneys, we found 180 upregulated and 104 downregulated transcripts compared with normal kidneys. Among the increased transcripts in the dysplastic kidneys were matrix-degrading enzymes (MMP7, MMP19, TIMP1), inflammation- and immunity-related genes, and growth factors. Expression of genes known to be essential for normal kidney development, such as WT1, BMP7, renin, angiotensin receptor 2 (AGTR2), SAL-like 1 (SALL1) and glypican 3 (GPC3), were decreased in dysplastic kidneys. Expression of selected gene products (BMP7, renin, and MMP7) was further confirmed in parallel sections and in several normal and human dysplastic kidneys, supporting the role of these genes as putative RD biomarkers. These results are among the first to reveal disrupted expression profiles during gestation in human RD patients.

Insertional mutagenesis in zebrafish: genes for development, genes for disease.

In order to rapidly identify a substantial fraction of the genes with a unique and essential role in vertebrate development, the laboratory of Nancy Hopkins at MIT has performed a large insertional mutagenesis screen in zebrafish using a pseudotyped retroviral vector as the mutagen. We have recovered mutations in about one-quarter of the embryonic essential genes in this organism, and have identified the mutated genes in nearly all of these (333). As the ease of gene identification allowed us to clone the mutated genes for nearly all of the mutants rather than prioritizing based upon the initially observed phenotypes, this has provided an unbiased view of the diversity of genes required for vertebrate development as well as a large collection of mutants to be screened for more specific phenotypes. In collaboration with other labs, we have screened the insertional mutant for the development of a variety of organs and cell types, as well as phenotypes that could represent disease models, such as cystic kidney and hepatomegaly. Furthermore, while all of these mutants are embryonic lethal in their homozygous state, we are investigating the heterozygous adults for additional phenotypes, such as cancer predisposition.

Meckel Gruber Syndrome--a case report.

Meckel Gruber Syndrome is a rare syndrome inherited as Mendelian autosomal recessive condition. The affected infant usually has a large occipital encephalocoele associated with renal cysts and sometimes polydactyly. The prognosis is poor. The affected child is still born or dies early in infancy. If diagnosis is done by prenatal ultrasound examination termination of pregnancy can be done.

Fetal obstructive uropathy: patterns of renal pathology.

Fetal obstructive uropathy (FOU) is characterized by obstruction of the urethra, renal anomalies, ureterovesical dilatation, oligohydramnios, cryptorchidism, and abdominal muscle wall changes. The main objective of the present study was to better understand the relationship between FOU and renal pathology using a series of 15 male autopsy cases. A total of 11 cases with patent anus and 4 with imperforate anus were analyzed. Of the first group, most cases showed obstruction at the level of prostatic urethra. Seven cases showed obstruction at the level of the prostatic urethra and histologic study revealed scarring and partial or complete absence of the prostate, while in the remaining four cases the prostate was present. Of the cases with imperforate anus, two showed obstruction at the level of prostatic urethra, one showed posterior urethral valves, and one was obstructed at the proximal urethra. In all cases the kidneys showed mixed (dysplastic and cystic) changes with no significant differences between the two groups. An inverse correlation was observed between degree of renal dysplasia and gestational age, whereas the opposite was true for cystic changes. Distal and collecting tubules were more intensely immunoreactive to the anti-cytokeratin antibody when compared to proximal tubules. Moreover, anti-cytokeratin immunoreactivity was more prominent in tubules displaying cystic dilatation. DNA fragmentation analysis of renal tissue revealed a higher apoptosis of mesenchymal and tubular cells in the FOU cases, compared to gestational aged-matched controls. These results suggest that renal anomalies in FOU might be related to the gestational age at which the injury occurred and to the duration of the obstruction.

Prenatal collapse of cysts in a dysplastic kidney.

Postnatal regression of prenatally or neonatally detected multicystic dysplastic kidney disease has been widely documented. However, renal cysts can regress during gestation, although they usually become larger in utero. We present a case of prenatally detected multicystic dysplastic kidney with an atypical course. During the third trimester, unilateral multicystic renal lesions in the fetus first enlarged and later involuted; by the second postpartum year, the kidney had become hypoplastic and nonfunctional. Our case also shows that before birth, blood flow in the affected kidney, measured with Doppler imaging, was normal until the cysts involuted.

Short-term urinary flow impairment deregulates PAX2 and PCNA expression and cell survival in fetal sheep kidneys.

Renal malformations account for most children with chronic renal failure and are often associated with urinary tract anatomical obstruction. We examined cellular and molecular events after experimental urinary flow impairment in fetal sheep. Ovine gestation lasts 144 to 150 days with the metanephros appearing at 27 to 30 days. We generated complete unilateral ureteric anatomical obstruction at 90 days when a few layers of glomeruli had formed. After 10 days, we recorded ureteric and pelvic dilatation with renal parenchymal weight greater than contralateral organs or those from unoperated fetuses. The nephrogenic cortex was replaced by disorganized cells separated by edema and prominent vascular spaces. Cortical histology was dominated by cysts associated with malformed glomerular tufts. Cystic epithelia expressed PAX2, a growth-stimulating transcription factor down-regulated during normal maturation, and proliferating cell nuclear antigen, a surrogate marker of cycling cells. Detection of apoptosis using propidium iodide and in situ end labeling showed a significant increase of the point prevalence of death in the obstructed cortex. Hence, PAX2 and proliferating cell nuclear antigen expression as well as death were deregulated, as we previously reported in human kidney malformations. Medullary collecting ducts and loops of Henle were also disrupted, correlating with impaired urinary dilution and sodium reabsorption. Therefore, complex aberrations of morphogenesis, gene expression, cell turnover, and urine composition occur relatively early after experimental impairment of fetal urinary flow.