Ashleigh E. Schaffer

Sox9+ ductal cells are multipotent progenitors throughout development but do not produce new endocrine cells in the normal or injured adult pancreas.

One major unresolved question in the field of pancreas biology is whether ductal cells have the ability to generate insulin-producing β-cells. Conclusive examination of this question has been limited by the lack of appropriate tools to efficiently and specifically label ductal cells in vivo. We generated Sox9CreERT2 mice, which, during adulthood, allow for labeling of an average of 70% of pancreatic ductal cells, including terminal duct/centroacinar cells. Fate-mapping studies of the Sox9+ domain revealed endocrine and acinar cell neogenesis from Sox9+ cells throughout embryogenesis. Very small numbers of non-β endocrine cells continue to arise from Sox9+ cells in early postnatal life, but no endocrine or acinar cell neogenesis from Sox9+ cells occurs during adulthood. In the adult pancreas, pancreatic injury by partial duct ligation (PDL) has been suggested to induce β-cell regeneration from a transient Ngn3+ endocrine progenitor cell population. Here, we identify ductal cells as a cell of origin for PDL-induced Ngn3+ cells, but fail to observe β-cell neogenesis from duct-derived cells. Therefore, although PDL leads to activation of Ngn3 expression in ducts, PDL does not induce appropriate cues to allow for completion of the entire β-cell neogenesis program. In conclusion, although endocrine cells arise from the Sox9+ ductal domain throughout embryogenesis and the early postnatal period, Sox9+ ductal cells of the adult pancreas no longer give rise to endocrine cells under both normal conditions and in response to PDL.

Exome Sequencing Can Improve Diagnosis and Alter Patient Management

Exome sequencing of 118 patients with neurodevelopmental disorders shows that this technique is useful for identifying new pathogenic mutations and for correcting diagnosis in ~10% of cases. A Needle in a Haystack Exome sequencing enables evaluation of all protein-coding variants in an individual genome and promises to revolutionize the practice of clinical genetics as it moves from the lab into the clinic. Bringing this technology to the clinic affords the opportunity not just to identify new disease-causing mutations but also to clarify disease presentation and diagnosis. There are many challenges to implementing this technology, however, including which patients to select for analysis, how to rank and prioritize the genetic variants, and how to align the data with the clinical record. In new work, Dixon-Salazar et al. studied a cohort of 118 probands with genetic forms of neurodevelopmental disease, all derived from consanguineous unions, using exome sequencing. All patients were previously excluded for genes most likely to cause their disease. The authors analyzed the exome sequences with a standardized bioinformatic pipeline. They found mutations in known disease-causing genes that in about 10% of cases led to a change in the underlying diagnosis. In 19% of cases, they identified mutations in genes not previously linked to disease. In the remaining cases, the genetic causes remained elusive. Thus, exome sequencing may both improve diagnosis and lead to alterations in patient management in some patients with neurodevelopmental disorders. However, analysis of more than one individual will be required to increase the success rate of identifying the causative mutation in most cases. The translation of “next-generation” sequencing directly to the clinic is still being assessed but has the potential for genetic diseases to reduce costs, advance accuracy, and point to unsuspected yet treatable conditions. To study its capability in the clinic, we performed whole-exome sequencing in 118 probands with a diagnosis of a pediatric-onset neurodevelopmental disease in which most known causes had been excluded. Twenty-two genes not previously identified as disease-causing were identified in this study (19% of cohort), further establishing exome sequencing as a useful tool for gene discovery. New genes identified included EXOC8 in Joubert syndrome and GFM2 in a patient with microcephaly, simplified gyral pattern, and insulin-dependent diabetes. Exome sequencing uncovered 10 probands (8% of cohort) with mutations in genes known to cause a disease different from the initial diagnosis. Upon further medical evaluation, these mutations were found to account for each proband’s disease, leading to a change in diagnosis, some of which led to changes in patient management. Our data provide proof of principle that genomic strategies are useful in clarifying diagnosis in a proportion of patients with neurodevelopmental disorders.

Nkx6.1 controls a gene regulatory network required for establishing and maintaining pancreatic Beta cell identity.

All pancreatic endocrine cell types arise from a common endocrine precursor cell population, yet the molecular mechanisms that establish and maintain the unique gene expression programs of each endocrine cell lineage have remained largely elusive. Such knowledge would improve our ability to correctly program or reprogram cells to adopt specific endocrine fates. Here, we show that the transcription factor Nkx6.1 is both necessary and sufficient to specify insulin-producing beta cells. Heritable expression of Nkx6.1 in endocrine precursors of mice is sufficient to respecify non-beta endocrine precursors towards the beta cell lineage, while endocrine precursor- or beta cell-specific inactivation of Nkx6.1 converts beta cells to alternative endocrine lineages. Remaining insulin+ cells in conditional Nkx6.1 mutants fail to express the beta cell transcription factors Pdx1 and MafA and ectopically express genes found in non-beta endocrine cells. By showing that Nkx6.1 binds to and represses the alpha cell determinant Arx, we identify Arx as a direct target of Nkx6.1. Moreover, we demonstrate that Nkx6.1 and the Arx activator Isl1 regulate Arx transcription antagonistically, thus establishing competition between Isl1 and Nkx6.1 as a critical mechanism for determining alpha versus beta cell identity. Our findings establish Nkx6.1 as a beta cell programming factor and demonstrate that repression of alternative lineage programs is a fundamental principle by which beta cells are specified and maintained. Given the lack of Nkx6.1 expression and aberrant activation of non-beta endocrine hormones in human embryonic stem cell (hESC)–derived insulin+ cells, our study has significant implications for developing cell replacement therapies.

CLP1 founder mutation links tRNA splicing and maturation to cerebellar development and neurodegeneration.

Neurodegenerative diseases can occur so early as to affect neurodevelopment. From a cohort of more than 2,000 consanguineous families with childhood neurological disease, we identified a founder mutation in four independent pedigrees in cleavage and polyadenylation factor I subunit 1 (CLP1). CLP1 is a multifunctional kinase implicated in tRNA, mRNA, and siRNA maturation. Kinase activity of the CLP1 mutant protein was defective, and the tRNA endonuclease complex (TSEN) was destabilized, resulting in impaired pre-tRNA cleavage. Germline clp1 null zebrafish showed cerebellar neurodegeneration that was rescued by wild-type, but not mutant, human CLP1 expression. Patient-derived induced neurons displayed both depletion of mature tRNAs and accumulation of unspliced pre-tRNAs. Transfection of partially processed tRNA fragments into patient cells exacerbated an oxidative stress-induced reduction in cell survival. Our data link tRNA maturation to neuronal development and neurodegeneration through defective CLP1 function in humans.

The transcription factors Nkx6.1 and Nkx6.2 possess equivalent activities in promoting beta-cell fate specification in Pdx1+ pancreatic progenitor cells.

Despite much progress in identifying transcriptional regulators that control the specification of the different pancreatic endocrine cell types, the spatiotemporal aspects of endocrine subtype specification have remained largely elusive. Here, we address the mechanism by which the transcription factors Nkx6.1 (Nkx6-1) and Nkx6.2 (Nkx6-2) orchestrate development of the endocrine alpha- and beta-cell lineages. Specifically, we assayed for the rescue of insulin-producing beta-cells in Nkx6.1 mutant mice upon restoring Nkx6 activity in select progenitor cell populations with different Nkx6-expressing transgenes. Beta-cell formation and maturation was restored when Nkx6.1 was expressed in multipotential Pdx1+ pancreatic progenitors, whereas no rescue was observed upon expression in committed Ngn3+ (Neurog3+) endocrine progenitors. Although not excluding additional roles downstream of Ngn3, this finding suggests a first requirement for Nkx6.1 in specifying beta-cell progenitors prior to Ngn3 activation. Surprisingly, although Nkx6.2 only compensates for Nkx6.1 in alpha-but not in beta-cell development in Nkx6.1-/- mice, a Pdx1-promoter-driven Nkx6.2 transgene had the same ability to rescue beta-cells as the Pdx1-Nkx6.1 transgene. This demonstrates that the distinct requirements for Nkx6.1 and Nkx6.2 in endocrine differentiation are a consequence of their divergent spatiotemporal expression domains rather than their biochemical activities and implies that both Nkx6.1 and Nkx6.2 possess alpha- and beta-cell-specifying activities.

Biallelic mutations in SNX14 cause a syndromic form of cerebellar atrophy and lysosome-autophagosome dysfunction

Pediatric-onset ataxias often present clinically as developmental delay and intellectual disability, with prominent cerebellar atrophy as a key neuroradiographic finding. Here we describe a new clinically distinguishable recessive syndrome in 12 families with cerebellar atrophy together with ataxia, coarsened facial features and intellectual disability, due to truncating mutations in the sorting nexin gene SNX14, encoding a ubiquitously expressed modular PX domain–containing sorting factor. We found SNX14 localized to lysosomes and associated with phosphatidylinositol (3,5)-bisphosphate, a key component of late endosomes/lysosomes. Patient-derived cells showed engorged lysosomes and a slower autophagosome clearance rate upon autophagy induction by starvation. Zebrafish morphants for snx14 showed dramatic loss of cerebellar parenchyma, accumulation of autophagosomes and activation of apoptosis. Our results characterize a unique ataxia syndrome due to biallelic SNX14 mutations leading to lysosome-autophagosome dysfunction.

An AKT3-FOXG1-reelin network underlies defective migration in human focal malformations of cortical development

Focal malformations of cortical development (FMCDs) account for the majority of drug-resistant pediatric epilepsy. Postzygotic somatic mutations activating the phosphatidylinositol-4,5-bisphosphate-3-kinase (PI3K)–protein kinase B (AKT)–mammalian target of rapamycin (mTOR) pathway are found in a wide range of brain diseases, including FMCDs. It remains unclear how a mutation in a small fraction of cells disrupts the architecture of the entire hemisphere. Within human FMCD-affected brain, we found that cells showing activation of the PI3K-AKT-mTOR pathway were enriched for the AKT3E17K mutation. Introducing the FMCD-causing mutation into mouse brain resulted in electrographic seizures and impaired hemispheric architecture. Mutation-expressing neural progenitors showed misexpression of reelin, which led to a non–cell autonomous migration defect in neighboring cells, due at least in part to derepression of reelin transcription in a manner dependent on the forkhead box (FOX) transcription factor FOXG1. Treatments aimed at either blocking downstream AKT signaling or inactivating reelin restored migration. These findings suggest a central AKT-FOXG1-reelin signaling pathway in FMCD and support pathway inhibitors as potential treatments or therapies for some forms of focal epilepsy.

Mutations in KATNB1 Cause Complex Cerebral Malformations by Disrupting Asymmetrically Dividing Neural Progenitors

Exome sequencing analysis of over 2,000 children with complex malformations of cortical development identified five independent (four homozygous and one compound heterozygous) deleterious mutations in KATNB1, encoding the regulatory subunit of the microtubule-severing enzyme Katanin. Mitotic spindle formation is defective in patient-derived fibroblasts, a consequence of disrupted interactions of mutant KATNB1 with KATNA1, the catalytic subunit of Katanin, and other microtubule-associated proteins. Loss of KATNB1 orthologs in zebrafish (katnb1) and flies (kat80) results in microcephaly, recapitulating the human phenotype. In the developing Drosophila optic lobe, kat80 loss specifically affects the asymmetrically dividing neuroblasts, which display supernumerary centrosomes and spindle abnormalities during mitosis, leading to cell cycle progression delays and reduced cell numbers. Furthermore, kat80 depletion results in dendritic arborization defects in sensory and motor neurons, affecting neural architecture. Taken together, we provide insight into the mechanisms by which KATNB1 mutations cause human cerebral cortical malformations, demonstrating its fundamental role during brain development.

A homozygous IER3IP1 mutation causes microcephaly with simplified gyral pattern, epilepsy, and permanent neonatal diabetes syndrome (MEDS).

Wolcott–Rallison syndrome (WRS) and the recently delineated microcephaly with simplified gyration, epilepsy, and permanent neonatal diabetes syndrome (MEDS) are clinically overlapping autosomal recessive disorders characterized by early onset diabetes, skeletal defects, and growth retardation. While liver and renal symptoms are more severe in WRS, neurodevelopmental characteristics are more pronounced in MEDS patients, in which microcephaly and uncontrolled epilepsy are uniformly present. Mutations in the EIF2AK3 gene were described in patients with WRS and defects in this gene lead to increased susceptibility to apoptotic cell death. Mutations in IER3IP1 have been reported in patients with MEDS and similarly, loss of activity results in apoptosis of neurons and pancreatic beta cells in patients. Here we report on a homozygous mutation of the IER3IP1 gene in four patients from two unrelated consanguineous Egyptian families presenting with MEDS who display burst suppression patterns on EEG. All patients presented with mildly elevated liver enzymes, microalbuminuria, and skeletal changes such as scoliosis and osteopenia, leading to repeated bone fractures. We expand the phenotypic spectrum of MEDS caused by IER3IP1 gene mutations and propose that WRS and MEDS are overlapping clinical syndromes, displaying significant gene‐dependent clinical variability.

Biallelic mutations in the 3′ exonuclease TOE1 cause pontocerebellar hypoplasia and uncover a role in snRNA processing

Deadenylases are best known for degrading the poly(A) tail during mRNA decay. The deadenylase family has expanded throughout evolution and, in mammals, consists of 12 Mg2+-dependent 3′-end RNases with substrate specificity that is mostly unknown. Pontocerebellar hypoplasia type 7 (PCH7) is a unique recessive syndrome characterized by neurodegeneration and ambiguous genitalia. We studied 12 human families with PCH7, uncovering biallelic, loss-of-function mutations in TOE1, which encodes an unconventional deadenylase. toe1-morphant zebrafish displayed midbrain and hindbrain degeneration, modeling PCH-like structural defects in vivo. Surprisingly, we found that TOE1 associated with small nuclear RNAs (snRNAs) incompletely processed spliceosomal. These pre-snRNAs contained 3′ genome-encoded tails often followed by post-transcriptionally added adenosines. Human cells with reduced levels of TOE1 accumulated 3′-end-extended pre-snRNAs, and the immunoisolated TOE1 complex was sufficient for 3′-end maturation of snRNAs. Our findings identify the cause of a neurodegenerative syndrome linked to snRNA maturation and uncover a key factor involved in the processing of snRNA 3′ ends.