Downregulation of Endothelin Receptor B Contributes to Defective B Cell Lymphopoiesis in Trisomy 21 Pluripotent Stem Cells.

Individuals with Trisomy 21 (T21) exhibit numerous hematological abnormalities, including reductions in numbers of circulating B and T lymphocytes. To elucidate molecular mechanisms underlying these phenotypes, we differentiated human isogenic disomic and trisomic pluripotent cells, and observed that trisomic cells showed defects in B cell, but not T cell differentiation. Global gene expression of differentiated, trisomic B cells revealed reduced expression of genes encoding endothelin signaling components, namely the Endothelin Receptor B (EDNRB), and its ligand Endothelin1 (EDN1). Depletion of EDNRB mRNA in cord blood-derived CD34+ cells led to defective B cell differentiation, supporting a hypothesis that low EDNRB expression in T21 contributes to intrinsic lymphoid defects. Further evidence for the role of the EDNRB pathway in B cell differentiation was obtained through CRISPR/Cas9 gene targeting in disomic and trisomic iPS cells. Knockout of EDNRB in both cell backgrounds reduced the capacity for B cell differentiation. Collectively, this work identifies downregulation of EDNRB as a causative factor for impaired B lymphocyte generation in trisomic cells, which may contribute to defects in immune function associated with T21. Furthermore, a novel role for endothelin signaling in regulation of B cell development has been identified.

Gene correction of HAX1 reversed Kostmann disease phenotype in patient-specific induced pluripotent stem cells.

Severe congenital neutropenia (SCN, Kostmann disease) is a heritable disorder characterized by a granulocytic maturation arrest. Biallelic mutations in HCLS1 associated protein X-1 (HAX1) are frequently detected in affected individuals, including those of the original pedigree described by Kostmann in 1956. To date, no faithful animal model has been established to study SCN mediated by HAX1 deficiency. Here we demonstrate defective neutrophilic differentiation and compensatory monocyte overproduction from patient-derived induced pluripotent stem cells (iPSCs) carrying the homozygous HAX1W44X nonsense mutation. Targeted correction of the HAX1 mutation using the CRISPR-Cas9 system and homologous recombination rescued neutrophil differentiation and reestablished an HAX1 and HCLS1-centered transcription network in immature myeloid progenitors, which is involved in the regulation of apoptosis, apoptotic mitochondrial changes, and myeloid differentiation. These findings made in isogenic iPSC-derived myeloid cells highlight the complex transcriptional changes underlying Kostmann disease. Thus, we show that patient-derived HAX1W44X -iPSCs recapitulate the Kostmann disease phenotype in vitro and confirm HAX1 mutations as the disease-causing monogenic lesion. Finally, our study paves the way for nonvirus-based gene therapy approaches in SCN.

Hematopoietic stem cells develop in the absence of endothelial cadherin 5 expression.

Rare endothelial cells in the aorta-gonad-mesonephros (AGM) transition into hematopoietic stem cells (HSCs) during embryonic development. Lineage tracing experiments indicate that HSCs emerge from cadherin 5 (Cdh5; vascular endothelial-cadherin)(+) endothelial precursors, and isolated populations of Cdh5(+) cells from mouse embryos and embryonic stem cells can be differentiated into hematopoietic cells. Cdh5 has also been widely implicated as a marker of AGM-derived hemogenic endothelial cells. Because Cdh5(-/-) mice embryos die before the first HSCs emerge, it is unknown whether Cdh5 has a direct role in HSC emergence. Our previous genetic screen yielded malbec (mlb(bw306)), a zebrafish mutant for cdh5, with normal embryonic and definitive blood. Using time-lapse confocal imaging, parabiotic surgical pairing of zebrafish embryos, and blastula transplantation assays, we show that HSCs emerge, migrate, engraft, and differentiate in the absence of cdh5 expression. By tracing Cdh5(-/-)green fluorescent protein (GFP)(+/+) cells in chimeric mice, we demonstrated that Cdh5(-/-)GFP(+/+) HSCs emerging from embryonic day 10.5 and 11.5 (E10.5 and E11.5) AGM or derived from E13.5 fetal liver not only differentiate into hematopoietic colonies but also engraft and reconstitute multilineage adult blood. We also developed a conditional mouse Cdh5 knockout (Cdh5(flox/flox):Scl-Cre-ER(T)) and demonstrated that multipotent hematopoietic colonies form despite the absence of Cdh5. These data establish that Cdh5, a marker of hemogenic endothelium in the AGM, is dispensable for the transition of hemogenic endothelium to HSCs.

Altered hematopoiesis in trisomy 21 as revealed through in vitro differentiation of isogenic human pluripotent cells.

Trisomy 21 is associated with hematopoietic abnormalities in the fetal liver, a preleukemic condition termed transient myeloproliferative disorder, and increased incidence of acute megakaryoblastic leukemia. Human trisomy 21 pluripotent cells of various origins, human embryonic stem (hES), and induced pluripotent stem (iPS) cells, were differentiated in vitro as a model to recapitulate the effects of trisomy on hematopoiesis. To mitigate clonal variation, we isolated disomic and trisomic subclones from the same parental iPS line, thereby generating subclones isogenic except for chromosome 21. Under differentiation conditions favoring development of fetal liver-like, γ-globin expressing, definitive hematopoiesis, we found that trisomic cells of hES, iPS, or isogenic origins exhibited a two- to fivefold increase in a population of CD43(+)(Leukosialin)/CD235(+)(Glycophorin A) hematopoietic cells, accompanied by increased multilineage colony-forming potential in colony-forming assays. These findings establish an intrinsic disturbance of multilineage myeloid hematopoiesis in trisomy 21 at the fetal liver stage.

A human stem cell model of early Alzheimer's disease pathology in Down syndrome.

Human cellular models of Alzheimer's disease (AD) pathogenesis would enable the investigation of candidate pathogenic mechanisms in AD and the testing and developing of new therapeutic strategies. We report the development of AD pathologies in cortical neurons generated from human induced pluripotent stem (iPS) cells derived from patients with Down syndrome. Adults with Down syndrome (caused by trisomy of chromosome 21) develop early-onset AD, probably due to increased expression of a gene on chromosome 21 that encodes the amyloid precursor protein (APP). We found that cortical neurons generated from iPS cells and embryonic stem cells from Down syndrome patients developed AD pathologies over months in culture, rather than years in vivo. These cortical neurons processed the transmembrane APP protein, resulting in secretion of the pathogenic peptide fragment amyloid-ß42 (Aß42), which formed insoluble intracellular and extracellular amyloid aggregates. Production of Aß peptides was blocked by a γ-secretase inhibitor. Finally, hyperphosphorylated tau protein, a pathological hallmark of AD, was found to be localized to cell bodies and dendrites in iPS cell-derived cortical neurons from Down syndrome patients, recapitulating later stages of the AD pathogenic process.

The role of CYP26 enzymes in defining appropriate retinoic acid exposure during embryogenesis.

Retinoic acid (RA) is a pleiotropic derivative of vitamin A, or retinol, which is responsible for all of the bioactivity associated with this vitamin. The teratogenic influences of vitamin A deficiency and excess RA in rodents were first observed more than 50 years ago. Efforts over the last 15-20 years have refined these observations by defining the molecular mechanisms that control RA availability and signaling during murine embryonic development. This review will discuss our current understanding of the role of RA in teratogenesis, with specific emphasis on the essential function of the RA catabolic CYP26 enzymes in preventing teratogenic consequences caused by uncontrolled distribution of RA. Particular focus will be paid to the RA-sensitive tissues of the caudal and cranial regions, the limb, and the testis, and how genetic mutation of factors controlling RA distribution have revealed important roles for RA during embryogenesis.

Analysis of Cyp26b1/Rarg compound-null mice reveals two genetically separable effects of retinoic acid on limb outgrowth.

The role of retinoic acid (RA) in limb development is unclear, although it has been suggested to be a proximalizing factor which plays a morphogenetic role in pattern formation. Exogenous RA produces a teratogenic effect on limb morphology; similarly, changes in the endogenous distribution of RA following genetic ablation of the RA-metabolizing enzyme, CYP26B1, result in phocomelia accompanied by changes in expression of proximo-distal (P-D) patterning genes, increased cell death, and delayed chondrocyte maturation. Here we show that disruption of RA receptor (RAR) gamma in a Cyp26b1(-/-) background is able to partially rescue limb skeletal morphology without restoring normal expression of proximo-distal patterning genes. We further show that embryos deficient in CYP26B1 exhibit early localized domains of mesenchymal cell death, which are reduced in compound-null animals. This model reveals two genetically separable effects of RA in the limb: an apoptotic effect mediated by RARgamma in the presence of ectopic RA, and a P-D patterning defect which is uncovered following the loss of both CYP26B1 and RARgamma. These data provide genetic evidence to clarify the roles of both RA and CYP26B1 in limb outgrowth and proximo-distal patterning.

Cyp26b1 expression in murine Sertoli cells is required to maintain male germ cells in an undifferentiated state during embryogenesis.

In mammals, germ cells within the developing gonad follow a sexually dimorphic pathway. Germ cells in the murine ovary enter meiotic prophase during embryogenesis, whereas germ cells in the embryonic testis arrest in G0 of mitotic cell cycle and do not enter meiosis until after birth. In mice, retinoic acid (RA) signaling has been implicated in controlling entry into meiosis in germ cells, as meiosis in male embryonic germ cells is blocked by the activity of a RA-catabolizing enzyme, CYP26B1. However, the mechanisms regulating mitotic arrest in male germ cells are not well understood. Cyp26b1 expression in the testes begins in somatic cells at embryonic day (E) 11.5, prior to mitotic arrest, and persists throughout fetal development. Here, we show that Sertoli cell-specific loss of CYP26B1 activity between E15.5 and E16.5, several days after germ cell sex determination, causes male germ cells to exit from G0, re-enter the mitotic cell cycle and initiate meiotic prophase. These results suggest that male germ cells retain the developmental potential to differentiate in meiosis until at least at E15.5. CYP26B1 in Sertoli cells acts as a masculinizing factor to arrest male germ cells in the G0 phase of the cell cycle and prevents them from entering meiosis, and thus is essential for the maintenance of the undifferentiated state of male germ cells during embryonic development.

Genetic disruption of CYP26B1 severely affects development of neural crest derived head structures, but does not compromise hindbrain patterning.

Cyp26b1 encodes a cytochrome-P450 enzyme that catabolizes retinoic acid (RA), a vitamin A derived signaling molecule. We have examined Cyp26b1(-/-) mice and report that mutants exhibit numerous abnormalities in cranial neural crest cell derived tissues. At embryonic day (E) 18.5 Cyp26b1(-/-) animals exhibit a truncated mandible, abnormal tooth buds, reduced ossification of calvaria, and are missing structures of the maxilla and nasal process. Some of these abnormalities may be due to defects in formation of Meckel's cartilage, which is truncated with an unfused distal region at E14.5 in mutant animals. Despite the severe malformations, we did not detect any abnormalities in rhombomere segmentation, or in patterning and migration of anterior hindbrain derived neural crest cells. Abnormal migration of neural crest cells toward the posterior branchial arches was observed, which may underlie defects in larynx and hyoid development. These data suggest different periods of sensitivity of anterior and posterior hindbrain neural crest derivatives to elevated levels of RA in the absence of CYP26B1.

Apoptotic extinction of germ cells in testes of Cyp26b1 knockout mice.

Cyp26b1 encodes a retinoic acid (RA) metabolizing cytochrome P450 enzyme that is expressed in embryonic tissues undergoing morphogenesis, including the testes. We have generated transgenic mice lacking Cyp26b1 and have observed increased RA levels in embryonic testes. Cyp26b1(-/-) germ cells prematurely enter meiosis at embryonic d 13.5 and appear to arrest at pachytene stage. Furthermore, after embryonic d 13.5, a rapid increase in apoptosis is observed in male germ cells derived from Cyp26b1(-/-) embryos; germ cells are essentially absent in mutant male neonates. In contrast, testicular somatic cells appear to develop normally in the absence of Cyp26b1. Moreover, ovarian germ and somatic cells appear unaffected by the lack of CYP26B1. We also show that the synthetic retinoid Am580, which is resistant to CYP26 metabolism, induces meiosis of male germ cells in cultured gonads, suggesting that abnormal development of germ cells in the Cyp26b1(-/-) testes results from excess RA rather than the absence of CYP26B1-generated metabolites of RA. These results provide evidence that CYP26B1 maintains low levels of RA in the developing testes that blocks entry into meiosis and acts as a survival factor to prevent apoptosis of male germ cells.

Transcriptional co-operativity between distant retinoic acid response elements in regulation of Cyp26A1 inducibility.

Cyp26A1 encodes an RA (retinoic acid)-catabolizing CYP (cytochrome P450) protein that plays a critical role in regulating RA distribution in vivo. Cyp26A1 expression is inducible by RA, and the locus has previously been shown to contain a RARE (RA response element), R1, within the minimal promoter [Loudig, Babichuk, White, Abu-Abed, Mueller and Petkovich (2000) Mol. Endocrinol. 14, 1483-1497]. In the present study, we report the identification of a second functional RARE (R2) located 2.0 kb upstream of the Cyp26A1 transcriptional start site. Constructs containing murine sequences encompassing both R1 and R2 showed that these elements work together to generate higher transcriptional activity upon treatment with RA than those containing R1 alone. Inclusion of R2 also dramatically enhanced the sensitivity of reporter constructs to RA, as even treatment with 10(-8) M RA resulted in a 5-fold induction of reporter activity. Mutational analysis identified R2 as the functional element responsible for the increased RA inducibility of promoter constructs. The element was shown to bind RARgamma (RA receptor gamma)/RXRalpha (retinoid X receptor alpha) heterodimers in vitro, and inclusion of nuclear receptors in transfections boosted the transcriptional response. A construct containing both R1 and R2 was used to generate a stable luciferase reporter cell line that can be used as a tool to identify factors regulating Cyp26A1 expression. The analysis of R1 and R2 has led to the proposal that the two elements work synergistically to provide a maximal response to RA and that R2 is an upstream enhancer.

Developing with lethal RA levels: genetic ablation of Rarg can restore the viability of mice lacking Cyp26a1.

We have previously reported that the retinoic acid (RA) catabolizing enzyme CYP26A1 plays an important role in protecting tail bud tissues from inappropriate exposure to RA generated in the adjacent trunk tissues by RALDH2, and that Cyp26a1-null animals exhibit spina bifida and caudal agenesis. We now show that, in the absence of Cyp26a1, retinoic acid receptor gamma (RARgamma) mediates ectopic RA-signaling in the tail bud. We also show that activated RARgamma results in downregulation of Wnt3a and Fgf8, which integrate highly conserved signaling pathways known for their role in specifying caudal morphogenesis. Ablation of the gene for RARgamma (Rarg) rescues Cyp26a1-null mutant animals from caudal regression and embryonic lethality, thus demonstrating that CYP26A1 suppresses the RA-mediated downregulation of WNT3A and FGF8 signaling pathways by eliminating ectopic RA in gastrulating tail bud mesoderm.

Differential expression of the retinoic acid-metabolizing enzymes CYP26A1 and CYP26B1 during murine organogenesis.

We recently cloned the murine homologue of Cyp26B1, a novel retinoic acid (RA)-metabolizing enzyme and showed that its gene expression pattern is unique from that of Cyp26A1 during early embryogenesis. Here, we complete this comparative expression analysis from embryonic day (E) 12 to postnatal stages. Cyp26B1 expression was found in developing tendons and precartilaginous elements and in perichondrium by E14.5, while Cyp26A1 expression was restricted to extremities of rib and vertebral cartilage. Cyp26A1 and Cyp26B1 were expressed, in the distal epithelium and mesenchyme of the limbs and genital tubercle, respectively. High Cyp26B1 expression was found in craniofacial areas undergoing morphogenetic growth, whereas Cyp26A1 message was restricted to the mouth and dental epithelium. Cyp26A1 alone was expressed in the developing neural retina, while both genes were co-expressed in the retinal pigment epithelium. Cyp26B1 was specifically expressed in the developing hindbrain (pons, cerebellum) and forebrain (striatum, hippocampus), with forebrain expression persisting postnatally. In addition, Cyp26B1 was expressed at specific levels of the differentiating upper and lower thoracic spinal cord, adjacent to the cervical and lumbar regions that express the RA-synthesizing enzyme RALDH-2. In viscera, Cyp26B1 transcripts were detected in the developing lung, kidney, spleen, thymus and testis, whereas Cyp26A1 transcripts were found in the diaphragm and outer stomach mesenchyme. Cyp26B1 was also specifically expressed in dermis surrounding the developing hair follicles. Regulated RA metabolism may therefore be required in many developing systems.