Eya1 is essential for branchial arch segmentation and branchial epithelium development through regulating Notch signaling pathway

Student Oral Presentation Session 3Craniofacial anomalies are common features of Branchio-Oto-Renal (BOR) syndrome patients. Mutations in the Eya1 gene have been found in around half of the BOR patients, but the pathogenic mechanisms mediated by Eya1 in the craniofacial malformations remain unknown. In this study, we use Eya1 mutant mice as a disease model to study the abnormal early branchial arch (BA) development. Eya1-/- mutant embryos have hypoplastic BA2. The formation of branchial cleft was severely affected. Interestingly, Notch signaling was down-regulated in the mutant branchial ...postprin

Statistical issues and approaches in endophenotype research

This special topic comprises 9 papers which were presented at the Symposium. (Chinese Science Bulletin, 2011, v. 56 n. 32 Editorial. doi: 10.1007/s11434-011-4716-4)This journal issue entitled: SPECIAL TOPIC Endophenotype Strategies for the Study of Neuropsychiatric DisordersThe endophenotype concept was initially proposed to enhance the power of genetic studies of complex disorders. It is closely related to the genetic component in a liability-threshold model; a perfect endophenotype should have a correlation of 1 with the genetic component of the liability to disease. In reality, a putative endophenotype is unlikely to be a perfect representation of the genetic component of disease liability. The magnitude of the correlation between a putative endophenotype and the genetic component of disease liability can be estimated by fitting multivariate genetic models to twin data. A number of statistical methods have been developed for incorporating endophenotypes in genetic linkage and association analyses with the aim of improving statistical power. The most recent of such methods can handle multiple endophenotypes simultaneously for the greatest increase in power. In addition to increasing statistical power, endophenotype research plays an important role in helping to understand the mechanisms which connect the associated genetic variants with disease occurrence. Novel statistical approaches may be required for the analysis of the complex relationships between endophenotypes at different levels and how they converge to cause the occurrence of disease. © 2011 Science China Press and Springer-Verlag Berlin Heidelberg.published_or_final_versio

Sufu defines the balance of hindbrain progenitor cells maintenance and differentiation

Suppressor of fused (Sufu) was identified as a regulator in Hedgehog signalling. Study shown that Sufu knockout mice were embryonic lethal at E9.5, exhibiting cephalic deformities, open neural tube and ventralized spinal cord resulting from ectopic Shh signalling, implying indispensable role of Sufu during development of central nervous system. Aiming to investigate the functions of Sufu in hindbrain neurogenesis, we used B2‐r4‐Cre to knock‐out Sufu in rhombomere4 (r4). We observed significant enlargement of mutant r4 size from E10.5, exhibiting more profound expansion in the dorsal region at E12.5. Accordingly, BrdU pulse labelling and sox2 staining showed region specific increased accumulation of proliferative progenitor cells, indicating differential maintenance of progenitor pools along the dorsoventral axis of r4. Tuj1 staining also showed impaired differentiation of the ectopic progenitor cells. Further analysis revealed dramatic dorsal expansion of pMN and p2 progenitor domains in mutant r4. Surprisingly, the FoxA2 positive floor plate, and the dorsal p0 domain were not severely affected, suggesting a novel domain specific regulation of neural progenitor pools by Sufu. Intriguingly, we observed spatial upregulation Gli1 and Gli2 transcription factors, selectively at the region that resides highly proliferative cells, implying that the increased cell proliferation could be caused by the changes of Gli transcription factors. Indeed, concomitant deletion of Gli2 in the Sufu mutant largely rescued the aberrant phenotypes. These findings clearly showed the requirement of Sufu to suppress Gli2 to conduct a domain specific regulation of hindbrain progenitor maintenance and differentiation. Our study demonstrates novel function of Sufu to ensure a tightly controlled progenitor pools maintenance and differentiation, mainly achieve by suppressing Gli2 activation.postprin

Development of a micromanipulator-based loading device for mechanoregulation study of human mesenchymal stem cells in three-dimensional collagen constructs

Mechanical signal is important for regulating cellular activities, including proliferation, metabolism, matrix production, and orientation. Bioreactors with loading functions can be used to precondition cells in three-dimensional (3D) constructs so as to study the cellular responses to mechanical stimulation. However, full-scale bioreactor is not always an affordable option considering the high cost of equipments and the liter-sized medium with serum and growth factor supplements. In this study, a custom-built loading system was developed by coupling a conventional camera-equipped inverted research microscope with two micromanipulators. The system was programmed to deliver either cyclic compressive loading with different frequencies or static compressive loading for 1 week to investigate the cellular responses of human mesenchymal stem cells (hMSCs) entrapped in a 3D construct consists of reconstituted collagen fibers. Cellular properties, including their alignment, cytoskeleton, and cell metabolism, and properties of matrix molecules, such as collagen fiber alignment and glycosaminoglycan deposition, were evaluated. Using a MatLab-based image analysis program, reorientation of the entrapped cells from a random distribution to a preferred alignment along the loading direction in constructs with both static and cyclic compression has been demonstrated, but no such alignment was found in the free-floating controls. Fluorescent staining on filamentous actin cytoskeleton also confirmed the finding. Nevertheless, the collagen fiber meshwork entrapping the hMSCs remained randomly distributed, and no change in cellular metabolism and glycosaminoglycans production was noted. The current study provides a simple and affordable option toward setting up a mechanoregulation facility based on existing laboratory equipments and sheds new insights on the effect of mechanical loading on the alignment of hMSCs in 3D collagen constructs. Copyright © 2010, Mary Ann Liebert, Inc.published_or_final_versio

Sox10 regulates enteric neural crest cell migration in the developing gut

Concurrent Sessions 1: 1.3 - Organs to organisms: Models of Human Diseases: abstract no. 1417th ISDB 2013 cum 72nd Annual Meeting of the Society for Developmental Biology, VII Latin American Society of Developmental Biology Meeting and XI Congreso de la Sociedad Mexicana de Biologia del Desarrollo. The Conference's web site is located at http://www.inb.unam.mx/isdb/Sox10 is a HMG-domain containing transcription factor which plays important roles in neural crest cell survival and differentiation. Mutations of Sox10 have been identified in patients with Waardenburg-Hirschsprung syndrome, who suffer from deafness, pigmentation defects and intestinal aganglionosis. Enteric neural crest cells (ENCCs) with Sox10 mutation undergo premature differentiation and fail to colonize the distal hindgut. It is unclear, however, whether Sox10 plays a role in the migration of ENCCs. To visualize the migration behaviour of mutant ENCCs, we generated a Sox10NGFP mouse model where EGFP is fused to the N-terminal domain of Sox10. Using time-lapse imaging, we found that ENCCs in Sox10NGFP/+ mutants displays lower migration speed and altered trajectories compared to normal controls. This behaviour was cell-autonomous, as shown by organotypic grafting of Sox10NGFP/+ gut segments onto control guts and vice versa. ENCCs encounter different extracellular matrix (ECM) molecules along the developing gut. We performed gut explant culture on various ECM and found that Sox10NGFP/+ ENCCs tend to form aggregates, particularly on fibronectin. Time-lapse imaging of single cells in gut explant culture indicated that the tightly-packed Sox10 mutant cells failed to exhibit contact inhibition of locomotion. We determined the expression of adhesion molecule families by qPCR analysis, and found integrin expression unaffected while L1-cam and selected cadherins were altered, suggesting that Sox10 mutation affects cell adhesion properties of ENCCs. Our findings identify a de novo role of Sox10 in regulating the migration behaviour of ENCCs, which has important implications for the treatment of Hirschsprung disease.postprin

Distribution of carbon monoxide-producing neurons in human colon and in Hirschsprung's disease patients

Hirschsprung's disease (HSCR) is characterized by the absence of ganglion cells and impaired relaxation of the gut. Nitric oxide (NO) and, more recently, carbon monoxide (CO) have been identified as inhibitory neurotransmitters causing relaxation. A deficiency in NO has been reported in aganglionic gut; we hypothesized that CO could also be involved in impaired gut motility in HSCR. The aim of the study was to determine the distribution of CO-and NO-producing enzymes in the normal and aganglionic gut. We performed laser capture microdissection, reverse transcription-polymerase chain reaction, and immunohistochemistry on colon biopsies of normal controls (n = 9) and patients with HSCR (n = 10). The mRNA expression of heme oxygenase-2 (HO-2), immunoreactivities of HO-2 and NO synthase, was determined and compared. Results show a high level of expression of HO-2 mRNA localized in the myenteric plexus. Expression of HO-2 mRNA was also detected in the mucosa, submucosa, and muscular layer. Down-regulation of HO-2 mRNA expression was detected in the aganglionic colon. Immunoreactivities of HO-2 and NO synthase were localized mainly to the ganglion plexus and to nerve fibers within the muscle in the control colons and normoganglionic colons. HO-2-containing neurons were more abundant than NO synthase-containing neurons in the myenteric plexus. Nearly all of the NO synthase-containing neurons also contained HO-2. HO-2 and NO synthase were selectively absent in the myenteric and submucosal regions and in the muscle of the aganglionic colon. Our findings suggest involvement of both CO and NO in the pathophysiology of HSCR. Copyright 2002, Elsevier Science (USA). All rights reserved.postprin

Sonic hedgehog regulates the proliferation, differentiation, and migration of enteric neural crest cells in gut

Enteric neural crest cells (NCCs) migrate and colonize the entire gut and proliferate and differentiate into neurons and glia of the enteric nervous system in vertebrate embryos. We have investigated the mitogenic and morphogenic functions of Sonic hedgehog (Shh) on enteric NCCs in cell and organ culture. Enteric NCCs expressed Shh receptor Patched and transcripts encoding the Shh signal transducer (Gli1). Shh promoted the proliferation and inhibited the differentiation of NCCs. The pro-neurogenic effect of glial cell line-derived neurotrophic factor (GDNF) on NCCs was abolished by Shh. In gut explants, NCCs migrated from the explants onto the adjacent substratum if GDNF was added, whereas addition of Shh abolished this migration. Neuronal differentiation and coalescence of neural crest-derived cells into myenteric plexuses in explants was repressed by the addition of Shh. Our data suggest that Shh controls the proliferation and differentiation of NCCs and modulates the responsiveness of NCCs toward GDNF inductions.published_or_final_versio

Analysis of craniofacial defects in Six1/Eya1-associated Branchio-Oto-Renal Syndrome

Poster Session I - Morphogenesis: 205/B10117th ISDB 2013 cum 72nd Annual Meeting of the Society for Developmental Biology, 7th Latin American Society of Developmental Biology Meeting and 11th Congreso de la Sociedad Mexicana de Biologia del Desarrollo.Branchio-Oto-Renal (BOR) syndrome patients exhibit craniofacial and renal anomalies as well as deafness. BOR syndrome is caused by mutations in Six1 or Eya1, both of which regulate cell proliferation and differentiation. The molecular mechanism underlying the craniofacial and branchial arch (BA) defects in BOR syndrome is unclear. We have found that Hoxb3 is up-regulated in the second branchial arch (BA2) of Six1-/- mutants. Moreover, Hoxb3 over-expression in transgenic mice leads to BA abnormalities which are similar to the BA defects in Six1-/- or Eya1-/- mutants, suggesting a regulatory relationship among Six1, Eya1 and Hoxb3 genes. The aim of this study is to investigate the molecular mechanism underlying abnormal BA development in BOR syndrome using Six1 and Eya1 mutant mice. Two potential Six1 binding sites were identified on the Hoxb3 gene. In vitro and in vivo Chromatin IP assays showed that Six1 could directly bind to one of the sites specifically. Furthermore, using a chick in ovo luciferase assay we showed that Six1 could suppress gene expression through one of the specific binding sites. On the other hand, in Six1-/- mutants, we found that the Notch ligand Jag1 was up-regulated in BA2. Similarly, in Hoxb3 transgenic mice, ectopic expression of Jag1 could be also detected in BA2. To investigate the activation of Notch signaling pathway, we found that Notch intracellular domain (NICD), a direct indicator of Notch pathway activation, was up-regulated in BAs of Six1-/-; Eya1-/- double mutants. Our results indicate that Hoxb3 and Notch signaling pathway are involved in mediating the craniofacial defects of Six1/Eya1-associated Branchio-Oto-Renal Syndrome.postprin

Cyp26b1 mediates differential regulation of RA signaling in neural progenitor populations along the anteriorposterior axis of the adult spinal cord

Poster Session - Neural Regeneration: no. 64DMM 2011 entitled: Re-engineering Regenerative MedicineNeural stem cells from the adult subventricular zone (SVZ) are highly heterogeneous, with their position of origin being a key factor in determining the neuronal subtype they can give rise to. Whether this diversity extends to other regions in the adult CNS has not been demonstrated. In vitro studies with directed neuronal differentiation of ES cells suggest that subtype specification may be regulated by the positional identity present in the ES‐derived cell, since altering the positional identity leads to corresponding changes in motor neuron subtype. This limited plasticity suggests the position identity of the original stem cell source is a critical factor for the generation of the desired neuronal subtype. The adult spinal cord consists of endogenous stem/progenitor cells which are …postprin

Cyp26b1 mediates differential neurogenicity in axial-specific populations of adult spinal cord progenitor cells

Utilization of endogenous adult spinal cord progenitor cells (SCPCs) for neuronal regeneration is a promising strategy for spinal cord repair. To mobilize endogenous SCPCs for injury repair, it is necessary to understand their intrinsic properties and to identify signaling factors that can stimulate their neurogenic potential. In this study, we demonstrate that adult mouse SCPCs express distinct combinatorial Hox genes and exhibit axial-specific stem cell properties. Lumbar-derived neurospheres displayed higher primary sphere formation and greater neurogenicity compared with cervical- and thoracic-derived neurospheres. To further understand the mechanisms governing neuronal differentiation of SCPCs from specific axial regions, we examined the neurogenic responses of adult SCPCs to retinoic acid (RA), an essential factor for adult neurogenesis. Although RA is a potent inducer of neuronal differentiation, we found that RA enhanced the generation of neurons specifically in cervical- but not lumbar-derived cells. We further demonstrate that the differential RA response was mediated by the RA-degrading enzyme cytochrome P450 oxidase b1 Cyp26b1. Lumbar cells express high levels of Cyp26b1 and low levels of the RA-synthesizing enzyme retinaldehyde dehydrogenase Raldh2, resulting in limited activation of the RA signaling pathway in these cells. In contrast, low Cyp26b1 expression in cervical spinal cord progenitor cells allows RA signaling to be readily activated upon RA treatment. The intrinsic heterogeneity and signaling factor regulation among adult SCPCs suggest that different niche factor regimens are required for site-specific mobilization of endogenous SCPCs from distinct spatial regions of the spinal cord for injury repair.published_or_final_versio