Frontonasal Dysplasia: Towards an Understanding of Molecular and Developmental Aetiology.

The complex anatomy of the skull and face arises from the requirement to support multiple sensory and structural functions. During embryonic development, the diverse component elements of the neuro- and viscerocranium must be generated independently and subsequently united in a manner that sustains and promotes the growth of the brain and sensory organs, while achieving a level of structural integrity necessary for the individual to become a free-living organism. While each of these individual craniofacial components is essential, the cranial and facial midline lies at a structural nexus that unites these disparately derived elements, fusing them into a whole. Defects of the craniofacial midline can have a profound impact on both form and function, manifesting in a diverse array of phenotypes and clinical entities that can be broadly defined as frontonasal dysplasias (FNDs). Recent advances in the identification of the genetic basis of FNDs along with the analysis of developmental mechanisms impacted by these mutations have dramatically altered our understanding of this complex group of conditions.

GAPTrap: A Simple Expression System for Pluripotent Stem Cells and Their Derivatives.

The ability to reliably express fluorescent reporters or other genes of interest is important for using human pluripotent stem cells (hPSCs) as a platform for investigating cell fates and gene function. We describe a simple expression system, designated GAPTrap (GT), in which reporter genes, including GFP, mCherry, mTagBFP2, luc2, Gluc, and lacZ are inserted into the GAPDH locus in hPSCs. Independent clones harboring variations of the GT vectors expressed remarkably consistent levels of the reporter gene. Differentiation experiments showed that reporter expression was reliably maintained in hematopoietic cells, cardiac mesoderm, definitive endoderm, and ventral midbrain dopaminergic neurons. Similarly, analysis of teratomas derived from GT-lacZ hPSCs showed that ß-galactosidase expression was maintained in a spectrum of cell types representing derivatives of the three germ layers. Thus, the GAPTrap vectors represent a robust and straightforward tagging system that enables indelible labeling of PSCs and their differentiated derivatives.

Cauli: a mouse strain with an Ift140 mutation that results in a skeletal ciliopathy modelling Jeune syndrome.

Cilia are architecturally complex organelles that protrude from the cell membrane and have signalling, sensory and motility functions that are central to normal tissue development and homeostasis. There are two broad categories of cilia; motile and non-motile, or primary, cilia. The central role of primary cilia in health and disease has become prominent in the past decade with the recognition of a number of human syndromes that result from defects in the formation or function of primary cilia. This rapidly growing class of conditions, now known as ciliopathies, impact the development of a diverse range of tissues including the neural axis, craniofacial structures, skeleton, kidneys, eyes and lungs. The broad impact of cilia dysfunction on development reflects the pivotal position of the primary cilia within a signalling nexus involving a growing number of growth factor systems including Hedgehog, Pdgf, Fgf, Hippo, Notch and both canonical Wnt and planar cell polarity. We have identified a novel ENU mutant allele of Ift140, which causes a mid-gestation embryonic lethal phenotype in homozygous mutant mice. Mutant embryos exhibit a range of phenotypes including exencephaly and spina bifida, craniofacial dysmorphism, digit anomalies, cardiac anomalies and somite patterning defects. A number of these phenotypes can be attributed to alterations in Hedgehog signalling, although additional signalling systems are also likely to be involved. We also report the identification of a homozygous recessive mutation in IFT140 in a Jeune syndrome patient. This ENU-induced Jeune syndrome model will be useful in delineating the origins of dysmorphology in human ciliopathies.

Phenotypic variability of distal 22q11.2 copy number abnormalities.

The availability of microarray technology has led to the recent recognition of copy number abnormalities of distal chromosome 22q11.2 that are distinct from the better-characterized deletions and duplications of the proximal region. This report describes five unrelated individuals with copy number abnormalities affecting distal chromosome 22q11.2. We report on novel phenotypic features including diaphragmatic hernia and uterine didelphys associated with the distal microdeletion syndrome; and frontomedial polymicrogyria and callosal agenesis associated with the distal microduplication syndrome. We describe the third distal chromosome 22q11.2 microdeletion patient with Goldenhar syndrome. Patients with distal chromosome 22q11.2 copy number abnormalities exhibit inter- and intra-familial phenotypic variability, and challenge our ability to draw meaningful genotype-phenotype correlations.

The RCAS retroviral expression system in the study of skeletal development.

RCAS is a replication-competent retroviral vector system that allows sustained misexpression of a gene of interest in avian cells. This tool has been used to gain fundamental insights into skeletal development in the chick embryo, and consequently into broader principles of morphogenesis. In this review, we discuss a range of RCAS-based strategies that have been employed to examine gene function during early patterning of the limb bud, later skeletal differentiation, and craniofacial morphogenesis. The potential for the RCAS system as a tool for loss-of-function studies is also discussed.

Bone regeneration in a rabbit critical-sized skull defect using autologous adipose-derived cells.

Repair of substantial cranial defects in adults and children may be compromised due to limitations in donor bone stocks for autologous grafts. We evaluated the capability of autologous adipose-derived mesenchymal cells (ADCs) in combination with polylactic acid (PLA) scaffolds to regenerate bone in a critical-sized skull defect. Thirty adult New Zealand White rabbits were divided into six groups of five animals each: (1) PLA alone (control), (2) fibronectin-coated PLA, (3) PLA with ADCs, (4) fibronectin-coated PLA with ADCs, (5) PLA with osteogenically induced ADCs (osADCs), and (6) fibronectin-coated PLA with osADCs. All the animals were humanely killed after 6 weeks. X-ray, histology, and histomorphometric analysis were performed to evaluate the new bone formation inside the PLA scaffold. Radiographically and histomorphometrically, the groups in which the PLA was not fibronectin coated showed no bone formation in contrast to the fibronectin-coated groups (Gp1 vs. Gp2, p < 0.0005); the group treated with osteo-induced ADCs and fibronectin (Gp6) showed significantly more bone formation than the group treated with undifferentiated ADCs (Gp4) and the group treated without cells (Gp5, p < 0.0005, in both cases). These data indicate that the surface treatment with fibronectin promotes bone formation within the scaffold, and that autologous, osteo-induced adipose-derived cells enhance bone formation if seeded into a fibronectin-treated PLA scaffold.

Isolated Anxa5+/Sca-1+ perivascular cells from mouse meningeal vasculature retain their perivascular phenotype in vitro and in vivo.

Pericytes are closely associated with endothelial cells, contribute to vascular stability and represent a potential source of mesenchymal progenitor cells. Using the specifically expressed annexin A5-LacZ fusion gene (Anxa5-LacZ), it became possible to isolate perivascular cells (PVC) from mouse tissues. These cells proliferate and can be cultured without undergoing senescence for multiple passages. PVC display phenotypic characteristics of pericytes, as they express pericyte-specific markers (NG2-proteoglycan, desmin, alphaSMA, PDGFR-beta). They also express stem cell marker Sca-1, whereas endothelial (PECAM), hematopoietic (CD45) or myeloid (F4/80, CD11b) lineage markers are not detectable. These characteristics are in common with the pericyte-like cell line 10T1/2. PVC also display a phagocytoic activity higher than 10T1/2 cells. During coculture with endothelial cells both cell types stimulate angiogenic processes indicated by an increased expression of PECAM in endothelial cells and specific deposition of basement membrane proteins. PVC show a significantly increased induction of endothelial specific PECAM expression compared to 10T1/2 cells. Accordingly, in vivo grafts of PVC aggregates onto chorioallantoic membranes of quail embryos recruit endothelial cells, get highly vascularized and deposit basement membrane components. These data demonstrate that isolated Anxa5-LacZ(+) PVC from mouse meninges retain their capacity for differentiation to pericyte-like cells and contribute to angiogenic processes.

Polyalanine expansion mutations in the X-linked hypopituitarism gene SOX3 result in aggresome formation and impaired transactivation.

Polyalanine expansion mutations have been identified in eight transcription factors that are associated with a range of congenital disorders. While some of these mutant proteins have been shown to generate cellular aggregates in heterologous cell lines, little is known about the mechanism by which these aggregates cause disease. Here we examine the aggregation and functional properties of the two known polyalanine expansion mutations associated with X-linked Hypopituitarism (XH), SOX3(22Ala) and SOX3(26Ala), which contain an additional seven and eleven alanine residues, respectively. SOX3(22Ala) and SOX3(26Ala) proteins form cytoplasmic aggregates and nuclear inclusions in transiently transfected COS-7 and CHO K1 cells, and in transfected explant cultures of chick neural epithelium. SOX3(26Ala) exhibits a more potent aggregation phenotype, resulting in significantly more cells with dispersed cytoplasmic and large perinuclear aggregates. SOX3(22Ala) and SOX3(26Ala) protein aggregates exhibit the key properties of aggresomes including vimentin redistribution, colocalisation with the Microtubule Organising Centre and sensitivity to microtubule disruption. This is the first time that aggresomes have been implicated in the aetiology of a polyalanine expansion disorder, suggesting that XH and protein conformation disorders may become manifest through similar pathological mechanisms. Further, we show that mutant SOX3 proteins have impaired transcriptional activity and reduced capacity to inhibit beta-catenin/TCF-mediated transcription. These data suggest that deregulation of SOX3 target genes and inappropriate canonical Wnt signaling in central nervous system (CNS) progenitors may also contribute to dysfunction of the hypothalamic-pituitary axis in XH patients.

Sox10 overexpression induces neural crest-like cells from all dorsoventral levels of the neural tube but inhibits differentiation.

SoxE genes (Sox8, Sox9, and Sox10) are early response genes to neural crest induction. Although the early role of Sox9 has been examined in chick and frog, later roles in neural crest migration and differentiation remain largely unexplored. We first examined which SoxE genes were expressed in trunk neural crest cells and then investigated their function using in ovo electroporation. The results of this analysis reveal that Sox10 is present in migrating neural crest cells, whereas other SoxE genes are only expressed transiently after induction. Ectopic expression of Sox10 in the neural tube at trunk level induced expression of HNK-1 in neuroepithelial cells followed by extensive emigration from all levels of the dorsoventral neuraxis, including the floor plate. Sox10-expressing cells failed to express neuronal, Schwann, or melanocyte markers up to 6 days posttransfection (E8), suggesting these cells were maintained in an undifferentiated state. Overexpression of Sox8 or Sox9 had similar but not identical effects on neuroepithelial cells. These results show that high levels of Sox10, Sox9, or Sox8 expression in the neural tube are capable of inducing a migratory neural crest-like phenotype even in the absence of dorsal signals and can maintain these cells in an undifferentiated state.

Neurovascular congruence results from a shared patterning mechanism that utilizes Semaphorin3A and Neuropilin-1.

Peripheral nerves and blood vessels have similar patterns in quail forelimb development. Usually, nerves extend adjacent to existing blood vessels, but in a few cases, vessels follow nerves. Nerves have been proposed to follow vascular smooth muscle, endothelium, or their basal laminae. Focusing on the major axial blood vessels and nerves, we found that when nerves grow into forelimbs at E3.5-E5, vascular smooth muscle was not detectable by smooth muscle actin immunoreactivity. Additionally, transmission electron microscopy at E5.5 confirmed that early blood vessels lacked smooth muscle and showed that the endothelial cell layer lacks a basal lamina, and we did not observe physical contact between peripheral nerves and these endothelial cells. To test more generally whether lack of nerves affected blood vessel patterns, forelimb-level neural tube ablations were performed at E2 to produce aneural limbs; these had completely normal vascular patterns up to at least E10. To test more generally whether vascular perturbation affected nerve patterns, VEGF(165), VEGF(121), Ang-1, and soluble Flt-1/Fc proteins singly and in combination were focally introduced via beads implanted into E4.5 forelimbs. These produced significant alterations to the vascular patterns, which included the formation of neo-vessels and the creation of ectopic avascular spaces at E6, but in both under- and overvascularized forelimbs, the peripheral nerve pattern was normal. The spatial distribution of semaphorin3A protein immunoreactivity was consistent with a negative regulation of neural and/or vascular patterning. Semaphorin3A bead implantations into E4.5 forelimbs caused failure of nerves and blood vessels to form and to deviate away from the bead. Conversely, semaphorin3A antibody bead implantation was associated with a local increase in capillary formation. Furthermore, neural tube electroporation at E2 with a construct for the soluble form of neuropilin-1 caused vascular malformations and hemorrhage as well as altered nerve trajectories and peripheral nerve defasciculation at E5-E6. These results suggest that neurovascular congruency does not arise from interdependence between peripheral nerves and blood vessels, but supports the hypothesis that it arises by a shared patterning mechanism that utilizes semaphorin3A.