Notochordal Markers Research Articles

Alcohol induces neural tube defects by reducing retinoic acid signaling and promoting neural plate expansion.

Introduction: Neural tube defects (NTDs) are among the most debilitating and common developmental defects in humans. The induction of NTDs has been attributed to abnormal folic acid (vitamin B9) metabolism, Wnt and BMP signaling, excess retinoic acid (RA), dietary components, environmental factors, and many others. In the present study we show that reduced RA signaling, including alcohol exposure, induces NTDs. Methods: Xenopus embryos were exposed to pharmacological RA biosynthesis inhibitors to study the induction of NTDs. Embryos were treated with DEAB, citral, or ethanol, all of which inhibit the biosynthesis of RA, or injected to overexpress Cyp26a1 to reduce RA. NTD induction was studied using neural plate and notochord markers together with morphological analysis. Expression of the neuroectodermal regulatory network and cell proliferation were analyzed to understand the morphological malformations of the neural plate. Results: Reducing RA signaling levels using retinaldehyde dehydrogenase inhibitors (ethanol, DEAB, and citral) or Cyp26a1-driven degradation efficiently induce NTDs. These NTDs can be rescued by providing precursors of RA. We mapped this RA requirement to early gastrula stages during the induction of neural plate precursors. This reduced RA signaling results in abnormal expression of neural network genes, including the neural plate stem cell maintenance genes, geminin, and foxd4l1.1. This abnormal expression of neural network genes results in increased proliferation of neural precursors giving rise to an expanded neural plate. Conclusion: We show that RA signaling is required for neural tube closure during embryogenesis. RA signaling plays a very early role in the regulation of proliferation and differentiation of the neural plate soon after the induction of neural progenitors during gastrulation. RA signaling disruption leads to the induction of NTDs through the mis regulation of the early neuroectodermal network, leading to increased proliferation resulting in the expansion of the neural plate. Ethanol exposure induces NTDs through this mechanism involving reduced RA levels.

Head organizer: Cerberus and IGF cooperate in brain induction in Xenopus embryos

Neural induction by cell-cell signaling was discovered a century ago by the organizer transplantations of Spemann and Mangold in amphibians. Spemann later found that early dorsal blastopore lips induced heads and late organizers trunk-tail structures. Identifying region-specific organizer signals has been a driving force in the progress of animal biology. Head induction in the absence of trunk is designated archencephalic differentiation. Two specific head inducers, Cerberus and Insulin-like growth factors (IGFs), that induce archencephalic brain but not trunk-tail structures have been described previously. However, whether these two signals interact with each other had not been studied to date and was the purpose of the present investigation. It was found that Cerberus, a multivalent growth factor antagonist that inhibits Nodal, BMP and Wnt signals, strongly cooperated with IGF2, a growth factor that provides a positive signal through tyrosine kinase IGF receptors that activate MAPK and other pathways. The ectopic archencephalic structures induced by the combination of Cerberus and IGF2 are of higher frequency and larger than either one alone. They contain brain, a cyclopic eye and multiple olfactory placodes, without trace of trunk structures such as notochord or somites. A dominant-negative secreted IGF receptor 1 blocked Cerberus activity, indicating that endogenous IGF signals are required for ectopic brain formation. In a sensitized embryonic system, in which embryos were depleted of β-catenin, IGF2 did not by itself induce neural tissue while in combination with Cerberus it greatly enhanced formation of circular brain structures expressing the anterior markers Otx2 and Rx2a, but not spinal cord or notochord markers. The main conclusion of this work is that IGF provides a positive signal initially uniformly expressed throughout the embryo that potentiates the effect of an organizer-specific negative signal mediated by Cerberus. The results are discussed in the context of the history of neural induction.

Pinhead antagonizes Admp to promote notochord formation

SummaryDorsoventral patterning of a vertebrate embryo critically depends on the activity of Smad1 that mediates signaling by BMP proteins, anti-dorsalizing morphogenetic protein (Admp), and their antagonists. Pinhead (Pnhd), a cystine-knot-containing secreted protein, is expressed in the ventrolateral mesoderm during Xenopus gastrulation; however, its molecular targets and signaling mechanisms have not been fully elucidated. Our mass spectrometry-based screen of the gastrula secretome identified Admp as Pnhd-associated protein. We show that Pnhd binds Admp and inhibits its ventralizing activity by reducing Smad1 phosphorylation and its transcriptional targets. Importantly, Pnhd depletion further increased phospho-Smad1 levels in the presence of Admp. Furthermore, Pnhd synergized with Chordin and a truncated BMP4 receptor in the induction of notochord markers in ectoderm cells, and Pnhd-depleted embryos displayed notochord defects. Our findings suggest that Pnhd binds and inactivates Admp to promote notochord development. We propose that the interaction between Admp and Pnhd refines Smad1 activity gradients during vertebrate gastrulation.

Vitamin E is necessary for zebrafish nervous system development

Vitamin E (VitE) deficiency results in embryonic lethality. Knockdown of the gene ttpa encoding for the VitE regulatory protein [α-tocopherol transfer protein (α-TTP)] in zebrafish embryos causes death within 24 h post-fertilization (hpf). To test the hypothesis that VitE, not just α-TTP, is necessary for nervous system development, adult 5D strain zebrafish, fed either VitE sufficient (E+) or deficient (E−) diets, were spawned to obtain E+ and E− embryos, which were subjected to RNA in situ hybridization and RT-qPCR. Ttpa was expressed ubiquitously in embryos up to 12 hpf. Early gastrulation (6 hpf) assessed by goosecoid expression was unaffected by VitE status. By 24 hpf, embryos expressed ttpa in brain ventricle borders, which showed abnormal closure in E− embryos. They also displayed disrupted patterns of paired box 2a (pax2a) and SRY-box transcription factor 10 (sox10) expression in the midbrain-hindbrain boundary, spinal cord and dorsal root ganglia. In E− embryos, the collagen sheath notochord markers (col2a1a and col9a2) appeared bent. Severe developmental errors in E− embryos were characterized by improper nervous system patterning of the usually carefully programmed transcriptional signals. Histological analysis also showed developmental defects in the formation of the fore-, mid- and hindbrain and somites of E− embryos at 24 hpf. Ttpa expression profile was not altered by the VitE status demonstrating that VitE itself, and not ttpa, is required for development of the brain and peripheral nervous system in this vertebrate embryo model.

Aging aggravates intervertebral disc degeneration by regulating transcription factors toward chondrogenesis.

Osterix is a critical transcription factor of mesenchymal stem cell fate, where its loss or loss of Wnt signaling diverts differentiation to a chondrocytic lineage. Intervertebral disc (IVD) degeneration activates the differentiation of prehypertrophic chondrocyte-like cells and inactivates Wnt signaling, but its interactive role with osterix is unclear. First, compared to young-adult (5 mo), mechanical compression of old (18 mo) IVD induced greater IVD degeneration. Aging (5 vs 12 mo) and/or compression reduced the transcription of osterix and notochordal marker T by 40-75%. Compression elevated the transcription of hypertrophic chondrocyte marker MMP13 and pre-osterix transcription factor RUNX2, but less so in 12 mo IVD. Next, using an Ai9/td reporter and immunohistochemical staining, annulus fibrosus and nucleus pulposus cells of young-adult IVD expressed osterix, but aging and compression reduced its expression. Lastly, in vivo LRP5-deficiency in osterix-expressing cells inactivated Wnt signaling in the nucleus pulposus by 95%, degenerated the IVD to levels similar to aging and compression, reduced the biomechanical properties by 45-70%, and reduced the transcription of osterix, notochordal markers and chondrocytic markers by 60-80%. Overall, these data indicate that age-related inactivation of Wnt signaling in osterix-expressing cells may limit regeneration by depleting the progenitors and attenuating the expansion of chondrocyte-like cells.

Human iPSCs can be differentiated into notochordal cells that reduce intervertebral disc degeneration in a porcine model.

Introduction: As many as 80% of the adult population experience back pain at some point in their lifetimes. Previous studies have indicated a link between back pain and intervertebral disc (IVD) degeneration. Despite decades of research, there is an urgent need for robust stem cell therapy targeting underlying causes rather than symptoms. It has been proposed that notochordal cells (NCs) appear to be the ideal cell type to regenerate the IVD: these cells disappear in humans as they mature, are replaced by nucleus pulposus (NP) cells, and their disappearance correlates with the initiation of degeneration of the disc. Human NCs are in short supply, thus here aimed for generation of notochordal-like cells from induced pluripotent cells (iPSCs).Methods: Human iPSCs were generated from normal dermal fibroblasts by transfecting plasmids encoding for six factors: OCT4, SOX2, KLF4, L-MYC, LIN28, and p53 shRNA. Then the iPSCs were treated with GSK3i to induce differentiation towards Primitive Streak Mesoderm (PSM). The differentiation was confirmed by qRT-PCR and immunofluorescence. PSM cells were transfected with Brachyury (Br)-encoding plasmid and the cells were encapsulated in Tetronic-tetraacrylate-fibrinogen (TF) hydrogel that mimics the NP environment (G'=1kPa), cultured in hypoxic conditions (2% O2) and with specifically defined growth media. The cells were also tested in vivo in a large animal model. IVD degeneration was induced after an annular puncture in pigs, 4 weeks later the cells were injected and IVDs were analyzed at 12 weeks after the injury using MRI, gene expression analysis and histology.Results: After short-term exposure of iPSCs to GSK3i there was a significant change in cell morphology, Primitive Streak Mesoderm (PSM) markers (Brachyury, MIXL1, FOXF1) were upregulated and markers of pluripotency (Nanog, Oct4, Sox2) were downregulated, both compared to the control group. PSM cells nucleofected with Br (PSM-Br) cultured in TF hydrogels retained the NC phenotype consistently for up to 8 weeks, as seen in the gene expression analysis. PSM-Br cells were co-cultured with bone marrow (BM)-derived mesenchymal stem cells (MSCs) which, with time, expressed the NC markers in higher levels, however the levels of expression in BM-MSCs alone did not change. Higher expression of NC and NP marker genes in human BM-MSCs was found to be induced by iNC-condition media (iNC-CM) than porcine NC-CM. The annular puncture induced IVD degeneration as early as 2 weeks after the procedure. The injected iNCs were detected in the degenerated discs after 8 weeks in vivo. The iNC-treated discs were found protected from degeneration. This was evident in histological analysis and changes in the pH levels, indicative of degeneration state of the discs, observed using qCEST MRI. Immunofluorescence stains show that their phenotype was consistent with the in vitro study, namely they still expressed the notochordal markers Keratin 18, Keratin 19, Noto and Brachyury.Conclusion: In the present study, we report a stepwise differentiation method to generate notochordal cells from human iPSCs. These cells not only demonstrate a sustainable notochordal cell phenotype in vitro and in vivo, but also show the functionality of notochordal cells and have protective effect in case of induced disc degeneration and prevent the change in the pH level of the injected IVDs. The mechanism of this effect could be suggested via the paracrine effect on resident cells, as it was shown in the in vitro studies with MSCs.

An optimized culture system for notochordal cell expansion with retention of phenotype.

BackgroundNotochordal (NC) cells display therapeutic potential in treating degeneration of the intervertebral disc. However, research on their phenotype and function is limited by low‐cell yields and a lack of appropriate methodology for cell expansion. Utilizing porcine cells, this study aimed to develop an optimized culture system which allows expansion of NC cell populations with retention of phenotype.MethodsPost‐natal porcine and foetal human nucleus pulposus tissue was compared histologically and expression of known NC cell marker genes by porcine NC cells was analyzed. Porcine NC cells were isolated from six‐week post‐natal discs and cultured in vitro under varied conditions: (1) DMEM vs αMEM; (2) laminin‐521, fibronectin, gelatin and uncoated tissue culture‐treated polystyrene (TCP); (3) 2% O2 vs normoxia; (4) αMEM (300 mOsm/L) vs αMEM (400 mOsm/L); (5) surface stiffness of 0.5 and 4 kPa and standard TCP. Adherence, proliferation, morphology and expression of NC cell markers were assessed over a 14‐day culture period.ResultsNative porcine nucleus pulposus tissue demonstrated similar morphology to human foetal tissue and porcine NC cells expressed known notochordal markers (CD24, KRT8, KRT18, KRT19, and T). Use of αMEM media and laminin‐521‐coated surfaces showed the greatest cell adherence, proliferation and retention of NC cell morphology and phenotype. Proliferation of NC cell populations was further enhanced in hypoxia (2%) and phenotypic retention was improved on 0.5 kPa culture surfaces.DiscussionOur model has demonstrated an optimized system in which NC cell populations may be expanded while retaining a notochordal phenotype. Application of this optimized culture system will enable NC cell expansion for detailed phenotypic and functional study, a major advantage over current culture methods described in the literature. Furthermore, the similarities identified between porcine and human NC cells suggest this system will be applicable in human NC cell culture for investigation of their therapeutic potential.

Butyl benzyl phthalate (BBP) induces caudal defects during embryonic development

Butyl benzyl phthalate (BBP) is commonly added during the manufacturing of plastics to increase flexibility and elasticity. However, BBP leaches off of plastic and environment presence has been detected in soil, groundwater and sediment potentially effecting organisms in the environment. Given the widespread uses of BBP in household, consumer goods and the presence of BBP in the environment, studies on developmental toxicity are needed. Here, we use a zebrafish model to investigate the early developmental toxicity of BBP. We treated gastrula staged embryos with increasing concentrations of BBP and noted concentration-dependent defects in caudal tail development, but the effect was caudal specific with no other developmental defects noted. In situ hybridization studies using muscle and notochord markers show alterations in muscle development and non-linear, kinked notochord staining. A more detailed antibody staining using a myosin specific marker shows disorganized myofibrils and a loss of chevron shaped somites. Furthermore, vascular development in the tail was also disrupted in a concentration dependent manner. We conclude that BBP is toxic to caudal development in zebrafish. The sensitivity of zebrafish during development to environmental toxins and chemicals has been useful in assessing the health of the aquatic environment. The results presented here are a useful early warning system for contamination that could affect human health.

Notochordal and nucleus pulposus marker expression is maintained by sub-populations of adult human nucleus pulposus cells through aging and degeneration

The nucleus pulposus (NP) of the intervertebral disc (IVD) demonstrates substantial changes in cell and matrix composition with both ageing and degeneration. While recent transcriptomic profiling studies have helped define human NP cell phenotype, it remains unclear how expression of these markers is influenced by ageing or degeneration. Furthermore, cells of the NP are thought to derive from the notochord, although adult NP lacks identifiable notochordal (NC) cells. This study aimed to confirm expression of previously identified NP and NC marker genes in adult human NP cells from a range of ages and degenerate states. Importantly, using gene expression analysis (N = 60) and immunohistochemistry (N = 56) the study demonstrates expression of NP markers FoxF1, Pax-1, keratin-8/18, carbonic anhydrase-12, and NC markers brachyury, galectin-3 and CD24 in cells of the NP irrespective of age or degeneration. Our immunohistochemical data, combined with flow cytometry (N = 5) which identified a small number of CA12+Gal3+T+CD24+ cells, suggests the possible presence of a sub-population of cells with an NC-like phenotype in adult NP tissue. These findings suggest that the NP contains a heterogeneous population of cells, which may possess varied phenotypic and functional profiles and thus warrant further investigation to improve our understanding of IVD homeostasis and repair.

The Expression of Keratin 19 in Intervertebral Disc Degeneration and Aging

Introduction Low back pain (LBP) affects 80% of the whole population around the world and causes great social-economic burden. Intervertebral disc (IVD) degenerates with aging and is a major contribution to LBP. Mature NP (nucleus pulposus, the center part of IVD) cells are similar to, but not the same as, chondrocytes. The search for NP markers has prompted the proposal of keratin-19 as one of the NP markers mainly based on its transcriptional level.1 In our previous study, among all the investigated. keratin-19 is the most downregulated gene in degenerated human NP. 2 In this study, we aim to track the changes in the protein expression of keratin-19 in disc degeneration and aging process with human clinical specimens and rodent models of induced degeneration and natural aging. Material and Methods The corresponding informed patient consent and institutional review board (IRB) approval and animal ethics approval was obtained from the relevant committee. Human IVD were collected from patients with disc degeneration (graded IV-V at the Schneiderman scale) undergoing discectomy or during corrective scoliosis surgery. Rat induced degeneration model was established by needle puncture as described previously.3 Mice ranged from date of birth to 2 years old were used to represent natural aging in rodents. The expression of keratin-19 was investigated by immunochemical staining. Keratin-19 positivity was determined as the number of keratin-19 positive cells divided by the total number of cells per whole section. Results Keratin-19 expression was detected in both NP and AF (annulus fibrosus) of degenerated and non-degenerated human IVD. The signal of Keratin-19 was exclusively intracellular. Cells within a cluster showed heterogeneous expression for Keratin -19. Degenerated human NP contains significantly less Keratin-19 positive cells when compared with non-degenerated human NP (D-NP versus ND-NP: 13.87 ± 1.27% versus 29.99 ± 4.68%, p = 0.016). In healthy rat, Keratin-19 was found to be strongly expressed in the NP but not in the AF. A minor portion of cells in the cartilaginous EP (endplate) was also Keratin-19 positive. Keratin-19 positivity remained at a similar level post injury in the NP, but were induced in the AF after puncture. In aging mice, at P0, Keratin-19 showed strong expression in the vacuolated notochordal NP cells and in the cartilage anlage of vertebrae bodies, with no expression in the AF. From P0 to 18 months of age, Keratin-19 remained expressed at a relatively constant level in the NP constituted by large vacuolated notochordal cells. By 24 months of age, Keratin-19 expression decreased in the NP. Conclusion We found that although Keratin-19 was significantly decreased in human degenerated NP, it only showed a mild decrease or otherwise unchanged expression in rodent models of progressive disc degeneration and aging. Since Keratin-19 is also a notochordal cell marker,4 it may reflect that notochordal cells are still present in the aged mouse NP and during the mild stage of degeneration in the rat model. To our knowledge, this is the first investigation into the expression and location of keratin-19 in different IVD aging/degeneration systems. Acknowledgments This work was supported by General Funding from National Science Foundation of China (NSFC, 81371993), Small Project Funding of The University of Hong Kong (201409176191), and General Research Fund of RGC in Hong Kong (17104815). References Lv F, Leung VY, Huang S, Huang Y, Sun Y, Cheung KM. In search of nucleus pulposus-specific molecular markers. Rheumatology (Oxford) 2014;53(4):600–610 Lv F, et al. Global Spine Congress, Hong Kong; 2013 Leung VY, et al. Stem Cells 2014;218(1):113 Weiler C, Nerlich AG, Schaaf R, Bachmeier BE, Wuertz K, Boos N. Immunohistochemical identification of notochordal markers in cells in the aging human lumbar intervertebral disc. Eur Spine J 2010;19(10):1761–1770

Spatiotemporal analysis of putative notochordal cell markers reveals CD24 and keratins 8, 18, and 19 as notochord‐specific markers during early human intervertebral disc development

ABSTRACTIn humans, the nucleus pulposus (NP) is composed of large vacuolated notochordal cells in the fetus but, soon after birth, becomes populated by smaller, chondrocyte‐like cells. Although animal studies indicate that notochord‐derived cells persist in the adult NP, the ontogeny of the adult human NP cell population is still unclear. As such, identification of unique notochordal markers is required. This study was conducted to determine the spatiotemporal expression of putative human notochordal markers to aid in the elucidation of the ontogeny of adult human NP cells. Human embryos and fetuses (3.5–18 weeks post‐conception (WPC)) were microdissected to isolate the spine anlagens (notochord and somites/sclerotome). Morphology of the developing IVD was assessed using hematoxylin and eosin. Expression of keratin (KRT) 8, KRT18, KRT19, CD24, GAL3, CD55, BASP1, CTGF, T, CD90, Tie2, and E‐cadherin was assessed using immunohistochemistry. KRT8, KRT18, KRT19 were uniquely expressed by notochordal cells at all spine levels at all stages studied; CD24 was expressed at all stages except 3.5 WPC. While GAL3, CD55, BASP1, CTGF, and T were expressed by notochordal cells at specific stages, they were also co‐expressed by sclerotomal cells. CD90, Tie2, and E‐cadherin expression was not detectable in developing human spine cells at any stage. This study has identified, for the first time, the consistent expression of KRT8, KRT18, KRT19, and CD24 as human notochord‐specific markers during early IVD development. Thus, we propose that these markers can be used to help ascertain the ontogeny of adult human NP cells. © 2016 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. J Orthop Res 34:1327–1340, 2016.

Notochord manipulation does not impact oesophageal and tracheal formation from isolated foregut in 3D explant culture.

Tracheo-oesophageal malformations result from disturbed foregut separation during early development. The notochord, a specialised embryonic structure, forms immediately adjacent to the dividing foregut. In the Adriamycin mouse model of oesophageal atresia, foregut and notochord abnormalities co-exist, and the site and severity of foregut malformations closely correlate to the position and extent of the notochord defects. Notochord and foregut abnormalities also co-exist in the Noggin Knockout mouse as well in a small number of human cases. The notochord is a source of powerful molecular signals during early embryogenesis, being particularly important for neural crest development. The influence of notochord signaling on the adjacent foregut is not known. The purpose of this study was to examine the impact of notochord manipulation on foregut separation using a robust 3D explant method for culturing isolated foregut which permits oeosphageal and tracheal formation in vitro. Foregut was micro-dissected from embryonic day 9 mice (License B100/4447 Irish Medicines Board), embedded in collagen and cultured for 48h with native notochord intact (n=6), notochord removed (n=10) or additional notochord transplanted from stage matched controls (n=8). Specimens were analysed for foregut morphology and molecular patterning using immunohistochemistry for Hnf3b (an endoderm marker) and Sox2 (a notochord and oesophageal marker) on cryosections. Foregut separation into distinct oesophagus and trachea was observed in isolated foregut specimens with or without their native notochord. In specimens with additional notochord transplants, foregut morphology and molecular patterning were comparable to controls whether or not the native notochord was maintained. In particular foregut separation was not disrupted by the transplantation of additional notochord at the dorsal foregut endoderm. The relationship between the embryonic foregut and notochord is complex and ill-defined; however, the notochord does not contribute essentially to oesophagus and trachea formation beyond E9 in the mouse, and the transplantation of additional notochord does not disrupt foregut separation in 3D explant culture.

Native nucleus pulposus tissue matrix promotes notochordal differentiation of human induced pluripotent stem cells with potential for treating intervertebral disc degeneration.

Native porcine nucleus pulposus (NP) tissue harbors a number of notochordal cells (NCs). Whether the native NP matrix supports the homeostasis of notochordal cells is poorly understood. We hypothesized the NP matrix alone may contain sufficient regulatory factors and can serve as stimuli to generate notochordal cells (NCs) from human pluripotent stem cells. NCs are a promising cell sources for cell-based therapy to treat some types of intervertebral disc (IVD) degeneration. One major limitation of this emerging technique is the lack of available NCs as a potential therapeutic cell source. Human pluripotent stem cells derived from reprogramming or somatic cell nuclear transfer technique may yield stable and unlimited source for therapeutic use. We devised a new method to use porcine NP matrix to direct notochordal differentiation of human induced pluripotent stem cells (hiPSCs). The results showed that hiPSCs successfully differentiated into NC-like cells under the influence of devitalized porcine NP matrix. The NC-like cells expressed typical notochordal marker genes including brachyury (T), cytokeratin-8 (CK-8) and cytokeratin-18 (CK-18), and they displayed the ability to generate NP-like tissue in vitro, which was rich in aggrecan and collagen type II. These findings demonstrated the proof of concept for using native NP matrix to direct notochordal differentiation of hiPSCs. It provides a foundation for further understanding the biology of NCs, and eventually towards regenerative therapies for disc degeneration.

An understanding of intervertebral disc development, maturation and cell phenotype provides clues to direct cell-based tissue regeneration therapies for disc degeneration.

Cell-based regenerative medicine therapies have been proposed for repairing the degenerated intervertebral disc (a major cause of back pain). However, for this approach to be successful, it is essential to characterise the phenotype of its native cells to guarantee that implanted cells differentiate and maintain the correct phenotype to ensure appropriate cell and tissue function. While recent studies have increased our knowledge of the human nucleus pulposus (NP) cell phenotype, their ontogeny is still unclear. The expression of notochordal markers by a subpopulation of adult NP cells suggests that, contrary to previous reports, notochord-derived cells are retained in the adult NP, possibly coexisting with a second population of cells originating from the annulus fibrosus or endplate. It is not known, however, how these two cell populations interact and their specific role(s) in disc homeostasis and disease. In particular, notochordal cells are proposed to display both anabolic and protective roles; therefore, they may be the ideal cells to repair the degenerate disc. Thus, understanding the ontogeny of the adult NP cells is paramount, as it will inform the medical and scientific communities as to the ideal phenotype to implant into the degenerate disc and the specific pathways involved in stem cell differentiation towards such a phenotype.

Early left‐right asymmetries during axial morphogenesis in the chick embryo

The primitive node is the "hub" of early left-right patterning in the chick embryo: (1) it undergoes asymmetrical morphogenesis immediately after its appearance at Stage 4; (2) it is closely linked to the emerging asymmetrical expression of nodal and shh at Stage 5; and (3) its asymmetry is spatiotemporally related to the emerging notochord, the midline barrier maintaining molecular left-right patterning from Stage 6 onward. Here, we study the correlation of node asymmetry to notochord marker expression using high-resolution histology, and we test pharmacological inhibition of shh signaling using cyclopamine at Stages 4 and 5. Just as noggin expression mirrors an intriguing structural continuity between the right node shoulder and the notochord, shh expression in the left node shoulder confirms a similar continuity with the future floor plate. Shh inhibition at Stage 4 or 5 suppressed nodal in both its paraxial or lateral plate mesoderm domains, respectively, and resulted in randomized heart looping. Thus, the "primordial" paraxial nodal asymmetry at Stage 4/5 (1) appears to be dependent on, but not instructed by, shh signaling and (2) may be fixed by asymmetrical roots of the notochord and the floor plate, thereby adding further twists to the node's pivotal role during left-right patterning.

Intradural chordoma of the Meckel's cave: A challenging differential diagnosis

Chordomas are midline tumors that arise from embryonic remnants of the notochord and are considered to be malignant tumors because of their tendency to invade and destroy the involved bone. Cases of intradural chordomas without bone involvement have been rarely described with a predilection for prepontine location. The absence of bony invasion renders the complete excision of these tumors more feasible and is related to their better prognosis in comparison to conventional chordomas. Herein we report the first intradural chordoma arising in the Meckel's cave. The intradural location of the lesion, outside midline structures, in the absence of bone infiltration, made the differential diagnosis versus other meningeal lesions such as chordoid meningioma challenging. The intense and strong immunohistochemical expression of pan-cytokeratins, S100, cytokeratin-19 and of the notochordal marker brachyury allowed differential diagnosis toward other tumors showing chordoid morphology. The expression of brachyury, which had not been previously analyzed in intradural chordoma, definitely links the histogenesis of this neoplasia to the notochord, similar to that of conventional chordoma. We also show that, different from conventional chordoma, intradural chordoma does not express the metallo-proteinases (MMPs) -2 and -9, which may account for its indolent biological behavior.

Foxa2 mediates critical functions of prechordal plate in patterning and morphogenesis and is cell autonomously required for early ventral endoderm morphogenesis

Summary Axial mesendoderm is comprised of prechordal plate and notochord. Lack of a suitable Cre driver has hampered the ability to genetically dissect the requirement for each of these components, or genes expressed within them, to anterior patterning. Here, we have utilized Isl1-Cre to investigate roles of the winged helix transcription factor Foxa2 specifically in prechordal plate and ventral endoderm. Foxa2loxP/loxP; Isl1-Cre mutants died at 13.5 dpc, exhibiting aberrations in anterior neural tube and forebrain patterning, and in ventral foregut morphogenesis and cardiac fusion. Molecular analysis of Foxa2loxP/loxP; Isl1-Cre mutants indicated that Foxa2 is required in Isl1 lineages for expression of notochord and dorsal foregut endoderm markers, Shh. Brachyury, and Hlxb9. Our results support a requirement for Foxa2 in prechordal plate for notochord morphogenesis, axial patterning, and patterning of dorsal foregut endoderm. Loss of Foxa2 in ventral endoderm resulted in reduced expression of Sox17, Gata4, and ZO proteins, accounting at least in part for observed lack of foregut fusion, cardia bifida, and increased apoptosis of ventral endoderm.

Canonical Wnt signaling in the notochordal cell is upregulated in early intervertebral disk degeneration

The notochordal cell (NC) of the nucleus pulposus (NP) is considered a potential NP progenitor cell, and early intervertebral disk (IVD) degeneration involves replacement of NCs by chondrocyte-like cells (CLCs). Wnt/β-catenin signaling plays a crucial role in maintaining the notochordal fate during embryogenesis, but is also involved in tissue degeneration and regeneration. The canine species, which can be subdivided into non-chondrodystrophic and chondrodystrophic breeds, is characterized by differential maintenance of the NC: in non-chondrodystrophic dogs, the NC remains the predominant cell type during the majority of life, with IVD degeneration only occurring at old age; conversely, in chondrodystrophic dogs the NC is lost early in life, with concurrent degeneration of all IVDs. This study investigated Wnt/β-catenin signaling in the healthy, NC-rich NP and early degenerated, CLC-rich NP of both breed types by immunohistochemistry of β-catenin and relative gene expression of brachyury and cytokeratin 8 (notochordal markers) and Wnt targets axin2, cyclin D1, and c-myc. Both NCs and CLCs showed nuclear and cytoplasmic β-catenin protein expression and axin2 gene expression, but β-catenin signal intensity and Wnt target gene expression were higher in the CLC-rich NP. Primary NCs in monolayer culture (normoxic conditions) showed Wnt/β-catenin signaling comparable to the in vivo situation, with increased cyclin D1 and c-myc gene expression. In conclusion, Wnt/β-catenin signaling activity in the NC within the NC-rich NP and in culture supports the role of this cell as a potential progenitor cell; increased Wnt/β-catenin signaling activity in early IVD degeneration may be a reflection of its dual role.

Xenopus Zic3 controls notochord and organizer development through suppression of the Wnt/β-catenin signaling pathway

Zic3 controls neuroectodermal differentiation and left–right patterning in Xenopus laevis embryos. Here we demonstrate that Zic3 can suppress Wnt/β-catenin signaling and control development of the notochord and Spemann's organizer. When we overexpressed Zic3 by injecting its RNA into the dorsal marginal zone of 2-cell-stage embryos, the embryos lost mesodermal dorsal midline structures and showed reduced expression of organizer markers (Siamois and Goosecoid) and a notochord marker (Xnot). Co-injection of Siamois RNA partially rescued the reduction of Xnot expression caused by Zic3 overexpression. Because the expression of Siamois in the organizer region is controlled by Wnt/β-catenin signaling, we subsequently examined the functional interaction between Zic3 and Wnt signaling. Co-injection of Xenopus Zic RNAs and β-catenin RNA with a reporter responsive to the Wnt/β-catenin cascade indicated that Zic1, Zic2, Zic3, Zic4, and Zic5 can all suppress β-catenin-mediated transcriptional activation. In addition, co-injection of Zic3 RNA inhibited the secondary axis formation caused by ventral-side injection of β-catenin RNA in Xenopus embryos. Zic3-mediated Wnt/β-catenin signal suppression required the nuclear localization of Zic3, and involved the reduction of β-catenin nuclear transport and enhancement of β-catenin degradation. Furthermore, Zic3 co-precipitated with Tcf1 (a β-catenin co-factor) and XIC (I-mfa domain containing factor required for dorsoanterior development). The findings in this report produce a novel system for fine-tuning of Wnt/β-catenin signaling.

The maternal genes Ci-p53/p73-a and Ci-p53/p73-b regulate zygotic ZicL expression and notochord differentiation in Ciona intestinalis embryos

I isolated a Ciona intestinalis homolog of p53, Ci-p53/p73-a, in a microarray screen of rapidly degraded maternal mRNA by comparing the transcriptomes of unfertilized eggs and 32-cell stage embryos. Higher expression of the gene in eggs and lower expression in later embryonic stages were confirmed by whole-mount in situ hybridization (WISH) and quantitative reverse transcription-PCR (qRT-PCR); expression was ubiquitous in eggs and early embryos. Knockdown of Ci-p53/p73-a by injection of antisense morpholino oligonucleotides (MOs) severely perturbed gastrulation cell movements and expression of notochord marker genes. A key regulator of notochord differentiation in Ciona embryos is Brachyury ( Ci-Bra), which is directly activated by a zic-like gene ( Ci-ZicL). The expression of Ci-ZicL and Ci-Bra in A-line notochord precursors was downregulated in Ci-p53/p73-a knockdown embryos. Maternal expression of Ci-p53/p73-b, a homolog of Ci-p53/p73-a, was also detected. In Ci-p53/p73-b knockdown embryos, gastrulation cell movements, expression of Ci-ZicL and Ci-Bra in A-line notochord precursors, and expression of notochord marker gene at later stages were perturbed. The upstream region of Ci-ZicL contains putative p53-binding sites. Cis-regulatory analysis of Ci-ZicL showed that these sites are involved in expression of Ci-ZicL in A-line notochord precursors at the 32-cell and early gastrula stages. These results suggest that p53 genes are maternal factors that play a crucial role in A-line notochord differentiation in C. intestinalis embryos by regulating Ci-ZicL expression.