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The notochord is a defining feature of all chordates. The transcription factors Zic and ETS regulate enhancer activity within the notochord. We conduct high-throughput screens of genomic elements within developing Ciona embryos to understand how Zic and ETS sites encode notochord activity. Our screen discovers an enhancer located near Lama, a gene critical for notochord development. Reversing the orientation of an ETS site within this enhancer abolishes expression, indicating that enhancer grammar is critical for notochord activity. Similarly organized clusters of Zic and ETS sites occur within mouse and human Lama1 introns. Within a Brachyury (Bra) enhancer, FoxA and Bra, in combination with Zic and ETS binding sites, are necessary and sufficient for notochord expression. This binding site logic also occurs within other Ciona and vertebrate Bra enhancers. Collectively, this study uncovers the importance of grammar within notochord enhancers and discovers signatures of enhancer logic and grammar conserved across chordates.

The cell type-specific expression of key transcription factors is central to development and disease. Brachyury/T/TBXT is a major transcription factor for gastrulation, tailbud patterning, and notochord formation; however, how its expression is controlled in the mammalian notochord has remained elusive. Here, we identify the complement of notochord-specific enhancers in the mammalian Brachyury/T/TBXT gene. Using transgenic assays in zebrafish, axolotl, and mouse, we discover three conserved Brachyury-controlling notochord enhancers, T3, C, and I, in human, mouse, and marsupial genomes. Acting as Brachyury-responsive, auto-regulatory shadow enhancers, in cis deletion of all three enhancers in mouse abolishes Brachyury/T/Tbxt expression selectively in the notochord, causing specific trunk and neural tube defects without gastrulation or tailbud defects. The three Brachyury-driving notochord enhancers are conserved beyond mammals in the brachyury/tbxtb loci of fishes, dating their origin to the last common ancestor of jawed vertebrates. Our data define the vertebrate enhancers for Brachyury/T/TBXTB notochord expression through an auto-regulatory mechanism that conveys robustness and adaptability as ancient basis for axis development.

Chondroitin sulfate (CS) and undenatured type II collagen (Col II) are the two major, biological macromolecules of cartilage-related tissues. In this study, a new extraction. process was developed to obtain CS and Col II simultaneously. By this process, CS. and undenatured Col II were extracted from sturgeon notochord with the yields of 5.34, ± 0.74% and 45.25 ± 5.25%, respectively. The SEC-RI-MALLS result showed that the, average molecular weight of notochord CS was 38.4 kDa. FTIR NMR, and SAX-HPLC, results indicated the notochord CS was mainly composed of CS-A. The new extraction, process had no effect on the triple helical structure of Col II. To analyze the interaction, between the two macromolecules, the effect of CS on Col II fibril formation was, examined using turbidity assay and SEM observation. CS accelerated the completion, of Col II self-assembly and inhibited the lateral aggregation of fibrils. The results of this, study suggested that the sturgeon notochord is a valuable source of CS and Col II. The. new extraction method not only improves the utilization rate of sturgeon notochord, but,also reduces the waste of aquatic resources. CS and Col II derived from sturgeon, notochord have the potential for use in biomedical materials

Embryonic development across all vertebrates begins upon the fertilization of an egg by a sperm cell to become a single-celled zygote. Embryogenesis continues with various stages of division to eventually make up an entire organism. The processes governing development are finely orchestrated and include many participants, such as genes involved in gene regulatory networks and non-coding regions of DNA, or enhancers, to regulate the expression of those genes. Defects or perturbations to this strictly regulated machinery can lead to various clinical conditions, such as congenital heart disease. Thus, deepening our understanding of embryogenesis may help us understand the mechanisms driving congenital abnormalities as well as the evolution of developmental pathways. One defining characteristic of all chordate embryos is the presence of a notochord during development. The notochord is a long, semi-rigid fibrous rod of mesodermal origin that provides structural support and serves as a signaling center to pattern the neighboring neural tube, paraxial mesoderm, and gut. A complete understanding of notochord structure and function during early and late life stages is thus essential to better understand congenital vertebral defects. For example, failure of vertebral notochord cells to transition to the nucleus pulposus, the cushioning between intervertebral discs of the spine, is associated with chordomas, slow-growing tumors formed from notochord cell remnants within the spine or the base of the skull. The ascidian Ciona intestinalis Type A (Ciona) is a marine organism that is evolutionarily similar to vertebrates. Through electroporation, Ciona is readily amenable to high-throughput, high-resolution functional studies of cis-regulatory elements like enhancers in their native, whole-embryo context. To identify key notochord enhancers, I analyzed the importance of enhancer grammar–the transcription factor order, orientation, spacing, and binding affinity–in modulating notoch

Among chordate taxa, the cephalochordates diverged earlier than urochordates and vertebrates; thus, they retain unique, primitive developmental features. In particular, the amphioxus notochord has muscle-like properties, a feature not seen in urochordates or vertebrates. Amphioxus contains two Brachyury genes, Bra1 and Bra2. Bra2 is reportedly expressed in the blastopore, notochord, somites, and tail bud, in contrast to a low level of Bra1 expression only in notochord. To distinguish the expression profiles of the two Brachyury genes at the single-cell level, we carried out single-cell RNA-seq (scRNA-seq) analysis using the amphioxus, Branchiostoma japonicum. This scRNA-seq analysis classified B. japonicum embryonic cells into 15 clusters at developmental stages from midgastrula to early swimming larva. Brachyury was expressed in cells of clusters 4, 5, 8, and 9. We first confirmed that cluster 8 comprises cells that form somites since this cluster specifically expresses four myogenic factor genes. Cluster 9 contains a larger number of cells with high levels of Bra2 expression and a smaller number of cells with Bra1 expression. Simultaneous expression in cluster 9 of tool-kit genes, including FoxA, Goosecoid, and hedgehog, showed that this cluster comprises cells that form the notochord. Expression of Bra2, but not Bra1, in cells of clusters 4 and 5 at the gastrula stage together with expression of Wnt1 and Caudal indicates that clusters 4 and 5 comprise cells of the blastopore, which contiguously form the tail bud. In addition, Hox1, Hox3, and Hox4 were highly expressed in Bra2-expressing clusters 4, 5, 8, and 9 in a temporally coordinated manner, suggesting roles of anterior Hox genes in specification of mesodermal organs, including somites, notochord, and tail bud. This scRNA-seq analysis therefore highlights differences between the two Brachyury genes in relation to embryonic regions in which they are expressed and their levels of expression. Bra2 is the ancestr, source:https://www.frontiersin.org/article/10.3389/fcell.2021.696875

The mammalian intervertebral disc (IVD) consists of a gel-like, disordered nucleus pulposus (NP) surrounded by a highly ordered collagen structure, the annulus fibrosus (AF). While this concentric array of lamellae has been amply studied, its physical origin is poorly understood. The notochord is a rod-like organ located in the mid-line of the growing embryo and plays an essential role in IVD development. The aim of this study was to elucidate the effect of notochord development on the collagen fiber arrangement evolution in the AF. To that end, we studied IVD development in mouse embryos and compared these observations to those from chicken embryos, which do not form the typical laminar structure around the NP. In mouse, cross-aligned collagen arrangement of the AF forms from the sclerotome upon bulging of the notochord to become NP. By contrast, the notochord in the chicken embryo swells substantially without the physical restrictions of the future vertebrae and thus do not bulge. From these observations, we conclude that physical and geometrical constrictions are essential for the formation of the highly structured AF. (C) 2018 Elsevier Inc. All rights reserved.

The mammalian intervertebral disc (IVD) consists of a gel-like, disordered nucleus pulposus (NP) surrounded by a highly ordered collagen structure, the annulus fibrosus (AF). While this concentric array of lamellae has been amply studied, its physical origin is poorly understood. The notochord is a rod-like organ located in the mid-line of the growing embryo and plays an essential role in IVD development. The aim of this study was to elucidate the effect of notochord development on the collagen fiber arrangement evolution in the AF. To that end, we studied IVD development in mouse embryos and compared these observations to those from chicken embryos, which do not form the typical laminar structure around the NP. In mouse, cross-aligned collagen arrangement of the AF forms from the sclerotome upon bulging of the notochord to become NP. By contrast, the notochord in the chicken embryo swells substantially without the physical restrictions of the future vertebrae and thus do not bulge. From these observations, we conclude that physical and geometrical constrictions are essential for the formation of the highly structured AF. (C) 2018 Elsevier Inc. All rights reserved.

Chordoma, an aggressive tumor derived from notochordal remnants, is difficult to treat due to its proximity to the spinal cord and brain stem and its resistance to conventional treatments, such as radiation and chemotherapy. The development of effective treatments requires research at the molecular level, which presumably due to its rare diagnosis, is lacking for chordoma. Recent studies have identified potential targets for systemic therapy; however, there are currently no drugs approved by the Food and Drug Administration (FDA) to treat chordoma. One promising approach is to target the cytoskeleton, in order to stall progression and sensitize cells to chemotherapeutics. Similar to other cancers, chordoma cells co-express vimentin and cytokeratin intermediate filaments (IFs), which have both been found to play roles in cell mechanical properties and behaviors and their expression has been associated with cancer metastasis, chemoresistance, and poor prognosis. Therefore, we investigated the functional roles of vimentin and cytokeratin IFs in chordoma cells using RNA interference (RNAi). First, we examined whether cytoskeletal disruption by siRNA-mediated silencing of vimentin or cytokeratin-8 altered the chordoma phenotype. We determined that the vacuolated cytoplasm, a distinguishing feature of chordoma, was dependent on cytokeratin-8 IFs. Next, we examined the effects of vimentin and cytokeratin-8 knockdown on chordoma cell mechanics. We found that chordoma cell stiffness, traction forces, and mechanosensitivity to substrate stiffness were all dependent on vimentin IFs. These results suggest that vimentin, rather than cytokeratin, IFs play a predominant role in chordoma cell mechanobiology. Finally, we analyzed the roles of vimentin and cytokeratin-8 IFs in cellular behaviors associated with cancer progression. We demonstrated that chordoma cell invasion and expression of the biomarker sonic hedgehog were dependent on vimentin. Further, we found that decreasing vi

Background: Chordoma are malignant tumors of the axial skeleton, which arise from remnants of the notochord. The Notochord (chorda dorsalis) is an essential embryonic structure involved in the development of the nervous system and axial skeleton. Therefore, the notochord seems to be the most biologically relevant control tissue to study chordoma in molecular biology research. Nevertheless, up to now mainly different tissues but not the notochord have been used as control for chordoma, due to difficulty of isolating notochordal tissue. Here, we describe a fast and precise method of isolating notochordal cells.Methods: Examination of human fetuses, with a gestation of 9, 11 and 13 weeks, using (immuno) histochemical methods was performed. To isolate pure notochord cells for further molecular biology investigation five flash frozen fetuses between 9 and 10 weeks of gestation were dissected by microtome slicing. Thereafter pure notochord cells for further molecular biology investigation where harvested by using laser capture microdissection (LCM). RNA was extracted from these samples and used in quantitative PCR.Results: This study illustrates notochord of embryonic spines in three different stages of gestation (9-1113 weeks). Immunohistochemical staining with brachyury showed strong staining of the notochord, but also weak staining of the intervertebral disc and vertebral body. LCM of notochord slices and subsequent total RNA extraction resulted in a good yield of total RNA. qPCR analysis of two housekeeping genes confirmed the quality of the RNA.Conclusion: LCM is a fast and precise method to isolate notochord and the quality and yield RNA extracted from this tissue is sufficient for qPCR analysis. Therefore early embryo notochord isolated by LCM is suggested to be the gold standard for future research in chordoma development, classification and diagnosis. (C) 2016 Elsevier B.V. All rights reserved.

Background: Chordoma are malignant tumors of the axial skeleton, which arise from remnants of the notochord. The Notochord (chorda dorsalis) is an essential embryonic structure involved in the development of the nervous system and axial skeleton. Therefore, the notochord seems to be the most biologically relevant control tissue to study chordoma in molecular biology research. Nevertheless, up to now mainly different tissues but not the notochord have been used as control for chordoma, due to difficulty of isolating notochordal tissue. Here, we describe a fast and precise method of isolating notochordal cells.Methods: Examination of human fetuses, with a gestation of 9, 11 and 13 weeks, using (immuno) histochemical methods was performed. To isolate pure notochord cells for further molecular biology investigation five flash frozen fetuses between 9 and 10 weeks of gestation were dissected by microtome slicing. Thereafter pure notochord cells for further molecular biology investigation where harvested by using laser capture microdissection (LCM). RNA was extracted from these samples and used in quantitative PCR.Results: This study illustrates notochord of embryonic spines in three different stages of gestation (9-1113 weeks). Immunohistochemical staining with brachyury showed strong staining of the notochord, but also weak staining of the intervertebral disc and vertebral body. LCM of notochord slices and subsequent total RNA extraction resulted in a good yield of total RNA. qPCR analysis of two housekeeping genes confirmed the quality of the RNA.Conclusion: LCM is a fast and precise method to isolate notochord and the quality and yield RNA extracted from this tissue is sufficient for qPCR analysis. Therefore early embryo notochord isolated by LCM is suggested to be the gold standard for future research in chordoma development, classification and diagnosis. (C) 2016 Elsevier B.V. All rights reserved.

Lower back pain is the leading cause of disability worldwide. Discogenic pain secondary to intervertebral disc degeneration is a significant cause of low back pain. Disc degeneration is a complex multifactorial process. Animal models are essential to furthering understanding of the degenerative process and testing potential therapies. The adult human lumbar intervertebral disc is characterized by the loss of notochordal cells, relatively large size, essentially avascular nature, and exposure to biomechanical stresses influenced by bipedalism. Animal models are compared with regard to the above characteristics. Numerous methods of inducing disc degeneration are reported. Broadly these can be considered under the categories of spontaneous degeneration, mechanical and structural models. The purpose of such animal models is to further our understanding and, ultimately, improve treatment of disc degeneration. The role of animal models of disc degeneration in translational research leading to clinical trials of novel cellular therapies is explored.Copyright © 2016 Chris Daly et al.

Lower back pain is the leading cause of disability worldwide. Discogenic pain secondary to intervertebral disc degeneration is a significant cause of low back pain. Disc degeneration is a complex multifactorial process. Animal models are essential to furthering understanding of the degenerative process and testing potential therapies. The adult human lumbar intervertebral disc is characterized by the loss of notochordal cells, relatively large size, essentially avascular nature, and exposure to biomechanical stresses influenced by bipedalism. Animal models are compared with regard to the above characteristics. Numerous methods of inducing disc degeneration are reported. Broadly these can be considered under the categories of spontaneous degeneration, mechanical and structural models. The purpose of such animal models is to further our understanding and, ultimately, improve treatment of disc degeneration. The role of animal models of disc degeneration in translational research leading to clinical trials of novel cellular therapies is explored.Copyright © 2016 Chris Daly et al.

BACKGROUND: The T gene (brachyury gene) is the founding member of the T-box family of transcription factors and is vital for the formation and differentiation of the mesoderm and the axial development of all vertebrates.RESULTS: We report here on four patients from three consanguineous families exhibiting sacral agenesis, a persistent notochordal canal and abnormal ossification of the vertebral bodies, and the identification and characterisation of their underlying genetic defect. Given the consanguineous nature and the similarity of the phenotypes between the three families, we performed homozygosity mapping and identified a common 4.1 Mb homozygous region on chromosome 6q27, containing T, brachyury homologue (mouse) or T. Sequencing of T in the affected individuals led to the identification of a homozygous missense mutation, p.H171R, in the highly conserved T-box. The homozygous mutation results in diminished DNA binding, increased cell growth, and interferes with the normal expression of genes involved in ossification, notochord maintenance and axial mesoderm development.CONCLUSIONS: We have identified a shared homozygous mutation in three families in T and linked it to a novel syndrome consisting of sacral agenesis, a persistent notochordal canal and abnormal ossification of the vertebral bodies. We suggest that screening for the ossification of the vertebrae is warranted in patients with sacral agenesis to evaluate the possible causal involvement of T.

INTRODUCTION: Sacrococcigeal cordoma represents more than 50 percent of the cordomas. The total resection in block combined with radiotherapy in high dose has shown to prolong the recurrence.CLINICAL CASE: Male, 58 years old operated of sacrococcigeal cordoma is presented by means of total resection in block for posterior approach. The postoperative image studies confirmed the total removal. It presented cerebrospinal fistulae and local infection treated with spinal drainage and antibiotics. Also, transient urinary incontinence occurred for 6 months. After that he received cobalt radiotherapy. Until now he stayed asymptomatic and free of illness in resonance magnetic images.CONCLUSIONS: The best treatment options are obtained with a focus multidisciplinary, in specialized centers and with advanced neurosurgical and radiotherapy techniques., INTRODUCCIÓN: El cordoma sacrococcígeo representa más del 50 por ciento de los cordomas. La resección total en bloque combinada con radioterapia en altas dosis ha mostrado prolongar la recurrencia.CASO CLÍNICO: Se presenta un paciente masculino de 58 años operado de cordoma sacrococcígeo mediante resección total en bloque por vía posterior. Los estudios de imagen postoperatorios confirmaron la remoción total. Como complicaciones posquirúrgicas mediatas el paciente presentó una fístula de líquido cefalorraquídeo y una infección local que remitieron con drenaje espinal y antibioticoterapia. Además, presentó trastornos transitorios del control del esfínter vesical que desaparecieron a los 6 meses. Posteriormente recibió radioterapia con cobalto. Se ha mantenido asintomático y sin evidencia de recidiva en los estudios de imágenes por resonancia magnética.CONCLUSIONES: Las mejores opciones de tratamiento se obtienen con un enfoque multidisciplinario, en centros especializados y con avanzadas técnicas neuroquirúrgicas asociada a la radioterapia.

INTRODUCTION: Sacrococcigeal cordoma represents more than 50 percent of the cordomas. The total resection in block combined with radiotherapy in high dose has shown to prolong the recurrence.CLINICAL CASE: Male, 58 years old operated of sacrococcigeal cordoma is presented by means of total resection in block for posterior approach. The postoperative image studies confirmed the total removal. It presented cerebrospinal fistulae and local infection treated with spinal drainage and antibiotics. Also, transient urinary incontinence occurred for 6 months. After that he received cobalt radiotherapy. Until now he stayed asymptomatic and free of illness in resonance magnetic images.CONCLUSIONS: The best treatment options are obtained with a focus multidisciplinary, in specialized centers and with advanced neurosurgical and radiotherapy techniques., INTRODUCCIÓN: El cordoma sacrococcígeo representa más del 50 por ciento de los cordomas. La resección total en bloque combinada con radioterapia en altas dosis ha mostrado prolongar la recurrencia.CASO CLÍNICO: Se presenta un paciente masculino de 58 años operado de cordoma sacrococcígeo mediante resección total en bloque por vía posterior. Los estudios de imagen postoperatorios confirmaron la remoción total. Como complicaciones posquirúrgicas mediatas el paciente presentó una fístula de líquido cefalorraquídeo y una infección local que remitieron con drenaje espinal y antibioticoterapia. Además, presentó trastornos transitorios del control del esfínter vesical que desaparecieron a los 6 meses. Posteriormente recibió radioterapia con cobalto. Se ha mantenido asintomático y sin evidencia de recidiva en los estudios de imágenes por resonancia magnética.CONCLUSIONES: Las mejores opciones de tratamiento se obtienen con un enfoque multidisciplinario, en centros especializados y con avanzadas técnicas neuroquirúrgicas asociada a la radioterapia.

BACKGROUND: The T gene (brachyury gene) is the founding member of the T-box family of transcription factors and is vital for the formation and differentiation of the mesoderm and the axial development of all vertebrates.RESULTS: We report here on four patients from three consanguineous families exhibiting sacral agenesis, a persistent notochordal canal and abnormal ossification of the vertebral bodies, and the identification and characterisation of their underlying genetic defect. Given the consanguineous nature and the similarity of the phenotypes between the three families, we performed homozygosity mapping and identified a common 4.1 Mb homozygous region on chromosome 6q27, containing T, brachyury homologue (mouse) or T. Sequencing of T in the affected individuals led to the identification of a homozygous missense mutation, p.H171R, in the highly conserved T-box. The homozygous mutation results in diminished DNA binding, increased cell growth, and interferes with the normal expression of genes involved in ossification, notochord maintenance and axial mesoderm development.CONCLUSIONS: We have identified a shared homozygous mutation in three families in T and linked it to a novel syndrome consisting of sacral agenesis, a persistent notochordal canal and abnormal ossification of the vertebral bodies. We suggest that screening for the ossification of the vertebrae is warranted in patients with sacral agenesis to evaluate the possible causal involvement of T.

Much discussion about benign notochordal cell tissue in vertebrae has centered on the nature of its relationship, if any, to chordoma. Often referred to as benign notochordal cell tumors (BNCTs), these lesions have unique morphological features, however, differentiating between notochordal cells in discs, BNCT, and chordoma can be difficult. They are described as radiologically distinct from chordoma, with lysis, contrast enhancement, and a soft tissue mass indicating chordoma.

Los cordomas son neoplasias que derivan de restos embrionarios de la notocorda, siendo su principal asiento en la región sacrococcigea; los de localización cervical son poco frecuentes, suponen menos del 10% de todos los cordomas. Su diagnóstico se ve retrasado hasta que alcanzan un gran tamaño debido a su lento crecimiento, pese a lo cual son tumores localmente agresivos por su relación con estructuras neurovasculares críticas, presentando una alta tasa de recurrencia local. El tratamiento de elección es la cirugía radical y la radioterapia con protones se emplea para la persistencia tumoral y las recidivas. Presentamos el caso de un paciente con cordoma cervical, dada la rareza de esta entidad., Chordomas are neoplasms that arise from notochord embryonic remnants, been the sacrococcygeal spine the main site of involvement; the cervical spine site is uncommon and it account for less than 10% of chordomas. Because of their slow growth, the diagnosis is delayed until they reach a large size, despite which they are locally aggressive tumours due to their relation to critical neurovascular structures and present a high rate of local recurrence. Radical surgery is the elective treatment and proton radiotherapy is used when residual tumour tissue and recurrences. Because of the uncommonness of this pathology, we report a case of a patient with cervical chordoma.

Los cordomas son neoplasias que derivan de restos embrionarios de la notocorda, siendo su principal asiento en la región sacrococcigea; los de localización cervical son poco frecuentes, suponen menos del 10% de todos los cordomas. Su diagnóstico se ve retrasado hasta que alcanzan un gran tamaño debido a su lento crecimiento, pese a lo cual son tumores localmente agresivos por su relación con estructuras neurovasculares críticas, presentando una alta tasa de recurrencia local. El tratamiento de elección es la cirugía radical y la radioterapia con protones se emplea para la persistencia tumoral y las recidivas. Presentamos el caso de un paciente con cordoma cervical, dada la rareza de esta entidad., Chordomas are neoplasms that arise from notochord embryonic remnants, been the sacrococcygeal spine the main site of involvement; the cervical spine site is uncommon and it account for less than 10% of chordomas. Because of their slow growth, the diagnosis is delayed until they reach a large size, despite which they are locally aggressive tumours due to their relation to critical neurovascular structures and present a high rate of local recurrence. Radical surgery is the elective treatment and proton radiotherapy is used when residual tumour tissue and recurrences. Because of the uncommonness of this pathology, we report a case of a patient with cervical chordoma.

The mouse transcription factor Noto is expressed in the notochord and involved in its development. Noto mouse mutants, Noto(tc/tc)(truncate) and Noto(GFP/GFP) (Noto null mutant), exhibit a segmental lack of the notochord in the caudal part of the embryo and subsequent tail truncation in adult animals. In order to address the relationship between the tail bud (the undifferentiated mesenchymal cells in the tip of the embryo tail) and the caudal notochord, Noto(GFP/GFP), a loss- of-function mutant, was analyzed. Taking advantage of Noto(GFP/+) heterozygotes, we could track Noto-GFP-expressing cells from the tail bud over the tail cord to the caudal notochord, and confirm a morphological continuum from the tail bud mesenchyme to the caudal notochord. Loss of Noto affected tail bud morphogenesis: Noto-GFP-expressing cells were scattered in the tail bud mesenchyme. They segregated in the notochord-like structure within the medullary cord instead at the tail cord, which subsequently disappeared. In the tail cord, instead of the notochord, additional lumen of the tail gut was formed. These findings suggest that Noto is involved in both rearrangement and morphogenesis of the tail bud during notochord formation., JOURNAL ARTICLE, SCOPUS: ar.j, info:eu-repo/semantics/published

The mouse transcription factor Noto is expressed in the notochord and involved in its development. Noto mouse mutants, Noto(tc/tc)(truncate) and Noto(GFP/GFP) (Noto null mutant), exhibit a segmental lack of the notochord in the caudal part of the embryo and subsequent tail truncation in adult animals. In order to address the relationship between the tail bud (the undifferentiated mesenchymal cells in the tip of the embryo tail) and the caudal notochord, Noto(GFP/GFP), a loss- of-function mutant, was analyzed. Taking advantage of Noto(GFP/+) heterozygotes, we could track Noto-GFP-expressing cells from the tail bud over the tail cord to the caudal notochord, and confirm a morphological continuum from the tail bud mesenchyme to the caudal notochord. Loss of Noto affected tail bud morphogenesis: Noto-GFP-expressing cells were scattered in the tail bud mesenchyme. They segregated in the notochord-like structure within the medullary cord instead at the tail cord, which subsequently disappeared. In the tail cord, instead of the notochord, additional lumen of the tail gut was formed. These findings suggest that Noto is involved in both rearrangement and morphogenesis of the tail bud during notochord formation., JOURNAL ARTICLE, SCOPUS: ar.j, info:eu-repo/semantics/published

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SOX9 is an evolutionary conserved transcription factor that is expressed in a variety of tissues, with essential functions in cartilage, testis, heart, glial cell, inner ear and neural crest development. By comparing human and pufferfish genomic sequences, we previously identified eight highly conserved sequence elements between 290 kb 5' and 450 kb 3' to human SOX9. In this study, we assayed the regulatory potential of elements E1 to E7 in transgenic mice using a lacZ reporter gene driven by a 529 bp minimal mouse Sox9 promoter. We found that three of these elements and the Sox9 promoter control distinct subsets of the tissue-specific expression pattern of Sox9. E3, located 251 kb 5' to SOX9, directs lacZ expression to cranial neural crest cells and to the inner ear. E1 is located 28 kb 5' to SOX9 and controls expression in the node, notochord, gut, bronchial epithelium and pancreas. Transgene expression in the neuroectoderm is mediated by E7, located 95 kb 3' to SOX9, which regulates expression in the telencephalon and midbrain, and by the Sox9 minimal promoter which controls expression in the ventral spinal cord and hindbrain. We show that E3-directed reporter gene expression in neural crest cells of the first but not of the second and third pharyngeal arch is dependent on beta-catenin, revealing a complex regulation of Sox9 in cranial neural crest cells. Moreover, we identify and discuss highly conserved transcription factor binding sites within enhancer E3 that are in good agreement with current models for neural crest and inner ear development. Finally, we identify enhancer E1 as a cis-regulatory element conserved between vertebrates and invertebrates, indicating that some cis-regulatory sequences that control developmental genes in vertebrates might be phylogenetically ancient. © 2005 Elsevier Inc. All rights reserved.

SOX9 is an evolutionary conserved transcription factor that is expressed in a variety of tissues, with essential functions in cartilage, testis, heart, glial cell, inner ear and neural crest development. By comparing human and pufferfish genomic sequences, we previously identified eight highly conserved sequence elements between 290 kb 5' and 450 kb 3' to human SOX9. In this study, we assayed the regulatory potential of elements E1 to E7 in transgenic mice using a lacZ reporter gene driven by a 529 bp minimal mouse Sox9 promoter. We found that three of these elements and the Sox9 promoter control distinct subsets of the tissue-specific expression pattern of Sox9. E3, located 251 kb 5' to SOX9, directs lacZ expression to cranial neural crest cells and to the inner ear. E1 is located 28 kb 5' to SOX9 and controls expression in the node, notochord, gut, bronchial epithelium and pancreas. Transgene expression in the neuroectoderm is mediated by E7, located 95 kb 3' to SOX9, which regulates expression in the telencephalon and midbrain, and by the Sox9 minimal promoter which controls expression in the ventral spinal cord and hindbrain. We show that E3-directed reporter gene expression in neural crest cells of the first but not of the second and third pharyngeal arch is dependent on beta-catenin, revealing a complex regulation of Sox9 in cranial neural crest cells. Moreover, we identify and discuss highly conserved transcription factor binding sites within enhancer E3 that are in good agreement with current models for neural crest and inner ear development. Finally, we identify enhancer E1 as a cis-regulatory element conserved between vertebrates and invertebrates, indicating that some cis-regulatory sequences that control developmental genes in vertebrates might be phylogenetically ancient. © 2005 Elsevier Inc. All rights reserved.

Atrophins are evolutionarily conserved proteins that are thought to act as transcriptional co-repressors. Mammalian genomes contain two atrophin genes. Dominant polyglutamine-expanded alleles of atrophin 1 have been identified as the cause of dentatorubralpallidoluysian atrophy, an adult-onset human neurodegenerative disease with similarity to Huntington's. In a screen for recessive mutations that disrupt patterning of the early mouse embryo, we identified a line named openmind carrying a mutation in atrophin 2. openmind homozygous; embryos exhibit a variety of patterning defects that first appear at E8.0. Defects include a specific failure in ventralization of the anterior neural plate, loss of heart looping and irregular partitioning of somites. In mutant embryos, Shh expression fails to initiate along the anterior midline at E8.0, and Fgf8 is delocalized from the anterior neural ridge at E8.5, revealing a crucial role for atrophin 2 in the formation and function of these two signaling centers. Atrophin 2 is also required for normal organization of the apical ectodermal ridge, a signaling center that directs limb pattern. Elevated expression of atrophin 2 in neurons suggests it may interact with atrophin 1 in neuronal development or function. We further show that atrophin 2 associates with histone deacetylase 1 in mouse embryos, providing a biochemical link between Atr2 and a chromatin-modifying enzyme. Based on our results, and on those of others, we propose that atrophin proteins act as transcriptional co-repressors during embryonic development.

Atrophins are evolutionarily conserved proteins that are thought to act as transcriptional co-repressors. Mammalian genomes contain two atrophin genes. Dominant polyglutamine-expanded alleles of atrophin 1 have been identified as the cause of dentatorubralpallidoluysian atrophy, an adult-onset human neurodegenerative disease with similarity to Huntington's. In a screen for recessive mutations that disrupt patterning of the early mouse embryo, we identified a line named openmind carrying a mutation in atrophin 2. openmind homozygous; embryos exhibit a variety of patterning defects that first appear at E8.0. Defects include a specific failure in ventralization of the anterior neural plate, loss of heart looping and irregular partitioning of somites. In mutant embryos, Shh expression fails to initiate along the anterior midline at E8.0, and Fgf8 is delocalized from the anterior neural ridge at E8.5, revealing a crucial role for atrophin 2 in the formation and function of these two signaling centers. Atrophin 2 is also required for normal organization of the apical ectodermal ridge, a signaling center that directs limb pattern. Elevated expression of atrophin 2 in neurons suggests it may interact with atrophin 1 in neuronal development or function. We further show that atrophin 2 associates with histone deacetylase 1 in mouse embryos, providing a biochemical link between Atr2 and a chromatin-modifying enzyme. Based on our results, and on those of others, we propose that atrophin proteins act as transcriptional co-repressors during embryonic development.

Neural tube closure is a fundamental embryonic event whose molecular regulation is poorly understood. As mouse neurulation progresses along the spinal axis, there is a shift from midline neural plate bending to dorsolateral bending. Here, we show that midline bending is not essential for spinal closure since, in its absence, the neural tube can close by a 'default' mechanism involving dorsolateral bending, even at upper spinal levels. Midline and dorsolateral bending are regulated by mutually antagonistic signals from the notochord and surface ectoderm. Notochordal signaling induces midline bending and simultaneously inhibits dorsolateral bending. Sonic hedgehog is both necessary and sufficient to inhibit dorsolateral bending, but is neither necessary nor sufficient to induce midline bending, which seems likely to be regulated by another notochordal factor. Attachment of surface ectoderm cells to the neural plate is required for dorsolateral bending, which ensures neural tube closure in the absence of sonic hedgehog signaling.

It is well established that Hensen's nodes can induce the formation of supernumerary digits after grafting into the anterior margin of the developing limb bud. The recent finding that distinct mesodermal cell populations are segregated within the node has made it possible to isolate different prospective cell types in an attempt to correlate digit-inducing ability with cell fate. We find that the prospective notochord cells contained within Hensen's node are able to induce supernumerary digits, whereas presumptive somite cells cannot. This early difference in inducing ability persists into later stages of development: epithelial somites are unable to induce while notochord from all lengths of the neuraxis continues to induce. Using probes to retinoic acid receptor-beta 2 and sonic hedgehog (shh) we find no evidence to support the idea that inducing tissues generate extra digits by releasing retinoic acid into adjacent limb tissue but find that the inducing ability of a tissue correlates with its expression of shh.

It is well established that Hensen's nodes can induce the formation of supernumerary digits after grafting into the anterior margin of the developing limb bud. The recent finding that distinct mesodermal cell populations are segregated within the node has made it possible to isolate different prospective cell types in an attempt to correlate digit-inducing ability with cell fate. We find that the prospective notochord cells contained within Hensen's node are able to induce supernumerary digits, whereas presumptive somite cells cannot. This early difference in inducing ability persists into later stages of development: epithelial somites are unable to induce while notochord from all lengths of the neuraxis continues to induce. Using probes to retinoic acid receptor-beta 2 and sonic hedgehog (shh) we find no evidence to support the idea that inducing tissues generate extra digits by releasing retinoic acid into adjacent limb tissue but find that the inducing ability of a tissue correlates with its expression of shh.

Thesis (M.S.)--Florida Atlantic University, 1977., Summary: Paramyosin fibers were observed in newly born Gambusia affinis larvae. Extraction procedures specific for paramyosin apparently removed the protein as evidence by electron microscopic examination. Recrystallization and polyacrylamide gel electrophoresis have identified the fibers as relatively homogenous paramyosin protein. The midstage embryo did not possess paramyosin fibers; however, the notochord and adjacent tissues were observed to be in a state of rapid protein synthesis. Mitochondrial association with nuclei and 'semi-rough' endoplasmic reticulum were seen. Mitochondria were shown to possess lamellar bodies, which may be a form of degeneration. Lamellar body formation observed in other species of animals is discussed., Charles E. Schmidt College of Science