Publications by authors named "Martín I Garcia-Castro"

23 Publications

  • Page 1 of 1

Distinct molecular profile and restricted stem cell potential defines the prospective human cranial neural crest from embryonic stem cell state.

Stem Cell Res 2020 12 11;49:102086. Epub 2020 Nov 11.

School of Medicine Division of Biomedical Sciences, University of California, Riverside, USA. Electronic address:

Neural crest cells are an embryonic multipotent stem cell population. Recent studies in model organisms have suggested that neural crest cells are specified earlier than previously thought, at blastula stages. However, the molecular dynamics of early neural crest specification, and functional changes from pluripotent precursors to early specified NC, remain to be elucidated. In this report, we utilized a robust human model of cranial neural crest formation to address the distinct molecular character of the earliest stages of neural crest specification and assess the functional differences from its embryonic stem cell precursor. Our human neural crest model reveals a rapid change in the epigenetic state of neural crest and pluripotency genes, accompanied by changes in gene expression upon Wnt-based induction from embryonic stem cells. These changes in gene expression are directly regulated by the transcriptional activity of β-catenin. Furthermore, prospective cranial neural crest cells are characterized by restricted stem cell potential compared to embryonic stem cells. Our results suggest that human neural crest induced by Wnt/β-catenin signaling from human embryonic stem cells rapidly acquire a prospective neural crest cell state defined by a unique molecular signature and endowed with limited potential compared to pluripotent stem cells.
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http://dx.doi.org/10.1016/j.scr.2020.102086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7932500PMC
December 2020

RNA-based CRISPR-Mediated Loss-of-Function Mutagenesis in Human Pluripotent Stem Cells.

J Mol Biol 2020 06 25;432(13):3956-3964. Epub 2020 Apr 25.

Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, Viikinkaari 5 E, 00014 Helsinki, Finland. Electronic address:

Current approaches for Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-Associated-9 (Cas9)-mediated genome editing in human pluripotent stem (PS) cells mainly employ plasmids or ribonucleoprotein complexes. Here, we devise an improved transfection protocol of in vitro transcribed Cas9 mRNA and crRNA:tracrRNA duplex that can effectively generate indels in four genetic loci (two active and two inactive) and demonstrate utility in four human PS cell lines (one embryonic and three induced PS cell lines). Our improved protocol incorporating a Cas9-linked selection marker and a staggered transfection strategy promotes targeting efficiency up to 85% and biallelic targeting efficiency up to 76.5% of total mutant clones. The superior targeting efficiency and the non-integrative nature of our approach underscore broader applications in high-throughput arrayed CRISPR screening and in generating custom-made or off-the-shelf cell products for human therapy.
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http://dx.doi.org/10.1016/j.jmb.2020.04.017DOI Listing
June 2020

Disrupted ER membrane protein complex-mediated topogenesis drives congenital neural crest defects.

J Clin Invest 2020 02;130(2):813-826

Pediatric Genomics Discovery Program, Department of Pediatrics and Genetics, Yale University School of Medicine, New Haven, Connecticut, USA.

Multipass membrane proteins have a myriad of functions, including transduction of cell-cell signals, ion transport, and photoreception. Insertion of these proteins into the membrane depends on the endoplasmic reticulum (ER) membrane protein complex (EMC). Recently, birth defects have been observed in patients with variants in the gene encoding a member of this complex, EMC1. Patient phenotypes include congenital heart disease, craniofacial malformations, and neurodevelopmental disease. However, a molecular connection between EMC1 and these birth defects is lacking. Using Xenopus, we identified defects in neural crest cells (NCCs) upon emc1 depletion. We then used unbiased proteomics and discovered a critical role for emc1 in WNT signaling. Consistent with this, readouts of WNT signaling and Frizzled (Fzd) levels were reduced in emc1-depleted embryos, while NCC defects could be rescued with β-catenin. Interestingly, other transmembrane proteins were mislocalized upon emc1 depletion, providing insight into additional patient phenotypes. To translate our findings back to humans, we found that EMC1 was necessary for human NCC development in vitro. Finally, we tested patient variants in our Xenopus model and found the majority to be loss-of-function alleles. Our findings define molecular mechanisms whereby EMC1 dysfunction causes disease phenotypes through dysfunctional multipass membrane protein topogenesis.
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http://dx.doi.org/10.1172/JCI129308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994125PMC
February 2020

Blastula stage specification of avian neural crest.

Dev Biol 2020 02 11;458(1):64-74. Epub 2019 Oct 11.

Division of Biomedical Sciences, School of Medicine, University of California, Riverside, USA. Electronic address:

Cell fate specification defines the earliest steps towards a distinct cell lineage. Neural crest, a multipotent stem cell population, is thought to be specified from the ectoderm, but its varied contributions defy canons of segregation potential and challenges its embryonic origin. Aiming to resolve this conflict, we have assayed the earliest specification of neural crest using blastula stage chick embryos. Specification assays on isolated chick epiblast explants identify an intermediate region specified towards the neural crest cell fate. Furthermore, low density culture suggests that the specification of intermediate cells towards the neural crest lineage is independent of contact mediated induction and Wnt-ligand induced signaling, but is, however, dependent on transcriptional activity of β-catenin. Finally, we have validated the regional identity of the intermediate region towards the neural crest cell fate using fate map studies. Our results suggest a model of neural crest specification within a restricted epiblast region in blastula stage chick embryos.
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http://dx.doi.org/10.1016/j.ydbio.2019.10.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7050198PMC
February 2020

WNT/β-catenin modulates the axial identity of embryonic stem cell-derived human neural crest.

Development 2019 08 29;146(16). Epub 2019 Aug 29.

School of Medicine Division of Biomedical Sciences, University of California, Riverside, CA 92521, USA

WNT/β-catenin signaling is crucial for neural crest (NC) formation, yet the effects of the magnitude of the WNT signal remain ill-defined. Using a robust model of human NC formation based on human pluripotent stem cells (hPSCs), we expose that the WNT signal modulates the axial identity of NCs in a dose-dependent manner, with low WNT leading to anterior OTX HOX NC and high WNT leading to posterior OTX HOX NC. Differentiation tests of posterior NC confirm expected derivatives, including posterior-specific adrenal derivatives, and display partial capacity to generate anterior ectomesenchymal derivatives. Furthermore, unlike anterior NC, posterior NC exhibits a transient TBXT/SOX2 neuromesodermal precursor-like intermediate. Finally, we analyze the contributions of other signaling pathways in posterior NC formation, which suggest a crucial role for FGF in survival/proliferation, and a requirement of BMP for NC maturation. As expected retinoic acid (RA) and FGF are able to modulate HOX expression in the posterior NC. Surprisingly, early RA supplementation prohibits NC formation. This work reveals for the first time that the amplitude of WNT signaling can modulate the axial identity of NC cells in humans.
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http://dx.doi.org/10.1242/dev.175604DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6737904PMC
August 2019

FGF Modulates the Axial Identity of Trunk hPSC-Derived Neural Crest but Not the Cranial-Trunk Decision.

Stem Cell Reports 2019 05;12(5):920-933

University of California Riverside, Department of Biomedical Sciences, Riverside, CA 92521, USA. Electronic address:

The neural crest is a transient embryonic tissue that gives rise to a multitude of derivatives in an axially restricted manner. An in vitro counterpart to neural crest can be derived from human pluripotent stem cells (hPSCs) and can be used to study neural crest ontogeny and neurocristopathies, and to generate cells for therapeutic purposes. In order to successfully do this, it is critical to define the specific conditions required to generate neural crest of different axial identities, as regional restriction in differentiation potential is partly cell intrinsic. WNT and FGF signaling have been implicated as inducers of posterior fate, but the exact role that these signals play in trunk neural crest formation remains unclear. Here, we present a fully defined, xeno-free system for generating trunk neural crest from hPSCs and show that FGF signaling directs cells toward different axial identities within the trunk compartment while WNT signaling is the primary determinant of trunk versus cranial identity.
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http://dx.doi.org/10.1016/j.stemcr.2019.04.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6524753PMC
May 2019

Human neural crest induction by temporal modulation of WNT activation.

Dev Biol 2019 05 1;449(2):99-106. Epub 2019 Mar 1.

Division of Biomedical Sciences, School of Medicine, University of California Riverside, Riverside, California, USA. Electronic address:

The developmental biology of neural crest cells in humans remains unexplored due to technical and ethical challenges. The use of pluripotent stem cells to model human neural crest development has gained momentum. We recently introduced a rapid chemically defined approach to induce robust neural crest by WNT/β-CATENIN activation. Here we investigate the temporal requirements of ectopic WNT activation needed to induce neural crest cells. By altering the temporal activation of canonical WNT/β-CATENIN with a GSK3 inhibitor we find that a 2 Day pulse of WNT/β-CATENIN activation via GSK3 inhibition is optimal to generate bona fide neural crest cells, as shown by their capacity to differentiate to neural crest specific fates including peripheral neurons, glia, melanoblasts and ectomesenchymal osteocytes, chondrocytes and adipocytes. Although a 2 Day pulse can impart neural crest character when GSK3 is inhibited days after seeding, optimal results are obtained when WNT is activated from the beginning, and we find that the window of competence to induce NCs from non-neural ectodermal/placodal precursors closes by day 3 of culture. The reduced requirement for exogenous WNT activation offers an approach that is cost-effective, and we show that this adherent 2-dimensional approach is efficient in a broad range of culture platforms ranging from 96-well vessels to 10 cm dishes.
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http://dx.doi.org/10.1016/j.ydbio.2019.02.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6685424PMC
May 2019

Specification and formation of the neural crest: Perspectives on lineage segregation.

Genesis 2019 01 15;57(1):e23276. Epub 2019 Jan 15.

Division of Biomedical Sciences, School of Medicine, University of California, Riverside, California.

The neural crest is a fascinating embryonic population unique to vertebrates that is endowed with remarkable differentiation capacity. Thought to originate from ectodermal tissue, neural crest cells generate neurons and glia of the peripheral nervous system, and melanocytes throughout the body. However, the neural crest also generates many ectomesenchymal derivatives in the cranial region, including cell types considered to be of mesodermal origin such as cartilage, bone, and adipose tissue. These ectomesenchymal derivatives play a critical role in the formation of the vertebrate head, and are thought to be a key attribute at the center of vertebrate evolution and diversity. Further, aberrant neural crest cell development and differentiation is the root cause of many human pathologies, including cancers, rare syndromes, and birth malformations. In this review, we discuss the current findings of neural crest cell ontogeny, and consider tissue, cell, and molecular contributions toward neural crest formation. We further provide current perspectives into the molecular network involved during the segregation of the neural crest lineage.
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http://dx.doi.org/10.1002/dvg.23276DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6570420PMC
January 2019

Electroporation and in vitro culture of early rabbit embryos.

Data Brief 2018 Dec 4;21:316-320. Epub 2018 Oct 4.

School of Medicine Division of Biomedical Sciences, University of California, Riverside, CA 92521, USA.

The functional interrogation of factors underlying early mammalian development is necessary for the understanding and amelioration of human health conditions. The associated article [1] reports on the molecular characterization of markers of neural crest cells in gastrula and neurula stage rabbit embryos. This article presents survival data of rabbit embryos cultured in vitro, as well as immunofluorescence data for molecular markers of neural crest cells following approximately 24-h of culture. Lastly, towards the functional analysis of early neural crest and other developmental genes, this article provides data on the introduction of exogenous DNA into early stage rabbit embryos using electroporation.
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http://dx.doi.org/10.1016/j.dib.2018.09.131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6197720PMC
December 2018

Early specification and development of rabbit neural crest cells.

Dev Biol 2018 12 20;444 Suppl 1:S181-S192. Epub 2018 Jun 20.

School of Medicine Division of Biomedical Sciences, University of California, Riverside, CA 92521, USA. Electronic address:

The phenomenal migratory and differentiation capacity of neural crest cells has been well established across model organisms. While the earliest stages of neural crest development have been investigated in non-mammalian model systems such as Xenopus and Aves, the early specification of this cell population has not been evaluated in mammalian embryos, of which the murine model is the most prevalent. Towards a more comprehensive understanding of mammalian neural crest formation and human comparative studies, we have used the rabbit as a mammalian system for the study of early neural crest specification and development. We examine the expression profile of well-characterized neural crest markers in rabbit embryos across developmental time from early gastrula to later neurula stages, and provide a comparison to markers of migratory neural crest in the chick. Importantly, we apply explant specification assays to address the pivotal question of mammalian neural crest ontogeny, and provide the first evidence that a specified population of neural crest cells exists in the rabbit gastrula prior to the overt expression of neural crest markers. Finally, we demonstrate that FGF signaling is necessary for early rabbit neural crest formation, as SU5402 treatment strongly represses neural crest marker expression in explant assays. This study pioneers the rabbit as a model for neural crest development, and provides the first demonstration of mammalian neural crest specification and the requirement of FGF signaling in this process.
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http://dx.doi.org/10.1016/j.ydbio.2018.06.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6685428PMC
December 2018

Top-Down Inhibition of BMP Signaling Enables Robust Induction of hPSCs Into Neural Crest in Fully Defined, Xeno-free Conditions.

Stem Cell Reports 2017 10 14;9(4):1043-1052. Epub 2017 Sep 14.

University of Sheffield, Department of Biomedical Science, Sheffield S10 2TN, UK.

Defects in neural crest development have been implicated in many human disorders, but information about human neural crest formation mostly depends on extrapolation from model organisms. Human pluripotent stem cells (hPSCs) can be differentiated into in vitro counterparts of the neural crest, and some of the signals known to induce neural crest formation in vivo are required during this process. However, the protocols in current use tend to produce variable results, and there is no consensus as to the precise signals required for optimal neural crest differentiation. Using a fully defined culture system, we have now found that the efficient differentiation of hPSCs to neural crest depends on precise levels of BMP signaling, which are vulnerable to fluctuations in endogenous BMP production. We present a method that controls for this phenomenon and could be applied to other systems where endogenous signaling can also affect the outcome of differentiation protocols.
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http://dx.doi.org/10.1016/j.stemcr.2017.08.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5639211PMC
October 2017

WNT/β-catenin signaling mediates human neural crest induction via a pre-neural border intermediate.

Development 2016 Feb;143(3):398-410

Kline Biology Tower, Department of Molecular, Cellular and Developmental Biology, Yale University, 266 Whitney Avenue, New Haven, CT 06511, USA 203 School of Medicine Research Building, School of Medicine, University of California Riverside, Riverside, CA 92521, USA

Neural crest (NC) cells arise early in vertebrate development, migrate extensively and contribute to a diverse array of ectodermal and mesenchymal derivatives. Previous models of NC formation suggested derivation from neuralized ectoderm, via meso-ectodermal, or neural-non-neural ectoderm interactions. Recent studies using bird and amphibian embryos suggest an earlier origin of NC, independent of neural and mesodermal tissues. Here, we set out to generate a model in which to decipher signaling and tissue interactions involved in human NC induction. Our novel human embryonic stem cell (ESC)-based model yields high proportions of multipotent NC cells (expressing SOX10, PAX7 and TFAP2A) in 5 days. We demonstrate a crucial role for WNT/β-catenin signaling in launching NC development, while blocking placodal and surface ectoderm fates. We provide evidence of the delicate temporal effects of BMP and FGF signaling, and find that NC development is separable from neural and/or mesodermal contributions. We further substantiate the notion of a neural-independent origin of NC through PAX6 expression and knockdown studies. Finally, we identify a novel pre-neural border state characterized by early WNT/β-catenin signaling targets that displays distinct responses to BMP and FGF signaling from the traditional neural border genes. In summary, our work provides a fast and efficient protocol for human NC differentiation under signaling constraints similar to those identified in vivo in model organisms, and strengthens a framework for neural crest ontogeny that is separable from neural and mesodermal fates.
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http://dx.doi.org/10.1242/dev.130849DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4760313PMC
February 2016

Pax7 is regulated by cMyb during early neural crest development through a novel enhancer.

Development 2013 Sep;140(17):3691-702

Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520-8103, USA.

The neural crest (NC) is a migratory population of cells unique to vertebrates that generates many diverse derivatives. NC cells arise during gastrulation at the neural plate border (NPB), which is later elevated as the neural folds (NFs) form and fuse in the dorsal region of the closed neural tube, from where NC cells emigrate. In chick embryos, Pax7 is an early marker, and necessary component of NC development. Unlike other early NPB markers, which are co-expressed in lateral ectoderm, medial neural plate or posterior-lateral mesoderm, Pax7 early expression seems more restricted to the NPB. However, the molecular mechanisms controlling early Pax7 expression remain poorly understood. Here, we identify a novel enhancer of Pax7 in avian embryos that replicates the expression of Pax7 associated with early NC development. Expression from this enhancer is found in early NPB, NFs and early emigrating NC, but unlike Pax7, which is also expressed in mesodermal derivatives, this enhancer is not active in somites. Further analysis demonstrates that cMyb is able to interact with this enhancer and modulates reporter and endogenous early Pax7 expression; thus, cMyb is identified as a novel regulator of Pax7 in early NC development.
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http://dx.doi.org/10.1242/dev.088328DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3742149PMC
September 2013

SUMOylation of Pax7 is essential for neural crest and muscle development.

Cell Mol Life Sci 2013 May 18;70(10):1793-806. Epub 2012 Dec 18.

Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520-8103, USA.

Regulatory transcription factors of the Pax family play fundamental roles in the function of multipotent cells during vertebrate development, post-natal regeneration, and cancer. Pax7 and its homologue Pax3 are important players in neural crest and muscle development. Both genes are coexpressed in various tissues and are thought to provide similar, but not identical, functions. The mechanisms that allow specific regulation of Pax7 remain largely unknown. Here, we report for the first time that Pax7 is regulated by SUMOylation. We identify the interaction of Pax7 with Ubc9, the SUMO conjugating enzyme, and reveal that SUMOylation machinery is enriched in neural crest precursors and plays a critical role in NC development. We demonstrate that Pax7 becomes SUMOylated and identify an essential role for lysine 85 (K85) in Pax7-SUMOylation. Despite high conservation surrounding K85 amongst Pax genes, we were unable to identify SUMOylation of other Pax proteins tested, including Pax3. Using a non-SUMOylatable Pax7 variant (K85 X R), we demonstrate that SUMOylation is essential for the function of Pax7 in neural crest development, C2C12 myogenic differentiation, and transcriptional transactivation. Our study provides new mechanistic insight into the molecular regulation of Pax7's function by SUMOylation in neural crest and muscle development.
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http://dx.doi.org/10.1007/s00018-012-1220-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3628956PMC
May 2013

FGF signaling transforms non-neural ectoderm into neural crest.

Dev Biol 2012 Dec 19;372(2):166-77. Epub 2012 Sep 19.

Department of Molecular, Cellular, and Developmental Biology, KBT 1100, Yale University, P.O. Box 208103, New Haven, CT 06520-8103, USA.

The neural crest arises at the border between the neural plate and the adjacent non-neural ectoderm. It has been suggested that both neural and non-neural ectoderm can contribute to the neural crest. Several studies have examined the molecular mechanisms that regulate neural crest induction in neuralized tissues or the neural plate border. Here, using the chick as a model system, we address the molecular mechanisms by which non-neural ectoderm generates neural crest. We report that in response to FGF the non-neural ectoderm can ectopically express several early neural crest markers (Pax7, Msx1, Dlx5, Sox9, FoxD3, Snail2, and Sox10). Importantly this response to FGF signaling can occur without inducing ectopic mesodermal tissues. Furthermore, the non-neural ectoderm responds to FGF by expressing the prospective neural marker Sox3, but it does not express definitive markers of neural or anterior neural (Sox2 and Otx2) tissues. These results suggest that the non-neural ectoderm can launch the neural crest program in the absence of mesoderm, without acquiring definitive neural character. Finally, we report that prior to the upregulation of these neural crest markers, the non-neural ectoderm upregulates both BMP and Wnt molecules in response to FGF. Our results provide the first effort to understand the molecular events leading to neural crest development via the non-neural ectoderm in amniotes and present a distinct response to FGF signaling.
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http://dx.doi.org/10.1016/j.ydbio.2012.09.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3541687PMC
December 2012

Pax7 lineage contributions to the mammalian neural crest.

PLoS One 2012 27;7(7):e41089. Epub 2012 Jul 27.

Biology Department, Eastern Connecticut State University, Willimantic, Connecticut, United States of America.

Background: Neural crest cells are vertebrate-specific multipotent cells that contribute to a variety of tissues including the peripheral nervous system, melanocytes, and craniofacial bones and cartilage. Abnormal development of the neural crest is associated with several human maladies including cleft/lip palate, aggressive cancers such as melanoma and neuroblastoma, and rare syndromes, like Waardenburg syndrome, a complex disorder involving hearing loss and pigment defects. We previously identified the transcription factor Pax7 as an early marker, and required component for neural crest development in chick embryos. In mammals, Pax7 is also thought to play a role in neural crest development, yet the precise contribution of Pax7 progenitors to the neural crest lineage has not been determined.

Methodology/principal Findings: Here we use Cre/loxP technology in double transgenic mice to fate map the Pax7 lineage in neural crest derivates. We find that Pax7 descendants contribute to multiple tissues including the cranial, cardiac and trunk neural crest, which in the cranial cartilage form a distinct regional pattern. The Pax7 lineage, like the Pax3 lineage, is additionally detected in some non-neural crest tissues, including a subset of the epithelial cells in specific organs.

Conclusions/significance: These results demonstrate a previously unappreciated widespread distribution of Pax7 descendants within and beyond the neural crest. They shed light regarding the regionally distinct phenotypes observed in Pax3 and Pax7 mutants, and provide a unique perspective into the potential roles of Pax7 during disease and development.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0041089PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3407174PMC
April 2013

Current perspectives of the signaling pathways directing neural crest induction.

Cell Mol Life Sci 2012 Nov 1;69(22):3715-37. Epub 2012 May 1.

Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520-8103, USA.

The neural crest is a migratory population of embryonic cells with a tremendous potential to differentiate and contribute to nearly every organ system in the adult body. Over the past two decades, an incredible amount of research has given us a reasonable understanding of how these cells are generated. Neural crest induction involves the combinatorial input of multiple signaling pathways and transcription factors, and is thought to occur in two phases from gastrulation to neurulation. In the first phase, FGF and Wnt signaling induce NC progenitors at the border of the neural plate, activating the expression of members of the Msx, Pax, and Zic families, among others. In the second phase, BMP, Wnt, and Notch signaling maintain these progenitors and bring about the expression of definitive NC markers including Snail2, FoxD3, and Sox9/10. In recent years, additional signaling molecules and modulators of these pathways have been uncovered, creating an increasingly complex regulatory network. In this work, we provide a comprehensive review of the major signaling pathways that participate in neural crest induction, with a focus on recent developments and current perspectives. We provide a simplified model of early neural crest development and stress similarities and differences between four major model organisms: Xenopus, chick, zebrafish, and mouse.
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http://dx.doi.org/10.1007/s00018-012-0991-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3478512PMC
November 2012

FGF/MAPK signaling is required in the gastrula epiblast for avian neural crest induction.

Development 2012 Jan 30;139(2):289-300. Epub 2011 Nov 30.

Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520-8103, USA.

Neural crest induction involves the combinatorial inputs of the FGF, BMP and Wnt signaling pathways. Recently, a two-step model has emerged where BMP attenuation and Wnt activation induces the neural crest during gastrulation, whereas activation of both pathways maintains the population during neurulation. FGF is proposed to act indirectly during the inductive phase by activating Wnt ligand expression in the mesoderm. Here, we use the chick model to investigate the role of FGF signaling in the amniote neural crest for the first time and uncover a novel requirement for FGF/MAPK signaling. Contrary to current models, we demonstrate that FGF is required within the prospective neural crest epiblast during gastrulation and is unlikely to operate through mesodermal tissues. Additionally, we show that FGF/MAPK activity in the prospective neural plate prevents the ectopic expression of lateral ectoderm markers, independently of its role in neural specification. We then investigate the temporal participation of BMP/Smad signaling and suggest a later involvement in neural plate border development, likely due to widespread FGF/MAPK activity in the gastrula epiblast. Our results identify an early requirement for FGF/MAPK signaling in amniote neural crest induction and suggest an intriguing role for FGF-mediated Smad inhibition in ectodermal development.
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http://dx.doi.org/10.1242/dev.070276DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3243094PMC
January 2012

Embryonic Pax7-expressing progenitors contribute multiple cell types to the postnatal olfactory epithelium.

J Neurosci 2010 Jul;30(28):9523-32

Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06520-8103, USA.

Prolonged neurogenesis driven by stem/progenitor cells is a hallmark of the olfactory epithelium (OE), beginning at the placodal stages in the embryo and continuing throughout adult life. Despite the progress made to identify and study the regulation of adult OE progenitors, our knowledge of embryonic OE precursors and their cellular contributions to the adult OE has been stalled by the lack of markers able to distinguish individual candidate progenitors. Here we identify embryonic OE Pax7+ progenitors, detected at embryonic day 10.5 (E10.5) in the olfactory pit with an antigen profile and location previously assigned to presumptive OE stem cells. Using Cre-loxP technology (Pax7-cre/ROSA YFP mice), we expose a wide range of derivatives, including CNS and olfactory neurons, non-neuronal cells, and olfactory ensheathing glia, all made from embryonic Pax7+ cells. Importantly, the expression of Pax7 in the embryonic OE is downregulated from E15.5, such that after birth, no Pax7+ cells are found in the OE, and thus the progenitor population here identified is restricted to embryonic stages. Our results provide the first evidence for a population of Pax7-expressing embryonic progenitors that contribute to multiple OE lineages and demonstrate novel insights into the unique spatiotemporal patterning of the postnatal OE.
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http://dx.doi.org/10.1523/JNEUROSCI.0867-10.2010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2920205PMC
July 2010

Analysis of early human neural crest development.

Dev Biol 2010 Aug 15;344(2):578-92. Epub 2010 May 15.

Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520-8103, USA.

The outstanding migration and differentiation capacities of neural crest cells (NCCs) have fascinated scientists since Wilhelm His described this cell population in 1868. Today, after intense research using vertebrate model organisms, we have gained considerable knowledge regarding the origin, migration and differentiation of NCCs. However, our understanding of NCC development in human embryos remains largely uncharacterized, despite the role the neural crest plays in several human pathologies. Here, we report for the first time the expression of a battery of molecular markers before, during, or following NCC migration in human embryos from Carnegie Stages (CS) 12 to 18. Our work demonstrates the expression of Sox9, Sox10 and Pax3 transcription factors in premigratory NCCs, while actively migrating NCCs display the additional transcription factors Pax7 and AP-2alpha. Importantly, while HNK-1 labels few migrating NCCs, p75(NTR) labels a large proportion of this population. However, the broad expression of p75(NTR) - and other markers - beyond the neural crest stresses the need for the identification of additional markers to improve our capacity to investigate human NCC development, and to enable the generation of better diagnostic and therapeutic tools.
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http://dx.doi.org/10.1016/j.ydbio.2010.05.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2927129PMC
August 2010

Specification of the neural crest occurs during gastrulation and requires Pax7.

Nature 2006 May;441(7090):218-22

Division of Biology 139-74, California Institute of Technology, Pasadena, California 91125, USA.

The neural crest is a stem population critical for development of the vertebrate craniofacial skeleton and peripheral ganglia. Neural crest cells originate along the border between the neural plate and epidermis, migrate extensively and generate numerous derivatives, including neurons and glia of the peripheral nervous system, melanocytes, bone and cartilage of the head skeleton. Impaired neural crest development is associated with human defects, including cleft palate. Classically, the neural crest has been thought to form by interactions at the border between neural and non-neural ectoderm or mesoderm, and defined factors such as bone morphogenetic proteins (BMPs) and Wnt proteins have been postulated as neural crest-inducers. Although competence to induce crest cells declines after stage 10 (ref. 14), little is known about when neural crest induction begins in vivo. Here we report that neural crest induction is underway during gastrulation and well before proper neural plate appearance. We show that a restricted region of chick epiblast (stage 3-4) is specified to generate neural crest cells when explanted under non-inducing conditions. This region expresses the transcription factor Pax7 by stage 4 + and later contributes to neural folds and migrating neural crest. In chicken embryos, Pax7 is required for neural crest formation in vivo, because blocking its translation inhibits expression of the neural crest markers Slug, Sox9, Sox10 and HNK-1. Our results indicate that neural crest specification initiates earlier than previously assumed, independently of mesodermal and neural tissues, and that Pax7 has a crucial function during neural crest development.
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http://dx.doi.org/10.1038/nature04684DOI Listing
May 2006

Molecular mechanisms of neural crest induction.

Birth Defects Res C Embryo Today 2004 Jun;72(2):109-23

Division of Biology, California Institute of Technology, Pasadena 91125, USA.

The neural crest is an embryonic cell population that originates at the border between the neural plate and the prospective epidermis. Around the time of neural tube closure, neural crest cells emigrate from the neural tube, migrate along defined paths in the embryo and differentiate into a wealth of derivatives. Most of the craniofacial skeleton, the peripheral nervous system, and the pigment cells of the body originate from neural crest cells. This cell type has important clinical relevance, since many of the most common craniofacial birth defects are a consequence of abnormal neural crest development. Whereas the migration and differentiation of the neural crest have been extensively studied, we are just beginning to understand how this tissue originates. The formation of the neural crest has been described as a classic example of embryonic induction, in which specific tissue interactions and the concerted action of signaling pathways converge to induce a multipotent population of neural crest precursor cells. In this review, we summarize the current status of knowledge on neural crest induction. We place particular emphasis on the signaling molecules and tissue interactions involved, and the relationship between neural crest induction, the formation of the neural plate and neural plate border, and the genes that are upregulated as a consequence of the inductive events.
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http://dx.doi.org/10.1002/bdrc.20015DOI Listing
June 2004

Ectodermal Wnt function as a neural crest inducer.

Science 2002 Aug;297(5582):848-51

Division of Biology 139-74, California Institute of Technology, Wilson and California, Pasadena, CA 91125, USA.

Neural crest cells, which generate peripheral nervous system and facial skeleton, arise at the neural plate/ectodermal border via an inductive interaction between these tissues. Wnts and bone morphogenetic proteins (BMPs) play roles in neural crest induction in amphibians and zebrafish. Here, we show that, in avians, Wnt6 is localized in ectoderm and in vivo inhibition of Wnt signaling perturbs neural crest formation. Furthermore, Wnts induce neural crest from naive neural plates in vitro in a defined medium without added factors, whereas BMPs require additives. Our data suggest that Wnt molecules are necessary and sufficient to induce neural crest cells in avian embryos.
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August 2002