Neural Crest Differentiation Research Articles

Expression of actin‐binding proteins and requirement for actin‐depolymerizing factor in chick neural crest cells

Neural crest cells are multipotent cells that migrate extensively throughout vertebrate embryos to form diverse lineages. Cell migration requires polarized, organized actin networks that provide the driving force for motility. Actin-binding proteins that regulate neural crest cell migration are just beginning to be defined. We recently identified a number of actin-associated factors through proteomic profiling of methylated proteins in migratory neural crest cells. Here, we report the previously undocumented expression pattern of three of these proteins in chick early neural crest development: doublecortin (DCX), tropomyosin-1 (TPM-1), and actin depolymerizing factor (ADF). All three genes are expressed with varying degrees of specificity and intensity in premigratory and migratory neural crest cells, and their resulting proteins exhibit distinct subcellular localization in migratory neural crest cells. Morpholino knock down of ADF reveals it is required for Sox10 gene expression, but minimally important during neural crest migration. Neural crest cells express DCX, TPM-1, and ADF. ADF is necessary during neural crest specification, but largely dispensable for migration.

Transcriptome analysis reveals novel players in the cranial neural crest gene regulatory network

The neural crest is an embryonic stem cell population that gives rise to a multitude of derivatives. In particular, the cranial neural crest (CNC) is unique in its ability to contribute to both facial skeleton and peripheral ganglia. To gain further insight into the molecular underpinnings that distinguish the CNC from other embryonic tissues, we have utilized a CNC-specific enhancer as a tool to isolate a pure, region-specific NC subpopulation for transcriptional profiling. The resulting data set reveals previously unknown transcription factors and signaling pathways that may influence the CNC's ability to migrate and/or differentiate into unique derivatives. To elaborate on the CNC gene regulatory network, we evaluated the effects of knocking down known neural plate border genes and early neural crest specifier genes on selected neural crest-enriched transcripts. The results suggest that ETS1 and SOX9 may act as pan-neural crest regulators of the migratory CNC. Taken together, our analysis provides unprecedented characterization of the migratory CNC transcriptome and identifies new links in the gene regulatory network responsible for development of this critical cell population.

Notch signaling regulates neural crest differentiation from human pluripotent stem cells

Neural crest cells are specified at the border between the neural plate and the epiderm. They are capable of differentiating into various somatic cell types, including craniofacial and peripheral nerve tissues. Notch signaling plays important roles during neurogenesis; however, its function during human neural crest development is poorly understood. Here, we generated self-renewing premigratory neural-crest-like cells (pNCCs) from human pluripotent stem cells (hPSCs) and investigated the roles of Notch signaling during neural crest differentiation. pNCCs expressed various neural-crest-specifier genes, including SLUG (also known as SNAI2), SOX10 and TWIST1, and were able to differentiate into most neural crest derivatives. Blocking Notch signaling during the pNCC differentiation suppressed the expression of neural-crest-specifier genes. By contrast, ectopic expression of activated Notch1 intracellular domain (NICD1) augmented the expression of neural-crest-specifier genes, and NICD1 was found to bind to their promoter regions. Notch activity was also required for the maintenance of the premigratory neural crest state, and the suppression of Notch signaling led to the generation of neural-crest-derived neurons. Taken together, we provide a protocol for the generation of pNCCs and show that Notch signaling regulates the formation, migration and differentiation of neural crest from hPSCs.

Serotonin is required for pharyngeal arch morphogenesis in zebrafish

Abstract Serotonin (5-HT) is not only a neurotransmitter but also a mediator of developmental processes in vertebrates. In this study, we analyzed the importance of 5-HT during zebrafish development. The expression patterns of three zebrafish tryptophan hydroxylase isoforms (Tph1A, Tph1B, Tph2), the rate-limiting enzymes in 5-HT synthesis, were analyzed and compared to the appearance and distribution of 5-HT. 5-HT was found in the raphe nuclei correlating with tph2 expression and in the pineal gland correlating with tph1a and tph2 expressions. Tph2-deficient fish generated with antisense morpholino oligonucleotides exhibited morphogenesis defects during pharyngeal arch development. The correct specification of neural crest (NC) cells was not affected in tph2 morphants as shown by the expression of early markers, but the survival and differentiation of pharyngeal arch progenitor cells were impaired. An organizing role of 5-HT in pharyngeal arch morphogenesis was suggested by a highly regular pattern of 5-HT positive cells in this tissue. Moreover, the 5-HT2B receptor was expressed in the pharyngeal arches and its pharmacological inhibition also induced defects in pharyngeal arch morphogenesis. These results support an important role of Tph2-derived serotonin as a morphogenetic factor in the development of NC-derived tissues.

Diagnostic utility of SOX10 to distinguish malignant peripheral nerve sheath tumor from synovial sarcoma, including intraneural synovial sarcoma

Synovial sarcoma and malignant peripheral nerve sheath tumor pose a significant diagnostic challenge given similar histomorphology. The distinction is further complicated by similar immunophenotype and especially by occasional synovial sarcomas that present as intraneural tumors. Although the presence of a t(X;18) rearrangement or expression of TLE1 can help confirm the diagnosis of synovial sarcoma, negative results for these tests are not diagnostic of malignant peripheral nerve sheath tumor. The SOX10 transcription factor, a putative marker of neural crest differentiation, may have diagnostic utility in this differential, but immunohistochemical data are limited. The goal of the present study was to determine the diagnostic utility of SOX10 to discriminate between synovial sarcoma and malignant peripheral nerve sheath tumor. Forty-eight cases of malignant peripheral nerve sheath tumor, all from patients with documented neurofibromatosis, and 97 cases of genetically confirmed synovial sarcoma, including 4 intraneural synovial sarcomas, were immunohistochemically stained for SOX10. The stain was scored for intensity and fraction of cells staining. Thirty-two of 48 malignant peripheral nerve sheath tumors (67%) were SOX10-positive. The majority of malignant peripheral nerve sheath tumors showed ≥2+ staining, but staining did not correlate with grade. By contrast, only 7/97 (7%) synovial sarcomas were SOX10-positive. Only three synovial sarcomas showed ≥2+ staining but, importantly, two of these were intraneural synovial sarcoma. Therefore, SOX10 is a specific (93%), albeit not very sensitive (67%), diagnostic marker to support a diagnosis of malignant peripheral nerve sheath tumor over synovial sarcoma. Furthermore, the stain needs to be interpreted with caution in intraneural tumors in order to avoid a potential diagnostic pitfall. It remains to be determined whether SOX10-positive cells in intraneural synovial sarcoma represent entrapped Schwann cells, synovial sarcoma cells or both.

IN VITRO PROPERTIES OF NEURAL CREST-DERIVED MULTIPOTENT STEM CELLS FROM A BULGE REGION OF WHISKER FOLLICLE

71 The neural crest (NC), a transient structure in embryo development, is unique to vertebrates. NC is derived from the ectoderm and formed at the border of neuroectoderm and somatic ectoderm. Originally NC is represented by a cell population lying in the form of epithelial cords emerging from both sides of the neural tube. At the neurulation process end the NC cells undergo an epithelialmesenchymal transition (EMT) and migrate distantly in various tissues and organs of the developing embryo, where they give rise to the multiple differentiated cell types [1, 2]. In adult vertebrate organism the NC derivatives are represented by the following cell types: neurons and glial cells of the peripheral nervous system, the majority of sensory neurons, melanocytes, Merkel cells, odontoblasts, stromal and endothelial cells of cornea, C cells of thyroid gland, the adrenal medulla, a part of thymic stroma [1, 2]. The most plastic are the cells of cranial NC. In addition to the above mentioned differentiated cell types, they form a part of the bone, cartilage and connective tissues of the face, forehead and ventral neck [3, 4]. Unusually wide differentiation potential of the NC cells suggests an existence of multipotent stem cells. Stemple and Anderson firstly revealed the existence of multipotent stem cells in the NC in early embryonic stage of mammalian development [5]. A long time it was thought that, like NC per se, neural crestderived multipotent stem cells (NC-MSCs) have a transient nature. Consequently, the NCMSCs exist only at the premigratory (from the NC specification moment until the beginning of its cell migration) and migratory stages (from the NC cell delamination until their incoming into the sites of final destination), and further, within migration to sites of its final localization, undergo a process of commitment, turning into the progenitor cells with limited proliferative and/or differentiation potential [6, 7]. An example is the transition of NC-MSCs into the melanocyte precursors — melanoblasts. In the last decades, NC-derived cells have been isolated from a number of tissues and organs of the adult mammalians. These cells demonstrate the ability to self-renewal and multilineage differentiation, at least in vitro, i.e., they are multipotent stem cells [8–11]. The origin of these cells from NC was proved by creating special lines of transgenic mice [12, 13], as well as by means of their transplantation in the developing chick embryo, where the NC-MSCs migrated via the UDC 576.3+612.014.2/3

Developmental expression and role of Kinesin Eg5 during Xenopus laevis embryogenesis

The neural crest is a transient multipotent migratory cell population unique to vertebrates. These cells undergo an epithelial-to-mesenchymal transition and migrate extensively through the embryo. They differentiate into numerous diverse derivatives including the peripheral nervous system, melanocytes,and craniofacial cartilages. The development of the neural crest is mediated by complex interactions of multiple signals and transcription factors. The kinesin Eg5 is a plus end-directed microtubule-based motor protein that is essential for bipolar spindle formation during mitosis and meiosis, axon growth, and mammal embryonic development. We analyzed in detail the expression pattern of eg5 and established that it is expressed at the prospective neural fold, in the premigratory and migratory neural crest. Functional analysis revealed that in Xenopus, early embryogenesis eg5 function is required during neural crest induction, specification, and maintenance. eg5 is also required during neural crest migration and for derivatives formation. Moreover, we demonstrated a hierarchical relationship with the Indian Hedgehog signaling pathway. Our results show that eg5 is essential for the specification and maintenance of neural crest progenitors during Xenopus early embryogenesis rather than cell proliferation and survival.

Pax3 and Zic1 trigger the early neural crest gene regulatory network by the direct activation of multiple key neural crest specifiers

Neural crest development is orchestrated by a complex and still poorly understood gene regulatory network. Premigratory neural crest is induced at the lateral border of the neural plate by the combined action of signaling molecules and transcription factors such as AP2, Gbx2, Pax3 and Zic1. Among them, Pax3 and Zic1 are both necessary and sufficient to trigger a complete neural crest developmental program. However, their gene targets in the neural crest regulatory network remain unknown. Here, through a transcriptome analysis of frog microdissected neural border, we identified an extended gene signature for the premigratory neural crest, and we defined novel potential members of the regulatory network. This signature includes 34 novel genes, as well as 44 known genes expressed at the neural border. Using another microarray analysis which combined Pax3 and Zic1 gain-of-function and protein translation blockade, we uncovered 25 Pax3 and Zic1 direct targets within this signature. We demonstrated that the neural border specifiers Pax3 and Zic1 are direct upstream regulators of neural crest specifiers Snail1/2, Foxd3, Twist1, and Tfap2b. In addition, they may modulate the transcriptional output of multiple signaling pathways involved in neural crest development (Wnt, Retinoic Acid) through the induction of key pathway regulators (Axin2 and Cyp26c1). We also found that Pax3 could maintain its own expression through a positive autoregulatory feedback loop. These hierarchical inductions, feedback loops, and pathway modulations provide novel tools to understand the neural crest induction network.

Biphasic influence of Miz1 on neural crest development by regulating cell survival and apical adhesion complex formation in the developing neural tube

Myc interacting zinc finger protein-1 (Miz1) is a transcription factor known to regulate cell cycle- and cell adhesion-related genes in cancer. Here we show that Miz1 also plays a critical role in neural crest development. In the chick, Miz1 is expressed throughout the neural plate and closing neural tube. Its morpholino-mediated knockdown affects neural crest precursor survival, leading to reduction of neural plate border and neural crest specifier genes Msx-1, Pax7, FoxD3, and Sox10. Of interest, Miz1 loss also causes marked reduction of adhesion molecules (N-cadherin, cadherin6B, and α1-catenin) with a concomitant increase of E-cadherin in the neural folds, likely leading to delayed and decreased neural crest emigration. Conversely, Miz1 overexpression results in up-regulation of cadherin6B and FoxD3 expression in the neural folds/neural tube, leading to premature neural crest emigration and increased number of migratory crest cells. Although Miz1 loss effects cell survival and proliferation throughout the neural plate, the neural progenitor marker Sox2 was unaffected, suggesting a neural crest-selective effect. The results suggest that Miz1 is important not only for survival of neural crest precursors, but also for maintenance of integrity of the neural folds and tube, via correct formation of the apical adhesion complex therein.

Sip1 mediates an E-cadherin-to-N-cadherin switch during cranial neural crest EMT

The neural crest, an embryonic stem cell population, initially resides within the dorsal neural tube but subsequently undergoes an epithelial-to-mesenchymal transition (EMT) to commence migration. Although neural crest and cancer EMTs are morphologically similar, little is known regarding conservation of their underlying molecular mechanisms. We report that Sip1, which is involved in cancer EMT, plays a critical role in promoting the neural crest cell transition to a mesenchymal state. Sip1 transcripts are expressed in premigratory/migrating crest cells. After Sip1 loss, the neural crest specifier gene FoxD3 was abnormally retained in the dorsal neuroepithelium, whereas Sox10, which is normally required for emigration, was diminished. Subsequently, clumps of adherent neural crest cells remained adjacent to the neural tube and aberrantly expressed E-cadherin while lacking N-cadherin. These findings demonstrate two distinct phases of neural crest EMT, detachment and mesenchymalization, with the latter involving a novel requirement for Sip1 in regulation of cadherin expression during completion of neural crest EMT.

Role ofSp5as an essential early regulator of neural crest specification inxenopus

The neural crest (NC) is a multipotent embryonic cell population, which is induced by an integration of secreted signals including BMP, Wnt, and FGF and, subsequently, NC cell fates are specified by a regulatory network of specific transcription factors. This study was undertaken to identify a role of Sp5 transcription factor in vertebrates. Xenopus Sp5 is expressed in the prospective neural crest regions from gastrulation through the tadpole stages in early development. Knockdown of Sp5 caused severe defects in craniofacial cartilage, pigmentation, and dorsal fin. Gain- and loss-of-function of Sp5 led to up- and down-regulation of the expression of NC markers in the neural fold, respectively. In contrast, Sp5 had no effect on neural induction and patterning. Sp5 regulated the expression of neural plate border (NPB) specifiers, Msx1 and Pax3, and these regulatory factors recovered the expression of NC marker in the Sp5-deficient embryos. Depletion of Sp5 impaired NC induction by Wnt/β-catenin or FGF signal, whereas its co-expression rescued NC markers in embryos in which either signal was blocked. These results suggest that Sp5 functions as a critical early factor in the genetic cascade to regulate NC induction downstream of Wnt and FGF pathways.

In This Issue…

HomeCirculation ResearchVol. 113, No. 8In This Issue… Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticlePDF/EPUBIn This Issue… Originally published27 Sep 2013https://doi.org/10.1161/01.res.0000435046.80748.fdCirculation Research. 2013;113:945Smad2 in VSMC Differentiation of Neural Crest (p e76)Smad2 and MRTFB cooperate to turn neural crest cells into vascular smooth muscle, report Xie et al.Download figureDownload PowerPointProper differentiation of vascular smooth muscle cells (VSMCs) is not only essential for building blood vessels in the embryo, but also in adulthood, where abnormal VSMC differentiation is implicated in atherosclerosis, hypertension, and restenosis after angioplasty. Interestingly, VSMCs derive from 8 different progenitor cell types and exhibit differing characteristics depending on their origins. Xie and colleagues studied the neural crest cell (NCC) derived VSMCs, which ultimately locate to the aortic arch arteries, ascending aorta and carotid arteries. In vitro studies indicate that growth factor TGFβ regulates differentiation of NCC to VSMC, but in vivo experiments have given contrasting results. Xie and colleagues thus turned to a downstream target of TGFβ—Smad2—to resolve the issue. They deleted Smad2 from NCCs in mice and found that while NCCs still migrated to the aortic arch, they failed to differentiate to VSMCs. The VSMC layer in carotid arteries was also diminished. The team also showed that, in a mouse neural crest cell line, Smad2 interacted with transcription factor MRTFB—an important regulator of NCC to VSMC differentiation—which led to upregulation of VSMC markers. The authors therefore suggest that TGFβ signaling, via Smad2, is indeed important for NCC to VSMC differentiation.Myocardial Efferocytosis Heals the Heart (p 1004)Wan et al identify a macrophage receptor responsible for the disposal of dead heart cells after myocardial infarction.Download figureDownload PowerPointAfter a heart attack, macrophages gather at the injury site to gobble-up dying cardiomyocytes. This feasting on cellular corpses, or efferocytosis, is essential for efficient resolution of inflammation, which in turn limits the degree of adverse cardiac remodeling and the risk of progression to heart failure. Indeed, it has been shown that defective cardiomyocyte clearance in older mice is associated with impaired heart repair, and that macrophages from older animals exhibit reduced efferocytosis. To learn more, Wan and colleagues examined macrophages with deletions of known efferocytosis receptors. They found only one—MERTK—that was essential for removal of necrotic cardiomyocytes through phagocytosis. They also found that mice lacking MERTK still recruited macrophages to their hearts following myocardial infarction. However, inflammation was slow to resolve, there were more dying and dead cardiomyocytes, infarct size was larger, and heart function was worse. Interestingly, while levels of the MERTK receptor increased in the hearts of wild-type mice 3 to 7 days after a myocardial infarction, the authors also found that some of the MERTK was cleaved into an inactive form. Why this inactivation occurs, whether it worsens with age, and whether it can be prevented to improve recovery after myocardial infarction are all avenues the authors plan to investigate.CEACAM1 in Ischemic Stroke (p 1013)Ludewig et al identify an inflammation inhibitor in mice that, after a stroke, limits the breakdown of the blood brain barrier and thus further injury.Download figureDownload PowerPointAfter the initial hypoxic insult of an ischemic stroke, the problems are far from over. The resulting tissue damage leads to recruitment of immune cells, which cause inflammation with possible deterioration of the blood brain barrier and further brain damage. Immune cells express adhesion molecules that allow the cells to stick at the injury site and infiltrate the tissue. Blocking certain adhesion proteins has been shown to reduce cerebral damage after stroke in mice. But Ludewig and colleagues hypothesized that blocking the adhesion molecule CEACAM1 might have the opposite effect—because although it is also expressed on immune cells, it suppresses inflammation. They were right: Mice that lacked CEACAM1 faired worse after experimentally induced stroke. Their infarct sizes were larger, they had more pronounced cerebral inflammation, and their motility and neurological performance were more impaired. It turned out that without the anti-inflammatory action of CEACAM1, more immune cells that produced MMP-9—a secreted protease that promoted the breakdown of the blood brain barrier—were recruited to the ischemic brain. Together the results suggest that boosting CEACAM1 or inhibiting MMP-9 might be attractive ways to limit brain damage after stroke. Previous Back to top Next FiguresReferencesRelatedDetails September 27, 2013Vol 113, Issue 8 Advertisement Article InformationMetrics © 2013 American Heart Association, Inc.https://doi.org/10.1161/01.res.0000435046.80748.fd Originally publishedSeptember 27, 2013 PDF download Advertisement

An epigenetic signature of developmental potential in neural stem cells and early neurons

A cardinal property of neural stem cells (NSCs) is their ability to adopt multiple fates upon differentiation. The epigenome is widely seen as a read-out of cellular potential and a manifestation of this can be seen in embryonic stem cells (ESCs), where promoters of many lineage-specific regulators are marked by a bivalent epigenetic signature comprising trimethylation of both lysine 4 and lysine 27 of histone H3 (H3K4me3 and H3K27me3, respectively). Bivalency has subsequently emerged as a powerful epigenetic indicator of stem cell potential. Here, we have interrogated the epigenome during differentiation of ESC-derived NSCs to immature GABAergic interneurons. We show that developmental transitions are accompanied by loss of bivalency at many promoters in line with their increasing developmental restriction from pluripotent ESC through multipotent NSC to committed GABAergic interneuron. At the NSC stage, the promoters of genes encoding many transcriptional regulators required for differentiation of multiple neuronal subtypes and neural crest appear to be bivalent, consistent with the broad developmental potential of NSCs. Upon differentiation to GABAergic neurons, all non-GABAergic promoters resolve to H3K27me3 monovalency, whereas GABAergic promoters resolve to H3K4me3 monovalency or retain bivalency. Importantly, many of these epigenetic changes occur before any corresponding changes in gene expression. Intriguingly, another group of gene promoters gain bivalency as NSCs differentiate toward neurons, the majority of which are associated with functions connected with maturation and establishment and maintenance of connectivity. These data show that bivalency provides a dynamic epigenetic signature of developmental potential in both NSCs and in early neurons.

Identification and dissection of a key enhancer mediating cranial neural crest specific expression of transcription factor, Ets-1

Neural crest cells form diverse derivatives that vary according to their level of origin along the body axis, with only cranial neural crest cells contributing to facial skeleton. Interestingly, the transcription factor Ets-1 is uniquely expressed in cranial but not trunk neural crest, where it functions as a direct input into neural crest specifier genes, Sox10 and FoxD3. We have isolated and interrogated a cis-regulatory element, conserved between birds and mammals, that drives reporter expression in a manner that recapitulates that of endogenous Ets-1 expression in the neural crest. Within a minimal Ets-1 enhancer region, mutation of putative binding sites for SoxE, homeobox, Ets, TFAP2 or Fox proteins results in loss or reduction of neural crest enhancer activity. Morpholino-mediated loss-of-function experiments show that Sox9, Pax7, Msx1/2, Ets-1, TFAP2A and FoxD3, all are required for enhancer activity. In contrast, mutation of a putative cMyc/E-box sequence augments reporter expression, consistent with this being a repressor binding site. Taken together, these results uncover new inputs into Ets-1, revealing critical links in the cranial neural crest gene regulatory network.

Endothelin regulates neural crest deployment and fate to form great vessels through Dlx5/Dlx6-independent mechanisms

Endothelin-1 (Edn1), originally identified as a vasoconstrictor peptide, is involved in the development of cranial/cardiac neural crest-derived tissues and organs. In craniofacial development, Edn1 binds to Endothelin type-A receptor (Ednra) to induce homeobox genes Dlx5/Dlx6 and determines the mandibular identity in the first pharyngeal arch. However, it remains unsolved whether this pathway is also critical for pharyngeal arch artery development to form thoracic arteries. Here, we show that the Edn1/Ednra signaling is involved in pharyngeal artery development by controlling the fate of neural crest cells through a Dlx5/Dlx6-independent mechanism. Edn1 and Ednra knock-out mice demonstrate abnormalities in pharyngeal arch artery patterning, which include persistent first and second pharyngeal arteries, resulting in additional branches from common carotid arteries. Neural crest cell labeling with Wnt1-Cre transgene and immunostaining for smooth muscle cell markers revealed that neural crest cells abnormally differentiate into smooth muscle cells at the first and second pharyngeal arteries of Ednra knock-out embryos. By contrast, Dlx5/Dlx6 knockout little affect the development of pharyngeal arch arteries and coronary arteries, the latter of which is also contributed by neural crest cells through an Edn-dependent mechanism. These findings indicate that the Edn1/Ednra signaling regulates neural crest differentiation to ensure the proper patterning of pharyngeal arch arteries, which is independent of the regional identification of the pharyngeal arches along the dorsoventral axis mediated by Dlx5/Dlx6.

Cardiac progenitor cells from the left atrial appendage may originate from a resident non-hematopoietic myeloid progenitor population

Purpose: We recently identified three distinct cardiac progenitor cell (CPC) populations from the left atrial appendages (LAAs) of adult murine hearts. These populations have distinct phenotypes and characteristics. Type A CPCs are c-kit+/Sca-1+, Type B CPCs are c-kit+/CD45+, and more mature Type C CPCs can be derived from Type B CPCs in vitro. The purpose of this study was to analyze the embryologic origin and the connection between the different CPC populations. Methods: We performed a whole-genome expression array to the cell sorted Type A, Type B and Type C CPC populations. The LAAs were analyzed using immunohistochemistry. Tissue culture derived cells were analyzed using immunostaining and FACS. Neural crest lineage tracing was performed with Pax3-cre eYFP reporter mice. We investigated the spontaneous cardiac differentiation process from Type B to Type C using live microscopy and FACS. Results: Transcriptome comparison demonstrated a significant up-regulation of the neural crest differentiation –pathway genes in Type A and C CPCs. Type B CPCs expressed a gene profile, which matches that of non-hematopoietic, resident macrophage –like cells found in other tissues. Pax3 lineage tracing demonstrated traced cells in the subepicardial zone of the LAA, co-stained with F4/80 macrophage marker and c-kit. Closely attached were epicardially lined F4/80+ cells with no eYFP signal. In tissue culture, eYFP signal was strongest in the Type B CPCs and lower in the Type A CPCs. Fibroblasts in the culture were eYFP negative. The CD45 expression in the Type B CPCs was stable (∼90% of the cells) up to five passages folllowed by a rapid decline of CD45, c-kit and Flk-1 expression. In live microscopy, formation of giant Sca-1+ and Gata-4+ cells by cell fusion was followed by asymmetric divisions creating Type C CPCs, which expressed cardiac myosin heavy chain and atrial natriuretic factor. The transcriptome was significantly skewed toward a cardiomyocyte phenotype with the spontaneous differentiation, although this differentiation was not complete, as seen by electron microscopy. The partially differentiated Type C CPC population could be grown for >60 passages. Conclusion: Our results suggest that rather than having distinct embryonic origins, the different types of CPCs in the LAA originate from a resident, non-hematopoietic myeloid progenitor cell population. The neural crest differentiation pathway can be activated in these cells during adulthood, possibly in cardiac stress situations, enabling differentiation to cardiac lineage with a novel mechanism consisting of cell fusion and asymmetric division.

Prdm1a directly activates foxd3 and tfap2a during zebrafish neural crest specification

The neural crest comprises multipotent precursor cells that are induced at the neural plate border by a series of complex signaling and genetic interactions. Several transcription factors, termed neural crest specifiers, are necessary for early neural crest development; however, the nature of their interactions and regulation is not well understood. Here, we have established that the PR/SET domain-containing transcription factor Prdm1a is co-expressed with two essential neural crest specifiers, foxd3 and tfap2a, at the neural plate border. Through rescue experiments, chromatin immunoprecipitation and reporter assays, we have determined that Prdm1a directly binds to and transcriptionally activates enhancers for foxd3 and tfap2a and that they are functional, direct targets of Prdm1a at the neural plate border. Additionally, analysis of dominant activator and dominant repressor Prdm1a constructs suggests that Prdm1a is required both as a transcriptional activator and transcriptional repressor for neural crest development in zebrafish embryos.

Expression and Functional Characterization of Xhmg-at-hook Genes in Xenopus laevis

High Mobility Group A proteins (HMGA1 and HMGA2) are architectural nuclear factors involved in development, cell differentiation, and cancer formation and progression. Here we report the cloning, developmental expression and functional analysis of a new multi-AT-hook factor in Xenopus laevis (XHMG-AT-hook) that exists in three different isoforms. Xhmg-at-hook1 and 3 isoforms, but not isoform 2, are expressed throughout the entire development of Xenopus, both in the maternal and zygotic phase. Localized transcripts are present in the animal pole in the early maternal phase; during the zygotic phase, mRNA can be detected in the developing central nervous system (CNS), including the eye, and in the neural crest. We show evidence that XHMG-AT-hook proteins differ from typical HMGA proteins in terms of their properties in DNA binding and in protein/protein interaction. Finally, we provide evidence that they are involved in early CNS development and in neural crest differentiation.

SP8 regulates signaling centers during craniofacial development

Much of the bone, cartilage and smooth muscle of the vertebrate face is derived from neural crest (NC) cells. During craniofacial development, the anterior neural ridge (ANR) and olfactory pit (OP) signaling centers are responsible for driving the outgrowth, survival, and differentiation of NC populated facial prominences, primarily via FGF. While much is known about the functional importance of signaling centers, relatively little is understood of how these signaling centers are made and maintained. In this report we describe a dramatic craniofacial malformation in mice mutant for the zinc finger transcription factor gene Sp8. At E14.5 they show facial prominences that are reduced in size and underdeveloped, giving an almost faceless phenotype. At later times they show severe midline defects, excencephaly, hyperterlorism, cleft palate, and a striking loss of many NC and paraxial mesoderm derived cranial bones. Sp8 expression was primarily restricted to the ANR and OP regions during craniofacial development. Analysis of an extensive series of conditional Sp8 mutants confirmed the critical role of Sp8 in signaling centers, and not directly in the NC and paraxial mesoderm cells. The NC cells of the Sp8 mutants showed increased levels of apoptosis and decreased cell proliferation, thereby explaining the reduced sizes of the facial prominences. Perturbed gene expression in the Sp8 mutants was examined by laser capture microdissection coupled with microarrays, as well as in situ hybridization and immunostaining. The most dramatic differences included striking reductions in Fgf8 and Fgf17 expression in the ANR and OP signaling centers. We were also able to achieve genetic and pharmaceutical partial rescue of the Sp8 mutant phenotype by reducing Sonic Hedgehog (SHH) signaling. These results show that Sp8 primarily functions to promote Fgf expression in the ANR and OP signaling centers that drive the survival, proliferation, and differentiation of the NC and paraxial mesoderm that make the face.

The neural crest.

The neural crest (NC) is a highly migratory multipotent cell population that forms at the interface between the neuroepithelium and the prospective epidermis of a developing embryo. Following extensive migration throughout the embryo, NC cells eventually settle to differentiate into multiple cell types, ranging from neurons and glial cells of the peripheral nervous system to pigment cells, fibroblasts to smooth muscle cells, and odontoblasts to adipocytes. NC cells migrate in large numbers and their migration is regulated by multiple mechanisms, including chemotaxis, contact-inhibition of locomotion and cell sorting. Here, we provide an overview of NC formation, differentiation and migration, highlighting the molecular mechanisms governing NC migration.