Post-embryonic nerve-associated precursors to adult pigment cells: genetic requirements and dynamics of morphogenesis and differentiation

The pigment cells of vertebrates serve a variety of functions and generate a stunning variety of patterns. These cells are also implicated in human pathologies including melanoma. Whereas the events of pigment cell development have been studied extensively in the embryo, much less is known about morphogenesis and differentiation of these cells during post-embryonic stages. Previous studies of zebrafish revealed genetically distinct populations of embryonic and adult melanophores, the ectotherm homologue of amniote melanocytes. Here, we use molecular markers, vital labeling, time-lapse imaging, mutational analyses, and transgenesis to identify peripheral nerves as a niche for precursors to adult melanophores that subsequently migrate to the skin to form the adult pigment pattern. We further identify genetic requirements for establishing, maintaining, and recruiting precursors to the adult melanophore lineage and demonstrate novel compensatory behaviors during pattern regulation in mutant backgrounds. Finally, we show that distinct populations of latent precursors having differential regenerative capabilities persist into the adult. These findings provide a foundation for future studies of post-embryonic pigment cell precursors in development, evolution, and neoplasia.     

Cell dynamics and gene expression control in tissue homeostasis and development

During tissue and organ development and maintenance, the dynamic regulation of cellular proliferation and differentiation allows cells to build highly elaborate structures. The development of the vertebrate retina or the maintenance of adult intestinal crypts, for instance, involves the arrangement of newly created cells with different phenotypes, the proportions of which need to be tightly controlled. While some of the basic principles underlying these processes developing and maintaining these organs are known, much remains to be learnt from how cells encode the necessary information and use it to attain those complex but reproducible arrangements. Here, we review the current knowledge on the principles underlying cell population dynamics during tissue development and homeostasis. In particular, we discuss how stochastic fate assignment, cell division, feedback control and cellular transition states interact during organ and tissue development and maintenance in multicellular organisms. We propose a framework, involving the existence of a transition state in which cells are more susceptible to signals that can affect their gene expression state and influence their cell fate decisions. This framework, which also applies to systems much more amenable to quantitative analysis like differentiating embryonic stem cells, links gene expression programmes with cell population dynamics.     

Rod photoreceptor development in vitro: intrinsic properties of proliferating neuroepithelial cells change as development proceeds in the rat retina

We describe a reaggregated cell culture system in which retinal neuroepithelial cells from embryonic rats proliferate extensively and give rise to rod photoreceptors on the same schedule in vitro as they do in vivo. Both the proliferative potential of the embryonic neuroepithelial cells and the timing of their differentiation into rods are not changed by the presence of a 50-fold excess of neonatal neural retinal cells, although many more of the embryonic cells develop into rods in these circumstances. In such mixed-age cultures, dividing neonatal cells proliferate much less and give rise to rods much sooner than do dividing embryonic cells, suggesting that the dividing cells at the two ages are intrinsically different. These and other findings suggest that both cell-cell interactions and an intrinsic program in neuroepithelial cells determine cell fate in the developing rat retina.     

Cell Labeling and Injection in Developing Embryonic Mouse Hearts

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect their in vivo potential. Preferably, these cells should be placed in a proper embryonic environment in order to acquire their definite phenotype. Furthermore, cell lineage tracing studies in the mouse after labeling cells with dyes or retroviral vectors has remained mostly limited to early stage mouse embryos with still poorly developed organs. To overcome these limitations, we designed standard and ultrasound-mediated microinjection protocols to inject various agents in targeted regions of the heart in mouse embryos at E9.5 and later stages of development.  Embryonic explant or embryos are then cultured or left to further develop in utero. These agents include fluorescent dyes, virus, shRNAs, or stem cell-derived progenitor cells. Our approaches allow for preservation of the function of the organ while monitoring migration and fate of labeled and/or injected cells. These technologies can be extended to other organs and will be very helpful to address key biological questions in biology of development.     

The endoderm of the mouse embryo arises by dynamic widespread intercalation of embryonic and extraembryonic lineages

The cell movements underlying the morphogenesis of the embryonic endoderm, the tissue that will give rise to the respiratory and digestive tracts, are complex and not well understood. Using live imaging combined with genetic labeling, we investigated the cell behaviors and fate of the visceral endoderm during gut endoderm formation in the mouse gastrula. Contrary to the prevailing view, our data reveal no mass displacement of visceral endoderm to extraembryonic regions concomitant with the emergence of epiblast-derived definitive endoderm. Instead, we observed dispersal of the visceral endoderm epithelium and extensive mixing between cells of visceral endoderm and epiblast origin. Visceral endoderm cells remained associated with the epiblast and were incorporated into the early gut tube. Our findings suggest that the segregation of extraembryonic and embryonic tissues within the mammalian embryo is not as strict as believed and that a lineage previously defined as exclusively extraembryonic contributes cells to the embryo.     

Regulation of neural crest cell populations: occurrence, distribution and underlying mechanisms

Regulation is a significant developmental event because successful cell proliferation and migration are critical to shaping young embryos. Regulation -- the replacement of undifferentiated embryonic cells by other cells in response to signals received from the environment -- is distinct from wound healing and regeneration. Investigations on regulation of neural crest cells span all vertebrates and have revealed that regulative ability varies both among classes (even species), and spatially and temporally within individuals. In general, there is greatest regulation for cranial neural crest cells, less for trunk, and virtually none forcardiac. Regulation also appears to be more complete at early embryonic stages. Fate-mapping studies have demonstrated that large regions of neural crest cells must be removed to generate missing or morphologically reduced structures. Recent studies reveal that less extensive neural crest cell extirpations result in normal morphology of cartilaginous and neuronal elements in the head, and normal development of pigmentation in the trunk. Ablation of cardiac neural crest cells frequently generates abnormalities of the heart, great vessels and parasympathetic nerve innervation. Decreased cell death, increased division, change in fate and altered migration are possible cellular mechanisms of regulation. In mostcases, the specific mechanisms of regulation are unknown, but a major premise underlying regulation is that cell potential is greater than cell fate. This concept was born from studies which demonstrated that some cells were able to express alternative fates if transplanted to a new environment. Among the potential cellular mechanisms for regulation, cell migration has received the most attention. Following ablation of neural crest cells, replacement neural crest cells migrate into gaps, most frequently from anterior/posterior locations. Cells from surrounding epidermal and neural ectoderm may have limited regulative ability, while compensation by cells from the ventral neural tube has been demonstrated to an even lesser extent. Regulation by such non-crest cells would require their transformation into neural crest cells. The potential for regulation of neural crest by placodal cells supports a closer relationship between neural crest and placodal ectoderm than previously recognized. Decreased cell death has been discussed primarily with reference to (1) cranial ganglia that have dual contributions from neural crest and placodal cells and (2) programmed cell death in rhombomeres three and five. Increased cell division in response to neural crest ablation is likely more common than has been reported, but this mechanism is difficult to interpret without a 3-D context for viewing how patterns of division differ from normal. Lastly, changes in cell fate may be the driving factor in regulation of embryonic cells. It has been repeatedly demonstrated thatcell potential is greaterthan cell fate. Once reliable mechanisms for assessing cell potential are established, we may find that fates are commonly altered in response to environmental signals. Regulation is therefore significant both as a basic developmental mechanism and as a mechanism for evolutionary change. The more labile the fate of embryonic cells, the more potential there is for maintaining existing characters and for generating new ones. According to Ettensohn (1992, p. 50), further analysis of such systems might . With regard to the neural crest, studies on regulation of this vital population of cells provide insight to the origin of the neural crest, to embryonic repair, and to the source of many craniofacial malformations, heart and other embryonic defects. (ABSTRACT TRUNCATED)     

Germline stem cells in the Drosophila ovary descend from pole cells in the anterior region of the embryonic gonad

A fundamental yet unexplored question in stem cell biology is how the fate of tissue stem cells is initially determined during development. In Drosophila, germline stem cells (GSCs) descend from a subset of primordial germ cells (PGCs) at the onset of oogenesis. GSC determination may occur at the onset of oogenesis when a subset of PGCs is induced to become GSCs by contacting niche cells. Alternatively, the GSC fate could be predetermined for a subset of PGCs before oogenesis, due to either their interaction with specific somatic cells in the embryonic/larval gonads, or their inherently heterogeneous potential in becoming GSCs, or both. Here, we show that anterior somatic cells in the embryonic gonad already differ from posterior somatic cells and are likely to be the precursors of niche cells in the adult ovary. Furthermore, only pole cells in the anterior half of the embryonic gonad give rise to the PGCs that frequently acquire contact with nascent niche cells in the late larval ovary. Eventually, only these contacting PGCs become GSCs, whereas non-contacting PGCs directly differentiate into cystoblasts. The strong preference of these 'anterior PGCs' towards contacting niche cells does not require DE-cadherin-mediated adhesion and is not correlated with either orientation or rate of their divisions. These data suggest that the GSC fate is predetermined before oogenesis. The predetermination probably involves soma/pole-cell interaction in the anterior half of the embryonic gonad, followed by an active homing mechanism during PGC proliferation to maintain the contact between the 'anterior PGCs' and anterior somatic cells.     

Neural crest cells: From developmental biology to clinical interventions

Neural crest cells are multipotent cells, which are specified in embryonic ectoderm in the border of neural plate and epiderm during early development by interconnection of extrinsic stimuli and intrinsic factors. Neural crest cells are capable of differentiating into various somatic cell types, including melanocytes, craniofacial cartilage and bone, smooth muscle, and peripheral nervous cells, which supports their promise for cell therapy. In this work, we provide a comprehensive review of wide aspects of neural crest cells from their developmental biology to applicability in medical research. We provide a simplified model of neural crest cell development and highlight the key external stimuli and intrinsic regulators that determine the neural crest cell fate. Defects of neural crest cell development leading to several human disorders are also mentioned, with the emphasis of using human induced pluripotent stem cells to model neurocristopathic syndromes. Birth Defects Research (Part C) 102:263–274, 2014. © 2014 Wiley Periodicals, Inc.     

Cytoskeletal polarity mediates localized induction of the heart progenitor lineage

Cells must make appropriate fate decisions within a complex and dynamic environment. In vitro studies indicate that the cytoskeleton acts as an integrative platform for this environmental input. External signals regulate cytoskeletal dynamics and the cytoskeleton reciprocally modulates signal transduction. However, in vivo studies linking cytoskeleton/signalling interactions to embryonic cell fate specification remain limited. Here we show that the cytoskeleton modulates heart progenitor cell fate. Our studies focus on differential induction of heart fate in the basal chordate Ciona intestinalis. We have found that differential induction does not simply reflect differential exposure to the inductive signal. Instead, pre-cardiac cells employ polarized, invasive protrusions to localize their response to an ungraded signal. Through targeted manipulation of the cytoskeletal regulator CDC42, we are able to depolarize protrusive activity and generate uniform heart progenitor fate specification. Furthermore, we are able to restore differential induction by repolarizing protrusive activity. These findings illustrate how bi-directional interactions between intercellular signalling and the cytoskeleton can influence embryonic development. In particular, these studies highlight the potential for dynamic cytoskeletal changes to refine cell fate specification in response to crude signal gradients.     

Keeping things quiet: roles of NuRD and Sin3 co-repressor complexes during mammalian development

Gene inactivation studies of mammalian histone and DNA-modifying proteins have demonstrated a role for many such proteins in embryonic development. Post-implantation embryonic lethality implies a role for epigenetic factors in differentiation and in development of specific lineages or tissues. However a handful of chromatin-modifying enzymes have been found to be required in pre- or peri-implantation embryos. This is significant as implantation is the time when inner cell mass cells of the blastocyst exit pluripotency and begin to commit to form the various lineages that will eventually form the adult animal. These observations indicate a critical role for chromatin-modifying proteins in the earliest lineage decisions of mammalian development, and/or in the formation of the first embryonic cell types. Recent work has shown that the two major class I histone deacetylase-containing co-repressor complexes, the NuRD and Sin3 complexes, are both required at peri-implantation stages of mouse development, demonstrating the importance of histone deacetylation in cell fate decisions. Over the past 10 years both genetic and biochemical studies have revealed surprisingly divergent roles for these two co-repressors in mammalian cells. In this review we will summarise the evidence that the two major class I histone deacetylase complexes in mammalian cells, the NuRD and Sin3 complexes, play important roles in distinct aspects of embryonic development.     

Early Cell Fate Decisions of Human Embryonic Stem Cells and Mouse Epiblast Stem Cells Are Controlled by the Same Signalling Pathways

Human embryonic stem cells have unique value for regenerative medicine, as they are capable of differentiating into a broad variety of cell types. Therefore, defining the signalling pathways that control early cell fate decisions of pluripotent stem cells represents a major task. Moreover, modelling the early steps of embryonic development in vitro may provide the best approach to produce cell types with native properties. Here, we analysed the function of key developmental growth factors such as Activin, FGF and BMP in the control of early cell fate decisions of human pluripotent stem cells. This analysis resulted in the development and validation of chemically defined culture conditions for achieving specification of human embryonic stem cells into neuroectoderm, mesendoderm and into extra-embryonic tissues. Importantly, these defined culture conditions are devoid of factors that could obscure analysis of developmental mechanisms or render the resulting tissues incompatible with future clinical applications. Importantly, the growth factor roles defined using these culture conditions similarly drove differentiation of mouse epiblast stem cells derived from post implantation embryos, thereby reinforcing the hypothesis that epiblast stem cells share a common embryonic identity with human pluripotent stem cells. Therefore the defined growth factor conditions described here represent an essential step toward the production of mature cell types from pluripotent stem cells in conditions fully compatible with clinical use ant also provide a general approach for modelling the early steps of mammalian embryonic development.     

Wnt/Notch signalling and information processing during development

The Wnt and Notch signalling pathways represent two major channels of communication used by animal cells to control their identities and behaviour during development. A number of reports indicate that their activities are closely intertwined during embryonic development. Here, we review the evidence for this relationship and suggest that Wnt and Notch ('Wntch') signalling act as components of an integrated device that, rather than defining the fate of a cell, determines the probability that a cell will adopt that fate.     

Photomodulatable fluorescent proteins for imaging cell dynamics and cell fate

An organism arises from the coordinate generation of different cell types and the stereotypical organization of these cells into tissues and organs. Even so, the dynamic behaviors, as well as the ultimate fates, of cells driving the morphogenesis of an organism, or even an individual organ, remain largely unknown. Continued innovations in optical imaging modalities, along with the discovery and evolution of improved genetically-encoded fluorescent protein reporters in combination with model organism, stem cell and tissue engineering paradigms are providing the means to investigate these unresolved questions. The emergence of fluorescent proteins whose spectral properties can be photomodulated is one of the most significant new developments in the field of cell biology where they are primarily used for studying protein dynamics in cells. Likewise, the use of photomodulatable fluorescent proteins holds great promise for use in developmental biology. Photomodulatable fluorescent proteins also represent attractive and emergent tools for studying cell dynamics in complex populations by facilitating the labeling and tracking of individual or defined groups of cells. Here, we review the currently available photomodulatable fluorescent proteins and their application in model organisms. We also discuss prospects for their use in mice, and by extension in embryonic stem cell and tissue engineering paradigms.Key words: fluorescent protein, photomodulation, photoactivation, photoconversion, mouse, live imaging, embryonic development, organogenesis, GFP, PA-GFP, PS-CFP, Kaede, KikGR     

Sequential specification of neurons and glia by developmentally regulated extracellular factors

Cortical progenitor cells give rise to neurons during embryonic development and to glia after birth. While lineage studies indicate that multipotent progenitor cells are capable of generating both neurons and glia, the role of extracellular signals in regulating the sequential differentiation of these cells is poorly understood. To investigate how factors in the developing cortex might influence cell fate, we developed a cortical slice overlay assay in which cortical progenitor cells are cultured over cortical slices from different developmental stages. We find that embryonic cortical progenitors cultured over embryonic cortical slices differentiate into neurons and those cultured over postnatal cortical slices differentiate into glia, suggesting that the fate of embryonic progenitors can be influenced by developmentally regulated signals. In contrast, postnatal progenitor cells differentiate into glial cells when cultured over either embryonic or postnatal cortical slices. Clonal analysis indicates that the postnatal cortex produces a diffusible factor that induces progenitor cells to adopt glial fates at the expense of neuronal fates. The effects of the postnatal cortical signals on glial cell differentiation are mimicked by FGF2 and CNTF, which induce glial fate specification and terminal glial differentiation respectively. These observations indicate that cell fate specification and terminal differentiation can be independently regulated and suggest that the sequential generation of neurons and glia in the cortex is regulated by a developmental increase in gliogenic signals.     

Parathyroid hormone-related protein maintains mammary epithelial fate and triggers nipple skin differentiation during embryonic breast development

Prior reports have demonstrated that both parathyroid hormone-related protein (PTHrP) and the type I PTH/PTHrP receptor are necessary for the proper development of the embryonic mammary gland in mice. Using a combination of loss-of-function and gain-of-function models, we now report that PTHrP regulates a series of cell fate decisions that are central to the survival and morphogenesis of the mammary epithelium and the formation of the nipple. PTHrP is made in the epithelial cells of the mammary bud and, during embryonic mammary development, it interacts with the surrounding mesenchymal cells to induce the formation of the dense mammary mesenchyme. In response, these mammary-specific mesenchymal cells support the maintenance of mammary epithelial cell fate, trigger epithelial morphogenesis and induce the overlying epidermis to form the nipple. In the absence of PTHrP signaling, the mammary epithelial cells revert to an epidermal fate, no mammary ducts are formed and the nipple does not form. In the presence of diffuse epidermal PTHrP signaling, the ventral dermis is transformed into mammary mesenchyme and the entire ventral epidermis becomes nipple skin. These alterations in cell fate require that PTHrP be expressed during development and they require the presence of the PTH/PTHrP receptor. Finally, PTHrP signaling regulates the epidermal and mesenchymal expression of LEF1 and (&bgr;)-catenin, suggesting that these changes in cell fate involve an interaction between the PTHrP and Wnt signaling pathways.     

Assessment of pluripotency and multilineage differentiation potential of NTERA-2 cells as a model for studying human embryonic stem cells

Embryonal carcinoma cells are pluripotent stem cells derived from teratocarcinomas and are considered to be the malignant counterparts of human embryonic stem cells. As there are few reliable experimental systems available to study the molecular mechanisms governing normal embryogenesis, well-characterized human embryonal carcinoma stem cell lines may provide a robust and simple model to study certain aspects of pluripotency and cellular differentiation. Here, we have analysed NTERA-2 cL.D1 cells at molecular and cellular levels during expansion and differentiation, via formation of cell aggregates similar to embryoid bodies in embryonic stem cells. Thus, human embryonal carcinoma cells may provide a valuable insight into cell fate determination, into the embryonic ectoderm, mesoderm and endoderm and their downstream derivatives.     

Osterix-Cre Labeled Progenitor Cells Contribute to the Formation and Maintenance of the Bone Marrow Stroma

We have carried out fate mapping studies using Osterix-EGFPCre and Osterix-CreERt animal models and found Cre reporter expression in many different cell types that make up the bone marrow stroma. Constitutive fate mapping resulted in the labeling of different cellular components located throughout the bone marrow, whereas temporal fate mapping at E14.5 resulted in the labeling of cells within a region of the bone marrow. The identity of cell types marked by constitutive and temporal fate mapping included osteoblasts, adipocytes, vascular smooth muscle, perineural, and stromal cells. Prolonged tracing of embryonic precursors labeled at E14.5dpc revealed the continued existence of their progeny up to 10 months of age, suggesting that fate mapped, labeled embryonic precursors gave rise to long lived bone marrow progenitor cells. To provide further evidence for the marking of bone marrow progenitors, bone marrow cultures derived from Osterix-EGFPCre/Ai9 mice showed that stromal cells retained Cre reporter expression and yielded a FACS sorted population that was able to differentiate into osteoblasts, adipocytes, and chondrocytes in vitro and into osteoblasts, adipocytes, and perivascular stromal cells after transplantation. Collectively, our studies reveal the developmental process by which Osterix-Cre labeled embryonic progenitors give rise to adult bone marrow progenitors which establish and maintain the bone marrow stroma.     

Organotypic slice cultures of embryonic ventral midbrain: a system to study dopaminergic neuronal development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages (1-2). Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain (3,4). It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase (5). We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system (6).