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Home , Cell growth, Developmental biology

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1 Developmental

Frank J. Dye Western Connecticut State University, Department of Biological & Environmental Sciences, 181 White Street, Danbury, CT 06810, USA

1Introduction6

2 Historical Perspective 6 2.1 Origins of 7 2.2 Origins of 7 2.3 Relationship between Cell and Developmental Biology 7 2.4 Nuclear Equivalence 8

3 Cell Activities Underlying 9 3.1 Intracellular 9 3.1.1 Receptors 9 3.1.2 Intracellular Signal Transduction 9 3.1.3 Factors 9 3.2 10 3.2.1 Juxtacrine Signaling 10 3.2.2 Autocrine Signaling 10 3.2.3 10 3.2.4 Endocrine Signaling 11 3.2.5 Growth Factors 11 3.3 Cell–Cell Interaction 11 3.3.1 Cell Junctions 11 3.3.2 Cell Signals 11 3.4 Cell–Matrix Interaction 12 3.4.1 Signaling 12 3.4.2 Cell Movement 13

4 Cell Differentiation 14

An older version of this chapter has previously been published in: Meyers, R.A. (Ed.) Stem Cells, 2013, 978-3-527-32925-0.

Frontiers in Developmental Biology: Advances in and Medicine First Edition. Edited by Robert A. Meyers. © 2019 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2019 by Wiley-VCH Verlag GmbH & Co. KGaA. 4 Developmental Cell Biology

5 The and Development 15

6 17

7 Stem Cells 20

8 Cloning 22

9Chimeras23

10 MicroRNAs (miRNAs) 24

11 In-Vitro Fertilization/Nuclear Equivalence 26

References 27

Keywords

Cell cycle A series of phases, designated G1, S, G2 and M, that a cell sequentially passes through between the time it is formed by and the time it itself divides.

Chimera An consisting of parts derived from more than one pair of parents. The chimera of Greek mythology consisted of a lion’s head, a goat’s body, and a serpent’s tail. Chimeras may be made in the laboratory by, in the case of mammals, removing the zona pellucida of each and pushing the sticky together, which merge together to form a single chimeric embryo.

Embryonic stem cells (ESCs) Stem cells derived from the of the stage embryo.

Germ layers The three traditional germ layers of the nineteenth century – , , and – were expanded during the twentieth century to include a fourth , the . Recent inductions of induced pluripotent stem (iPS) cells have shown that differentiated cells developmentally derived from one germ layer (e.g., fibroblasts derived from mesoderm) may be induced to form differentiated cells from another germ layer (e.g., nerve cells normally derived from ectoderm).

Growth factors Extracellular polypeptide signal molecules that can stimulate a cell to grow or proliferate.

Induced pluripotent stem (iPS) cells differentiated cells that have been converted to cells with the pluripotency of embry- onic stem cells, generally accomplished by the activation of specific transcription factors. Developmental Cell Biology 5

In- vitro fertilization (IVF) Human/mammalian fertilization occurring under artificial conditions outside of the body.

Intracellular signal transduction Cascades of chemical reactions used by cells to convey information from the cell surface to the interior of the cell (e.g., the nucleus) where the information is acted upon. The cell makes use of multiple and interacting intracellular signal transduction pathways. microRNAs (miRNAs) Post-transcriptional regulators that bind to complementary sequences of target messenger RNA (mRNA) transcripts, usually resulting in silencing.

Organogenesis Organ formation.

Pluripotent cell A cell that is capable of giving rise to many of the cell types of an organism.

Receptors that bind other molecules or ions and, as a result of the binding, influence cellular activity.

Stem cell A cell that undergoes mitotic cell division to give rise to the same type of cell. At some point, stem cells leave the pool of mitotically dividing cells to begin a process of cell differentiation.

STAP cells -triggered acquisition of pluripotency cells. During early 2014, STAP cells were reportedly created by simply immersing mouse cells briefly into a mild acid bath. About 7–9% of cells from newborn mice survived the acid treatment and took just a week to form STAP cells. Haruko Obokata and her colleagues, of the RIKEN Center for Developmental Biology in Kobe, Japan, and Harvard Medical School, reportedly transformed blood, skin, brain, muscle, fat, bone marrow, lung, and liver cells from newborn mice into STAP cells. When injected into mouse embryos, STAP cells reportedly not only incorporated into any body but also formed parts of the . According to news reports, later in 2014, Obokata, who led the researchers reporting that dipping adult cells in acid could change them into stem cells, agreed to retract one of the papers describing the result.

Transcription factors Proteins that attach to DNA at a gene regulatory site, and by so doing influence the rate of transcription of a specific gene.

It is said that Thomas Hunt Morgan, the recipient of a Nobel Prize in or Medicine, in 1933, for his discoveries concerning the role played by the in heredity, started out as an embryologist but switched to . At the time, the two fields of and genetics were separate endeavors. was 6 Developmental Cell Biology

awarded the first Nobel Prize in embryology, a Nobel Prize in Physiology or Medicine, in 1935, for his discovery of the organizer effect in . Sixty years later, in 1995, a Nobel Prize in Physiology or Medicine was awarded to Edward B. Lewis, Christiane Nusslein-Volhard, and Eric F. Wieschaus for their discoveries concerning the genetic control of early embryonic development. Ironically, Edmund Beecher Wilson’s book, The Cell in Development and Heredity, 3rd edition, published in 1925, is consid- ered to be a classic in cytology (cell biology), genetics and embryology. The point being made is that the twentieth century witnessed the dissolution of the boundaries between embryology (developmental biology) and genetics. The following chapter makes it clear that this integration continues at an unabated pace.

1 of their underlying molecular and cellular Introduction mechanisms, and to understand the devel- opmental consequences of their operation, Developmental cell biology is concerned makes the early twenty-first century an with the consequences of what cells do exciting time to be a developmental cell in the context of developing . . It is also a hopeful time for those Developmental are also con- people suffering the ravages of dreadful cerned with events before fertilization diseases or injuries, such as or spinal (e.g., gametogenesis) and after birth – or its cord injuries, and for those awaiting the equivalent in nonmammalian species (e.g., promise of regenerative medicine. and ). The range of species in the purview of the developmental biologist covers, essentially, 2 the entire living world – , , Historical Perspective , fungi, and even . In the creation of a human being, cell prolif- Cell biology involves the investigation of eration is required to create the trillions the molecular basis of how cells work, for of cells of which the body is composed. example, how cells move, how Cell differentiation provides the more than are moved within cells, and how 200 different types of cell identified to date are regulated. Developmental biology is in the human body, while concerned with understanding how these shapes the body form characteristic of the processes are integrated to create the human species. organism: for example, how cells move The present understanding of molecular collectively to accomplish ; and cellular mechanisms that underlie how cells are polarized as part of cell dif- these phenomena has been progressing at ferentiation; and how cell communication an ever-increasing pace, a testimonial to the affects the origin of different cells types army of cell and developmental biologists within close proximity to each other to at work across the planet. To understand create an organ composed of functionally the regulation of the cell cycle, apoptosis, integrated tissues. Midway through the cell differentiation, and second decade of the twenty-first century, cell movement (to mention but a few such the boundaries between , phenomena), to understand the integration molecular biology, genetics, cell biology, Developmental Cell Biology 7 and developmental biology have all but embryology. Also regarded as the father of vanished. comparative embryology, Von Baer was the first to make embryology truly comparative. 2.1 Together with Christian Pander (a Russian Origins of Cell Biology zoologist, regarded as a founder of the science of embryology), he formulated The of Schwann and Schleiden the germ layer concept as a structural (1838/1839) is a scientific milestone of the fact for vertebrate embryology (see also nineteenth century. Schwann and Muller Ref. [1]). Wilhelm Roux (1850–1924), a (Johannes Muller, German physiologist and German embryologist, is considered to be comparative anatomist) saw the implica- a pioneer in (and by some, the founder tions of the cell theory for human . of) experimental embryology. On the Rudolph Virchow, who stated the famous basis of his experimental results, Roux aphorism, “omnis cellula e cellula,” laid the concluded that are self-differentiating foundation for cellular pathology during the rather than being driven by external middle of the nineteenth century, replacing conditions. the long-lived humoral pathology concept of disease causation. The realization that 2.3 organisms are composed of cells, coupled to Relationship between Cell and Developmental thedevelopmentofimprovedmicroscopy, Biology made the study of early development possible. During the last quarter of the By the dawn of the twentieth century, nineteenth century, the fundamental details cell biology and developmental biol- of and were elucidated at ogy – although not generally referred to the level of the light microscope. as such at the time – were burgeoning disciplines of biology. Early in the twentieth 2.2 century, Wilson [2] (1856–1939), an Amer- Origins of Developmental Biology ican zoologist specializing in cytology and embryology, wrote The Cell in Development The nineteenth century – a century preg- and Heredity (3rd edition, 1925). This book nant with advances in cytology (cell is correctly considered to be a classic in, biology) and embryology (developmental at least, the fields of cytology (cell biology) biology) – saw the laying of the foundations and embryology (developmental biology). of descriptive and comparative embryology, According to Gilbert [3], the experiments while the end of this same century provided of Hans Spemann (1869–1941; a German the beginnings of experimental embryol- zoologist and 1935 recipient of the Nobel ogy. Karl Ernst von Baer (1792–1876), an Prize in Physiology or Medicine for his dis- Estonian naturalist and pioneer embry- covery of the organizer effect in embryonic ologist, is considered by some to be the development) and his students framed the father of modern embryology. He was questions that experimental embryologists the first to describe the mammalian asked for most of the twentieth century (1827), and became the first to trace the (see also Ref. Saha [4]). In the editorial development of the egg to produce the of the article, “Where Cell Biology and embryo; his Uber Entwickelungsgeschichte Developmental Biology Meet,” in the inau- der Tiere (1828) became a standard text of gural issue of the journal, Developmental 8 Developmental Cell Biology

Cell, 1 (1): 1 (2001), Siegel and Sweet experiment consistent with the notion proposed that it really does seem as though of qualitative division. we are beginning to understand how cells 3) In 1892, challenged that – both individually and as parts of interpretation when he microdissected multicellular organisms – and that there is and separated embryos a growing interaction and overlap between at the two-cell stage and observed cell biology and developmental biology. the formation of two equivalent sea During the second half of the twentieth cen- urchins, thereby extending the notion tury, the merging of cell and developmental of nuclear equivalence at least to the biology was reflected in the titles of books two-cell stage. and journals (e.g., Annual Review of Cell 4) Later, Hans Spemann tied tiny hairs Biology became Annual Review of Cell and from his daughter’s head around Developmental Biology, In Vitro became early-stage newt embryos, separating In Vitro Cell and Developmental Biology, early and observing the and, in 2001, Cell Press established a new formation of two normal newts (albeit journal, Developmental Cell). Just as cell one smaller than the other), proving biologists endeavored to explain cellular developmental equivalence up to the phenomena at the molecular level, during eight-cell stage. the twentieth century it became increas- 5) Spemann famously envisioned, but ingly clear that development needed to be never technically realized, a “fantastical explained at the cellular level. Attempts experiment” whereby the nucleus of to explain the mechanisms and control a highly differentiated cell might be of , cytodifferentiation, transplanted back to the egg, to test embryo patterning, and morphogenesis whether it would remain specialized or necessitated molecular understanding [5]. would manifest embryonic potential. 6) Reporting precisely that experiment 2.4 in 1952, Briggs and King suggested Nuclear Equivalence that cells lose the ability to support normal embryonic development as In a recent publication, Daley [6] traced development and cell specialization the well-known history of the concept of progresses (Rana pipiens). nuclear equivalence; namely: 7) Gurdon established that normal devel- opment to adulthood could be achieved 1) In 1885, August Weismann published by the transfer of fully differentiated the theory that development worked by nuclei from the intestinal cells of “qualitative divisions” among daughter feeding-stage larvae (, 1962). cells, which segregated subsets of her- Gurdon’s profound contribution rep- itable material to specify their unique resents the foundation of the current traits. assumptions about nuclear equiva- 2) In 1888, Wilhelm Roux pricked lence … theintellectualfoundation andablatedonecellofatwo-cellfrog of the excitement that has consumed embryo and observed the formation of a the last 15 years of biology “half-embryo,” and suggested that, even [6] (italics added for emphasis by at the two-cell stage, the embryonic the present author (see Sect. 7; Stem blastomeres were nonequivalent – an Cells). Developmental Cell Biology 9

3 for example, the conversion of a cAMP Cell Activities Underlying Development signal to a signal as an intracellular signal transduction pathway progresses into 3.1 the interior of a cell. The tyrosine Intracellular Signal Transduction kinase (RTK) signal transduction pathway is an intracellular signal transduction pathway Developmentally important cellular activ- that is important in development, and was ities include cell division and differential oneofthefirstpathwaystounitevarious . In a multicellular organ- areas of developmental biology. In the case ism, these activities are under the control of migrating neural crest cells of humans of the cell’s environment, including signals and mice, the RTK pathway is important in from other cells. Although some of these activating the microphthalmia transcrip- signals (e.g., steroid hormones) are non- tion factor (Mitf) to produce pigment cells. polar and able to pass through the plasma Homologous signal transduction pathways membrane of the cell, other signals are polar are composed of homologous proteins (e.g., hormone epinephrine) and attach to arranged in a homologous manner. Such receptors in the plasma membrane. In order pathways form the basic infrastructure to convey information carried by the signal of development, yet the targets of these from the cell surface to the interior of the pathways may differ among organisms; for cell (e.g., the nucleus), where the informa- example, the dorsal–cactus pathway used tion is acted upon, the cell makes use of in for specifying dorsal–ventral cascades of chemical reactions that make up polarity is also used by the mammalian intracellular signal transduction pathways. to activate inflammatory proteins. When homologous developmen- 3.1.1 Receptors tal pathways are used for the same function Receptor proteins are proteins that bind in both protostomes and deuterostomes, other molecules or ions and, as a result of they are said to have a “deep” . the binding, can influence cellular activity; Homologous signal transduction pathways examples include receptors in the illustrate the conservation of molecular cell surface and progesterone receptors in mechanisms across taxons. the . A is a substance that can direct the differentiation of cells 3.1.3 Transcription Factors since, along a gradient of morphogenetic A is a molecule that substances, the cells respond differently attaches to DNA at a gene regulatory site at different concentrations. A morphogen and by so doing influences the rate of receptor gradient is a gradient of those transcription of a specific gene; a molecules that recognize the morphogen; that regulates the transcription of genes, the expression of the Brachyury and goosec- often, but not exclusively, by binding to oid genes has been correlated with the cis-regulatory elements (promoters and number of activin receptors on each cell enhancers). Transcription factors exist as that binds activin. two general types: (i) basal transcription factors (e.g., TFIID, TFIIA) which attach to 3.1.2 Intracellular Signal Transduction gene promoters; and (ii) cell-specific tran- Signal transduction involves the conversion scription factors (e.g., Pax6, Mitf) which of a signal from one form to another form; bind to gene enhancers. Trans-regulatory 10 Developmental Cell Biology

factors are usually transcription factors. 3.2 There are four major families of transcrip- Cell Signaling tion factors based on DNA-binding motifs: homeodomain; basic helix-loop-helix Signaling between and among cells is (bHLH); basic zipper (bZip); and necessary for order, that is the organism, zinc finger. The trans-activating to emerge from potential chaos, which is of a transcription factor is concerned multicellularity. Signaling between cells with activating or suppressing the gene’s of the anterior pituitary gland and the transcription. gonads orchestrates the development of A transcription initiation complex is, , and signaling between cells may collectively, the RNA polymerase and result in the form of cell known as associated basal transcription factors that apoptosis. attach to a gene . A battery is a group of genes regulated by the same 3.2.1 Juxtacrine Signaling transcription factor. Zygotic genes are Juxtacrine signaling is a mode of cell–cell those genes expressed by the embryonic communication in which signaling ; the embryo’s genes as opposed to molecules are retained on the surface maternal genes expressed in the by of the signaling cell and interact with recep- the maternal genome. In early Drosophila tor proteins on adjacent cell surfaces. An development, most of the zygotic genes, example is the interaction between Bride of which first are activated along the antero- Sevenless protein and its receptor Seven- posterior and dorsoventral axes set up less, in fly eye photoreceptor specification by maternal genes, encode transcription (see Sections 3.2.2, 3.2.3 and 3.2.4). factors, which then activate more zygotic genes. 3.2.2 Autocrine Signaling Combinatorial regulation is the control Autocrine signaling is a mode of cell–cell of gene transcription by two or more communication in which signaling transcription factors; the spatial patterns molecules (autocrine factors) attach to of gene expression are often delimited receptors on the same cell that produced by the combined action of transcription them. An example is the explosive prolif- factors. Historically, the bicoid protein eration of placental cytotrophoblast cells gradient provided the first reliable evi- in response to platelet-derived growth dence for the existence of morphogen factor (PDGF), which these cells themselves gradients that had been postulated to produce. control pattern formation. The bicoid protein is a transcription factor that acts 3.2.3 Paracrine Signaling as a morphogen; it switches on certain Paracrine signaling is a mode of cell–cell zygotic genes at different threshold con- communication in which signaling centrations, thus initiating a new pattern of molecules (paracrine factors) act as gene expression along the axis. The bicoid local mediators and only affect cells protein is a member of the homeodomain in the immediate environment of the family of transcriptional activators and signaling cell. Paracrine factors are the activates the hunchback gene by binding protein molecules used in paracrine to regulatory sites within the promoter signaling; these factors are the induc- region. ing factors of the classical experimental Developmental Cell Biology 11 embryologists. Most paracrine factors fall 3.3 into one of four major families: Hedge- Cell–Cell Interaction hog; Wnt; transforming -β (TGF-β); and fibroblast growth factor Cells may interact with each other in a vari- (FGF). ety of ways. In addition to cells interacting through cell signaling of the juxtacrine, 3.2.4 Endocrine Signaling autocrine, paracrine and endocrine vari- Endocrine signaling is a mode of cell–cell eties, cells may make physical contact with communication in which signaling eachotherthroughavarietyofcelljunc- molecules (endocrine factors) are released tions, termed tight junctions, gap junctions, into the circulatory system and may affect and desmosomes. cells (target cells) that are some distance from the signaling cell. An example is the 3.3.1 Cell Junctions effect of anterior pituitary gland hormones Cell junctions are specialized regions of on the gonads. Endocrine factors are contact between cells, and generally fall molecules (hormones) that function in into three categories: tight junctions; gap endocrine signaling, and include estrogens, junctions; and desmosomes. , progesterone and polypeptide • Tight junctions are specialized contacts hormones such as the . formed between cells that establish parti- Endocrine mimics (endocrine disruptors) tions between isolated compartments of are exogenous substances that act like hor- the body. During the compaction of early mones in the and disrupt mammalian embryos, tight junctions the functions of endogenous hormones; form between the cells of the ; examples are DDT, polychlorobiphenyls these cells with their tight junctions (PCBs), bisphenol A, and phthalates. seal the blastocyst cavity off from the embryo’s environment. 3.2.5 Growth Factors • Gap junctions are specialized contacts Growth factors are extracellular polypep- formed between cells that establish tide signaling molecules that can promote cytoplasmic continuity between the cell proliferation, and regulate cytodif- cells. Cells with these junctions rapidly ferentiation, cell survival, and cell death. communicate with each other. During FGFs constitute a family of protein growth the compaction of early mammalian factors that were first identified as mitogens embryos, gap junctions form between for fibroblasts in tissue culture, and which the cells of the inner cell mass (ICM). stimulate the proliferation of many cell • Desmosomes are a type of cell junction types, inhibit the differentiation of various that join one epithelial cell to another and types of stem cells, and act as inductive provide structural integrity to an epithe- signals in embryonic development.For lium; hemidesmosomes join epithelial example, FGF – which plays a key role cells to the basal , a specialized in the induction of ventral mesoderm (ECM) on which in Xenopus embryos – is secreted by epithelial cells reside. vegetal cells. Apoptosis may be initiated by a withdrawal of growth factors from 3.3.2 Cell Signals the cell, or by an active response to a A pathway in the context of cell–cell sig- signal. naling consists of the components required 12 Developmental Cell Biology

Factors regulating stem cell fate

Soluble factors Mesenchymal Substrate elasticity stem cell 1 kPa (soft) 10 kPa 100 kPa (rigid)

Substrate composition and 3-dimensionality Lineage: neuronal muscle bone

Fig. 1 Effect of substrate elasticity on stem cell fate. Reproduced with permission from Ref. [8].

for the sending, receiving and transduction mechanobiology, maintaining that of a signal, including one or more ligands, mechanical forces are ubiquitous in vivo membrane-associated receptors, intracel- and that these forces directly impact cell lular signal transducers and, depending on function, and that such forces regulate mor- the type of pathway, transcription factors. phogenesis, cell migration, to the ECM, as well as cell proliferation and 3.4 differentiation. Although, Eyckmans and Cell–Matrix Interaction coworkers suggested that the beginnings of mechanobiology date back to 1892, and Growing, quiescent and dying cells may all that mechanobiology received relatively be found within the same microenviron- little attention for much of the twentieth ment during morphogenesis. Additionally, century, the recent renaissance in studies of normal stem cell biology depends on the mechanics – primarily in – has availability of the stem cell niche. The ECM been based on tools that enable the mea- forms one component of the cell’s microen- surement and manipulation of mechanical vironment. In fact, it has been shown that forces in vitro. The topics considered the tension exerted by a stem cell’s ECM by Eyckmans et al. were: (i) the current may determine the of that stem cell’s understanding of the role of mechanical differentiation. Engler et al. [7] examined forces in cell biology; (ii) techniques that are the effect of matrix elasticity on the dif- being developed to enable such studies; and ferentiation of human mesenchymal stem (iii) recent efforts to consider mechanical cells (MSCs), and showed that soft matrices forces in development. Mechanobiology favored the differentiation of mesenchy- constitutes yet another example of the mal stem cells into neuronal-like cells, a merging of cell and developmental biology. moderate elasticity promoted myogenic dif- ferentiation, and a rigid matrix stimulated 3.4.1 Integrin Signaling osteogenic differentiation (see Fig. 1). Cells reside in a protein network, the ECM, Recently, Eyckmans et al. [9] have which they secrete into the extracellular reviewed the burgeoning field of space. The ECM exerts a profound control Developmental Cell Biology 13 over cells, the effects being mediated pri- Well-developed aggregates detectable marily by , a family of cell-surface by immunofluorescence are receptors that attach cells to the matrix known as focal adhesions and ECM con- and mediate the mechanical and chemical tacts. Thus, integrins serve as integrators of signals from it. The integrins span the the ECM and the , the property plasma membrane, bind the fibronectin of for which integrins are named. the ECM, and provide anchorage sites for the microfilaments of the cytoskele- 3.4.2 Cell Movement ton; that is, they integrate the extracellular Cell-shape changes and cell movements and intracellular scaffolds. Most integrins underlie the morphogenetic movements recognize several ECM proteins, and that create the form of the developing individual ECM proteins (e.g., fibronectin, organism. Examples of the burgeoning laminins, collagens, and vitronectin) bind literature on this topic include reviews to several integrins. Integrins can signal by Keller [10], Adler [11], and Hall [12]. through the plasma membrane in either Polarized cell movements shape the major direction: the extracellular binding activity features of the vertebrate during of integrins is regulated from inside the cell, development. The head-to-tail body axis of while the binding of the ECM elicits signals vertebrates is elongated during the embry- that are transmitted into the cell. onic stages by “convergent extension” tissue Adherent cells must be anchored to an movements during which cells intercalate appropriate ECM to survive; depending between one another and transverse to the partly on the signals from the matrix, they elongating body axis to form a narrower, either proliferate or exit the cell cycle and longer, array. Recent studies have shown differentiate. This anchorage requirement that these polarized cell movements are is lost in neoplastic cells. The cytoplasmic controlled by homologs of genes that domains of integrins always lack enzymatic control the polarity of epithelial cells in the activity, and thus they transduce signals developing wing and eye of Drosophila. by associating with adaptor proteins that The actin cytoskeleton mediates a variety connect the integrin to the cytoskeleton, of essential biological functions in all cytoplasmic , and transmembrane eukaryotic cells. In addition to providing growth factor receptors. Integrin sig- a structural framework around which cell naling and assembly of the cytoskeleton shape and polarity are defined, its dynamic are intimately linked. As integrins bind properties provide the driving force for to the ECM, they become clustered in cells to move and to divide. Understanding the plane of the plasma membrane and the biochemical mechanisms that control associate with a cytoskeletal and signaling theorganizationofactinisthereforea complex that promotes the assembly of major goal of current cell biology, with actin filaments. The reorganization of implications for both health and disease. actin filaments into larger stress fibers, in Members of the Rho family of small guano- turn, causes more integrin clustering, thus sine triphosphatases have emerged as key enhancing the matrix binding and organi- regulators of the actin cytoskeleton, and, zation by integrins in a positive feedback furthermore, through their interaction system. As a result, ECM proteins, integrins with multiple target proteins, they ensure and cytoskeletal proteins assemble into a coordinated control of other cellular aggregates on each side of the membrane. activities such as gene transcription and 14 Developmental Cell Biology

adhesion. Observations have suggested that role of pRB in differentiation has been stud- members of the Rho GTPase family are ied in a number of cell lines inducible for key regulatory molecules that link surface differentiation. In each instance, an early receptors to the organization of the actin event in differentiation was the dephos- cytoskeleton. The JAK/STAT pathway is a phorylation of pRB, which correlated with

central component of the signal transduc- cell cycle arrest in G0/G1, a prerequisite to tion network that controls cell proliferation, enter the differentiation pathway. During fate, and movement. Despite the present the differentiation of muscle cells, pRB understanding of actomyosin function in accumulates in the nucleus and forms individual migrating cells, little is known complexes with muscle-forming transcrip- of the mechanisms by which actomyosin tion factors such as MyoD and , drives collective cell movement in verte- thus preventing pRB rephosphorylation brate embryos. The collective movements and locking the cell in the differentiated of convergent extension drive both the state. However, the inactivation of pRB global reorganization of the early embryo reverses the differentiated and and local remodeling during organogenesis. allows cells to re-enter the cell cycle. Ajioka Shindo and Wallingford [13] have reported et al. [14] reported a new mouse model that planar cell polarity (PCP) proteins of retinoblastoma, which bears on the control convergent extension by exploiting relationship between cell differentiation an evolutionarily ancient function of the and the cell cycle. These authors showed septin cytoskeleton. By directing septin- that retinoblastoma is not driven by uncon- mediated compartmentalization of cortical trolled expansion of retinal progenitor cells, actomyosin, PCP proteins coordinate butratheristheresultofcellcyclere-entry the specific shortening of mesenchymal and expansion of differentiated horizontal cell–cellcontacts,whichinturnpowers interneurons in the retina (see Fig. 2). cell interdigitation. The data obtained by Recent studies conducted by Bowles et al. Shindo and Wallingford have illuminated [16] have provided details about how the the interface between developmental determination of mammalian germ signaling systems and the fundamental cells occurs during fetal development and machinery of cell behavior, and should depends on signals from gonadal provide insights into the etiology of human cells. It had been established previously birth defects such as spina bifida and that RA triggers ovarian germ cells to congenital kidney cysts [13]. enter meiosis and thereby commit to oogenesis whereas, in the developing testis, the CYP26B1 degrades RA and 4 the germ cells are not induced to enter Cell Differentiation meiosis. Bowles et al. [16], using in-vitro and in-vivo models, showed that FGF9 As the field of cell differentiation has produced in the fetal testis acts directly generated vast literature, two models of cell on germ cells to inhibit meiosis; in addi- differentiation will be considered here: (i) tion, FGF9 maintains the expression of the role of (pRB) pluripotency-related genes and upregulates in differentiation; and (ii) a model of how markers associated with male fate. retinoic acid (RA) and FGF9 act antagonis- Based on these data, it was concluded that tically to determine germ cell fate.The active two independent and mutually antagonistic Developmental Cell Biology 15

Differentiation into outer nuclear Proliferation and death Death of outer layer and inner nuclear of outer nuclear layer nuclear layer layer precursors precursors precursors

OUTER NUCLEAR LAYER

Proliferating progenitors

INNER NUCLEAR LAYER

Ectopic proliferation of Cell-cycle arrest of inner Alleviation of cell- inner nuclear layer nuclear layer cycle arrest precursors precursors retinoblastoma Loss of pocket proteins

Loss of p53

Fig. 2 Retinoblastoma development in mice. other retinal cells (amacrine, horizontal, and Muller The loss of pocket proteins allows proliferating glia cells) survive but become arrested in the cell progenitor cells in the retina to differentiate into cycle. A second event that suppresses the p53 outer and inner nuclear layer precursor cells. pathway may alleviate cell-cycle arrest, leading to These cells continue to proliferate at least for a proliferation of differentiated cells. Reproduced while, but then certain retinal cell types (rods, with permission from Ref. [15]. cones, ganglion, and bipolar cells) die, whereas pathways involving RA and FGF9 act in a single change in NPC composition as concert to determine mammalian germ an essential step in cell differentiation, cell sexual fate commitment, and support a and established a role for Nup210 in model in which the mitosis/meiosis switch gene expression regulation and cell fate is robustly controlled by both positive and determination (see Fig. 4). negative regulatory factors (see Fig. 3). D’Angelo et al. [17] showed that a specific change in nuclear pore complex (NPC) 5 composition is required for both myo- The Cell Cycle and Development genic and neuronal differentiation. The transmembrane nucleoporin Nup210 is The relationship between the cell cycle absent from proliferating myoblasts and and development is a topic that is of great embryonic stem cells (ESCs), but becomes interest to developmental cell biologists. expressed and incorporated into NPCs Since its elucidation during the 1950s, the during cell differentiation. Furthermore, canonical cell cycle, G1,S,G2,M,hasbeen the prevention of Nup210 production by studied intensively by cell biologists. It RNAi blocks myogenesis and the differ- has long been known that the cell cycle entiation of ESCs into neuroprogenitors. of embryos undergoing is not D’Angelo and colleagues found that the classical, but rather is an abbreviated addition of Nup210 to NPCs did not affect cycle consisting of S alternating with M. nuclear transport, but was required for the This embryonic (before gastrulation) cell induction of genes that are essential for cycle, using maternal gene products and cell differentiation. These results identified not providing time (G1) for , 16 Developmental Cell Biology

Testis Ovary

Sry, Sox9, Cyp26b1, Fgf9

RA Germ cell FGF9

FGF9 Stra8 RA

Male fate fate Oct4, Sox2, Nanos2, Dnmt3L, P15 Meiosis

Fig. 3 Retinoic acid (RA) and fibroblast growth are low (one white cell shown) to upregulate Stra8 factor 9 (FGF9) act antagonistically to determine (RA) or to prevent its upregulation (FGF9). Stra8 germ cell fate. The germ cell sexual fate is deter- expression in gonadal germ cells is essential for mined by two signaling molecules produced by entryintomeiosis,byanunknownmechanism. the somatic cells of the gonad, FGF9, and RA. FGF9 acts directly on germ cells to antagonize Cyp26b1 and Fgf9 are highly expressed in the Stra8 expression, maintain expression of pluripo- testis, but are downregulated in the ovary. Since tency markers, Oct4 and Sox2, and to induce male CYP26B1 degrades endogenous RA, levels of RA germ cell fate markers, Nanos2, Dnmt3L, andP15. are low in the testis while FGF9 levels are high. Reproduced with permission from Ref. [16]. In the ovary, RA is not degraded and FGF9 levels

results in cleavage of the into an the connection of signaling pathways to ever-increasing number of blastomeres regulation of cell cycle progression. of progressively decreasing size; that is, The extent to which the cell cycle can be multicellularity is achieved at the expense of altered, to serve the changing needs of the cell size. The transition of early developing organism, is provided by study- Xenopus development has been used to ing Drosophila development. Drosophila

study the conversion of S/M to G1/S/G2/M. embryonic cells undergo minimal growth Cell cycle alteration and regulation dur- between divisions, and the key event during ing development is a fundamental topic this developmental phase is patterning. As in considering the relationship between development progresses to the larval phase, the cell cycle and development (also see thedevelopmentalobjectivechanges, Ref. [18]). and so too does the nature of the cell Xenopus and early embryos cycle. The major objective of Drosophila provide excellent systems for studying the larval tissues is cell growth rather than relationship between the cell cycle and cell proliferation: the larval cells undergo development. Xenopus embryos bracketing repeated rounds of endoreplication, an the midblastula transition provide a devel- effort that may be designed to increase the oping system for the study of the sequential totalgenedosageinagivencellinaneffort establishment of cell cycle checkpoints and to support drastically increased cell size. Developmental Cell Biology 17

Differentiation signal Wild type Nup210 shRNA

No Nup210 at NPCs Nup210 at NPCs No Nup210 at NPCs

Nucleus Nucleus Nucleus

Early myogenic/neuronal Direct? differentiation factors + + + + + +

Myogenic genes Neuronal genes Myogenic genes Neuronal genes E-box

Nup210 mRNA mRNA

Nup210 mRNA Myogenesis Neuronal Apoptosis differentiation Differentiation- committed cell Differentiating cell

Fig. 4 Schematic model of Nup210 regulation of to the NPC where it regulates the expression of cell differentiation. In undifferentiated myoblasts genes required for myogenic and neuronal dif- the expression of Nup210 is repressed. Early dif- ferentiation. Prevention of Nup210 addition to ferentiation signals activate Nup210 gene expres- the NPC by shRNAs prevents the activation of sion. In myoblasts, Nup210 induction is likely car- Nup210-regulated genes and leads to the death ried out by myogenin/MyoD binding to its pro- of the differentiation-committed cell by apoptosis. moter E boxes. Nup210 protein is then recruited Reproduced with permission from Ref. [17].

Imaginal disc cells in the developing complex (APC). Cell-based experiments must undergo both growth and patterning, and reconstituted reactions have estab- and exhibit a “classical” G1/S/G2/M cell lished that PSC and Lemming (LMG; cycle, with a growth-sensitive cell cycle also called APC11) associate and ubiq- checkpoint at the G1/S boundary and a uitylate CYC-B cooperatively, marking it patterning checkpoint at the G2/M bound- for proteosomal degradation. Thus, PSC ary. The developmental program thereby appears to mediate both developmental neatly adjusts the cell cycle to promote gene silencing and the post-translational patterning without growth, growth without control of mitosis. Direct regulation of cell patterning, or growth and patterning simul- cycle progression might be a crucial part of taneously, as each is required for proper the PcG system’s function in development development. and cancer [19]. The polycomb group (PcG) proteins control development and cell proliferation through -mediated transcrip- 6 tional repression. Mohd-Sarip et al. [19] Organogenesis described a transcription-independent function for the PcG proteins Posterior sex Tissues and organs are composed of dif- combs (PSCs) in regulating the destruction ferentiated groups of cells. Each organ has of cyclin B (CYC-B). A substantial portion a characteristic and function, of PSC was found outside canonical PcG which emerges during development of the complexes, instead of being associated embryo, and thus needs to be studied on an with CYC-B and the -promoting individual basis. The practical applications 18 Developmental Cell Biology

of a general understanding of organogenesis Drosophila imaginal discs, and Pit 1 which, will include the repair and/or replacement together with Gata2, controls pituitary of a patient’s compromised organs, most differentiation. Fang et al. [21] carried likely starting with the patient’s own stem out a genome-wide expression analysis cells [20]. of embryonic development and reported The process of organogenesis has a transcriptome profiles of human early prerequisite step, namely the specification embryos covering development during of spatially defined regulatory domains the first third of organogenesis. In this that promote the differentiation pro- case, two major categories of genes were grams. This commonplace mechanism identified that displayed gradually reduced enables the recognition and definition or gradually increased expression patterns of regulatory fields as discrete territories across this developmental window. The of specific gene activities. Selector genes decreasing group appeared to include control the formation and identity of the stemness-specific and differentiation- various fields, while field-specific genes specificgenesthatareimportantforthe areaspecialclassofselectorgenesthat initiation of organogenesis, whereas the have the unique property of directing the increasing group appeared to be largely formation of complex, specialized struc- differentiation-related and indicative of tures such as organs. Examples of such diverse organ formation (see Fig. 5). Based organ-identity genes include: Pax6/eyeless, on these findings, a putative molecular which is required for eye formation in network was devised that may provide

Stemness-relevant genes/module

Initiation of organogenesis

Various organ formation

Differentiation-relevant genes/module

Fig. 5 Transcriptome analysis of early organogenesis in human embryos. Reproduced with permis- sion from Ref. [21]. Developmental Cell Biology 19 a framework for the regulation of early disorders in model organisms, and has high- human organogenesis. lighted the need for an in-vitro model of The complexity of the human brain human brain development. Lancaster et al. has made it difficult to study many brain [22] have developed a human pluripotent

The cerebral

All cells Progenitor cells

Forebrain Forebrain (cortex)

Choroid plexus Ventricle

(a)

Cerebral Organoid cell culture Procedure

Suspension Suspension Matrigel droplet Spinning bioreactor Day 0 Day 6 Day 11 Day 15

Embryoid Expanded (b) hPSCs bodies neuroepithelium Cerebral tissue

Fig. 6 Cerebral introduce new meth- system begins with dissociated pluripotent stem ods of modeling defects in neural development, cells which are first directed to a neural fate. Neu- particularly those of the cerebral cortex. (a) roectoderm (early neural stem cells) are then Organoids are composed of neural progenitors embedded in Matrigel, after which they are cul- (red) and neurons (green), as well as other cell tured in a spinning bioreactor and direct their types (blue marks all cells). In addition, organoids own development in the absence of most exoge- develop tissues such as neocortex, choroid plexus, nous growth factors. Modified from Ref. [22] by and ventricle; (b) The cerebral organoid culture Mark Springel, SITN, Harvard University. 20 Developmental Cell Biology

stem cell-derived three-dimensional (3D) and pluripotent hematopoietic stem cells. organoid culture system, termed cerebral A lineage-restricted stem cell is a cell organoids, which develop various discrete, that can produce only one type of cell in although interdependent, brain regions addition to renewing itself; an example (see Fig. 6). These include a cerebral cortex is the burst-forming unit, erythroid containing progenitor populations that (BFU-E). organize and produce mature cortical Embryonic germ (EG) cells are derived subtypes. Cerebral organoids have from primordial germ cells (PGCs); these also been shown to recapitulate features pluripotent stem cells may be produced of human cortical development, namely in culture by treating PGCs with stem characteristic progenitor zone organization cell factor, leukemia inhibition factor, and with abundant outer radial glial stem basic FGF2. cells. Finally, RNA interference (RNAi) Embryonic stem cells (ESCs) are derived and patient-specific induced pluripotent from the embryo; for example, those stem (iPS) cells were used to model micro- derived from normal mouse inner mass cephaly, a disorder that has been difficult to cells cultured in vitro. recapitulate in mice, and also demonstrated The is a part of a char- premature neuronal differentiation in acterized by dividing cells; these regions patient organoids, a defect that could help of plants contain stem cell populations to explain the disease phenotype. Together, that produce cells, some of which go on to these data show that 3D organoids can differentiate into plant tissues and some recapitulate development and disease, even of which constantly renew the stem cell in this most complex human tissue [22]. population. iPS cells are a type of pluripotent stem cell artificially derived from a non-pluripotent 7 cell (typically an adult somatic cell) by Stem Cells inducing a “forced” expression of certain genes. The recent creation of iPS cells has A stem cell is a cell which undergoes mitotic raised the question of whether the cell division to give rise to the same type of these cells and those of human ESCs of cell and, at some point, leaves the pool have similar pluripotent states. Guenther of mitotically dividing cells to begin the et al. [23] suggested that a comparison of process of cell differentiation. Adult stem the global chromatin structure and the cellsare,ineffect,anembryonicpopulation gene expression programs of these two of cells, continually producing cells that can cell types would provide a robust means of undergo further development within an assessing whether the genomes of the cells adult organism. The path of differentiation have similar pluripotent states. The results that a stem cell descendant enters depends obtained showed that: (i) genome-wide on the molecular milieu in which it resides. maps of nucleosomes with certain histone It is noteworthy that most stem cells are modifications indicate that there is little relatively quiescent. difference between ESCs and iPS cells The term pluripotent describes a cell with respect to this criterion; and (ii) gene that is capable of giving rise to several of expression profiles confirmed that the tran- the cell types of an organism; examples scriptional programs of ESCs and iPS cells include pluripotent epidermal stem cells showed very few consistent differences. Developmental Cell Biology 21

Fibroblasts

Gata4/Mef2c/Tbx5 Cardiac Transplantation In Vivo progenitors

Induced cardiomyoyctes (iCMs)

Fig. 7 Direct reprogramming of fibroblasts into functional cardiomyocytes. Reproduced with permis- sion from Ref. [24].

This led to the conclusion that these varia- Ieda et al. [24] suggested that these findings tions did not serve to distinguish ESCs from showed that functional cardiomyocytes can iPS cells. The reprogramming of fibroblasts be directly reprogrammed from differen- to iPS cells raises the possibility that a tiated somatic cells by defined factors (see somatic cell could be reprogrammed to an Fig. 7). The same authors also speculated alternative differentiated fate, without first that the reprogramming of endogenous becoming a stem/progenitor cell. A large or explanted fibroblasts might provide a pool of fibroblasts exists in the postnatal source of cardiomyocytes for regenerative heart, yet no single “master regulator” of approaches. direct cardiac reprogramming has been As noted by Daley [6], the legacy of identified. Ieda et al. [24] reported that Gurdon had established the principle of a combination of three developmental conservation of the genome during cellular transcription factors (i.e., Gata4, Mef2c, differentiation. Gurdon’s success with and Tbx5) rapidly and efficiently repro- nuclear transfer had established that a grammed postnatal cardiac or dermal molecular machinery within the egg cyto- fibroblasts directly into differentiated plasm was sufficient to reprogram a somatic cardiomyocyte-like cells. The induced genome to a pluripotent state. Ultimately, cardiomyocytes expressed cardiac-specific in 2004, in a review entitled The First markers, had a global gene expression Half-Century of Nuclear Transplantation, profile similar to cardiomyocytes, and Gurdon surmised that “ … asecondhalf- contracted spontaneously. Fibroblasts century of nuclear transplantation should transplanted into mouse hearts at one day identify the molecules and mechanisms that after transduction of the three factors also achieve nuclear reprogramming” (italics differentiated into cardiomyocyte-like cells. added by present author). In just over a 22 Developmental Cell Biology

year after the landmark report on repro- repair and regeneration” (italics added by gramming from Takahashi and Yamanaka present author). [25] several groups described the successful reprogramming of human cells (in 2007). With this accomplishment, the field turned 8 its attention away from somatic cell nuclear Cloning transfer (SCNT) and converged on this new factor-based reprogramming technology, In 1996, Campbell and Wilmut succeeded to exploit its practical implications. Applied in deriving two cloned sheep from a reprogramming was quickly applied to differentiated cell line established from patient-derived fibroblasts and a large a nine-day-old embryo, and a year later collection of disease-specific iPS cell lines achieved worldwide acclaim by successfully for conditions as diverse as Parkinson’s, dia- deriving a single sheep, “Dolly,” from the betes and primary immune deficiency were mammary cells of an adult ewe. In their established. As Daly [6] points out, “ … the case, success necessitated of Yamanaka experiments have ushered in an the cell cycle of the donor nucleus to better era of cellular alchemy .... thistechnology match that of the recipient oocyte. These heralds an era during which any patient’s data extended Gurdon’s principle of nuclear cells represent the ingredients for tissue equivalence (see above) for all somatic cells

Fibroblast Oocyte

SCNT

Differentiation

Pancreatic islet cells

Hematopoietic cells

Cardiomyocytes

Hepatocytes Blastocyst Neurons NT-ESCs

Fig. 8 Cytoplasm of human oocytes reprograms transplanted somatic cell nuclei to pluripotency. NT- ESCs can be efficiently derived from high-quality human oocytes. Human NT-ESCs are similar to ESCs derived from fertilized embryos. Reproduced with permission from Ref. [26]. Developmental Cell Biology 23

Oocyte

Fibrobiasts from aduit

Enucleation

Neurons SCNT Retinal pigment epithelia

Cardiomyocytes

Pancreatic beta Biastocyst cets

Cartiage

Differentiation & Clinical Apptication NT-hESCs

Fig. 9 Human ESCs were derived via SCNT from 35- and 75-year-old males. Reproduced with permis- sion from Ref. [27]. to fully differentiated cells of an adult mam- of SCNT as a strategy for regenerative mal, an experiment that had implications medicine (Fig. 9). far beyond the scientific [6]. In 2013, a cloning technique was described for reprogramming human cells to create 9 stem cells; the technique – SCNT – fused Chimeras mature cells from an to egg cells that had their DNA-containing nuclei removed A chimera is an organism consisting of (Fig. 8) [26]. parts derived from more than one pair of During 2014, it was reported that human parents. The chimera of Greek mythology cloning to produce stem cells can function consisted of a lion’s head, a goat’s body, even with cells from middle-aged or elderly and a serpent’s tail. Chimeras may be made people.Inthisstudy,Chunget al. [27] in the laboratory by (in the case of mam- employed a recently developed technique mals) removing the zona pellucida of each to generate human ESCs via SCNT, using embryo and pushing the sticky embryos dermal fibroblasts from 35- and 75-year-old together, so that they merge to form a males. The results demonstrated the appli- single chimeric embryo. This embryo, if cability of SCNT for adult human cells, transferred into the uterus of a maternal and supported the further investigation host, may develop into a chimeric organism. 24 Developmental Cell Biology

Such constructs have been created with, organ complementation is achievable. for example, different strains of mice. ESCs In reviewing these studies, Solter [29] are stem cells derived from the embryo; suggested that, although xenogeneic organ for example, derived from normal mouse complementation is unlikely to be a viable inner mass cells cultured in vitro.Theuse strategy for regenerative medicine, the of these cells to create chimeras allows the elegant studies of Kobayashi et al. should introduction into ESCs of an engineered provide a wealth of information for research gene with a known mutation, such that groups seeking to better understand the mice can be bred which have the same biology of stem cells and mammalian mutation in all of their cells. development (see Fig. 10). , an American develop- Totipotent cells in early embryos are mental biologist, is especially well known progenitors of all stem cells and are capable for developing mouse chimeras. In one of developing into a whole organism, of her experiments, early embryos con- including extraembryonic tissues such as sisting of only a few cells were removed placenta. Pluripotent cells in the ICM are from pregnant mice and placed in close the descendants of totipotent cells, and contact with similar cells of genetically can differentiate into any cell type of a unrelated embryos to form a composite body except extraembryonic tissues. The that was then implanted in a mouse uterus ability to contribute to chimeric animals to create a cellular mosaic. This technique upon reintroduction into host embryos is enabled Mintz to trace the tissue site of the key feature of murine totipotent and specific genetic diseases. A recent report by pluripotent cells. Rhesus monkey ESCs Kobayashi et al. [28] described the creation and isolated inner cell mass cells failed to of viable rat–mouse chimeras that devel- incorporate into host embryos and develop oped to term and became fully functional into chimeras, although chimeric offspring . In order to show that xenogeneic were produced following the aggregation organ complementation could be achieved, of totipotent cells of the four-cell embryos these investigators injected fluorescently (Fig. 11). These results provide insights labeled mouse or rat iPS cells into rat or into the species-specific nature of primate mouse and returned them to embryos, and suggest that a chimera assay blastocyst-compatible (to avoid rejection) using pluripotent cells may not be feasible pseudopregnant . The donor cells [30] (Fig. 11). (i.e., the injected iPS cells) not only made major contributions to the tissues of the host, but it was also found that the host 10 blastocyst could control the size of the MicroRNAs (miRNAs) chimera, as well as imposing additional morphogenetic regulations (as exemplified MicroRNAs (miRNAs) are post-transcrip- by the presence or absence of a gallbladder). tional regulators that bind to complemen- The study by Kobayashi and coworkers tary sequences of target messenger RNA also showed that cells derived from rat transcripts (mRNAs), which usually results iPS cells were able to completely rescue in gene silencing. miRNAs are short RNA a genetic deficiency of the host mouse molecules (on average only 22 nucleotides blastocyst, resulting in a normal functional long), and the human genome may encode pancreas, and confirming that xenogeneic over 1000 miRNAs, that can target about Developmental Cell Biology 25

(a)Mouse Rat (b)

Rat iPS cells

HOST MOUSE HOST MOUSE BLASTOCYST BLASTOCYST Mouse iPS cells Rat iPS cells

Normal Pdx1% HOST MOUSE HOST RAT BLASTOCYST BLASTOCYST

Pancreas derived entirely from injected rat iPs cells

Mouse-like chimeraRat-like chimera Mouse-like chimera Mouse-like chimera

Fig. 10 The generation of rat–mouse chimeras. (b) Fluorescently labeled rat iPS cells (blue) were (a) Induced pluripotent stem (iPS) cells were injected into normal mouse blastocysts (left) or derived from adult mouse and rat cells and blastocysts lacking the Pdx1 gene (right), which labeled with different fluorescent proteins. Rat encodes the transcription factor pancreatic and (blue) and mouse (red) iPS cells were injected duodenal 1 that is required for pan- into reciprocal blastocysts (mouse into rat, and creas development. Chimeras derived from normal vice versa) to produce intergeneric chimeras. or Pdx1-deficient mouse blastocysts showed an From these blastocysts, several chimeras were extensive contribution of rat cells to all tissues. born and some survived to adulthood. The However, in the Pdx1-deficient chimeras, the entire contribution of injected donor stem cells was pancreas was derived from donor rat cells (inset, observed throughout the body of the host. The blue) and was fully functional, including the pro- size and morphology of the newborn and adult duction of insulin by β islet cells. Reproduced chimeras was determined by the host blastocyst; with permission from Ref. [29].

60% of mammalian genes and are abundant heart to variability necessitates especially in many human cell types. miRNAs may robust regulatory mechanisms to govern each repress hundreds of mRNAs, and are cardiacgeneexpression.Recentstudies well conserved in eukaryotic organisms; have revealed central roles for miRNAs they are thought to be a vital and evolu- as governors of gene expression during tionarily ancient component of genetic cardiovascular development and disease. regulation. The integration of miRNAs into the genetic The heart, more than any other organ, circuitry of the heart provides a rich and requires precise functionality on a second- robust array of regulatory interactions to to-second basis throughout the lifespan control cardiac gene expression. miRNA of the organism. Even subtle perturba- regulatory networks also offer opportuni- tions in cardiac structure or function ties for therapeutically modulating cardiac have catastrophic consequences, resulting function through the manipulation of in lethal forms of congenital and adult pathogenic and protective miRNAs. The heart disease. Such intolerance of the roles of miRNAs as regulators of cardiac 26 Developmental Cell Biology

ESC injection

Blastocyst injection ESCs No ESC contribution

Cleaving embryo injection ICM injection

Isclated ICM

Geneticaliy differnt mono-chorionic

Blastocyst injection ICM derived Whole embryo eggregation

Chimera

Fig. 11 Summary of chimera studies with mon- chimeras when injected into host blastocysts. key embryos and embryonic cells. Rhesus mon- However, ICMs developed into separate key ESCs as well as isolated inner cell mass cells with placental support from the host embryo. (ICMs), blastomeres, or whole embryos were tested Aggregating several four-cell embryos efficiently for their ability to incorporate into host embryos produced live chimeric offspring. Reproduced and generate chimeric offspring. Established with permission from Ref. [30]. ESCs and freshly isolated ICMs failed to produce

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