Ectoderm specification

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In Xenopus laevis , the specification of the three germ layers (endoderm, mesoderm and ectoderm) occurs at the blastula stage. [1] Great efforts have been made to determine the factors that specify the endoderm and mesoderm. On the other hand, only a few examples of genes that are required for ectoderm specification have been described in the last decade. The first molecule identified to be required for the specification of ectoderm was the ubiquitin ligase Ectodermin (Ecto, TIF1-γ, TRIM33); later, it was found that the deubiquitinating enzyme, FAM/USP9x, is able to overcome the effects of ubiquitination made by Ectodermin in Smad4 (Dupont et al., 2009). Two transcription factors have been proposed to control gene expression of ectodermal specific genes: POU91/Oct3/4 [2] and FoxIe1/Xema. [3] [4] A new factor specific for the ectoderm, XFDL156, has shown to be essential for suppression of mesoderm differentiation from pluripotent cells. [5]

Contents

Proteins required for ectodermal specification. EctodermalSpecification.png
Proteins required for ectodermal specification.

Ectodermin and FAM

Biological role of Ectodermin and FAM

The protein Ectodermin, firstly identified in Xenopus embryos, promotes ectodermal fate and suppresses the mesoderm formation mediated by the signaling of Transforming Growth Factor β (TGFβ) and Bone Morphogenic Proteins (BMP), members of the TGFβ-superfamily. [6] When the TGFβ ligands bind to TGFβ receptors, they cause the activation of the signal transducers R-Smads (Smad2, Smad3). Smad4 forms a complex with activated R-Smads and activates transcription of specific genes in response to TGFβ signal. The BMP pathway transmits its signals in a similar way but through other types of R-Smads (Smad1, Smad5 and Smad8). The transcription factor Smad4 is the only common mediator shared between both TGFβ and the BMP pathways. [7] During ectoderm specification, the function of Smad4 is regulated by ubiquitination and deubiquitination made by ectodermin and FAM, respectively. The ubiquitination state of Smad4 will determine if it is able to respond to signals derived from TGFβ and BMP. [6] [8] The equilibrium of the activity, localization and timing of TGFβ and BMP transducers, Smad4, FAM and of Ectodermin should be achieved in order to be able to modulate the gene expression of genes required for germ layer formation.

Identification of Ectodermin and FAM

A cDNA library from the blastula stage of a frog embryo was cloned into RNA expression plasmids to generate synthetic mRNA. The mRNA was then injected into several Xenopus embryos at a four-cell stage and looked in early blastula embryos for an expansion of the region of the ectodermal marker Sox2 and diminution of the expression of the mesodermal marker Xbra. Ectodermin was one out of 50 clones to present this phenotype when injected into embryos. [6] The identification of FAM was done through a siRNA screen to find deubiquitinases that regulate the response to TGFβ.

Ectodermin and FAM localization

Ectodermin mRNA is maternally deposited in the animal pole of the egg. In the early blastula stage of the embryo, Ectodermin mRNA and protein forms a gradient that goes from the animal pole (highest concentration) down to the marginal zone (lowest concentration) to prevent TGFβ and nodal signals that induce mesoderm originating from the vegetal pole. Ectodermin mRNA is enriched in the dorsal side of the embryo, and at the end of this stage the expression gradually disappears. [6] Smad4 is ubiquitinated by Ectodermin in the nucleus and exported to the cytoplasm where it can be deubiquitinated by FAM; this way Smad4 can be recycled and be functional again. Although there is no expression profile of FAM in early embryos in Xenopus, in the zebra fish, FAM homolog is expressed ubiquitously at a two-cell stage but as development proceeds then its only expressed in the cephalic central nervous system. [9]

Ectodermin and FAM functions

Ectodermin is a ubiquitin E3 ligase that inhibits the TGFβ and the BMP signaling pathways by inhibition of Smad4 via ubiquitination of Lysine 519 and also though direct binding to phospho-Smad2. [6] [8] Injection of Ecto mRNA in the marginal zone leads to an expansion of the early ectodermal marker, Sox2, and a reduction of mesodermal markers (Xbra, Eomes, Vent-1, Mix-1 and Mixer). The opposite happens in knockdown experiments of Ectodermin by using a morpholino strategy; embryos become more sensitive to Activin response, they show an increase and expansion of the expression of mesodermal specific genes and down-regulate the expression of neural plate and epidermis marker (Sox2 and cytokeratin respectively). In line with a RING-finger dependent ubiquitin-ligase activity of Ectodermin, an Ecto RING-finger mutant (C97A/C100A) is inactive in gain-of-function. [6] Gain-of-function of FAM increases the responses from BMP and TGFβ and its loss-of-function by mutation in a critical residue for its activity caused inhibition of TGFβ response.

Conservation of Ectodermin and FAM in other species

The molecular function of human ectodermin to act as a negative regulator of Smad4 suggests that this specific function is conserved among the vertebrate lineage. [6] The sequence identity between FAM homologs is higher than 90% when comparing the homologs of Xenopus, zebrafish, mouse, and human, suggesting that this might also be conserved among other organisms. [9] Indeed, knockout gene inactivation in mouse embryos showed that the function of ectodermin as inhibitor of TGF-beta signaling is conserved. [10] Embryos lacking of ectodermin show defective development of the anterior visceral endoderm (AVE), which is the first tissue that is induced by TGF-beta signals in mouse embryos; in accordance with loss of an inhibitor, ectodermin-/- embryos showed enlarged AVE induction. As the AVE is a natural source of secreted TGF-beta antagonists, this primary AVE expansion caused secondarily, at later stages, an inhibition of extracellular TGF-beta ligands, resulting in embryos lacking of mesoderm development. This model was confirmed by the finding that ectodermin-/- embryos were rescued to wild type (normal AVE, normal mesoderm development) by lowering the genetic dosage of the main TGF-beta ligand of the embryo, Nodal. Further supporting a role as TGF-beta inhibitor, tissue-selective deletion of ectodermin from the epiblast (from which the mesoderm, but not the AVE, derive) left the AVE untouched but caused this time an expansion of anterior mesodermal fates, indicative of increased responsiveness to TGF-beta signals. Collectively, these data confirmed with genetic tools a cell-autonomous role for ectodermin as inhibitor of Smad4 responses previously identified in Xenopus embryos and human cell lines.

FOXI1e

Biological role of FOXI1e

During early in development in Xenopus, the transcription factor FoxI1e/Xema activates epidermal differentiation and represses endoderm and mesoderm specific genes in animal caps (Suri et al., 2005). It is suggested that FoxI1e is active before the ectoderm differentiates into epidermis and the central nervous system.

Identification of FoxI1e

Mir et al., 2005 identified FoxI1e (Xema) by selecting genes that were down-regulated under mesoderm-inducing signals in the ectoderm compared to vegetal region of an early blastula embryo. Also, high expression of this gene was observed in animal caps in embryos that lack VegT compared to wild type.

Localization in the cell

FoxI1e mRNA is expressed zygotically (stage 8.5) and reaches higher level of expression early in gastrulation and maintains that level in neurula, tailbud until early tadpole stages. [4] FoxI1e has a peculiar mosaic expression pattern, it is expressed first in the dorsal ectoderm and while gastrula progresses, the expression goes through the ventral side and its expression is down-regulated in the dorsal side when the neural plate is forming. [11] FoxI1e is dependent on BMP signals in the neurula stage, limiting the localization of FoxI1e to the ventral side of the ectoderm.

Protein function and regulation

FoxI1e/Xema belongs to the FoxI1 class of fork head transcription factor family, known to participate in mesoderm formation, eye development [12] and ventral head specification. [13] It has been proposed that Notch and/or NODAL, expressed in the vegetal/mesoderm region of the early blastula embryo, could potentially be the inhibitors of FoxI1e. [3] [11]

Loss and gain-of-function

Inhibition of FoxI1e mRNA maturation by a splice-blocking morpholino shows malformations in the development of epidermis and pervious system and down-regulates of ectoderm specific genes, whereas FoxI1e over-expression inhibits the formation of mesoderm and endoderm. Vegetal structures form late blastula masses that normally would give rise to endoderm and mesoderm, when injected with FoxI1e mRNA, they are able to express ectodermal specific markers (pan-ectodermal E-cadherin, epithelial cytokeratin, neural crest marker Slug and neural marker Sox-2) while endodermal markers (endodermin, Xsox17a) decreased in expression. [3] [4]

XFDL156

Biological role of XFDL156

The p53 protein binds to the promoters of early mesodermal genes. [14] p53 is maternally deposited transcript that forms a transcriptional factor complex with Smad2 and drives the expression of genes involved in mesoderm induction and activation of TGFβ target genes. [15] The zinc (Zn)-finger nuclear protein XFDL159, expressed in the animal cap, acts as an ectoderm factor their specifies the ectoderm by inhibiting p53 from activating genes for mesoderm differentiation. [5]

Identification of XFDL156

Construction of a cDNA library from animal caps at a stage of 11.5, cloned into expression vector and generated mRNA. The synthetic RNA was then injected into embryos and the animal caps of these collected embryos were obtained and submitted to activin treatment. Xbra was recovered by selecting the clone that represses the mesodermal marker Xbra. [5]

Localization of XFDL156

Since XFDL156 is a factor that interacts with p53, the localization of this protein is in the nucleus (Sasai et al., 2008). The mRNA of XFDL156 is maternally deposited and then expressed zygotically. The gene expression timeline shows a higher level of expression at early gastrula and a half decrease in expression at mid-gastrula and by the stage 20 the expression fades. [5]

Protein function of XFDL156

XFDR zinc finger binds to the regulatory region of p53 located at the C-terminal domain and its expression is not affected by the presence of activin, FoxI1e or XLPOU91 transcription factors.

Loss and gain-of-function in XFDL156

Loss-of-function by morpholino, causes incorrect mesodermal differentiation in the ectoedermal regions; this is caused by the desuppression of mesodermal markers (Xbra, VegT and Mix.2). Gain-of-functions causes decrease in expression of mesodermal markers. [5]

Conservation of XFDL homologs in other species

The human and mouse homologs of XFDR156 are able to complement XFDR function of interacting with p53 and inhibiting it to act as a transcription factor. [5]

Related Research Articles

Mesoderm Middle germ layer that forms muscle, bone, blood vessels and more

The mesoderm is the middle layer of the three germ layers that develop during gastrulation in the very early development of the embryo of most animals. The outer layer is the ectoderm, and the inner layer is the endoderm.

Blastulation Sphere of cells formed during early embryonic development in animals

Blastulation is the stage in early animal embryonic development that produces the blastula. The blastula is a hollow sphere of cells (blastomeres) surrounding an inner fluid-filled cavity. Embryonic development begins with a sperm fertilizing an egg cell to become a zygote, which undergoes many cleavages to develop into a ball of cells called a morula. Only when the blastocoel is formed does the early embryo become a blastula. The blastula precedes the formation of the gastrula in which the germ layers of the embryo form.

Gastrulation Stage in embryonic development in which germ layers form

Gastrulation is the stage in the early embryonic development of most animals, during which the blastula is reorganized into a multilayered structure known as the gastrula. Before gastrulation, the embryo is a continuous epithelial sheet of cells; by the end of gastrulation, the embryo has begun differentiation to establish distinct cell lineages, set up the basic axes of the body, and internalized one or more cell types including the prospective gut.

Ectoderm Outer germ layer that forms the neurons of brain, skin, and more

The ectoderm is one of the three primary germ layers formed in early embryonic development. It is the outermost layer, and is superficial to the mesoderm and endoderm. It emerges and originates from the outer layer of germ cells. The word ectoderm comes from the Greek ektos meaning "outside", and derma meaning "skin".

Paracrine signaling

Paracrine signaling is a form of cell signaling, a type of cellular communication in which a cell produces a signal to induce changes in nearby cells, altering the behaviour of those cells. Signaling molecules known as paracrine factors diffuse over a relatively short distance, as opposed to cell signaling by endocrine factors, hormones which travel considerably longer distances via the circulatory system; juxtacrine interactions; and autocrine signaling. Cells that produce paracrine factors secrete them into the immediate extracellular environment. Factors then travel to nearby cells in which the gradient of factor received determines the outcome. However, the exact distance that paracrine factors can travel is not certain.

A germ layer is a primary layer of cells that forms during embryonic development. The three germ layers in vertebrates are particularly pronounced; however, all eumetazoans produce two or three primary germ layers. Some animals, like cnidarians, produce two germ layers making them diploblastic. Other animals such as bilaterians produce a third layer between these two layers, making them triploblastic. Germ layers eventually give rise to all of an animal’s tissues and organs through the process of organogenesis.

Neural plate

The neural plate is a key developmental structure that serves as the basis for the nervous system. Opposite the primitive streak in the embryo, ectodermal tissue thickens and flattens to become the neural plate. The region anterior to the primitive knot can be generally referred to as the neural plate. Cells take on a columnar appearance in the process as they continue to lengthen and narrow. The ends of the neural plate, known as the neural folds, push the ends of the plate up and together, folding into the neural tube, a structure critical to brain and spinal cord development. This process as a whole is termed primary neurulation.

Lateral plate mesoderm

The lateral plate mesoderm is the mesoderm that is found at the periphery of the embryo. It is to the side of the paraxial mesoderm, and further to the axial mesoderm. The lateral plate mesoderm is separated from the paraxial mesoderm by a narrow region of intermediate mesoderm. The mesoderm is the middle layer of the three germ layers, between the outer ectoderm and inner endoderm.

Bone morphogenetic protein 4

Bone morphogenetic protein 4 is a protein that in humans is encoded by BMP4 gene. BMP4 is found on chromosome 14q22-q23.

Smads comprise a family of structurally similar proteins that are the main signal transducers for receptors of the transforming growth factor beta (TGF-B) superfamily, which are critically important for regulating cell development and growth. The abbreviation refers to the homologies to the Caenorhabditis elegans SMA and MAD family of genes in Drosophila.

The transforming growth factor beta (TGFB) signaling pathway is involved in many cellular processes in both the adult organism and the developing embryo including cell growth, cell differentiation, cell migration. apoptosis, cellular homeostasis and other cellular functions. This TGFB signaling pathways are conserved. In spite of the wide range of cellular processes that the TGFβ signaling pathway regulates, the process is relatively simple. TGFβ superfamily ligands bind to a type II receptor, which recruits and phosphorylates a type I receptor. The type I receptor then phosphorylates receptor-regulated SMADs (R-SMADs) which can now bind the coSMAD SMAD4. R-SMAD/coSMAD complexes accumulate in the nucleus where they act as transcription factors and participate in the regulation of target gene expression.

Chordin or CHRD is a protein with a prominent role in dorsal–ventral patterning during vertebrate early embryonic development. Chordin is encoded by the chrd gene.

Apical ectodermal ridge

The apical ectodermal ridge (AER) is a structure that forms from the ectodermal cells at the distal end of each limb bud and acts as a major signaling center to ensure proper development of a limb. After the limb bud induces AER formation, the AER and limb mesenchyme—including the zone of polarizing activity (ZPA)—continue to communicate with each other to direct further limb development.

The cells that give rise to the gametes are often set aside during embryonic cleavage. During development, these cells will differentiate into primordial germ cells, migrate to the location of the gonad, and form the germline of the animal.

In the field of developmental biology, regional differentiation is the process by which different areas are identified in the development of the early embryo. The process by which the cells become specified differs between organisms.

Goosecoid protein Protein-coding gene in the species Homo sapiens

Goosecoid (GSC) is a homeobox protein that is coded by the GSC gene. Like other homeobox proteins, goosecoid functions as a transcription factor involved in morphogenesis. In Xenopus, the goosecoid homeobox gene is thought to play a crucial role in the phenomenon of Spemann's Organizer. Through lineage tracing and timelapse microscopy, the effects of GSC on neighboring cell fates could be observed. In an experiment that injected cells with GSC and observed the effects of uninjected cells, GSC recruited neighboring uninjected cells in the dorsal blastopore lip of the Xenopus gastrula to form a twinned dorsal axis, suggesting that the Goosecoid protein plays a role in the regulation and migration of cells during gastrulation.

The Nodal signaling pathway is a signal transduction pathway important in regional and cellular differentiation during embryonic development.

This article is about the role of Fibroblast Growth Factor Signaling in Mesoderm Formation.

Xbra is a homologue of Brachyury (T) gene for Xenopus. It is a transcription activator involved in vertebrate gastrulation which controls posterior mesoderm patterning and notochord differentiation by activating transcription of genes expressed throughout mesoderm. The effects of Xbra is concentration dependent where concentration gradient controls the development of specific types of mesoderm in Xenopus. Xbra results of the expression of the FGF transcription factor, synthesized by the ventral endoderm. So while ventral mesoderm is characterized by a high concentration of FGF and Xbra, the dorsal mesoderm is characterized by a reunion of two others transcription factors, Siamois and XnR, which activates the synthesis of Goosecoid Transcription Factor. Goosecoid enables the depletion of Xbra. In a nutshell, high concentrations of Xbra induce ventral mesoderm while low concentration stimulates the formation of a back.

A developmental signaling center is defined as a group of cells that release various morphogens which can determine the fates, or destined cell types, of adjacent cells. This process in turn determines what tissues the adjacent cells will form. Throughout the years, various development signaling centers have been discovered.

References

  1. ↑ Heasman, J., Quarmby, J., and Wylie, C.C. (1984). The mitochondrial cloud of Xenopus oocytes: the source of germinal granule material. Dev Biol 105, 458-469.
  2. ↑ Snir, M., Ofir, R., Elias, S., and Frank, D. (2006). Xenopus laevis POU91 protein, an Oct3/4 homologue, regulates competence transitions from mesoderm to neural cell fates. EMBO J 25, 3664-3674.
  3. 1 2 3 Mir, A., Kofron, M., Zorn, A.M., Bajzer, M., Haque, M., Heasman, J., and Wylie, C.C. (2007). FoxI1e activates ectoderm formation and controls cell position in the Xenopus blastula. Development 134, 779-788.
  4. 1 2 3 Suri, C., Haremaki, T., and Weinstein, D.C. (2005). Xema, a foxi-class gene expressed in the gastrula stage Xenopus ectoderm, is required for the suppression of mesendoderm. Development 132, 2733-2742.
  5. 1 2 3 4 5 6 Sasai, N., Yakura, R., Kamiya, D., Nakazawa, Y., and Sasai, Y. (2008). Ectodermal factor restricts mesoderm differentiation by inhibiting p53. Cell 133, 878-890.
  6. 1 2 3 4 5 6 7 Dupont, S., Zacchigna, L., Cordenonsi, M., Soligo, S., Adorno, M., Rugge, M., and Piccolo, S. (2005). Germ-layer specification and control of cell growth by Ectodermin, a Smad4 ubiquitin ligase. Cell 121, 87-99.
  7. ↑ Siegel, P.M., and Massague, J. (2003). Cytostatic and apoptotic actions of TGFβeta in homeostasis and cancer. Nature Reviews – Cancer 3, 807-821.
  8. 1 2 Dupont, S., Mamidi, A., Cordenonsi, M., Montagner, M., Zacchigna, L., Adorno, M., Martello, G., Stinchfield, M.J., Soligo, S., Morsut, L., et al. (2009). FAM/USP9x, a deubiquitinating enzyme essential for TGFβeta signaling, controls Smad4 monoubiquitination. Cell 136, 123-135.
  9. 1 2 Khut, P.Y., Tucker, B., Lardelli, M., and Wood, S.A. (2007). Evolutionary and expression analysis of the zebrafish deubiquitylating enzyme, usp9. Zebrafish 4, 95-101.
  10. ↑ Morsut L, Yan KP, Enzo E, Aragona M, Soligo SM, Wendling O, Mark M, Khetchoumian K, Bressan G, Chambon P, Dupont S, Losson R, Piccolo S (2010). "Negative control of Smad activity by ectodermin/Tif1gamma patterns the mammalian embryo". Development. 137 (15): 2571–8. doi: 10.1242/dev.053801 . PMID   20573697.
  11. 1 2 Mir, A., Kofron, M., Heasman, J., Mogle, M., Lang, S., Birsoy, B., and Wylie, C. (2008). Long- and short-range signals control the dynamic expression of an animal hemisphere-specific gene in Xenopus. Dev Biol 315, 161-172.
  12. ↑ Pohl, B.S., Rossner, A., and Knochel, W. (2005). The Fox gene family in Xenopus laevis:FoxI2, FoxM1 and FoxP1 in early development. Int J Dev Biol 49, 53-58.
  13. ↑ Matsuo-Takasaki, M., Matsumura, M., and Sasai, Y. (2005). An essential role of Xenopus Foxi1a for ventral specification of the cephalic ectoderm during gastrulation. Development 132, 3885-3894.
  14. ↑ Cordenonsi, M., Dupont, S., Maretto, S., Insinga, A., Imbriano, C., and Piccolo, S. (2003). Links between tumor suppressors: p53 is required for TGFβeta gene responses by cooperating with Smads. Cell 113, 301-314.
  15. ↑ Takebayashi-Suzuki, K., Funami, J., Tokumori, D., Saito, A., Watabe, T., Miyazono, K., Kanda, A., and Suzuki, A. (2003). Interplay between the tumor suppressor p53 and TGF beta signaling shapes embryonic body axes in Xenopus. Development 130, 3929-3939.
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