Cell fate, cell differentiation, Notch signaling and gastrulation in early embryo Semmelweis University, Department of Anatomy, Histology and Embryology P-Fejszák Nóra 2016.11.17.

Embryonic cells are able to follow more than one pathway of development. The decision, which pathway will be selected by a cell, depends on the interaction between genetic backgrand and environmental factors. Specialized cell types expresse a subset of their all genes. Differentiation process involves: -switch in the gene expression pattern of cell -restricition in the cell potency

Fate: the sum of all structures that the cell or its descendants will form at a later stage of normal development Potency: the total of all structures or cell types that a cell can form in an appropriate environment Totipotentcells: can form all the cell types of body and the extraembryonic structures Pluripotent cells: can form all of the cell types that make up of the body Multipotent cells can develop into more than one cell type, but are more limited than pluripotent cells Determination: is a stepwise process by which the potency of cell becomes limited to its fate . Differentiation follows determination, as the cell elaborates a cell-specific developmental program. Differentiation results in the presence of cell types with different gene expression pattern.

Autonomous and conditinal way of cell differentiation

Modes of cell specification and their characteristics Autonomous specification I. Characteristic of most invertebrates in their early development Specification by differential acquisition of certain cytoplasmatic molecules present in thy egg (zygote) Asymmetric segregation of cellular determinants is based on the asymmetric localization of cytoplasmic molecules (usually proteins or mRNAs) within the zygote before it divides. .During the zygote division, one daughter cell receives most or all of the localized molecules, while the other daughter cell receives less (or none) of these molecules causing differnt gene expression pattern between daughter cells. The localized cytoplasmic determinants are often mRNAs encoding transcription factors, or the transcription factors themselves

Modes of cell specification and their characteristics Autonomous specification II. Tunicate (sea squirt) During the first cell divisions (after fertilization) varient blastomeres will be created that have already committed in a certain fate.

If split the sea squirt in 8 cell stage, each dissociated blastomere pair forms only few structures. They are not able to form the whole body dissociated blastomeres

Modes of cell specification and their characteristics Conditional specification I. Characteristic of all vertebrates and few invertebrates Specification by cell-cell or cell-environment interaction Cell fates are based on the inductive signals coming from the cells’ environment Inductive signals can change the gene expression pattern of target cells inducing the differentiation process Inductive signalings can be classified according to how the signal can reach the target cells. → direct contact →through diffusible molecules

Notch mediated signaling An important example for differentiation by direct cell-cell interaction Notch mediated signaling Direct cell-cell interaction (between the neighboring cells) Notch is an transmembrane protein works as receptor; its ligands Delta/ Serrate are also transmembrane proteins The Notch receptor and its ligands were identified in flies almost 100 years ago. Neurogenic mutant fly: the loss of function condition of Notch signaling has been described. The Notch signaling inhibits neurogenesis in wild type drosophila embryo → inhibits the transformation of ectoderm to neuroectoderm. Notch signaling: control of cell communication and cell fate Eric C. Lai Development 2004 131: 965-973; doi: 10.1242/dev.01074 The Notch loss of function mutant flies produce a remarkable excess of neurons at the expense of epidermis → „neurogenic” mutant

How exactly does the Notch signaling work? Lateral inhibition means equivalent cells compete to attain a preferred state → one cell inhibits neightbours from attaining same fate During the normal development of many tissues begins with a population of developmentally equivalent cells. At some point, one of these cells begins to differentiate into dominant mature cell type such as neurons. The prospective neurons transmit to its neighboring cells a signal that prevents them from differentiating into that same cell type. The neighboring cells preserved their self-renewal function for a while so they have a choice to differentiate into a an another cell type such as glial cell in central nervous system -making balance between the amount of different cell types -help to maintain the tissue specific progenitor cells -create pattern within tissues

Elements of Notch signaling Both the Notch receptor and its ligands, which belong to the Delta/Serrate/Lag2 (DSL) family, are transmembrane proteins. The Notch signal is initiated when the Notch receptor protein comes into contact with its ligand (Delta) located on an adjacent cell. Due to the receptor and ligand interaction the intracellular portion of the Notch protein (Notch intracellular domain [NICD]) is cleaved from the extracellular portion of the Notch protein. The NICD then moves into the cell nucleus where it forms a complex with transcriptional coactivator to induce the expression of target genes The target genes are transcriptional repressor genes (Hes1 and Hes5) Hes1 and Hes5 repress the expression of tissue specific genes (proneural genes) theraby leading to the maintenence of progenitor state. Notch signaling: control of cell communication and cell fate Eric C. Lai Development 2004 131: 965-973; doi: 10.1242/dev.01074

Major cell fate decision in early embryo: gastrulation

Why is gastrulation so important? Generation of the basic body plan. Specification of the axes: Anterior and posterior Dorsal and ventral Left and right Generation of the three germ layers Ectoderm, mesoderm, and endoderm

Primitive streak, groove Anterior Posterior Gastrulation begins with the formation of primitive streak. Epiblast cells move toward the midline of the embryo → they are jammed in the midline forming the primitív streak that first appear in the posterior part of the embryo. Cells which are located in the middle of the primitív streak, start to migrate into the interior of the embryo resulting the formation of primitív groove in the middle of the primitív streak.

Primitive streak, groove The primitív streak with the primitív groove gradually grow anteriorly clearly identifying the right-left axes of embryo. At the anterior end of the primitív streak there is a small but well-defined accumulation of cells, called primitív node or Hensen’s node.

What ‘s happen with the cells in the primitív groove? The movements of the cells are accopamanied by major changes in their structure. When epiblast cells enter the primitív streak, they become elongated and lose their connection with the basal lamina. So their morphologes change and these cells are known as bottle cells. Within the primitív groove these bottle cells become free from the epiblast layer. Bottle cells undergo an epithelio-mesenchymel transformation within the primitív groove and the newly form mesenchymel cells are able to migrate as individual cells. Main characteriatic feathures of this epithelio-mesenchymel transformation are that the E-cadharine synthesis downregulate within the bottle cells, hyaluronic acid is produced between the epiblast end hypoblast. Hyaluronic acid support the migration of the cells. Epithelial cell E-cadherin epiblast „slug” FGF8 E-cadherin mesoderm endoderm

Development of the definitive entoderm Entoderm Bi-laminar embryonic disk Hypoblast cells develop only into extraembryonal mesoderm Primitive streak Epiblast Hypoblast First entering epiblast-cells migrate and replace the hypoblast-cells forming the definitive endoderm Entoderm Epiblast cells give rise to the three germ layer of the embryo!!

Development of the Intraembryonal Mesoderm Primitive streak D16 epithelio-mesenchymal transformation Intraembryonal mesoderm Definitive entoderm Epiblast-cells migrate in the interlaminar space and forming intraembryonal mesoderm

Which factors will be determined the developmental fate of epiblast cells? Before the gastrulation the epiblast cells are pluripotent cells! The allocation of cells to the ectodermal, mesodermal or entodermal lineage may occure during the movments of cells through the primitive streak. Several genes exhibits distinct expression boundaries along the A-P and D-V axis of primitive streak. The D-V diversification is determined by FgF8 Along the A-P axes differnt subsets of genes express at different levels of primitive streak It is important that the epiblast cells when and where pass through the primitive streak!

Fate of epiblast cells The earliest, most posterior epiblast cells give rise to the extraembryonic tissues, including the mesodermal layer of the chorion and the visceral yolk sac mesoderm and blood islands.The earliest, most anterior subpopulation located in the Hensen node ventral layer will form endoderm. The Hensen node dorsal layer cells will form the notochord and precordal plate. lateral plate, paraxial and cardiac mesoderm emerge slightly later from the intermediate and anterior levels of the streak. Finally, those epiblast cells that migrate through the anterior and intermediate levels of the streak will form lateral plate, paraxial and cardiac mesoderm.

Migration of the Mesodermal Cells The newly form mesenchymal cells migrate and spread bilaterally. Those cells, which pass through at the level of Hensen’s node, migrate directly cranially and form the precordal plate and later take part in the formation of the notochord. Epiblast Mesoderm Entoderm Epiblast Entoderm

Primitiv streak regression At the beginning of gastrulation the primitiv streak grows cranially The primitiv streak growing changes for its regression. → its length is decreasing toward caudally This regression process is related with the elongatoin of the notochord The notochord is formed by the addition of cells to its caudal end while the primitiv streak become shorter and shorter