Samenvatting Ontwikkelingsbiologie
en Genetica
HC 1 05-02-18
How can a single cell give rise to a well-defined and complex multicellular organism with many
specialized cell types, tissues and organs?
Totipotent proliferating cells become post-mitotic differentiated cells. Development:
- Cell proliferation
- Differentiation
- Migration
- Morphogenesis
- Growth
- Cell death
- Homeostasis
What makes cells different from one another? How can uncommitted proliferating cells acquire
specific cell fates? What are stem cells and how are they maintained? Differentiation is usually
irreversible. Study animal systems to understand developmental processes; worms, zebrafish,
drosophila and mouse.
C. elegans: stem cell maintenance in the gonad, asymmetric cell division in the early embryo and fate
determination larval development.
Drosophila: stem cell maintenance in the gonad, asymmetric cell division nervous system and tissue
proliferation and growth.
Zebrafish: heart development and hormonal control reproductive system.
Chicken and tadpoles are not used since they are difficult to genetically change, they’re more for
comparison. Understanding stem cell biology, cell proliferation and differentiation decisions will
ultimately translate to regenerative therapy and cancer treatment.
Genetic variation (mutation) is an important tool to obtain molecular insight. Why use genetics?
- Discover which genes control a biological process
Identify mutants with defects in the process
Identify which genes are mutated (molecular cloning)
- Define how genes act together in regulatory networks
Are identified genes part of signal transduction pathways?
What is the order of gene activities (upstream-downstream)
- Determine the molecular mechanisms of biology
Combine genetics with molecular biology, cell biology, biochemistry and systems
biology
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,All cells have an identical genome; DNA RNA Protein. Many levels of regulation make cells different
from each other:
- DNA level
Chromatin state
Transcription factors, co-repressors and co-activators
RNA polymerases
- RNA level
Processing (splicing, polyadenylation, nuclear export)
Degradation/stabilization
Small RNA’s (degradation, translation, localization P bodies)
- Protein level:
Translation (biding factors, miRNA’s)
Processing (cleavage, secretion)
Localization, complex formation
Modification (phosphorylation, acetylation, methylation, ubiquitination)
Degradation/stabilization
External and cell intrinsic signals determine cell fate:
- Extrinsic regulation: the niche of a stem cell will provide signals to keep the stem cell
undifferentiated, so if the cell exits the niche if will start to differentiate.
- Intrinsic regulation: by asymmetric cell division one cell stays a stem cell and the other
becomes differentiated.
Stem cells can use asymmetric cell division in which one cell becomes differentiated, or it can divide
into two daughter cells, one of which receives signals to differentiate while the other one receives
signals to stay a stem cell. Stem cells combine self-renewal and production of differentiating
daughter cells. What defines stem cells?
- Self renewal?
- Totipotency/pluripotency?
Precursors of the germline are totipotent and self-renew (very few)
Mammalian embryonic stem cells (ES cells) are pluripotent
Tissue specific (adult, somatic) stem cells are multipotent or unipotent, may be
maintained by asymmetric cell division
C. elegans: simple animal, efficient genetics, transparent, cell lineage entirely known. Development
proceeds through highly reproducible division patterns. The formation and differentiation of each cell
type can be followed and studied with singe-cell resolution.
Two sexes: hermaphrodites and males (rare). Hermaphrodites produce sperm for a short time and
stores it in spermathecae, after which it switches to a female state and forms oocytes. Males are very
rare, but can easily maintained since crossing of a male with a hermaphrodite gives 50% males. By
mating with a male, the self-sperm of the hermaphrodite is replaced with the male sperm.
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,Development begins with the fusion of two gametes; the germline produces gametes and is a special
tissue:
- Maintenance of the totipotent state all cells and tissue of the next generation develop from
germ cells. Germline stem cells are truly totipotent
- Special cell cycle control: alteration between mitotic and meiotic cells division
- Immortal, germ cell DNA can be transmitted “forever” maintenance of genetic integrity is
very important! Efficient DNA repair pathways, suppression/silencing of foreign DNA.
Stem cells, germ cells and somatic gonad formation during C. elegans germline development
- Proliferation phase: two primordial germ cells proliferate two cells become migratory cells
and germline proliferation takes place in between them germ cells proliferate distally (distal
tip cells DTCs) and enter the meiotic cell cycle proximally DTCs move backwards, causing a
U-shaped gonad germ cells undergo spermatogenesis proximally (4 larval stages)
- Maintenance phase: GSCs self-renew; oogenesis proceeds in the proximal germ line (adult,
continues to grow)
Cell division is all completed in the
four larval stages. Formation of the
germ precursor cells and zygote; in
the distal end mitosis takes place,
then meiosis more towards the tail,
then oocyte formation and embryo
formation (after fertilization).
There is a meiotic cell cycle (two
times an M-phase), an embryonic
cell cycle (an S-phase and an M-
phase) and an endoreduplication cycle (G1 phase, S-phase and G2).
Most germ-precursor cells are in meiosis I prophase (homolog pairing, synapsis, recombination). In
the tip, mitosis takes place (after the distal tip cell (DTC)). Then the transition zone follows, then
pachytene (meiosis) and oocyte formation. There is a close association of the DTC with the mitotic
stem cell population. Is the DTC needed for maintenance of the mitotic stem cell population? This
appeared to be true, when the DTC was lost the cells began meiosis (germline proliferation defective
(GLP) phenotype). To find which molecules are involved, mutants were found with the same
phenotype; loss of lag-2, glp-1 of lag-1 activity was needed for this phenotype. To order genes in
pathways, we usually make double mutants, but these mutation result in the same phenotype. We
first need an opposite phenotype mutation to order.
Mutant with activated GLP-1 (gain of function): germline tumor of proliferating mitotic cells opposite
phenotype. Combining the opposite phenotype mutant and the other mutant gives information on
the order of the genes (if the genes are in the same pathway). If there is a downstream interruption
(like loss of Lag-2), then the pathway can’t continue (genetic epistasis analysis). Everything that
happens upstream doesn’t matter. Order:
Lag-2 *GLP-1 Lag-1 ectopic proliferation.
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, The conserved GLP-1Notch receptor signaling pathway promotes proliferation of germline stem cells.
Lag-2Delta expression in the DTC promotes proliferation and/or prevents entry meiosis by binding to
GLP-1Notch on the germ cells. Binding of LAG-2 causes internal cleavage of GLP-1 which moves to the
nucleus, together with transcription factor LAG-1 CSL.
Gld-1 gld-2 double loss of function also creates a germline tumor phenotype, epistatic to glp-1.
- Glp-1(0) Glp (few germ cells made)
- Gld-2(0) gld-1(0) tumorous
- Gld-2(0) gld-1(0); glp-1(0) tumorous
LAG-1 inhibits GLD-1 and GLD-2, which both normally promote meiosis and inhibit proliferation.
There is also a feedback mechanism, in which GLD-1 inhibits GLP-1 Notch.
Inhibition of GLD-1 by GLP-1Notch leads to spatial regulation of GLD-1 and meiosis, since GLD-1 induces
meiosis. What is the function of GLD-1 and GLD-2?
- GLD-1: KH domain RNA-binding protein
- GLD-2: Poly-A RNA polymerase
GLD-1 is an mRNA translational repressor. Many target mRNAs are repressed by GLD-1, including
glp-1 and cye-1 cyclin E mRNA, which induce proliferation. GLD-2 may stabilize target mRNAs.
GLD-1 promotes meiotic germ precursor state
by preventing inappropriate mRNA translation.
GLD-1 and MEX-3 presumably are
complementary translation inhibitory proteins.
Meiotic germline cells start to resemble somatic
cells in mex-3 gld-1 mutants. “Trans-
differentiation” and teratoma formation in germline of mex-3 gld-1 mutants. Inhibition of mRNA
translation is needed to maintain undifferentiated germ cells.
Specific translation suppression and chromatin regulation maintains germline state. The neuronal
transcription factor CHE-1 induces a specific type of differentiated neurons (“ASE”) in C. elegans. CHE-
1 binds a DNA-sequence element (ASE-motif) in the promotor of ASE-specific genes. Experiment:
ASE-neuron specific reporter transgene with GFP behind it abnormal CHE-1 transcription factor
expression activates reporter gene, but not neuronal cell fate. CHE-1 is not the only thing that is
needed for differentiation. Regulators of “repressive chromatin” state prevent neuronal fate
induction in the germline, such as lin-53 repression. Upon inhibition of lin-53 or HDACs, CHE-1
induces neural fate in the germline.
Human LIN-53: Rb-associated protein pRbAP46/48, component of chromatin modifying complexes.
The retinoblastoma tumor suppressor protein family inhibits transcription of cell-cycle genes and
developmental genes. RbAP46/48 forms part of multiple repressive chromatin complexes. Direct
conversion of C. elegans germ cells into specific neurons: single neuronal transcription factor
combined with lin-53 or HDAC inactivation induces specific neuronal fate.
Summary:
- Maintenance of a Germline Stem Cell population by a glp-1 Notch signal transduction
pathway
- Translation inhibition prevents expression of maternal mRNAs
- Repressive chromatin maintains the uncommitted fate of germline stem cells and germ
precursor cells
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