Lecture 1 Basic Concepts
Forward genetics: Random mutagenesis, screen for phenotypes
Reverse genetics: Make mutation in gene of interest and then analyze the phenotype
Life cycle of Drosophila: Sperm enters the oocyte
through the micropyle on the anterior part of the
oocyte → nuclear divisions without cytokinesis;
syncytium allows for diffusion of proteins → Nuclei
migrate to periphery of the cytoplasm → Syncytial
blastoderm → Cellularization gives rise to cellular
blastoderm.
Xenopus and other vertebrates have holoblastic
divisions → complete cleavage of the cells.
Zebrafish have meroblastic divisions (partial
cleavage) → forming a yolk syncytial layer
Gastrula, early multicellular embryo, composed of two or more germinal layers of cells from
which the various organs later derive. The gastrula develops from the hollow, single-layered
ball of cells called a blastula which itself is the product of the repeated cell division, or
cleavage, of a fertilized egg.
Embryonic patterning is the process of establishing positional information
at the molecular level among similar cells. It starts with polarity or symmetry-
breaking events like asymmetric cell divisions and molecular gradients.
Drosophila: ~50 maternal-effect genes set up the anterior-posterior and
dorsal-ventral axes. Bicoid is mostly expressed on the anterior site of the
embryo and inhibits the caudal translation from the maternal mRNA.
,Fate map tells what the progeny of the cells will
be by using lineage tracing with fluorescent dye.
Fate ≠ Specified ≠ Determined
Fate is lineage tracing, prediction based on the
position → we know what the cells (most likely)
will give rise to, but the cells do not yet know.
Specified is that the cells know what they will be,
but can change their minds.
Determined is that the cells know what they will
be and will proceed no matter what.
If cells are not determined yet, they can adapt to
the new environment. When a determined cell
gets transplanted into a new environment, it will
still develop into the tissue it was “assigned” →
Ex. eye-like structure in the trunk region.
Detection of protein → immunohistochemistry
(with antibodies)
Detection of RNA → in situ hybridization (with
RNA probes) is a technique that is used to
detect nucleotide sequences in cells, tissue sections, and even whole tissue (whole mount in
situ hybridization). This method is based on the complementary binding of a nucleotide probe to
a specific target sequence of DNA or RNA. These probes can be labeled with either radio‐,
fluorescent‐, or antigen‐labeled bases. Depending on the probe used, autoradiography,
fluorescence microscopy, or immunohistochemistry, respectively, are used for visualization.
,Lecture 2 Axis formation
The maternal to zygotic transition (MZT) occurs at the mid-blastula stage in Xenopus and
zebrafish; maternal RNA is degraded and the zygotic genome is activated (ZGA).
The mid-blastula transition (MBT) is the stage where new embryonic transcription takes place
and cells lose their synchrony → cells become more motile.
, In Xenopus are the ectoderm, endoderm and animal-vegetal axis maternally derived. The
mesoderm, dorsal-ventral and anterior-posterior axis are specified after zygotic genome
activation.
The animal-vegetal polarity in fish and amphibia; in the animal pole are the dividing (pigmented
in Xenopus) cells and the vegetal pole is the yolk, mostly at the bottom because of the weight of
the yolk. Before fertilization, specific mRNAs are transported to the vegetal pole for radial
symmetry breaking and germ layer specification. These mRNAs become the dorsalizing factors.
Sperm entry gives rise to cortical rotation and relocation of the vegetal RNAs and animal
orientated pigment. The site of the sperm entry will become the ventral site of the embryo and
the new location of the dorsalizing factors become the future dorsal site.
This relocation activates the Wnt pathway → when
Wnt is present it can bind to the receptor that will
inhibit the β-catenin degradation. Free β-catenin will
enter the nucleus and bind to transcription factors that
will induce gene transcription.
Wnt11b is responsible for relocation of the
prospective dorsal region and for the accumulation of
dorsal β-catenin. This accumulation can induce dorsal
axis at ventral side, therefore there are Wnt inhibitors
expressed at the ventral side to prevent second dorsal
axis.
In zebrafish there is no sperm entry or cortical rotation
involved. There is relocation of the maternal
dorsalizing activity by microtubules, and there is also
accumulation of dorsal β-catenin.
Forward genetics: Random mutagenesis, screen for phenotypes
Reverse genetics: Make mutation in gene of interest and then analyze the phenotype
Life cycle of Drosophila: Sperm enters the oocyte
through the micropyle on the anterior part of the
oocyte → nuclear divisions without cytokinesis;
syncytium allows for diffusion of proteins → Nuclei
migrate to periphery of the cytoplasm → Syncytial
blastoderm → Cellularization gives rise to cellular
blastoderm.
Xenopus and other vertebrates have holoblastic
divisions → complete cleavage of the cells.
Zebrafish have meroblastic divisions (partial
cleavage) → forming a yolk syncytial layer
Gastrula, early multicellular embryo, composed of two or more germinal layers of cells from
which the various organs later derive. The gastrula develops from the hollow, single-layered
ball of cells called a blastula which itself is the product of the repeated cell division, or
cleavage, of a fertilized egg.
Embryonic patterning is the process of establishing positional information
at the molecular level among similar cells. It starts with polarity or symmetry-
breaking events like asymmetric cell divisions and molecular gradients.
Drosophila: ~50 maternal-effect genes set up the anterior-posterior and
dorsal-ventral axes. Bicoid is mostly expressed on the anterior site of the
embryo and inhibits the caudal translation from the maternal mRNA.
,Fate map tells what the progeny of the cells will
be by using lineage tracing with fluorescent dye.
Fate ≠ Specified ≠ Determined
Fate is lineage tracing, prediction based on the
position → we know what the cells (most likely)
will give rise to, but the cells do not yet know.
Specified is that the cells know what they will be,
but can change their minds.
Determined is that the cells know what they will
be and will proceed no matter what.
If cells are not determined yet, they can adapt to
the new environment. When a determined cell
gets transplanted into a new environment, it will
still develop into the tissue it was “assigned” →
Ex. eye-like structure in the trunk region.
Detection of protein → immunohistochemistry
(with antibodies)
Detection of RNA → in situ hybridization (with
RNA probes) is a technique that is used to
detect nucleotide sequences in cells, tissue sections, and even whole tissue (whole mount in
situ hybridization). This method is based on the complementary binding of a nucleotide probe to
a specific target sequence of DNA or RNA. These probes can be labeled with either radio‐,
fluorescent‐, or antigen‐labeled bases. Depending on the probe used, autoradiography,
fluorescence microscopy, or immunohistochemistry, respectively, are used for visualization.
,Lecture 2 Axis formation
The maternal to zygotic transition (MZT) occurs at the mid-blastula stage in Xenopus and
zebrafish; maternal RNA is degraded and the zygotic genome is activated (ZGA).
The mid-blastula transition (MBT) is the stage where new embryonic transcription takes place
and cells lose their synchrony → cells become more motile.
, In Xenopus are the ectoderm, endoderm and animal-vegetal axis maternally derived. The
mesoderm, dorsal-ventral and anterior-posterior axis are specified after zygotic genome
activation.
The animal-vegetal polarity in fish and amphibia; in the animal pole are the dividing (pigmented
in Xenopus) cells and the vegetal pole is the yolk, mostly at the bottom because of the weight of
the yolk. Before fertilization, specific mRNAs are transported to the vegetal pole for radial
symmetry breaking and germ layer specification. These mRNAs become the dorsalizing factors.
Sperm entry gives rise to cortical rotation and relocation of the vegetal RNAs and animal
orientated pigment. The site of the sperm entry will become the ventral site of the embryo and
the new location of the dorsalizing factors become the future dorsal site.
This relocation activates the Wnt pathway → when
Wnt is present it can bind to the receptor that will
inhibit the β-catenin degradation. Free β-catenin will
enter the nucleus and bind to transcription factors that
will induce gene transcription.
Wnt11b is responsible for relocation of the
prospective dorsal region and for the accumulation of
dorsal β-catenin. This accumulation can induce dorsal
axis at ventral side, therefore there are Wnt inhibitors
expressed at the ventral side to prevent second dorsal
axis.
In zebrafish there is no sperm entry or cortical rotation
involved. There is relocation of the maternal
dorsalizing activity by microtubules, and there is also
accumulation of dorsal β-catenin.
