186
Xenopus
step to understand the control of cell fate specif cation at
the transcriptional level. The diploid frog Xenopus tropicalis
is an ideal system to build and study developmental GRN.
Xenopus has long been used as a model for vertebrate early
development and has contributed greatly to the elucidation
of gene regulation. Xenopus embryos can be experimentally
manipulated at the earliest stages of development to allow
gain-of-function and loss-of-function analyses with relative
ease. The readily available genomic tools make the Xenopus
system ideally suited for the systematic identif cation of
genes affected by such manipulations. These advantages
point to the utility of the Xenopus system for the collection
of the large number of data sets to be used for GRN study.
Identifying the molecular parts encoded in the genome is
only a frst step towards understanding how context-specif c
biological processes are controlled. As we shall see, the knowledge of how these parts are combined and function together
is an equally important question. A little over a decade ago,
efforts were made to compile the available molecular data
into GRNs describing Xenopus mesendoderm and Spemann
organizer development (Loose and Patient, 2004; Koide et al.,
2005). In 2017, additional fndings were applied to update the
mesendoderm GRN from fertilization to the beginning of
gastrulation, linking TFs and critical signaling pathways with
transcriptional targets (Charney et al., 2017a). Close attention
was paid to focus on “direct” interactions between TFs and
target genes to build the GRN regulating mesendoderm.
An important consideration in creating GRNs is to identify direct links between a TF and its direct target genes to
understand the underlying network substructures regulating
gene expression. We recommend the following three criteria
to be used to create an interpretable GRN (Koide et al., 2005).
First, an obvious and critically important criterion is that a
putative direct target gene must be expressed temporally and
spatially in a manner consistent with the expression of the TF
proposed to control it. For example, if the TF is an activator,
the target gene should be coexpressed in the same or overlapping region where the TF is expressed. Conversely, if the TF
is a repressor, target gene expression should be excluded (or
reduced) from the region where the TF is expressed. Second,
a strong correlation must exist between perturbation of a
regulatory TF and the expression changes of the suspected
target genes. Regulation can be measured following gainand/or loss-of-function experiments (e.g., injection of mRNA
encoding a TF or a translation blocking antisense morpholino
oligonucleotide) by analyzing changes in target gene RNA
expression (e.g., RNA-seq, RT-PCR, northern blotting, in situ
hybridization). Third, a direct physical interaction between
the TF and a CRM controlling the proposed target needs to
be validated experimentally because perturbation experiments alone are insuffcient to distinguish between direct
and indirect effects. Methods for assessing direct interaction
include chromatin immunoprecipitation (ChIP), gel electrophoretic mobility shift assay (EMSA), DNase footprinting,
or reporter gene assays (evaluating appropriate binding site
mutations). This linkage of direct TF binding to regulated
genes is often lacking in the published literature, making it
diffcult to state with confdence whether the induction of a
proposed target gene is direct or indirect. Only connections
that satisfy all three criteria should be defned as direct.
However, we recognize that DNA binding is only suggestive of functional regulation and that the “gold standard”
evidence is to mutate the TF binding site and examine the
effect on gene expression in vivo. This is particularly important as ChIP-seq analysis fnds abundant neutral binding sites
(in addition to those involved in regulation) that don’t affect
gene expression (Bardet et al., 2013). These criteria should be
carefully considered whenever building GRNs.
12.2. PAST OBSERVATIONS
12.2.1. BRIEF OVERVIEW OF XENOPUS DEVELOPMENT
After fertilization, the Xenopus zygote undergoes multiple
rounds of cell division to give rise to an embryo with smaller
cells (blastomeres) but without increasing the overall volume
of the embryo. During the early stages of this process, some
maternally deposited mRNAs and proteins are asymmetrically inherited by individual blastomeres through cytokinesis
(see Chapter 3). These differentially localized materials specify the germ layer cell identities by initiating their respective
germ layer-specifc GRN programs along the animal-vegetal
axis. Other maternal components are asymmetrically distributed along the future dorsal-ventral axis, which is initiated
by the site of sperm entry. During gastrulation, the germ layers undergo patterning and are subdivided into smaller more
specifed cell fate territories that eventually def ne organ/
tissue primordia. The broadly defned mesendodermal territory (vegetal and equatorial regions of the embryo) forms
distinct endodermal and mesodermal lineages. Initially,
the mesoderm is broadly defned into dorsal mesoderm
(Spemann’s organizer) and ventral mesoderm territories.
Demarcation of the boundary is not distinct. Further interactions subdivide mesoderm into more distinct domains that
give rise to head mesoderm, notochord, somite, lateral plate,
and ventral mesenchyme. The ectoderm also subdivides into
neural and epidermal ectoderm territories via neuralizing
signals emanating from Spemann’s organizer, which secretes
Bmp, Wnt, and Nodal antagonists to promote naïve ectoderm
(presumptive epidermal tissue) into neural tissue (see Chapter
4). As embryogenesis progresses, neural subdivisions are
formed. Neural crest and pre-placodal ectoderm also arise
from the neural plate border region that is formed between
the neural plate and the epidermis. At the same time, endodermal regions subdivide, providing distinct anteroposterior
characteristics to give rise to endodermal organ precursors.
GRN structural features underlying germ layer specif cation,
Spemann’s organizer formation, and mesendoderm patterning in Xenopus are discussed in the following section.
12.2.2. GRNS DURING GERM LAYER FORMATION
Germ layer specifcation (the delineation of ectoderm, mesoderm, endoderm) is one of the frst cell lineage commitment
Xenopus
step to understand the control of cell fate specif cation at
the transcriptional level. The diploid frog Xenopus tropicalis
is an ideal system to build and study developmental GRN.
Xenopus has long been used as a model for vertebrate early
development and has contributed greatly to the elucidation
of gene regulation. Xenopus embryos can be experimentally
manipulated at the earliest stages of development to allow
gain-of-function and loss-of-function analyses with relative
ease. The readily available genomic tools make the Xenopus
system ideally suited for the systematic identif cation of
genes affected by such manipulations. These advantages
point to the utility of the Xenopus system for the collection
of the large number of data sets to be used for GRN study.
Identifying the molecular parts encoded in the genome is
only a frst step towards understanding how context-specif c
biological processes are controlled. As we shall see, the knowledge of how these parts are combined and function together
is an equally important question. A little over a decade ago,
efforts were made to compile the available molecular data
into GRNs describing Xenopus mesendoderm and Spemann
organizer development (Loose and Patient, 2004; Koide et al.,
2005). In 2017, additional fndings were applied to update the
mesendoderm GRN from fertilization to the beginning of
gastrulation, linking TFs and critical signaling pathways with
transcriptional targets (Charney et al., 2017a). Close attention
was paid to focus on “direct” interactions between TFs and
target genes to build the GRN regulating mesendoderm.
An important consideration in creating GRNs is to identify direct links between a TF and its direct target genes to
understand the underlying network substructures regulating
gene expression. We recommend the following three criteria
to be used to create an interpretable GRN (Koide et al., 2005).
First, an obvious and critically important criterion is that a
putative direct target gene must be expressed temporally and
spatially in a manner consistent with the expression of the TF
proposed to control it. For example, if the TF is an activator,
the target gene should be coexpressed in the same or overlapping region where the TF is expressed. Conversely, if the TF
is a repressor, target gene expression should be excluded (or
reduced) from the region where the TF is expressed. Second,
a strong correlation must exist between perturbation of a
regulatory TF and the expression changes of the suspected
target genes. Regulation can be measured following gainand/or loss-of-function experiments (e.g., injection of mRNA
encoding a TF or a translation blocking antisense morpholino
oligonucleotide) by analyzing changes in target gene RNA
expression (e.g., RNA-seq, RT-PCR, northern blotting, in situ
hybridization). Third, a direct physical interaction between
the TF and a CRM controlling the proposed target needs to
be validated experimentally because perturbation experiments alone are insuffcient to distinguish between direct
and indirect effects. Methods for assessing direct interaction
include chromatin immunoprecipitation (ChIP), gel electrophoretic mobility shift assay (EMSA), DNase footprinting,
or reporter gene assays (evaluating appropriate binding site
mutations). This linkage of direct TF binding to regulated
genes is often lacking in the published literature, making it
diffcult to state with confdence whether the induction of a
proposed target gene is direct or indirect. Only connections
that satisfy all three criteria should be defned as direct.
However, we recognize that DNA binding is only suggestive of functional regulation and that the “gold standard”
evidence is to mutate the TF binding site and examine the
effect on gene expression in vivo. This is particularly important as ChIP-seq analysis fnds abundant neutral binding sites
(in addition to those involved in regulation) that don’t affect
gene expression (Bardet et al., 2013). These criteria should be
carefully considered whenever building GRNs.
12.2. PAST OBSERVATIONS
12.2.1. BRIEF OVERVIEW OF XENOPUS DEVELOPMENT
After fertilization, the Xenopus zygote undergoes multiple
rounds of cell division to give rise to an embryo with smaller
cells (blastomeres) but without increasing the overall volume
of the embryo. During the early stages of this process, some
maternally deposited mRNAs and proteins are asymmetrically inherited by individual blastomeres through cytokinesis
(see Chapter 3). These differentially localized materials specify the germ layer cell identities by initiating their respective
germ layer-specifc GRN programs along the animal-vegetal
axis. Other maternal components are asymmetrically distributed along the future dorsal-ventral axis, which is initiated
by the site of sperm entry. During gastrulation, the germ layers undergo patterning and are subdivided into smaller more
specifed cell fate territories that eventually def ne organ/
tissue primordia. The broadly defned mesendodermal territory (vegetal and equatorial regions of the embryo) forms
distinct endodermal and mesodermal lineages. Initially,
the mesoderm is broadly defned into dorsal mesoderm
(Spemann’s organizer) and ventral mesoderm territories.
Demarcation of the boundary is not distinct. Further interactions subdivide mesoderm into more distinct domains that
give rise to head mesoderm, notochord, somite, lateral plate,
and ventral mesenchyme. The ectoderm also subdivides into
neural and epidermal ectoderm territories via neuralizing
signals emanating from Spemann’s organizer, which secretes
Bmp, Wnt, and Nodal antagonists to promote naïve ectoderm
(presumptive epidermal tissue) into neural tissue (see Chapter
4). As embryogenesis progresses, neural subdivisions are
formed. Neural crest and pre-placodal ectoderm also arise
from the neural plate border region that is formed between
the neural plate and the epidermis. At the same time, endodermal regions subdivide, providing distinct anteroposterior
characteristics to give rise to endodermal organ precursors.
GRN structural features underlying germ layer specif cation,
Spemann’s organizer formation, and mesendoderm patterning in Xenopus are discussed in the following section.
12.2.2. GRNS DURING GERM LAYER FORMATION
Germ layer specifcation (the delineation of ectoderm, mesoderm, endoderm) is one of the frst cell lineage commitment