A fundamental process during both embryo development and stem cell differentiation is the control... more A fundamental process during both embryo development and stem cell differentiation is the control of cell lineage determination. In developing skeletal muscle, many of the diffusible signaling molecules, transcription factors and more recently non-coding RNAs that contribute to this process have been identified. This has facilitated advances in our understanding of the molecular mechanisms underlying the control of cell fate choice. Here we will review the role of non-coding RNAs, in particular microRNAs (miR-NAs), in embryonic muscle development and differentiation, and in satellite cells of adult muscle, which are essential for muscle growth and regeneration. Some of these short post-transcriptional regulators of gene expression are restricted to skeletal muscle, but their expression can also be more widespread. In addition, we discuss a few examples of long non-coding RNAs, which are numerous but much less well understood.
The expression of the myogenic regulatory factors (MRFs), Myf5, MyoD, myogenin (Mgn) and MRF4 hav... more The expression of the myogenic regulatory factors (MRFs), Myf5, MyoD, myogenin (Mgn) and MRF4 have been analysed during the development of chicken embryo somites and limbs. In somites, Myf5 is expressed first in somites and paraxial mesoderm at HH stage 9 followed by MyoD at HH stage 12, and Mgn and MRF4 at HH stage 14. In older somites, Myf5 and MyoD are also expressed in the ventrally extending myotome prior to Mgn and MRF4 expression. In limb muscles a similar temporal sequence is observed with Myf5 expression detected first in forelimbs at HH stage 22, MyoD at HH stage 23, Mgn at HH stage 24 and MRF4 at HH stage 30. This report describes the precise time of onset of expression of each MRF in somites and limbs during chicken embryo development, and provides a detailed comparative timeline of MRF expression in different embryonic muscle groups.
Klhl31 is a member of the Kelch like family in vertebrates, which are characterized by an amino-t... more Klhl31 is a member of the Kelch like family in vertebrates, which are characterized by an amino-terminal broad complex tram-track, bric-a-brac/poxvirus and zinc finger (BTB/POZ)domain, carboxy terminal Kelch repeats and a central linker region (Backdomain). In developing somites Klhl31 is highly expressed in the myotome down stream of myogenic regulators (MRF), and it remains expressed in differentiated skeletal muscle. In vivo gain- and loss-of-function approaches in chick embryos reveal a role of Klhl31 in skeletal myogenesis. Targeted mis expression of Klhl31 led to a reduced size of dermomyotome and myotome as indicated by detection of relevant myogenic markers, Pax3, Myf5, myogenin and myosin heavy chain (MF20).The knock-down of Klhl31 in developing somites, using antisense morpholinos (MO), led to an expansion of Pax3, Myf5, MyoD and myogenin expression domains and an increase in the number of mitotic cells in the dermomyotome and myotome. The mechanism underlying this phenotype was examined using complementary approaches, which show that Klhl31 interferes with β-catenin dependent Wnt signaling. Klhl31 reduced the Wnt-mediated activation of a luciferase reporter in cultured cells. Furthermore, Klhl31 attenuated secondary axis formation in Xenopus embryos in response to Wnt1 or β-catenin. Klhl31mis expression in the developing neural tube affected its dorsoventral patterning and led to reduced dermomyotome and myotome size.Co-transfection of a Wnt3a expression vector withKlhl31 in somites or in the neural tube rescued the phenotype and restored the size of dermomyotome and myotome. Thus, Klhl31 is a novel modulator of canonical Wnt signaling, important for vertebrate myogenesis. We propose that Klhl31 acts in the myotome to support cell cycle withdrawal and differentiation.
The development of a complex organism from a
single cell, the fertilised egg, has fascinated peop... more The development of a complex organism from a single cell, the fertilised egg, has fascinated people for centuries. Embryo development is highly reproducible and exquisitely regulated. How is it that all tissues and organs form in the right places and at the right time? How is the development of different organ systems coordinated, so that they all fit together correctly at the end? It is challenging to study development, because many embryos are small or inaccessible. The chick embryo is a popular model system with many experimental advantages, which include classic ‘cut and paste’ experiments and mechanistic gene function analyses. The combination of micromanipulations with gain- or loss-of-function is particularly powerful. The recent development of transgenic lines and advanced imaging techniques ensure that the chicken remains an attractive model system, which will continue to make major contributions to our understanding of molecular and cellular mechanisms controlling developmental processes.
During limb development Pax3 positive myoblasts delaminate from the hypaxial dermomyotome of limb... more During limb development Pax3 positive myoblasts delaminate from the hypaxial dermomyotome of limb level somites and migrate into the limb bud where they form the dorsal and ventral muscle masses. Only then do they begin to differentiate and express markers of myogenic commitment and determination such as Myf5 and MyoD. However the signals regulating this process remain poorly characterised. We show that FGF18, which is expressed in the distal mesenchyme of the limb bud, induces premature expression of both Myf5 and MyoD and that blocking FGF signalling also inhibits endogenous MyoD expression. This expression is mediated by ERK MAP kinase but not PI3K signalling. We also show that retinoic acid (RA) can inhibit the myogenic activity of FGF18 and that blocking RA signalling allows premature induction of MyoD by FGF18 at HH19. We propose a model where interactions between FGF18 in the distal limb and retinoic acid in the proximal limb regulate the timing of myogenic gene expression during limb bud development.
Myogenesis involves the stable commitment of progenitor cells followed by the execution of myogen... more Myogenesis involves the stable commitment of progenitor cells followed by the execution of myogenic differentiation, processes that are coordinated by myogenic regulatory factors, microRNAs and BAF chromatin remodeling complexes. BAF60a, BAF60b and BAF60c are structural subunits of the BAF complex that bind to the core ATPase Brg1 to provide functional specificity. BAF60c is essential for myogenesis; however, the mechanisms regulating the subunit composition of BAF/Brg1 complexes, in particular the incorporation of different BAF60 variants, are not understood. Here we reveal their dynamic expression during embryo myogenesis and uncover the concerted negative regulation of BAF60a and BAF60b by the muscle-specific microRNAs (myomiRs) miR-133 and miR-1/206 during somite differentiation. MicroRNA inhibition in chick embryos leads to increased BAF60a or BAF60b levels, a concomitant switch in BAF/Brg1 subunit composition and delayed myogenesis. The phenotypes are mimicked by sustained BAF60a or BAF60b expression and are rescued by morpholino knockdown of BAF60a or BAF60b. This suggests that myomiRs contribute to select BAF60c for incorporation into the Brg1 complex by specifically targeting the alternative variants BAF60a and BAF60b during embryo myogenesis, and reveals that interactions between tissue-specific non-coding RNAs and chromatin remodeling factors confer robustness to mesodermal lineage determination.
Proceedings of the National Academy of Sciences of the United States of America, 2014
In vertebrate embryos, cardiac progenitor cells (CPCs) undergo long-range migration after emergin... more In vertebrate embryos, cardiac progenitor cells (CPCs) undergo long-range migration after emerging from the primitive streak during gastrulation. Together with other mesoderm progenitors, they migrate laterally and then toward the ventral midline, where they form the heart. Signals controlling the migration of different progenitor cell populations during gastrulation are poorly understood. Several pathways are involved in the epithelial-to-mesenchymal transition and ingression of mesoderm cells through the primitive streak, including fibroblast growth factors and wingless-type family members (Wnt). Here we focus on early CPC migration and use live video microscopy in chicken embryos to demonstrate a role for bone morphogenetic protein (BMP)/SMA and MAD related (Smad) signaling. We identify an interaction of BMP and Wnt/glycogen synthase kinase 3 beta (GSK3β) pathways via the differential phosphorylation of Smad1. Increased BMP2 activity altered migration trajectories of prospective cardiac cells and resulted in their lateral displacement and ectopic differentiation, as they failed to reach the ventral midline. Constitutively active BMP receptors or constitutively active Smad1 mimicked this phenotype, suggesting a cell autonomous response. Expression of GSK3β, which promotes the turnover of active Smad1, rescued the BMP-induced migration phenotype. Conversely, expression of GSK3β-resistant Smad1 resulted in aberrant CPC migration trajectories. De-repression of GSK3β by dominant negative Wnt3a restored normal migration patterns in the presence of high BMP activity. The data indicate the convergence of BMP and Wnt pathways on Smad1 during the early migration of prospective cardiac cells. Overall, we reveal molecular mechanisms that contribute to the emerging paradigm of signaling pathway integration in embryo development.
The development and differentiation of vertebrate skeletal muscle provide an important paradigm t... more The development and differentiation of vertebrate skeletal muscle provide an important paradigm to understand the inductive signals and molecular events controlling differentiation of specific cell types. Recent findings show that a core transcriptional network, initiated by the myogenic regulatory factors (MRFs; MYF5, MYOD, myogenin and MRF4), is activated by separate populations of cells in embryos in response to various signalling pathways. This review will highlight how cells from multiple distinct starting points can converge on a common set of regulators to generate skeletal muscle.
A fundamental process during both embryo development and stem cell differentiation is the control... more A fundamental process during both embryo development and stem cell differentiation is the control of cell lineage determination. In developing skeletal muscle, many of the diffusible signaling molecules, transcription factors and more recently non-coding RNAs that contribute to this process have been identified. This has facilitated advances in our understanding of the molecular mechanisms underlying the control of cell fate choice. Here we will review the role of non-coding RNAs, in particular microRNAs (miR-NAs), in embryonic muscle development and differentiation, and in satellite cells of adult muscle, which are essential for muscle growth and regeneration. Some of these short post-transcriptional regulators of gene expression are restricted to skeletal muscle, but their expression can also be more widespread. In addition, we discuss a few examples of long non-coding RNAs, which are numerous but much less well understood.
The expression of the myogenic regulatory factors (MRFs), Myf5, MyoD, myogenin (Mgn) and MRF4 hav... more The expression of the myogenic regulatory factors (MRFs), Myf5, MyoD, myogenin (Mgn) and MRF4 have been analysed during the development of chicken embryo somites and limbs. In somites, Myf5 is expressed first in somites and paraxial mesoderm at HH stage 9 followed by MyoD at HH stage 12, and Mgn and MRF4 at HH stage 14. In older somites, Myf5 and MyoD are also expressed in the ventrally extending myotome prior to Mgn and MRF4 expression. In limb muscles a similar temporal sequence is observed with Myf5 expression detected first in forelimbs at HH stage 22, MyoD at HH stage 23, Mgn at HH stage 24 and MRF4 at HH stage 30. This report describes the precise time of onset of expression of each MRF in somites and limbs during chicken embryo development, and provides a detailed comparative timeline of MRF expression in different embryonic muscle groups.
Klhl31 is a member of the Kelch like family in vertebrates, which are characterized by an amino-t... more Klhl31 is a member of the Kelch like family in vertebrates, which are characterized by an amino-terminal broad complex tram-track, bric-a-brac/poxvirus and zinc finger (BTB/POZ)domain, carboxy terminal Kelch repeats and a central linker region (Backdomain). In developing somites Klhl31 is highly expressed in the myotome down stream of myogenic regulators (MRF), and it remains expressed in differentiated skeletal muscle. In vivo gain- and loss-of-function approaches in chick embryos reveal a role of Klhl31 in skeletal myogenesis. Targeted mis expression of Klhl31 led to a reduced size of dermomyotome and myotome as indicated by detection of relevant myogenic markers, Pax3, Myf5, myogenin and myosin heavy chain (MF20).The knock-down of Klhl31 in developing somites, using antisense morpholinos (MO), led to an expansion of Pax3, Myf5, MyoD and myogenin expression domains and an increase in the number of mitotic cells in the dermomyotome and myotome. The mechanism underlying this phenotype was examined using complementary approaches, which show that Klhl31 interferes with β-catenin dependent Wnt signaling. Klhl31 reduced the Wnt-mediated activation of a luciferase reporter in cultured cells. Furthermore, Klhl31 attenuated secondary axis formation in Xenopus embryos in response to Wnt1 or β-catenin. Klhl31mis expression in the developing neural tube affected its dorsoventral patterning and led to reduced dermomyotome and myotome size.Co-transfection of a Wnt3a expression vector withKlhl31 in somites or in the neural tube rescued the phenotype and restored the size of dermomyotome and myotome. Thus, Klhl31 is a novel modulator of canonical Wnt signaling, important for vertebrate myogenesis. We propose that Klhl31 acts in the myotome to support cell cycle withdrawal and differentiation.
The development of a complex organism from a
single cell, the fertilised egg, has fascinated peop... more The development of a complex organism from a single cell, the fertilised egg, has fascinated people for centuries. Embryo development is highly reproducible and exquisitely regulated. How is it that all tissues and organs form in the right places and at the right time? How is the development of different organ systems coordinated, so that they all fit together correctly at the end? It is challenging to study development, because many embryos are small or inaccessible. The chick embryo is a popular model system with many experimental advantages, which include classic ‘cut and paste’ experiments and mechanistic gene function analyses. The combination of micromanipulations with gain- or loss-of-function is particularly powerful. The recent development of transgenic lines and advanced imaging techniques ensure that the chicken remains an attractive model system, which will continue to make major contributions to our understanding of molecular and cellular mechanisms controlling developmental processes.
During limb development Pax3 positive myoblasts delaminate from the hypaxial dermomyotome of limb... more During limb development Pax3 positive myoblasts delaminate from the hypaxial dermomyotome of limb level somites and migrate into the limb bud where they form the dorsal and ventral muscle masses. Only then do they begin to differentiate and express markers of myogenic commitment and determination such as Myf5 and MyoD. However the signals regulating this process remain poorly characterised. We show that FGF18, which is expressed in the distal mesenchyme of the limb bud, induces premature expression of both Myf5 and MyoD and that blocking FGF signalling also inhibits endogenous MyoD expression. This expression is mediated by ERK MAP kinase but not PI3K signalling. We also show that retinoic acid (RA) can inhibit the myogenic activity of FGF18 and that blocking RA signalling allows premature induction of MyoD by FGF18 at HH19. We propose a model where interactions between FGF18 in the distal limb and retinoic acid in the proximal limb regulate the timing of myogenic gene expression during limb bud development.
Myogenesis involves the stable commitment of progenitor cells followed by the execution of myogen... more Myogenesis involves the stable commitment of progenitor cells followed by the execution of myogenic differentiation, processes that are coordinated by myogenic regulatory factors, microRNAs and BAF chromatin remodeling complexes. BAF60a, BAF60b and BAF60c are structural subunits of the BAF complex that bind to the core ATPase Brg1 to provide functional specificity. BAF60c is essential for myogenesis; however, the mechanisms regulating the subunit composition of BAF/Brg1 complexes, in particular the incorporation of different BAF60 variants, are not understood. Here we reveal their dynamic expression during embryo myogenesis and uncover the concerted negative regulation of BAF60a and BAF60b by the muscle-specific microRNAs (myomiRs) miR-133 and miR-1/206 during somite differentiation. MicroRNA inhibition in chick embryos leads to increased BAF60a or BAF60b levels, a concomitant switch in BAF/Brg1 subunit composition and delayed myogenesis. The phenotypes are mimicked by sustained BAF60a or BAF60b expression and are rescued by morpholino knockdown of BAF60a or BAF60b. This suggests that myomiRs contribute to select BAF60c for incorporation into the Brg1 complex by specifically targeting the alternative variants BAF60a and BAF60b during embryo myogenesis, and reveals that interactions between tissue-specific non-coding RNAs and chromatin remodeling factors confer robustness to mesodermal lineage determination.
Proceedings of the National Academy of Sciences of the United States of America, 2014
In vertebrate embryos, cardiac progenitor cells (CPCs) undergo long-range migration after emergin... more In vertebrate embryos, cardiac progenitor cells (CPCs) undergo long-range migration after emerging from the primitive streak during gastrulation. Together with other mesoderm progenitors, they migrate laterally and then toward the ventral midline, where they form the heart. Signals controlling the migration of different progenitor cell populations during gastrulation are poorly understood. Several pathways are involved in the epithelial-to-mesenchymal transition and ingression of mesoderm cells through the primitive streak, including fibroblast growth factors and wingless-type family members (Wnt). Here we focus on early CPC migration and use live video microscopy in chicken embryos to demonstrate a role for bone morphogenetic protein (BMP)/SMA and MAD related (Smad) signaling. We identify an interaction of BMP and Wnt/glycogen synthase kinase 3 beta (GSK3β) pathways via the differential phosphorylation of Smad1. Increased BMP2 activity altered migration trajectories of prospective cardiac cells and resulted in their lateral displacement and ectopic differentiation, as they failed to reach the ventral midline. Constitutively active BMP receptors or constitutively active Smad1 mimicked this phenotype, suggesting a cell autonomous response. Expression of GSK3β, which promotes the turnover of active Smad1, rescued the BMP-induced migration phenotype. Conversely, expression of GSK3β-resistant Smad1 resulted in aberrant CPC migration trajectories. De-repression of GSK3β by dominant negative Wnt3a restored normal migration patterns in the presence of high BMP activity. The data indicate the convergence of BMP and Wnt pathways on Smad1 during the early migration of prospective cardiac cells. Overall, we reveal molecular mechanisms that contribute to the emerging paradigm of signaling pathway integration in embryo development.
The development and differentiation of vertebrate skeletal muscle provide an important paradigm t... more The development and differentiation of vertebrate skeletal muscle provide an important paradigm to understand the inductive signals and molecular events controlling differentiation of specific cell types. Recent findings show that a core transcriptional network, initiated by the myogenic regulatory factors (MRFs; MYF5, MYOD, myogenin and MRF4), is activated by separate populations of cells in embryos in response to various signalling pathways. This review will highlight how cells from multiple distinct starting points can converge on a common set of regulators to generate skeletal muscle.
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Papers by Gi Fay Mok
single cell, the fertilised egg, has fascinated people
for centuries. Embryo development is highly
reproducible and exquisitely regulated. How is it
that all tissues and organs form in the right places
and at the right time? How is the development of
different organ systems coordinated, so that they
all fit together correctly at the end? It is challenging
to study development, because many embryos
are small or inaccessible. The chick embryo is a
popular model system with many experimental
advantages, which include classic ‘cut and paste’
experiments and mechanistic gene function analyses.
The combination of micromanipulations with
gain- or loss-of-function is particularly powerful.
The recent development of transgenic lines
and advanced imaging techniques ensure that
the chicken remains an attractive model system,
which will continue to make major contributions
to our understanding of molecular and cellular
mechanisms controlling developmental processes.
single cell, the fertilised egg, has fascinated people
for centuries. Embryo development is highly
reproducible and exquisitely regulated. How is it
that all tissues and organs form in the right places
and at the right time? How is the development of
different organ systems coordinated, so that they
all fit together correctly at the end? It is challenging
to study development, because many embryos
are small or inaccessible. The chick embryo is a
popular model system with many experimental
advantages, which include classic ‘cut and paste’
experiments and mechanistic gene function analyses.
The combination of micromanipulations with
gain- or loss-of-function is particularly powerful.
The recent development of transgenic lines
and advanced imaging techniques ensure that
the chicken remains an attractive model system,
which will continue to make major contributions
to our understanding of molecular and cellular
mechanisms controlling developmental processes.