Dennis Summerbell

A BAC transgenic analysis of the Mrf4/Myf5 locus reveals interdigitated elements that control activation and maintenance of gene expression during muscle development

The muscle-specific transcription factors Myf5 and Mrf4 are two of the four myogenic regulatory factors involved in the transcriptional cascade responsible for skeletal myogenesis in the vertebrate embryo. Myf5 is the first of these four genes to be expressed in the mouse. We have previously described discrete enhancers that drive Myf5 expression in epaxial and hypaxial somites, branchial arches and central nervous system, and argued that additional elements are required for proper expression (Summerbell, D., Ashby, P. R., Coutelle, O., Cox, D., Yee, S. P. and Rigby, P. W. J. (2000) Development 127, 3745-3757). We have now investigated the transcriptional regulation of both Myf5 and Mrf4 using bacterial artificial chromosome transgenesis. We show that a clone containing Myf5 and 140 kb of upstream sequences is sufficient to recapitulate the known expression patterns of both genes. Our results confirm and reinforce the conclusion of our earlier studies, that Myf5 expression is regulated differently in each of a considerable number of populations of muscle progenitors, and they begin to illuminate the evolutionary origins of this complex regulation. We further show that separate elements are involved in the activation and maintenance of expression in the various precursor populations, reflecting the diversity of the signals that control myogenesis. Mrf4 expression requires at least four elements, one of which may be shared with Myf5, providing a possible explanation for the linkage of these genes throughout vertebrate phylogeny. Further complexity is revealed by the demonstration that elements which control Mrf4 and Myf5 are embedded in an unrelated neighbouring gene.

Analysis of a key regulatory region upstream of the Myf5 gene reveals multiple phases of myogenesis, orchestrated at each site by a combination of elements dispersed throughout the locus

Myf5 is the first myogenic regulatory factor to be expressed in the mouse embryo and it determines the entry of cells into the skeletal muscle programme. A region situated between -58 kb and -48 kb from the gene directs Myf5 transcription at sites where muscles will form. We now show that this region consists of a number of distinct regulatory elements that specifically target sites of myogenesis in the somite, limbs and hypoglossal cord, and also sites of Myf5 transcription in the central nervous system. Deletion of these sequences in the context of the locus shows that elements within the region are essential, and also reveals the combinatorial complexity of the transcriptional regulation of Myf5. Both within the -58 kb to -48 kb region and elsewhere in the locus, multiple sequences are present that direct transcription in subdomains of a single site during development, thus revealing distinct phases of myogenesis when subpopulations of progenitor cells enter the programme of skeletal muscle differentiation.

Retinoic acid, a developmental signalling molecule

Retinoic acid has been used as a tool both by embryologists studying the spatial organization of cells in the embryo and by molecular biologists studying the control of gene expression in the nucleus. Embryologists have shown that retinoic acid can modify the pattern of cell differentiation so as to duplicate complete parts of the embryo in a well-organized way; molecular biologists have shown that retinoic acid can act as the switch starting the sequence of differential gene expression that results in cell differentiation. In the past year these two approaches have converged so that there now seems a real possibility that we may soon for the first time understand how a particular vertebrate development system works.

Interplay between DNA Methylation and Transcription Factor Availability: Implications for Developmental Activation of the Mouse Myogenin Gene

ABSTRACT During development, gene activation is stringently regulated to restrict expression only to the correct cell type and correct developmental stage. Here, we present mechanistic evidence that suggests DNA methylation contributes to this regulation by suppressing premature gene activation. Using the mouse Myogenin promoter as an example of the weak CpG island class of promoters, we find that it is initially methylated but becomes demethylated as development proceeds. Full hypersensitive site formation of the Myogenin promoter requires both the MEF2 and SIX binding sites, but binding to only one site can trigger the partial chromatin opening of the nonmethylated promoter. DNA methylation markedly decreases hypersensitive site formation that now occurs at a detectable level only when binding to both MEF2 and SIX binding sites is possible. This suggests that the probability of activating the methylated promoter is low until two of the factors are coexpressed within the same cell. Consistent with this, the single-cell analysis of developing somites shows that the coexpression of MEF2A and SIX1, which bind the MEF2 and SIX sites, correlates with the fraction of cells that demethylate the Myogenin promoter. Taken together, these studies imply that DNA methylation helps to prevent inappropriate gene activation until sufficient activating factors are coexpressed.

Multiple levels of transcriptional and post-transcriptional regulation are required to define the domain of Hoxb4 expression

Hox genes are key determinants of anteroposterior patterning of animal embryos, and spatially restricted expression of these genes is crucial to this function. In this study, we demonstrate that expression of Hoxb4 in the paraxial mesoderm of the mouse embryo is transcriptionally regulated in several distinct phases, and that multiple regulatory elements interact to maintain the complete expression domain throughout embryonic development. An enhancer located within the intron of the gene (region C) is sufficient for appropriate temporal activation of expression and the establishment of the correct anterior boundary in the paraxial mesoderm (somite 6/7). However, the Hoxb4 promoter is required to maintain this expression beyond 8.5 dpc. In addition, sequences within the 3′ untranslated region (region B) are necessary specifically to maintain expression in somite 7 from 9.0 dpc onwards. Neither the promoter nor region B can direct somitic expression independently, indicating that the interaction of regulatory elements is crucial for the maintenance of the paraxial mesoderm domain of Hoxb4 expression. We further report that the domain of Hoxb4 expression is restricted by regulating transcript stability in the paraxial mesoderm and by selective translation and/or degradation of protein in the neural tube. Moreover, the absence of Hoxb4 3′-untranslated sequences from transgene transcripts leads to inappropriate expression of some Hoxb4 transgenes in posterior somites, indicating that there are sequences within region B that are important for both transcriptional and post-transcriptional regulation.

The segmentation of axons from the segmental nerve roots to the chick wing.

THE chick wing is innervated by adjacent spinal roots which meet at the brachial plexus and then redistribute their axons to the peripheral nerves. Both the anatomical arrangement of nerves within the wing and the innervation territories supplied by each root are constant among animals of the same strain1–3. We describe here the pathways taken by the axons from each of the main roots to their particular territories in the normal wing, and also in wings which have dorso-ventral and antero-posterior axes reversed relative to the host axes. Our observations suggest that the characteristic innervation territories of the segmental roots may arise from two basic rules: that axons travel in parallel over long distances without crossing, and that the branching pattern of nerves within the limb is determined by the local environment3,4.

Transcriptional regulation during somitogenesis.

Publisher Summary This chapter discusses the biochemical mechanisms involved in the regulation of transcription during the development of the mouse embryo. General approach for this study involves taking genes known to act at a fairly high level in a particular genetic pathway and then to try to understand how their transcription is controlled using the power of transgenic mouse technology. In each case the objective is to identify of each of the transcription factors that control the chosen regulatory gene. It is reported that segmental identity in the vertebrate embryo is controlled by the four clusters of Hox genes. Therefore, the chapter analyzes the regulation of one such gene, Hoxb-4. One of the earliest transcriptional responses in the epithelial somite is the activation of the cascade of basic helix-loop-helix (bHLH) transcription factors that together regulate the commitment of skeletal myoblasts and their subsequent differentiation. Although there is a considerable knowledge of genes that function during somitogenesis, it is clear that many more such genes remain unknown and one has therefore sought to develop techniques that may allow the discovery of novel genes that act during defined aspects of the process.

Theories of biological pattern formation

Abstract The Royal Society devoted the 25th and 26th of March to a discussion of theories of biological pattern formation 1 . The meeting reviewed the work of the last decade in a style that should make it intelligible to the general reader, and it provided encouragement that the next decade will be equally exciting. I review here not the individual papers, but the general theme. So many different competing ideas exist in the field that it is often difficult to arrive at any generalization or even to resolve the conflicting nomenclature.

The segmental precision of the motor projection to the intercostal muscles in the developing chicken embryo

Each skeletal muscle in the vertebrate is innervated by a group of motoneurons called a motoneuron pool. Retrograde labelling of single motoneuron pools has suggested that the arrangement of motoneuron pools innervating different limb muscles does not change during the embryonic period when more than 50% of the motoneurons die. In this study we retrogradely labelled neighbouring intercostal motoneuron pools differentially with latex microspheres or dextran amines coupled to fluorescent dyes. We then mapped the positions of the differentially labelled motoneurons in whole-mount preparations using a computer-aided drawing system. While the intercostal motoneuron pools are clearly segregated even at early stages, there is some intermingling at the rostral and caudal ends. We used a logistic regression to determine the extent of segmental overlap, and to facilitate a quantitative comparison of the overlap at different stages. Statistical analysis shows that the overlap (expressed as the percentage of the length of the overlapping motoneuron pools) decreases modestly during the period of motoneuron death. Computer simulations suggest that this decrease does not result from random motoneuron death alone; one alternative possibility is selective death of motoneurons in the overlap zone. Occasional “rogue” motoneurons, that is, motoneurons of one pool that scatter into the neighbouring pool, are still present at the end of the period of cell death, representing a potential source of “noise” in the establishment of segmental patterns of connectivity.

A Unique Population of Non-Dividing Cells in the Somites

“It is not birth, marriage or death, but gastrulation which is truly the most important time in your life.” Lewis Wolpert quoted in Slack, 1983. While one can hardly mistake childbirth, and while half the world has been known to stop for a wedding, gastrulation is neither obvious nor dramatic for the onlooker. The real show stopping events are the processes of neurulation and segmentation which produce recognisable living organisms from a featureless surface (Figure 1).