Astrid Buchberger

Chick NKx-2.3 represents a novel family member of vertebrate homologues to the Drosophila homeo☐ gene tinman: differential expression of cNKx-2.3 and cNKx-2.5 during heart and gut development

NKx homeodomain proteins are members of a growing family of vertebrate transcription factors with strong homology to the NK genes in Drosophila. Here, we describe the cloning of cNKx-2.3 and cNKx-2.5 cDNAs and their expression during chick development. Both genes are expressed in the developing heart with distinct but overlapping spatio-temporal patterns. While cNKx-2.5 is activated in early precardiac mesoderm and continues to be uniformly expressed throughout the mature heart, expression of NKx-2.3 starts later in differentiated myocardial cells with regional differences compared to NKx-2.5. Additionally, both genes are expressed in adjacent domains of the developing mid- and hindgut mesoderm as well as in branchial arches. The highly conserved structure of cNKx-2.5 and its similar expression to mouse and Xenopus NKx-2.5 genes and to the Drosophila gene tinman argue that it constitutes the chick homologue of these genes. Different temporal and spatial activity of cNKx-2.3 in heart and gut as well as in a regionally restricted expression domain in the neural tube suggest that cNKx-2.3 is a member of the NK-2 gene family which may be involved in specifying mesodermally and ectodermally derived cell types in the embryo.

Evidence for the participation of nerve growth factor and its low‐affinity receptor (p75NTR) in the regulation of the myogenic program

We have studied expression and function of neurotrophins and their receptors during myogenic differentiation of C2C12 cells, a clonal cell line derived from mouse muscle that is capable of in vitro differentiation. The genes coding for nerve growth factor (NGF), brain‐derived neurotrophic factor (BDNF) and their common low‐affinity receptor p75neurotrophin receptor (p75NTR) were shown to be expressed in C2C12 myoblasts and downregulated during myogenic differentiation and fusion into myotubes. Cocultures with dorsal root ganglia from day 8 chick embryos revealed neurite‐promoting activities of C2C12 cells that ceased with myogenic differentiation. These data suggest a temporal and developmental window for the effect of myogenic cell‐derived neurotrophins on neuronal as well as on myogenic cell populations. NGF was shown to increase DNA synthesis and cell growth of C2C12 myoblasts and to enhance myogenic differentiation in this cell line. We present evidence that NGF‐mediated processes take place at stages preceding myogenic differentiation. Enhanced muscle differentiation was also seen in p75NTR‐overexpressing C2C12 myoblasts which maintained high levels of receptors but ceased to produce NGF during differentiation. In contrast, when exogenous NGF was present at the onset of myogenic differentiation of receptor‐overexpressing cells, muscle cell development was strongly repressed. This indicates that downregulation of p75NTR is necessary for guiding myogenic cells towards terminal differentiation. Since none of the trk high‐affinity neurotrophin receptors could be demonstrated in C2C12 cells, we conclude that NGF mediates its nonneurotrophic effect via its low‐affinity receptor in an autocrine fashion. J. Cell. Physiol. 176:10–21, 1998.

Chicken NKx2–8, a novel homeobox gene expressed during early heart and foregut development

cNkx2-8 represents a novel member of the NK2-family transcription factors. The gene contains three highly conserved regions, the TN-, NK2-, and homeodomains which are diagnostic for this group of proteins. cNkx2-8 is expressed during chick embryogenesis in ventral foregut endoderm, myocardial mesoderm, epithelium of the branchial arches and the dorsal mesocardium. While cNkx2-8 expression partially overlaps with other NK genes, such as Nkx2-5 and Nkx2-3, its onset and aspects of its expression domains are specific. Thus, structural data and the expression profile suggest that cNkx2-8 constitutes a new homeobox protein which may cooperate with its known relatives in defining an antero-ventral field including the developing heart and pharyngeal endoderm.

Myf5 expression in somites and limb buds of mouse embryos is controlled by two distinct distal enhancer activities

The initiation of skeletal muscle development in the mouse embryo is strictly associated with the expression of the muscle-specific transcription factor Myf5, the first of four myogenic regulatory factors (MRFs) to be expressed in muscle progenitors, and ablation of the Myf5 gene prevents myogenesis. The complex spatiotemporal expression pattern of Myf5 depends on many discrete regulatory elements that are dispersed over long distances throughout the gene locus. These multiple control modules act differently in the various muscle precursor populations, presumably in response to diverse signals that control myogenesis. A potent enhancer region regulating Myf5 expression in limb muscles and somites has been identified previously at –58/–48 kb upstream of the transcriptional start site (Hadchouel et al., 2000). Here, we focus on the physical and functional dissection of this control region. We demonstrate that a conserved sequence of 270 bp located around –57 kb is required and sufficient to drive Myf5 expression in limbs and to maintain it in somites. A second enhancer nearby is responsible for Myf5 transcription in occipital/cranial somites. This enhancer activity also directs expression accurately to the myotome, preventing ectopic expression in the dermomyotome during the second phase of Myf5 gene activation in somites. Our data suggest that the enhancer identified here collaborates with other somitic enhancers to ensure correct myotomal Myf5 expression. Moreover, it constitutes an important element that mediates somitic expression after the initial and transient Myf5 activation through a previously described sonic hedgehog-dependent early epaxial enhancer.

Copy number variation of two separate regulatory regions upstream of SOX9 causes isolated 46,XY or 46,XX disorder of sex development

Background SOX9 mutations cause the skeletal malformation syndrome campomelic dysplasia in combination with XY sex reversal. Studies in mice indicate that SOX9 acts as a testis-inducing transcription factor downstream of SRY, triggering Sertoli cell and testis differentiation. An SRY-dependent testis-specific enhancer for Sox9 has been identified only in mice. A previous study has implicated copy number variations (CNVs) of a 78 kb region 517–595 kb upstream of SOX9 in the aetiology of both 46,XY and 46,XX disorders of sex development (DSD). We wanted to better define this region for both disorders. Results By CNV analysis, we identified SOX9 upstream duplications in three cases of SRY-negative 46,XX DSD, which together with previously reported duplications define a 68 kb region, 516–584 kb upstream of SOX9, designated XXSR (XX sex reversal region). More importantly, we identified heterozygous deletions in four families with SRY-positive 46,XY DSD without skeletal phenotype, which define a 32.5 kb interval 607.1–639.6 kb upstream of SOX9, designated XY sex reversal region (XYSR). To localise the suspected testis-specific enhancer, XYSR subfragments were tested in cell transfection and transgenic experiments. While transgenic experiments remained inconclusive, a 1.9 kb SRY-responsive subfragment drove expression specifically in Sertoli-like cells. Conclusions Our results indicate that isolated 46,XY and 46,XX DSD can be assigned to two separate regulatory regions, XYSR and XXSR, far upstream of SOX9. The 1.9 kb SRY-responsive subfragment from the XYSR might constitute the core of the Sertoli-cell enhancer of human SOX9, representing the so far missing link in the genetic cascade of male sex determination.

A homeo-paired domain-binding motif directs Myf5 expression in progenitor cells of limb muscle

Recruitment of multipotent mesodermal cells to the myogenic lineage is mediated by the transcription factor Myf5, the first of the myogenic regulatory factors to be expressed in most sites of myogenesis in the mouse embryo. Among numerous elements controlling the spatiotemporal pattern of Myf5 expression, the -58/-56 kb distal Myf5 enhancer directs expression in myogenic progenitor cells in limbs and in somites. Here, we show by site-directed mutagenesis within this enhancer that a predicted homeobox adjacent to a putative paired domain-binding site is required for the activity in muscle precursor cells in limbs and strongly contributes to expression in somites. By contrast, predicted binding sites for Tcf/Lef, Mef3 and Smad transcription factors play no apparent role for the expression in limbs but might participate in the control in somites. A 30mer oligonucleotide sequence containing and surrounding the homeo and paired domain-binding motifs directs faithful expression in myogenic cells in limbs and also enhances myotomal expression in somites. Pax3 and Meox2 transcription factors can bind to these consensus sites in vitro and therefore constitute potential regulators. However, genetic evidence in the Meox2-deficient mouse mutant argues against a role for Meox2 in the regulation of Myf5 expression. The data presented here demonstrate that a composite homeo and paired domain-binding motif within the -58/-56 enhancer is required and sufficient for activation of the Myf5 gene in muscle progenitor cells in the limb. Although Pax3 constitutes a potential cognate transcription factor for the enhancer, it fails to transactivate the site in transfection experiments.

Chicken winged‐helix transcription factor cFKH‐1 prefigures axial and appendicular skeletal structures during chicken embryogenesis

The cDNA cFKH‐1 encodes a chicken winged helix/forkhead domain transcription factor that presents a dynamic expression pattern during chicken embryogenesis. Transcripts accumulate predominantly in early paraxial mesoderm, developing somites, and within mesenchymal precursors of skeletal structures. cFKH‐1 RNA is first detected in the developing mesoderm of HH stage 6 embryos. During subsequent development cFKH‐1 RNA accumulates in a dorsal domain of the anterior presomitic mesoderm and later in all cells of the epithelial somites before it becomes limited to the sclerotome when somites compartmentalise. cFKH‐1 expression persists in the sclerotome, forming the vertebrae and in mesenchymal condensations in limb buds that will give rise later to the appendicular bones. In differentiated chondrocytes and definitive bone structures, however, cFKH‐1 expression is down‐regulated. Additional expression domains are found in mesenchyme of branchial arches and the head, in the dorsal aorta, and weakly in the endocardium. Based on its expression pattern and the structure of the forkhead DNA‐binding domain cFKH‐1 constitutes a chicken relative to the murine family of fkh‐1/MF1 and MFH‐1 factors. The embryonic expression of the cFKH‐1 gene defines distinct mesodermal domains and suggests that it may regulate gene expression in mesenchymal cell lineages that will form cartilage in trunk and limb buds. Dev. Dyn. 1998;212:94–101.

Expression of the novel basic-helix-loop-helix transcription factor cMespo in presomitic mesoderm of chicken embryos

We have identified a novel chicken gene, cMespo, which encodes a basic-helix-loop-helix (bHLH) protein with sequence homology to a subgroup of bHLH transcription factors that have been implicated in somitogenesis. cMespo transcripts are first found in the primitive streak of gastrulating chick embryos (HH stage 4) and continue to accumulate in presomitic mesoderm (PSM) until somite formation has been concluded. cMespo, however, is not expressed within somites or in tailbud mesoderm. The expression domain of cMespo in PSM largely overlaps with delta-1 but spares a region of several prospective somites at the rostral end of PSM in which c-Meso and Cek-8 are expressed.

The MADS domain containing transcription factor cMef2a is expressed in heart and skeletal muscle during embryonic chick development

Abstract Muscle enhancer factor 2 (MEF2) proteins are important transcription factors for muscle-specific gene activation. Four family members are known in mammals, referred to as MEF2A, MEF2B, MEF2C, and MEF2D. Here we report the isolation and expression pattern of the chick Mef2a gene (cMef2a). cMef2a expression starts in precardiac mesoderm of HH stage 8 embryos. During further embryonic development expression continues in the heart tube and later in atrium and ventricle. A second cMef2a expression domain appears in somites of stage 13 embryos. Somitic cMef2a expression is limited to the myotome and is not found in newly formed somites until the muscle-specific transcription factors MyoD and myogenin are present. This suggests that activation of the cMef2a gene in skeletal muscle is dependent on these basic helix-loop-helix transcription factors. cMef2a expression in heart and skeletal muscle continues into adulthood when it is also seen in intestinal mesenchyme and in brain.

Dynamic expression of chicken cMeso2 in segmental plate and somites

Abstract