Hans-Henning Arnold

MyoD or Myf-5 is required for the formation of skeletal muscle

Mice carrying null mutations in the myogenic regulatory factors Myf-5 or MyoD have apparently normal skeletal muscle. To address whether these two factors functionally substitute for one another in myogenesis, mice carrying mutant Myf-5 and MyoD genes were interbred. While mice lacking both MyoD and Myf-5 were born alive, they were immobile and died soon after birth. Northern blot and S1 nuclease analyses indicated that Myf-5(-1-);MyoD(-1-) mice expressed no detectable skeletal muscle-specific mRNAs. Histological examination of these mice revealed a complete absence of skeletal muscle. Immunohistochemical analysis indicated an absence of desmin-expressing myoblast-like cells. These observations suggest that either Myf-5 or MyoD is required for the determination of skeletal myoblasts, their propagation, or both during embryonic development and indicate that these factors play, at least in part, functionally redundant roles in myogenesis.

Know Your Neighbors: Three Phenotypes in Null Mutants of the Myogenic bHLH Gene MRF4

The data from the MRF4, Hox, and globin studies suggest that many other knockout experiments may be subject to similar effects. The examples we have discussed involve loci that contain two or more related genes, and these may be especially prone to the evolution of joint locus control elements and other interrelated cis-regulatory interactions. It is equally plausible that our current picture is skewed toward these cases by investigator knowledge of the identity of neighboring genes and that similar long-range effects will be seen for unrelated adjacent genes. We therefore feel that until substantially more is understood about cis-regulation over substantial regions of the genome it is prudent to consider these effects in design and in interpretation. One might think that direct complementation testing would clarify which knockouts are subject to neighborhood complications, but in the mouse this solution is not straightforward. It has proved frustratingly difficult to capture and reintroduce into mice pieces of DNA that faithfully recapitulate the full developmental pattern and correct level of expression of the wildtype gene. The culprits appear to be the very same aspects of gene structure that are at work in generating the neighborhood problem: the dispersal of pertinent regulatory elements over very large stretches of DNA that may also include additional genes. Moreover, minigenes that might be induced to express at will complementing protein coding sequences, in the manner of heat shock regulated constructions in Drosophila, are unreliable in current mouse transgenic formats. For these reasons the application of improved, if somewhat more complex, knockout strategies provides a more immediate solution to neighborhood uncertainties. To avoid deleting sequences that may have regulatory effects on adjacent genes, one can instead introduce an effectively positioned stop codon. This can be coupled with removal of the selection cassette by site-specific recombinases such as yeast Flp or phage P1 Cre. Alternatively, the so-called “hit-and-run” strategy, which provides for simultaneous introduction of subtle nonsense or missense mutations together with the elimination of all selection cassette residue (Ramirez-Solis et al. 1993xRamirez-Solis, R, Zheng, II, Whiting, J, Krumlauf, R, and Bradley, A. Cell. 1993; 73: 279–294Abstract | Full Text PDF | PubMed | Scopus (227)See all ReferencesRamirez-Solis et al. 1993), can be used. This design achieves the cleanest introduction of small and specific mutations, but calls for somewhat greater effort in establishing and identifying the desired ES cell line. Finally, for many existing knockouts, an immediate challenge is to consider their phenotypes with attention to the identities and activities of neighboring genes.

Muscle differentiation: more complexity to the network of myogenic regulators.

Recent genetic and biochemical approaches have advanced our understanding of control mechanisms underlying myogenesis in vertebrate organisms. In particular, systematic combinations of targeted gene disruptions in mice have revealed unique and overlapping functions of members of the MyoD family of transcription factors within the regulatory network that establishes skeletal muscle cell lineages. Moreover, Pax3 has been identified as a key regulator of myogenesis which seems to act genetically upstream of MyoD. In addition, novel genes have been discovered that modulate myogenesis and the activity of myogenic basic helix-loop-helix (bHLH) proteins in positive or negative ways. The molecular mechanisms of these interactions and cooperativity are being elucidated, most notably between the myogenic bHLH factors and MEF2 transcription factors.

BMP-2 induces ectopic expression of cardiac lineage markers and interferes with somite formation in chicken embryos

In Drosophila induction of the homeobox gene tinman and subsequent heart formation are dependent on dpp signaling from overlying ectoderm. In order to define vertebrate heart-inducing signals we screened for dpp-homologues expressed in HH stage 4 chicken embryos. The majority of transcripts were found to be BMP-2 among several other members of the BMP family. From embryonic HH stage 4 onwards cardiogenic mesoderm appeared to be in close contact to BMP-2 expressing cells which initially were present in lateral mesoderm and subsequently after headfold formation in the pharyngeal endoderm. In order to assess the role of BMP-2 for heart formation, gastrulating chick embryos in New culture were implanted with BMP-2 producing cells. BMP-2 implantation resulted in ectopic cardiac mesoderm specification. BMP-2 was able to induce Nkx2-5 expression ectopically within the anterior head domain, while GATA-4 was also induced more caudally. Cardiogenic induction by BMP-2, however remained incomplete, since neither Nkx2-8 nor the cardiac-restricted structural gene VMHC-1 became ectopically induced. BMP-2 expressing cells implanted adjacent to paraxial mesoderm resulted in impaired somite formation and blocked the expression of marker genes, such as paraxis, Pax-3, and the forkhead gene cFKH-1. These results suggest that BMP-2 is part of the complex of cardiogenic signals and is involved in the patterning of early mesoderm similar to the role of dpp in Drosophila.

BMP2 is required for early heart development during a distinct time period

BMP2, like its Drosophila homologue dpp, is an important signaling molecule for specification of cardiogenic mesoderm in vertebrates. Here, we analyzed the time-course of BMP2-requirement for early heart formation in whole chick embryos and in explants of antero-lateral plate mesoderm. Addition of Noggin to explants isolated at stage 4 and cultured for 24 h resulted in loss of NKX2.5, GATA4, eHAND, Mef2A and vMHC expression. At stages 5-8 the individual genes showed differential sensitivity to Noggin addition. While expression of eHAND, NKX2.5 and Mef2A was clearly reduced by Noggin vMHC was only marginally affected. In contrast, GATA4 expression was enhanced after Noggin treatment. The developmental period during which cardiac mesoderm required the presence of BMP signaling in vivo was assessed by implantation of Noggin expressing cells into stage 4-8 embryos which were then cultured until stage 10-11. Complete loss of NKX2.5 and eHAND expression was observed in embryos implanted at stages 4-6, and expression was still suppressed in stages 7 and 8 implanted embryos. GATA4 expression was also blocked by Noggin at stage 4, however increased at stages 5, 6 and 7. Explants of central mesendoderm, that normally do not form heart tissue were employed to study the time-course of BMP2-induced cardiac gene expression. The induction of cardiac lineage markers in central mesendoderm of stage 5 embryos was distinct for different genes. While GATA4, -5, -6 and MEF2A were induced to maximal levels within 6 h after BMP2 addition, eHAND and dHAND required 12 h to reach maximum levels of expression. NKX2.5 was induced by 6 h and accumulated over 48 h. vMHC and titin were induced at significant levels only after 48 h of BMP2 addition. These results indicate that cardiac marker genes display distinct expression kinetics after BMP2 addition and differential response to Noggin treatment suggesting complex regulation of myocardial gene expression in the early tubular heart.

4 Genetics of Muscle Determination and Development

Skeletal muscles in vertebrates develop from somites as the result of patterning and cell type specification events. Here, we review the current knowledge of genes and signals implicated in these processes. We discuss in particular the role of the myogenic determination genes as deduced from targeted gene disruptions in mice and how their expression may be controlled. We also refer to other transcription factors which collaborate with the myogenic regulators in positive or negative ways to control myogenesis. Moreover, we review experiments that demonstrate the influence of tissues surrounding the somites on the process of muscle formation and provide model views on the underlying mechanisms. Finally, we present recent evidence on genes that play a role in regeneration of muscle in adult organisms.

The Cell Adhesion Molecule M-Cadherin Is Not Essential for Muscle Development and Regeneration

ABSTRACT M-cadherin is a classical calcium-dependent cell adhesion molecule that is highly expressed in developing skeletal muscle, satellite cells, and cerebellum. Based on its expression pattern and observations in cell culture, it has been postulated that M-cadherin may be important for the fusion of myoblasts to form myotubes, the correct localization and function of satellite cells during muscle regeneration, and the specialized architecture of adhering junctions in granule cells of cerebellar glomeruli. In order to investigate the potential roles of M-cadherin in vivo, we generated a null mutation in mice. Mutant mice were viable and fertile and showed no gross developmental defects. In particular, the skeletal musculature appeared essentially normal. Moreover, muscle lesions induced by necrosis were efficiently repaired in mutant mice, suggesting that satellite cells are present, can be activated, and are able to form new myofibers. This was also confirmed by normal growth and fusion potential of mutant satellite cells cultured in vitro. In the cerebellum of M-cadherin-lacking mutants, typical contactus adherens junctions were present and similar in size and numbers to the equivalent junctions in wild-type animals. However, the adhesion plaques in the cerebellum of these mutants appeared to contain elevated levels of N-cadherin compared to wild-type animals. Taken together, these observations suggest that M-cadherin in the mouse serves no absolutely required function during muscle development and regeneration and is not essential for the formation of specialized cell contacts in the cerebellum. It seems that N-cadherin or other cadherins can largely compensate for the lack of M-cadherin.

The homeobox gene it NKX3.2 is a target of left–right signalling and is expressed on opposite sides in chick and mouse embryos

Vertebrate internal organs display invariant left-right (L-R) asymmetry. A signalling cascade that sets up L-R asymmetry has recently been identified (reviewed in [1]). On the right side of Hensens node, activin represses Sonic hedgehog (Shh) expression and induces expression of the genes for the activin receptor (ActRIIa) and fibroblast growth factor-8 (FGF8) [2] [3]. On the left side, Shh induces nodal expression in lateral plate mesoderm (LPM); nodal in turn upregulates left-sided expression of the bicoid-like homeobox gene Pitx2 [4] [5] [6]. Here, we found that the homeobox gene NKX3.2 is asymmetrically expressed in the anterior left LPM and in head mesoderm in the chick embryo. Misexpression of the normally left-sided signals Nodal, Lefty2 and Shh on the right side, or ectopic application of retinoic acid (RA), resulted in upregulation of NKX3.2 contralateral to its normal expression in left LPM. Ectopic application of FGF8 on the left side blocked NKX3.2 expression, whereas the FGF receptor-1 (FGFR-1) antagonist SU5402, implanted on the right side, resulted in bilateral NKX3.2 expression in the LPM, suggesting that FGF8 is an important negative determinant of asymmetric NKX3.2 expression. NKX3.2 expression was also found to be asymmetric in the mouse LPM but, unlike in the chick, it was expressed in the right LPM. In the inversion of embryonic turning (inv) mouse mutant, which has aberrant L-R development, NKX3.2 was expressed predominantly on the left side. Thus, NKX3.2 transcripts accumulate on opposite sides of mouse and chick embryos although, in both the mouse and chick, NKX3.2 expression is controlled by the L-R signalling pathways.

Targeted disruption of the Nkx3.1 gene in mice results in morphogenetic defects of minor salivary glands: parallels to glandular duct morphogenesis in prostate

To investigate functions of the homeodomain-containing transcription factor Nkx3.1 a null mutation was generated by targeted gene disruption introducing the bacterial LacZ gene as reporter into the locus. In addition to defects in duct morphogenesis of the prostate and bulbourethral gland displaying progressive epithelial hyperplasia and reduced ductal branching (Bhatia-Gaur, R., Donjacour, A.A., Sciavolino, P.J., Kim, M., Desai, N., Young, P., Norton, C.R., Gridley, T., Cardiff, R.D., Cunha, G.R., Abate-Shen, C., Shen, M.M., 1999. Genes Dev. 13, 966-977), we observed a novel phenotype in minor salivary glands of Nkx3.1 null mutants. Minor salivary glands in the oral cavity of mutant mice appeared reduced in size and exhibited severely altered duct morphology. Other Nkx3.1 expressing regions were unaffected by the mutation. The activity of the Nkx3. 1/LacZ allele faithfully reflected the known expression domains of Nkx3.1 in sclerotome, a subset of blood vessels, Rathkes pouch, and ductal epithelium in prostate and minor salivary glands during pre- and postnatal mouse development. However, it was additionally expressed in the heart, duodenum and lung. These ectopic expression domains resemble the pattern of the Nkx2.6 gene which is closely linked to Nkx3.1 in the mouse genome and its regulation may therefore be affected by the mutation. In Nkx3.1/Shh compound mutant mice we found that Nkx3.1 expression in sclerotome and prostate was strictly dependent on sonic hedgehog (Shh) signaling, while other expression domains including heart and gut were independent of Shh. Expression in lung appeared augmented in the absence of Shh. Our results suggest that Nkx3.1 plays a unique role in regulating proliferation of glandular epithelium and in the formation of ducts in prostate and minor salivary glands.

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.