Overexpression of Lis1 in different stages of spermatogenesis does not result in an aberrant phenotype.

Previous studies showed that in the mouse mutant Lis1(GT/GT) gene trap integration in intron 2 of Lis1 gene leads to male infertility in homozygous Lis1(GT/GT) mice. We further analyzed this line and could confirm the suggested downregulation of a testis-specific Lis1 transcript in mutant animals in a quantitative manner. Moreover, we analyzed the gene trap mutation on different genetic backgrounds in incipient congenic animals and could exclude a genetic background effect. To gain further insights into the role and requirement of LIS1 in spermatogenesis, 3 transgenic lines were generated, that overexpress Lis1 under control of the testis-specific promoters hEF-1α, which is exclusively active in spermatogonial cells, PGK2, which is active in pachytene spermatocytes and following stages of spermatogenesis, and Tnp2 which is active in round spermatids and following stages of spermatogenesis, respectively. All 3 transgenic lines remained fertile and testis sections displayed no abnormalities. To overcome the infertility of Lis1(GT/GT) males, these transgenic Lis1-overexpressing animals were mated with Lis1(GT/GT) mice to generate 'rescued' Lis1(GT/GT)/Lis1(Tpos) males. 'Rescued' animals from all transgenic lines remained infertile, thus overexpression of Lis1 in different stages of spermatogenesis could not rescue the infertility phenotype of homozygous gene trap males.

Chick CFC controls Lefty1 expression in the embryonic midline and nodal expression in the lateral plate.

Members of the EGF-CFC family of proteins have recently been implicated as essential cofactors for Nodal signaling. Here we report the isolation of chick CFC and describe its expression pattern, which appears to be similar to Cfc1 in mouse. During early gastrulation, chick CFC was asymmetrically expressed on the left side of Hensen's node as well as in the emerging notochord, prechordal plate, and lateral plate mesoderm. Subsequently, its expression became confined to the heart fields, notochord, and posterior mesoderm. Implantation experiments suggest that chick CFC expression in the lateral plate mesoderm is dependent on BMP signaling, while in the midline its expression depends on an Activin-like signal. The asymmetric expression domain within Hensen's node was not affected by application of FGF8, Noggin, or Shh antibody. Implantation of cells expressing human or mouse CFC2, or chick CFC on the right side of Hensen's node randomized heart looping without affecting expression of genes involved in left-right axis formation, including SnR, Nodal, Car, or Pitx2. Application of antisense oligodeoxynucleotides to the midline of Hamburger-Hamilton stage 4-5 embryos also randomized heart looping, but in contrast to the overexpression experiments, antisense oligodeoxynucleotide treatment resulted in bilateral expression of Nodal, Car, Pitx2, and NKX3.2, whereas Lefty1 expression in the midline was transiently lost. Application of the antisense oligodeoxynucleotides to the lateral plate mesoderm abolished Nodal expression. Thus, chick CFC seems to have a dual function in left-right axis formation by maintaining Nodal expression in the lateral plate mesoderm and controlling expression of Lefty1 expression in the midline territory.

Activated raf kinase inhibits muscle cell differentiation through a MEF2-dependent mechanism.

Muscle cell development is dependent on the activity of cell type-specific basic-helix-loop-helix transcription factors, MyoD, Myf-5, myogenin, and MRF4 which collaborate with myocyte enhancer factor 2 proteins to activate muscle-specific gene expression. Growth factors and activated Ras prevent differentiation of myoblasts in culture but the downstream signalling pathways are not well understood. Here, we demonstrate that active Raf kinase (Raf-BxB) completely inhibits myogenic conversion of 10T1/2 cells mediated by Myf-5 and differentiation of L6 myoblasts as indicated by the absence of myotubes, lack of myogenin expression, and markedly reduced expression of myosin heavy chain. However, activated Raf inhibits transcriptional activation by Myf-5 only partially suggesting that other potential targets of Ras/Raf signalling may be involved. Significantly, we observed that elevated Raf kinase activity in L6 muscle cells suppresses the accumulation of MEF2 protein in nuclei, while MEF2 transcription appears unaffected. Moreover, forced expression of MEF2A in 10T1/2 cells rescues MyoD dependent myogenic conversion in the presence of constitutively active Raf kinase and partially restores transactivation of a myogenin promoter-dependent reporter gene in L6 muscle cells containing activated Raf kinase. From these observations we conclude that persistent activation of Raf signalling affects nuclear MEF2 functions which may explain why myogenin expression and myoblast differentiation are inhibited.

Isolation and characterization of the novel popeye gene family expressed in skeletal muscle and heart.

We identified a novel gene family in vertebrates which is preferentially expressed in developing and adult striated muscle. Three genes of the Popeye (POP) family were detected in human and mouse and two in chicken. Chromosomal mapping indicates that Pop1 and Pop3 genes are clustered on mouse chromosome 10, whereas Pop2 maps to mouse chromosome 16. We found evidence that POP1 and POP3 in chicken may also be linked and multiple transcript isoforms are generated from this locus. The POP genes encode proteins with three potential transmembrane domains that are conserved in all family members. Individual POP genes exhibit specific expression patterns during development and postnatally. Chicken POP3 and mouse Pop1 are first preferentially expressed in atrium and later also in the subepicardial compact layer of the ventricles. Chicken POP1 and mouse Pop2 are expressed in the entire heart except the outflow tract. All three Pop genes are expressed in heart and skeletal muscle of the adult mouse and lower in lung. Pop1 and Pop2 expression is upregulated in uterus of pregnant mice. Like the mouse genes, human POP genes are predominantly expressed in skeletal and cardiac muscle. The strong conservation of POP genes during evolution and their preferential expression in heart and skeletal muscle suggest that these novel proteins may have an important function in these tissues in vertebrates.

NKX2.3 is required for MAdCAM-1 expression and homing of lymphocytes in spleen and mucosa-associated lymphoid tissue.

Targeted disruption of the transcription factor NKX2.3 gene in mice results in anatomical defects of intestine and secondary lymphoid organs. Here, we report that spleen and Peyer's patches of NKX2. 3-deficient mice are considerably reduced in size and lack the ordered tissue architecture. T and B cells are misplaced within the spleen and mesenteric lymph nodes and fail to segregate into the appropriate T and B cell areas. Furthermore, splenic marginal zones, characterized by specific B cells and various types of macrophage-derived cells around the marginal sinus, are absent in mutants. Homozygous NKX2.3 mutants lack the mucosal addressin cell adhesion molecule-1 (MAdCAM-1) that is normally expressed in mucosa-associated lymphoid tissue (MALT) and spleen. We provide evidence that NKX2.3 can activate MAdCAM-1 transcription directly, suggesting that MAdCAM-1 is at least partly responsible for the migration and homing defects of lymphocytes and macrophages in mutants. Therefore, expression of MAdCAM-1 seems to be required for building functional structures in spleen and MALT, a prerequisite for unimpaired migration and segregation of B and T cells to and within these organs.

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.

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.

NKX2 gene expression in neuroectoderm but not in mesendodermally derived structures depends on sonic hedgehog in mouse embryos.

NKX2 genes in vertebrates encode a sub- family of homeodomain-containing transcription factors which regulate morphogenetic events and cell differentiation during embryogenesis. In mouse embryos several NKX2 genes are expressed in the ventral midline domains of the neuroectoderm, while other NKX2 genes are primarily expressed in the mesendoderm and mesendodermally derived organs, such as heart and gut. Within several patterning centers for tissue organization sonic hedgehog (Shh) is an important signal in the formation of ventral midline structures in vertebrate embryos. Here, we investigated the role of Shh in the embryonic expression of six different but closely related NKX2 genes in Shh null mutant mice. We found that expression of NKX2.1, NKX2.2, and NKX2.9 in neural domains requires Shh signaling, whereas NKX2.3, NKX2.5 and NKX2.6 expression in endoderm and mesoderm is independent of Shh.

Transcriptional activation of the myogenin gene by MEF2-mediated recruitment of myf5 is inhibited by adenovirus E1A protein.

The basic helix-loop-helix (bHLH) transcription factor myogenin plays a crucial role in terminal differentiation of committed myoblasts into mature myocytes. Transcriptional activation of the myogenin gene requires coordinate action of myocyte enhancer factor 2 (MEF2) proteins and the myogenic bHLH regulators, MyoD or Myf5. Here we show that transcription of the myogenin gene in differentiated cells correlates with MEF2 and NF1 binding to their cognate sites in the proximal myogenin promoter but not with binding of Myf5 or MyoD to the E-box. The importance of MEF2 activity was further demonstrated by expression of antisense MEF2 RNA which repressed MEF2 and Myf5-mediated MEF2 site-dependent reporter gene activation and the synergistic transactivation of a myogenin CAT reporter by Myf5 and MEF2. Adenovirus E1A which has previously been shown to specifically interfere with myogenin gene transcription also inhibited the cooperative transactivation by Myf5/MEF2 and MEF2. Consistently, coimmunoprecipitation studies revealed impaired MEF2/Myf5 protein-protein interactions. These results support a model of transcriptional activation and stabilization of myogenin expression in which DNA-bound MEF2 recruits myogenic bHLH factors into an active but E1A-sensitive transcription factor complex.

Two highly related homeodomain proteins, Nkx5-1 and Nkx5-2, display different DNA binding specificities.

The mouse Nkx5-1 and Nkx5-2 genes are related to NK genes in Drosophila and encode proteins with very similar homeodomains. In higher vertebrates Nkx5 genes are specifically expressed in the inner ear. Inactivation of the mouse Nkx5-1 gene by homologous recombination revealed a critical role for the formation of vestibular inner ear structures. Here, we investigated biochemical properties of the proteins encoded by the Nkx5 genes. A similar consensus binding sequence was isolated for both Nkx5 proteins using binding site selection. This sequence is related to target sequences previously identified for other Nkx proteins and contains the conserved homeodomain binding core TAAT. An additional, novel and unrelated high affinity binding sequence could be identified for the Nkx5-2 protein.

The homeobox gene 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 Hensen's 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.

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

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.

Targeted disruption of the homeobox transcription factor Nkx2-3 in mice results in postnatal lethality and abnormal development of small intestine and spleen.

The homeodomain transcription factor Nkx2-3 is expressed in gut mesenchyme and spleen of embryonic and adult mice. Targeted inactivation of the Nkx2-3 gene results in severe morphological alterations of both organs and early postnatal lethality in the majority of homozygous mutants. Villus formation in the small intestine appears considerably delayed in Nkx2-3(-)/- foetuses due to reduced proliferation of the epithelium, while massively increased growth of crypt cells ensues in surviving adult mutants. Interestingly, differentiated cell types of the intestinal epithelium are present in homozygous mutants, suggesting that Nkx2-3 is not required for their cell lineage allocation or migration-dependent differentiation. Hyperproliferation of the gut epithelium in adult mutants is associated with markedly reduced expression of BMP-2 and BMP-4, suggesting that these signalling molecules may be involved in mediating non-cell-autonomous control of intestinal cell growth. Spleens of Nkx2-3 mutants are generally smaller and contain drastically reduced numbers of lymphatic cells. The white pulp appears anatomically disorganized, possibly owing to a homing defect in the spleen parenchyme. Moreover, some of the Nkx2-3 mutants exhibit asplenia. Taken together these observations indicate that Nkx2-3 is essential for normal development and functions of the small intestine and spleen.

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.

cMeso-1, a novel bHLH transcription factor, is involved in somite formation in chicken embryos.

The segmentation of somites from the paraxial mesoderm is a crucial event in vertebrate embryonic development; however, the mechanisms underlying this process are not well understood. In a yeast two-hybrid screen we have identified the novel basic-helix-loop-helix (bHLH) protein cMeso-1 which is expressed in the presomitic mesoderm of early chicken embryos. Initially the gene is activated in the epiblast and transcripts concentrate later in and around the primitive streak. When the segmental plate is laid down the cMeso-1 expression domain successively retracts toward the caudal end but a second domain appears in bilateral stripes in the anterior paraxial mesoderm. This highly dynamic domain of cMeso-1 transcripts demarcates the area immediately posterior to the next prospective pair of somites in cyclic waves which apparently correspond to the formation of new somites. Loss of cMeso-1 function by antisense RNA or oligonucleotides results in severe attenuation of somitogenesis suggesting that it plays an important role in setting up the segmentation process. The dynamic and periodically reiterated expression of cMeso-1 along the anteroposterior axis is not dependent on anterior structures or the propagation of a signal along the anteroposterior axis but seems to follow an intrinsic patterning program which is already set up in the segmental plate.

Nkx2-9 is a novel homeobox transcription factor which demarcates ventral domains in the developing mouse CNS.

Nkx homeobox transcription factors are expressed in diverse embryonic cells and presumably control cell-type specification and morphogenetic events. Nkx2-9 is a novel family member of NK2 genes which lacks the conserved TN-domain found in all hitherto known murine Nkx2 genes. The prominent expression of Nkx2-9 in ventral brain and neural tube structures defines a subset of neuronal cells along the entire neuraxis. During embryonic development, Nkx2-9-expressing cells shift from the presumptive floor plate into a more dorsolateral position of the neuroectoderm and later become limited to the ventricular zone. Nkx2-9 expression overlaps with that of Nkx2-2 but is generally broader. While initially Nkx2-9 is expressed in close proximity to sonic hedgehog, its expression domain clearly segregates from sonic hedgehog at later developmental stages. The dynamic expression pattern of Nkx2-9 in ventral domains of the CNS is consistent with a possible role in the specification of a distinct subset of neurons.

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.

Two regulatory genes, cNkx5-1 and cPax2, show different responses to local signals during otic placode and vesicle formation in the chick embryo.

The early stages of otic placode development depend on signals from neighbouring tissues including the hindbrain. The identity of these signals and of the responding placodal genes, however, is not known. We have identified a chick homeobox gene cNkx5-1, which is expressed in the otic placode beginning at stage 10 and exhibits a dynamic expression pattern during formation and further differentiation of the otic vesicle. In a series of heterotopic transplantation experiments, we demonstrate that cNkx5-1 can be activated in ectopic positions. However, significant differences in otic development and cNkx5-1 gene activity were observed when placodes were transplanted into the more rostral positions within the head mesenchyme or into the wing buds of older hosts. These results indicate that only the rostral tissues were able to induce and/or maintain ear development. Ectopically induced cNkx5-1 expression always reproduced the endogenous pattern within the lateral wall of the otocyst that is destined to form vestibular structures. In contrast, cPax2 which is expressed in the medial wall of the early otic vesicle later forming the cochlea never resumed its correct expression pattern after transplantation. Our experiments illustrate that only some aspects of gene expression and presumably pattern formation during inner ear development can be established and maintained ectopically. In particular, the dorsal vestibular structures seem to be programmed earlier and differently from the ventral cochlear part.

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.