Impaired skin wound healing in peroxisome proliferator-activated receptor (PPAR)alpha and PPARbeta mutant mice.

We show here that the alpha, beta, and gamma isotypes of peroxisome proliferator-activated receptor (PPAR) are expressed in the mouse epidermis during fetal development and that they disappear progressively from the interfollicular epithelium after birth. Interestingly, PPARalpha and beta expression is reactivated in the adult epidermis after various stimuli, resulting in keratinocyte proliferation and differentiation such as tetradecanoylphorbol acetate topical application, hair plucking, or skin wound healing. Using PPARalpha, beta, and gamma mutant mice, we demonstrate that PPARalpha and beta are important for the rapid epithelialization of a skin wound and that each of them plays a specific role in this process. PPARalpha is mainly involved in the early inflammation phase of the healing, whereas PPARbeta is implicated in the control of keratinocyte proliferation. In addition and very interestingly, PPARbeta mutant primary keratinocytes show impaired adhesion and migration properties. Thus, the findings presented here reveal unpredicted roles for PPARalpha and beta in adult mouse epidermal repair.

Spatial regulation of homeobox gene fusions in the embryonic central nervous system of transgenic mice.

The spatially restricted expression of mammalian homeobox genes in teh embryonic central nervous system (CNS) provides an opportunity to study the basis of spatial gene regulation in mammalian development. Here, we define a regulatory region of the murine Hox 1.3 gene that mediates such a region-specific expression pattern. The Hox 1.3 gene contains two exons, encodes a putative protein of 270 amino acids, and is expressed preferentially in the spinal cord at midgestation. We have analyzed transgenic mice containing various Hox 1.3 DNA fragments fused to reporter sequences, such as a human growth hormone gene fragment or the E. coli lacZ structural gene. As shown by RNAase protection assays or by in situ analyses of beta-galactosidase activity, several hybrid genes are expressed in the embryonic central nervous system in a spatially restricted manner, along both the rostrocaudal and dorsoventral axes. A 912 nucleotide sequence located immediately upstream of the Hox 1.3 coding sequence is sufficient to direct expression to the dorsolateral cells of the brachial spinal cord.

Genetic control of murine limb morphogenesis: relationships with human syndromes and evolutionary relevance.

Over the past ten years, the discovery and functional characterisation of murine Hox genes has led to a better understanding of some of the molecular mechanisms underlying limb development. It has also shed some light on the potential genetic events which have accompanied the fin-to-limb transition, an evolutionary step of critical importance which opened the way to the evolution of higher vertebrates. This convergence between developmental biology and the sciences of evolution is one of the synergistic interface that has been established recently thanks to the use of genetic engineering and transgenic animals. The increasing number of human genetic syndromes which are derived from mutations in developmental control genes remind us that many human genetic diseases are nothing else but alterations in our developmental programme. Here, we illustrate these various issues by discussing the function of Hox genes during limb development.

Synpolydactyly in mice with a targeted deficiency in the HoxD complex.

The morphogenesis of mammalian digits requires the function of several genes of the HoxD complex during development of limb buds. Using embryonic stem (ES) cells and a site-specific recombination system (loxP/Cre), we have induced a deficiency that eliminates the products of the Hoxd-13, Hoxd-12 and Hoxd-11 genes simultaneously. A Hoxd-11/lacz reporter gene replaced the deleted region in order to monitor the effect of this triple inactivation at the cellular level. Mice homozygous for this deficiency showed small digit primordia, a disorganized cartilage pattern and impaired skeletal mass. These alterations are similar to the defects seen in a human synpolydactyly, suggesting that this syndrome, which is associated with a subtle mutation in HOXD13 (ref. 8), may involve the loss of function of several Hoxd genes. These results indicate the existence of a functional hierarchy among these genes and provide us with an animal model to study human digit malformations.

Hox genes in digit development and evolution.

Homeobox genes located in the 5' part of the HoxA and HoxD complexes are required for proliferation of skeletal progenitor cells of the vertebrate limb. Specific combinations of gene products determine the length of the upper arm (genes belonging to groups 9 and 10), the lower arm (groups 10, 11 and 12) and the digits (groups 11, 12 and 13). In these different domains, individual gene products quantitatively contribute to an overall protein dose, with predominant roles for groups 11 and 13. Quantitative reduction in the gene dose in each set results in truncations of the corresponding anatomical regions. The physical order of the genes in the HoxA and HoxD complexes, as well as a unidirectional sequence in gene activation, allow for completion of the process in a precise order, which in turn makes possible the sequential outgrowth of the respective primordia. While the skeletal patterns of upper and lower limb are relatively stable throughout the tetrapods, more variation is seen in the digits. Molecular analysis of the underlying regulatory processes promises further exciting insights into the genetic control of development, pathology and the course of evolution.

Correlation of expression of Wnt-1 in developing limbs with abnormalities in growth and skeletal patterning.

The Wnt genes are members of a family of vertebrate genes related to the Drosophila gene wingless (wg). They encode secreted molecules that are thought to be important in patterning and growth control during ontogenesis. Several such genes are transcribed in localized domains during limb budding and morphogenesis. We report here a congenital limb malformation in a mouse transgenic line that ectopically expresses Wnt-1 in the developing limbs. The hemizygote phenotype, which is inherited as an autosomal dominant trait, presents extensive distal truncations of skeletal elements, skeletal fusions and interdigital webbing. The data shown here demonstrate that abnormal Wnt-1 expression is correlated with retarded mesenchymal condensations replaced by highly proliferative cells in the limb bud. This seems to lead to an inability of the affected cells to participate in normal skeletal development leading to the adult defects.

Effect of lentinan on the production of migration inhibitory factor induced by syngeneic tumor in mice.

Attempts were made to reveal the mechanism of the inhibitory effect of lentinan on methylcholanthrene-induced transplantable fibrosarcoma (A/Ph.MC.SI) isografts. Since the involvement of a delayed-type hypersensitivity response can be supposed to operate against this syngeneic tumor, the enlargement of regional lymph nodes, the DNA synthesis activity and the migration inhibitory factor producing capacity of the regional lymph-node cells were studied in the control and lentinan-treated, tumor-bearing mice at different times after tumor implantation. The only parameter affected until the first sign of tumor inhibition on the 8th day was the MIF-producing capacity. Regional lymph-node cells in the lentinan-treated mice showed a peak response at this time while practically no MIF was produced by cells of non-treated mice. The early phase of the developing anti-tumor reaction (enlargement of and proliferation in the lymph nodes) was initiated in all recipients but only some of them could reject the tumor in the non-treated animals that rejected the tumors in all cases. It can be supposed that a tumor-induced immunosuppression emerges in the tumor-bearers and can be overcome by lentinan treatment, most likely through enhancement of the MIF production. For measurement of MIF activity a continuously dividing in vitro macrophage-like cell line was established and employed in the micro-wound migration assay.

Cooperation of regulatory elements involved in the activation of the Hoxd-11 gene.

We have used lacZ reporter gene constructs to study the cis regulation of the murine Hoxd-11 gene (previously Hox-4.6) in transgenic mice. We identified a genomic region, which was able to simulate important aspects of the developmental regulation of the gene. A short regulatory region, located 3' to the Hoxd-11 transcription unit, was required to mimic initial activation. This regulatory region contains two stretches of DNA, that are highly conserved in the chicken Hoxd-11 3' flanking region. A chimeric construct containing these short homologous regions from the chicken gene could replace the complete murine fragment thus demonstrating that the conserved domains carry the main regulatory elements involved in this activation. The first half of this bipartite regulatory region has positive regulatory activity, while the second half is required to restrict gene expression to the proper posterior domain in the somitic mesoderm. Our results suggest that the control of the Hoxd-11 promoter involves tissue-specific cooperations between regulatory elements.

Structure and activity of regulatory elements involved in the activation of the Hoxd-11 gene during late gastrulation.

We have used reporter gene constructs to study the cis regulation of the Hoxd-11 gene (previously Hox-4.6) in transgenic mice. We identified a 5 kb regulatory unit, which was able to reproduce important aspects of the initial activation of the gene along the major body axis. The comparison of the nucleotide sequence of this DNA fragment with the corresponding avian genomic region revealed the presence of seven highly homologous stretches of DNA outside the protein coding regions. In particular, the 3' flanking region contained two such domains that are required to mediate the embryonic activation. A chimeric construct containing the two short homologous regions from the chicken gene could replace the complete murine fragment thus demonstrating that the conserved domains carry the main regulatory elements involved in this activation. The first half of this bipartite regulatory region has enhancer activity when tested with a heterologous promoter, while the second half is required to restrict the enhancer activity to the proper expression domain. These results suggest that stage- and tissue-specific cooperation between regulatory elements is required to control properly the activity of the Hoxd-11 promoter.

Interspecies exchange of a Hoxd enhancer in vivo induces premature transcription and anterior shift of the sacrum.

The precise activation, in space and time, of vertebrate Hox genes is an essential requirement for normal morphogenesis. In order to assess for the functional potential of evolutionary conserved Hox regulatory sequences, a phylogenetically conserved bipartite regulatory element necessary for proper spatial and temporal activation of the Hoxd-11 gene was replaced by its fish counterpart in the HoxD complex of mice, using an ES cell-based targeted exchange. Fetuses carrying this replacement activated Hoxd-11 transcription prematurely, which led to a rostral shift of its expression boundary and a consequent anterior transposition of the sacrum. These results demonstrate the high phylogenetic conservation of regulatory mechanisms acting over vertebrate Hox complexes and suggest that minor time difference (heterochronies) in Hox gene activation may have contributed to important morphological variations in the course of evolution.

Control of colinearity in AbdB genes of the mouse HoxD complex.

During development, vertebrate Hox genes are activated in a temporal and spatial sequence colinear with the position of the genes within their clusters. To investigate the mechanistic basis of this phenomenon, we used the ES cell technology and the loxP/Cre system to engineer a conditional fusion of the 5' exon of Hoxd-13 with the 3' exon of Hoxd-12. This hybrid transcription unit was regulated like Hoxd-11, with expression limits in the trunk, limbs, intestinal, and urogenital systems more anterior than those expected for either Hoxd-13 or Hoxd-12. An in vivo interspecies replacement by the fish homologous DNA fragment showed that anteriorization was not due to a distance effect, thus suggesting the presence of a regulatory element between Hoxd-13 and Hoxd-12 that may contribute to the establishment, early on, of a repressive state over these two genes.

Effect of lentinan on tumor growth in murine allogeneic and syngeneic hosts.

The effect of lentinan on retardation and regression of transplanted tumors was analysed in allogeneic and syngeneic tumor-host systems. The effect of lentinan treatment was most variable in random-bred Swiss albino mice bearing sarcoma 180. With inbred host strains, it was most effective in A/PH and less, or not at all, in others (A.BY, A.CA, A.SW, DBA/2, BALB/c, C3H/Di, AKR, BIO, BIO.1, BIO.BR, BIO.D2). In order to eliminate the allogeneic differences, a syngeneic transplantable 3-methylcholanthrene-induced sarcoma (A/PhMC.SI) has been developed in the A/Ph strain, previously found to be the most responsive to lentinan. The growth of this sarcoma was dramatically inhibited and regression was detected in all lentinan-treated syngeneic recipients. Both the lentinan-treated and untreated regressor animals exhibited a high degree of resistance to a secondary challenge. However, the growth of a spontaneous, transplantable syngeneic adenocarcinoma in A/Ph mice was not influenced by lentinan treatment. The results presented substantiate further the conclusion that the effect of lentinan is mediated through host mechanisms, and show that these mechanisms are able to act against a syngeneic tumor.

Spontaneous differentiation in the colonies of a nullipotent embryonal carcinoma cell line (F9).

Experiments were undertaken to reveal the spontaneous differentiation capacity of the nullipotent F9 embryonal-carcinoma (EC) cell line in colonies derived from single cells. Culture conditions which allowed the development of neuroblasts in colonies of the multipotent EC cell line (PCC3) were worked out, and comparative studies on neuroblast differentiation in PCC3 and F9 colonies were conducted. Neural-cell-specific silver impregnation, selective staining of cells having electrically excitable membranes with merocyanine 540 and the observation of nerve processes were considered as differentiation markers. The appearance of neuroblasts in F9 and PCC3 colonies could be detected from the 6th day after seeding. The development of neuroblasts was less prevalent in high-density cultures, especially in the case of F9. By the 8th day in differentiating colonies, PCC3 cells lost much of their colony-forming activity, while F9 cells preserved their original high plating efficiency, in spite of advanced differentiation. The determination of growth parameters during differentiation in colonies led to the conclusion that F9 cells had lost certain growth-control mechanisms which normally restrict the clonal growth of EC cells. It is suggested that the phenomenon of nullipotence may be analysed in terms of the coordinated regulation of proliferation and differentiation of EC cells.

The use of lacZ gene fusions in the studies of mammalian development: developmental regulation of mammalian homeobox genes in the CNS.

1. Mammalian cells can produce bacterial beta-galactosidase when they carry the lacZ gene under the control of mammalian regulatory elements. The single cell resolution of the beta-galactosidase histochemical detection method makes this molecule an excellent marker in studies of development at the cellular and molecular level. Different lacZ fusion genes can be engineered to study the histological diversification of cell lineages, the developmental regulation of isolated genes, or to recognize and clone genes with new expression profile. 2. Transgenic mice carrying lacZ gene fusions provide information on the cell type, developmental stage and spatial specificity of cis-acting regulatory regions linked to a mammalian homeobox gene. We describe our strategy for designing the gene fusions. 3. The scord region of the Hox 1.3 gene is sufficient to determine spatially restricted expression of a heterologous protein in the midgestational spinal cord. We propose to use this region to alter the expression pattern of other homeobox gene products. Developmental alterations due to a variant expression pattern would point to the function of the misexpressed gene.

Cytoskeletal characterization of a neuroectodermal cell line and embryonic mouse brain.

The cytoskeleton of a newly isolated mouse embryo brain-derived cell line and of dissected mesencephalon-rhombencephalon samples of the 10- to 11-day-old mouse embryo, constituted of a ventricular zone only, has been examined by immunocytochemistry, electrophoretic separation and Western blotting techniques. In accordance with their epithelial organization, the ventricular cells contain cytokeratins as main constituents of their cytoskeleton. Vimentin has been also revealed as early as day 10.5 of gestation. The complex cytokeratin polypeptide pattern puts this histological type of epithelia into the category of complex, rather than simple, epithelia and calls for explanations other than keratinization for the presence and functional role of the high-molecular weight, basic keratins. The cytoskeletal composition of the embryo brain-derived gliogenic cell line reflects the vimentin- and cytokeratin-positive character of the ventricular cells.

Function of posterior HoxD genes in the morphogenesis of the anal sphincter.

Vertebrate 5'-located HoxD genes are expressed in the most caudal part of the digestive tract and their potential functions during gut development have been assessed by gene disruptions. We have inserted reporter lacZ sequences within the Hoxd-12 gene and analysed the morphology of the gut in these mice as well as in Hoxd-13 mutant animals. When homozygous, both mutations induce an important disorganization of the anorectal region. In particular, severe alterations of the smooth muscle layers of the rectum led to defective morphogenesis of the internal anal sphincter. Similarly, Hoxd-12 and Hoxd-13 functionally overlap during digit development. The function of these genes in the morphogenesis of the digestive system as well as their functional evolution are discussed.

Deletion of a HoxD enhancer induces transcriptional heterochrony leading to transposition of the sacrum.

A phylogenetically conserved transcriptional enhancer necessary for the activation of Hoxd-11 was deleted from the HoxD complex of mice by targeted mutagenesis. While genetic and expression analyses demonstrated the role of this regulatory element in the activation of Hoxd-11 during early somitogenesis, the function of this gene in developing limbs and the urogenital system was not affected, suggesting that Hox transcriptional controls are different in different axial structures. In the trunk of mutant embryos, transcriptional activation of Hoxd-11 and Hoxd-10 was severely delayed, but subsequently resumed with appropriate spatial distributions. The resulting caudal transposition of the sacrum indicates that proper vertebral specification requires a precise temporal control of Hox gene expression, in addition to spatial regulation. A slight time delay in expression (transcriptional heterochrony) cannot be compensated for at a later developmental stage, eventually leading to morphological alterations.

Ulnaless (Ul), a regulatory mutation inducing both loss-of-function and gain-of-function of posterior Hoxd genes.

Ulnaless (Ul), an X-ray-induced dominant mutation in mice, severely disrupts development of forearms and forelegs. The mutation maps on chromosome 2, tightly linked to the HoxD complex, a cluster of regulatory genes required for proper morphogenesis. In particular, 5'-located (posterior) Hoxd genes are involved in limb development and combined mutations within these genes result in severe alterations in appendicular skeleton. We have used several engineered alleles of the HoxD complex to genetically assess the potential linkage between these two loci. We present evidence indicating that Ulnaless is allelic to Hoxd genes. Important modifications in the expression patterns of the posterior Hoxd-12 and Hoxd-13 genes at the Ul locus suggest that Ul is a regulatory mutation that interferes with a control mechanism shared by multiple genes to coordinate Hoxd function during limb morphogenesis.

Regulation of number and size of digits by posterior Hox genes: a dose-dependent mechanism with potential evolutionary implications.

The proper development of digits, in tetrapods, requires the activity of several genes of the HoxA and HoxD homeobox gene complexes. By using a variety of loss-of-function alleles involving the five Hox genes that have been described to affect digit patterning, we report here that the group 11, 12, and 13 genes control both the size and number of murine digits in a dose-dependent fashion, rather than through a Hox code involving differential qualitative functions. A similar dose-response is observed in the morphogenesis of the penian bone, the baculum, which further suggests that digits and external genitalia share this genetic control mechanism. A progressive reduction in the dose of Hox gene products led first to ectrodactyly, then to olygodactyly and adactyly. Interestingly, this transition between the pentadactyl to the adactyl formula went through a step of polydactyly. We propose that in the distal appendage of polydactylous short-digited ancestral tetrapods, such as Acanthostega, the HoxA complex was predominantly active. Subsequent recruitment of the HoxD complex contributed to both reductions in digit number and increase in digit length. Thus, transition through a polydactylous limb before reaching and stabilizing the pentadactyl pattern may have relied, at least in part, on asynchronous and independent changes in the regulation of HoxA and HoxD gene complexes.