Identification of a prx1 limb enhancer

Mice with a loss of function of prx1, a paired-related homeobox gene formerly called Mhox, showed craniofacial defects, limb shortening, and incompletely penetrant spina bifida. To investigate the mechanisms that regulate prx1 expression, we analyzed a 2.4-kb prx1 genomic flanking region in transgenic mice. This region of the prx1 gene contains an enhancer element that directs expression of a LacZ reporter gene in limb bud mesenchyme and a subset of craniofacial mesenchyme. Deletional analysis in transgenic founders identified a necessary 530-bp core element. Comparison of this core element with human Prx1 sequence showed two highly conserved cassettes that also contained a prx recognition element. Moreover, transgene expression was diminished in posterior handplate of prx1; prx2 double mutant mice. Our data reveal that the prx1 limb enhancer is proximally located within the prx1 gene and suggest that prx1 may have an autoregulatory function in limb mesenchyme.     

Cre‐mediated transgene activation in the developing and adult mouse brain

Summary: The neuron‐specific rat enolase (NSE) promoter was employed to establish transgenic mice expressing Cre recombinase in the central nervous system. Founders were crossed with dormant lacZ indicator mice and specificity as well as efficiency of Cre‐mediated transgene activation was determined by PCR and/or X‐gal staining. Whereas most transgenic lines exhibited Cre activity in early development resulting in widespread Cre activity, one line (NSE‐Cre26) expressed high levels of Cre in the developing and adult brain. With the exception of kidney, which showed occasionally low level of Cre activity, Cre recombination in double transgenics was restricted to the nervous system. Whole‐mount X‐gal staining of 9.5 dpc embryos indicated Cre‐mediated lacZ expression in forebrain, hindbrain, and along the midbrain flexure. A similar expression pattern was observed during later stages of embryogenesis (11.5–13.5 dpc). In adult mice, Cre recombinase was expressed in cerebral cortex and cerebellum and high levels of Cre‐mediated lacZ expression were observed in hippocampus, cortex, and septum. The NSE‐Cre26 transgenic mouse line thus provides a useful tool to specifically overexpress and/or inactivate genes in the developing and adult brain. genesis 31:118–125, 2001. © 2001 Wiley‐Liss, Inc.     

Deletion of Tgfbr2 in Prx1-cre expressing mesenchyme results in defects in development of the long bones and joints

In this study, we address the function of Transforming Growth Factor beta (TGF-beta) and its type II receptor (Tgfbr2) in limb development in vivo. Mouse embryos were generated in which the Tgfbr2 gene was deleted in early limb mesenchyme using Prx1Cre-mediated LoxP recombination. A high level of Tgfbr2 gene deletion was verified in limb mesenchyme by PCR between E9.5 and E10.5 days in Cre expressing mice. RT-PCR assays indicated a significant depletion of Tgfbr2 mRNA by E10.5 days as a result of Cre mediated gene deletion. Furthermore, limb mesenchyme from Cre(+);Tgfbr2(f/f) mice placed in micromass culture did not respond to exogenously added TGF-beta1 confirming the functional deletion of the receptor. However, there was an unexpected increase in the number and intensity of Alcian blue stained chondrogenic nodules in micromass cultures derived from Tgfbr2-deleted limbs relative to cultures from control limbs suggesting that Tgfbr2 normally limits chondrogenesis in vitro. In vivo, early limb development and chondrocyte differentiation occurred normally in Tgfbr2-depleted mice. Later in development, depletion of Tgfbr2 in limb mesenchyme resulted in short limbs and fusion of the joints in the phalanges. Alteration in the length of the long bones was primarily due to a decrease in chondrocyte proliferation after E13.5 days. In addition, the transition from prehypertrophic to hypertrophic cells was accelerated while there was a delay in late hypertrophic differentiation leading to a reduction in the length of the marrow cavity. In the joint, cartilage cells replaced interzone cells during development. Analysis of markers for joint development indicated that the joint was specified properly and that the interzone cells were initially formed but not maintained. The results suggest that Tgfbr2 is required for normal development of the skeleton and that Tgfbr2 can act to limit chondrogenesis in mesenchymal cells like the interzone.     

A Cre transgene active in developing endodermal organs, heart, limb, and extra-ocular muscle

Cre-mediated recombination, a method widely used in mice for tissue-specific inactivation of endogenous genes or activation of transgenes, is critically dependent on the availability of mouse lines in which Cre recombinase functions in the tissue of interest or its progenitors. Here we describe a transgenic mouse line, Osr1-cre, in which Cre is active from embryonic day (E)11.5 in a few specific tissues. These include the endoderm of the posterior foregut, midgut, hindgut, and developing urogenital system, the heart left atrium, extra-ocular muscle progenitors, and mesenchyme in particular regions of the limb. Furthermore, starting at E12.5, Cre functions in limb interdigital mesenchyme. Within the urogenital system, recombination appears to be virtually complete in the epithelium of the bladder and urethra just posterior to it by E14.5. In males, some of these urethral cells form the prostate. The spatiotemporal pattern of Cre activity in Osr1-cre makes it a unique resource among the lines available for Cre-mediated recombination experiments.     

Conditional inactivation of Fgfr1 in mouse defines its role in limb bud establishment, outgrowth and digit patterning

Previous studies have implicated fibroblast growth factor receptor 1 (FGFR1) in limb development. However, the precise nature and complexity of its role have not been defined. Here, we dissect Fgfr1 function in mouse limb by conditional inactivation of Fgfr1 using two different Cre recombinase-expressing lines. Use of the T (brachyury)-cre line led to Fgfr1 inactivation in all limb bud mesenchyme (LBM) cells during limb initiation. This mutant reveals FGFR1 function in two phases of limb development. In a nascent limb bud, FGFR1 promotes the length of the proximodistal (PD) axis while restricting the dimensions of the other two axes. It also serves an unexpected role in limiting LBM cell number in this early phase. Later on during limb outgrowth, FGFR1 is essential for the expansion of skeletal precursor population by maintaining cell survival. Use of mice carrying the sonic hedgehog(cre) (Shh(cre)) allele led to Fgfr1 inactivation in posterior LBM cells. This mutant allows us to test the role of Fgfr1 in gene expression regulation without disturbing limb bud growth. Our data show that during autopod patterning, FGFR1 influences digit number and identity, probably through cell-autonomous regulation of Shh expression. Our study of these two Fgfr1 conditional mutants has elucidated the multiple roles of FGFR1 in limb bud establishment, growth and patterning.     

Dlx5/6-enhancer directed expression of Cre recombinase in the pharyngeal arches and brain

Dlx5 and Dlx6, two members of the Distalless gene family, are required for development of numerous tissues during embryogenesis, including facial and limb development. This gene pair is expressed in tandem, transcribed toward each other and separated by a short intergenic region containing multiple putative enhancers. Targeted inactivation of Dlx5 and Dlx6 in mice results in multiple developmental defects in craniofacial and limb structures, suggesting that these genes are crucial for aspects of both neural crest and nonneural crest development. To further investigate potential developmental roles of Dlx5 and Dlx6, we used one of the Dlx5/6 intergenic enhancers to drive Cre recombinase expression in transgenic mice. Crossing Dlx5/6-Cre transgenic mice with mice from the R26R strain results in beta-galactosidase staining in the apical ectodermal ridge, brain, and neural crest-derived mesenchyme of the pharyngeal arches, with staining in term embryos observed in the facial skeleton and specific brain structures. However, in contrast to endogenous expression patterns of Dlx5 and Dlx6, Cre expression within the pharyngeal arches occurs during a very narrow window in early development. Our studies suggest that Dlx5/6-Cre mice may prove useful both in further understanding the function and regulation of Distalless genes during development and in studies of gene function in conditional knockout mice.     

Versican Facilitates Chondrocyte Differentiation and Regulates Joint Morphogenesis

Versican/PG-M is a large chondroitin sulfate proteoglycan in the extracellular matrix, which is transiently expressed in mesenchymal condensation areas during tissue morphogenesis. Here, we generated versican conditional knock-out mice Prx1-Cre/Vcan^flox/flox, in which Vcan is pruned out by site-specific Cre recombinase driven by the Prx1 promoter. Although Prx1-Cre/Vcan^flox/flox mice are viable and fertile, they develop distorted digits. Histological analysis of newborn mice reveals hypertrophic chondrocytic nodules in cartilage, tilting of the joint, and a slight delay of chondrocyte differentiation in digits. By immunostaining, whereas the joint interzone of Prx1-Cre/Vcan^+/+ shows an accumulation of TGF-β, concomitant with versican, that of Prx1-Cre/Vcan^flox/flox without versican expression exhibits a decreased incorporation of TGF-β. In a micromass culture system of mesenchymal cells from limb bud, whereas TGF-β and versican are co-localized in the perinodular regions of developing cartilage in Prx1-Cre/Vcan^+/+, TGF-β is widely distributed in Prx1-Cre/Vcan^flox/flox. These results suggest that versican facilitates chondrogenesis and joint morphogenesis, by localizing TGF-β in the extracellular matrix and regulating its signaling.     

Efficient gene-specific expression of cre recombinase in the mouse embryo by targeted insertion of a novel IRES-Cre cassette into endogenous loci.

Site specific recombinases have provided the experimental strategy necessary to modulate the expression of gene products in the mouse embryo. In this study we have exploited Cre recombinase to develop a widely applicable cell marking system which functions efficiently even at early post-implantation embryonic stages. Importantly, the techniques and reagents derived in this study are generally applicable to any recombinase driven approach, including strategies to temporally and spatially modulate endogenous or ectopic gene expression in the embryo. The cell marking scheme has two essential components which were derived as separate mouse lines. The first line carries a universal conditional lacZ reporter (UCR) locus which was prepared by using gene targeting in a novel approach to modify a ubiquitously expressed retroviral lacZ promoter trap insertion. The UCR locus is silent until it undergoes a Cre mediated DNA rearrangement to restore lacZ expression. To generate the Cre expressing allele, we outline a flexible strategy which requires the introduction of a novel IRES-Cre cassette into exon sequence of an endogenous locus by gene targeting. We successfully demonstrate this approach by generating a Cre expressing allele of the EphA2 gene, an Eph receptor protein tyrosine kinase expressed early in development. Analysis of double heterozygote embryos clearly demonstrates that Cre recombinase is expressed in vivo from the EphA2 IRES-Cre allele, and that the conditional reporter locus is efficiently restored in EphA2-expressing cells as early as 7.5 dpc. This cell marking experiment establishes the feasibility of expressing Cre recombinase from a single copy allele in the embryo and demonstrates the utility of the conditional reporter mouse which can be used in the analysis of any Cre expressing allele.     

5-Aza-2'-deoxycytidine-induced cytotoxicity and limb reduction defects in the mouse

BACKGROUND: 5-Aza-2'-deoxycytidine (dAZA), causes hindlimb phocomelia in CD-1 mice. Studies in our laboratory have examined the hypothesis that compound- induced changes in gene expression may uniquely affect hindlimb pattern formation. The present study tests the hypothesis that dAZA causes limb dysplasia by inducing cytotoxicity among rapidly proliferating cells in the limb bud mesenchyme. METHODS: Pregnant CD-1 mice were given a teratogenic dose of dAZA (i.p.) at different times on GD 10 and fetuses evaluated for skeletal development in both sets of limbs by standard methods. Using general histology and BrdU immunohistochemistry, limb mesenchymal cell death and cell proliferation were then assessed in embryos at various times post dosing, shortly after initial limb bud outgrowth. The effect of dAZA on early limb chondrogenesis was also studied using Northern analysis of scleraxis and Alcian blue staining of whole mount limb buds. RESULTS: Compound related hindlimb defects were not restricted to a specific set of skeletal elements but consisted of a range of temporally related limb anomalies. Modest defects of the radius were observed as well. These results are consistent with a general insult to the limb mesenchyme. Mesenchymal cell death and reduced cell proliferation were also observed in both sets of limbs. The timing and location of these effects indicate a role for cytotoxicity in the etiology of dAZA induced limb defects. These effects also agree with the greater teratogenicity of dAZA in the hindlimb because they were more pronounced in that limb. The expression of scleraxis, a marker of early chondrogenesis, was reduced 12 hr after dAZA exposure, a time coincident with maximal cell death, as was the subsequent emergence of Alcian blue stained long bone anlagen. CONCLUSIONS: These findings support the hypothesis that cytotoxic changes in the limb bud mesenchyme during early limb outgrowth can induce the proximal limb truncations characteristic of phocomelia after dAZA administration.     

Cdc42 is required for chondrogenesis and interdigital programmed cell death during limb development

Cdc42, a member of the Rho subfamily of small GTPases, is known to be a regulator of multiple cellular functions, including cytoskeletal organization, cell migration, proliferation, and apoptosis. However, its tissue-specific roles, especially in mammalian limb development, remain unclear. To investigate the physiological function of Cdc42 during limb development, we generated limb bud mesenchyme-specific inactivated Cdc42 (Cdc42(fl/fl); Prx1-Cre) mice. Cdc42(fl/fl); Prx1-Cre mice demonstrated short limbs and body, abnormal calcification of the cranium, cleft palate, disruption of the xiphoid process, and syndactyly. Severe defects were also found in long bone growth plate cartilage, characterized by loss of columnar organization of chondrocytes, and thickening and massive accumulation of hypertrophic chondrocytes, resulting in delayed endochondral bone formation associated with reduced bone growth. In situ hybridization analysis revealed that expressions of Col10 and Mmp13 were reduced in non-resorbed hypertrophic cartilage, indicating that deletion of Cdc42 inhibited their terminal differentiation. Syndactyly in Cdc42(fl/fl); Prx1-Cre mice was caused by fusion of metacarpals and a failure of interdigital programmed cell death (ID-PCD). Whole mount in situ hybridization analysis of limb buds showed that the expression patterns of Sox9 were ectopic, while those of Bmp2, Msx1, and Msx2, known to promote apoptosis in the interdigital mesenchyme, were down-regulated. These results demonstrate that Cdc42 is essential for chondrogenesis and ID-PCD during limb development.     

SF/HGF is a mediator between limb patterning and muscle development.

Scatter factor/hepatocyte growth factor (SF/HGF) is known to be involved in the detachment of myogenic precursor cells from the lateral dermomyotomes and their subsequent migration into the newly formed limb buds. As yet, however, nothing has been known about the role of the persistent expression of SF/HGF in the limb bud mesenchyme during later stages of limb bud development. To test for a potential role of SF/HGF in early limb muscle patterning, we examined the regulation of SF/HGF expression in the limb bud as well as the influence of SF/HGF on direction control of myogenic precursor cells in limb bud mesenchyme. We demonstrate that SF/HGF expression is controlled by signals involved in limb bud patterning. In the absence of an apical ectodermal ridge (AER), no expression of SF/HGF in the limb bud is observed. However, FGF-2 application can rescue SF/HGF expression. Excision of the zone of polarizing activity (ZPA) results in ectopic and enhanced SF/HGF expression in the posterior limb bud mesenchyme. We could identify BMP-2 as a potential inhibitor of SF/HGF expression in the posterior limb bud mesenchyme. We further demonstrate that ZPA excision results in a shift of Pax-3-positive cells towards the posterior limb bud mesenchyme, indicating a role of the ZPA in positioning of the premuscle masses. Moreover, we present evidence that, in the limb bud mesenchyme, SF/HGF increases the motility of myogenic precursor cells and has a role in maintaining their undifferentiated state during migration. We present a model for a crucial role of SF/HGF during migration and early patterning of muscle precursor cells in the vertebrate limb.     

Mice expressing GFP and CreER in osteochondro progenitor cells in the periosteum

We generated Prx1CreER-GFP transgenic mice that express tamoxifen-inducible Cre recombinase and GFP under the control of a 2.4 kb Prx1 promoter. The transgene is expressed in osteochondro progenitor cells in the developing limb buds and in a subpopulation of periosteal cells that is closely associated with the cortical bone. GFP-expressing cells isolated from the diaphyses of long bones by cell sorting express multiple markers of periosteal cells, including Prx1, Fgf18, Tenascin-W, Periostin, and Thrombospondin 2. In addition, these cells undergo chondrogenic and osteogenic differentiation in culture upon induction. Cell fate analysis using the Rosa26 LacZ reporter indicated that transgene-expressing cells give rise to some of the chondrocytes and osteoblasts in the fracture callus. Collectively, these observations strongly suggest that the transgene-expressing cells are osteochondro progenitor cells in the periosteum. The established Prx1CreER-GFP mice would offer novel approaches for analyzing the functions of periosteal cells in vitro and in vivo.     

A novel transgenic technique that allows specific marking of the neural crest cell lineage in mice.

Neural crest cells are embryonic, multipotent stem cells that give rise to various cell/tissue types and thus serve as a good model system for the study of cell specification and mechanisms of cell differentiation. For analysis of neural crest cell lineage, an efficient method has been devised for manipulating the mouse genome through the Cre-loxP system. We generated transgenic mice harboring a Cre gene driven by a promoter of protein 0 (P0). To detect the Cre-mediated DNA recombination, we crossed P0-Cre transgenic mice with CAG-CAT-Z indicator transgenic mice. The CAG-CAT-Z Tg line carries a lacZ gene downstream of a chicken beta-actin promoter and a "stuffer" fragment flanked by two loxP sequences, so that lacZ is expressed only when the stuffer is removed by the action of Cre recombinase. In three different P0-Cre lines crossed with CAG-CAT-Z Tg, embryos carrying both transgenes showed lacZ expression in tissues derived from neural crest cells, such as spinal dorsal root ganglia, sympathetic nervous system, enteric nervous system, and ventral craniofacial mesenchyme at stages later than 9.0 dpc. These findings give some insights into neural crest cell differentiation in mammals. We believe that P0-Cre transgenic mice will facilitate many interesting experiments, including lineage analysis, purification, and genetic manipulation of the mammalian neural crest cells.     

Notch1 and 2 cooperate in limb ectoderm to receive an early Jagged2 signal regulating interdigital apoptosis

Spontaneous and engineered mutations in the Notch ligand Jagged2 produced the Syndactylism phenotype (Jiang, R.L., Lan, Y., Chapman, H.D., Shawber, C., Norton, C.R., Serreze, D.V., Weinmaster, G., Gridley, T., 1998. Defects in limb, craniofacial, and thymic Development in Jagged2 mutant mice. Genes Dev. 12, 1046-1057; Sidow, A., Bulotsky, M.S., Kerrebrock, A.W., Bronson, R.T., Daly, M.J., Reeve, M.P., Hawkins, T.L., Birren, B.W., Jaenisch, R., Lander, E.S., 1997. Serrate2 is disrupted in the mouse limb-development mutant syndactylism. Nature 389, 722-725). Given that additional ligands may be expressed in the developing limb bud, it was possible that loss of Jagged2 disabled only part of Notch function in the limb. In addition, it is not clear from the expression pattern of Jagged2 in the apical ectodermal ridge (AER) whether the ectodermal or mesenchymal compartment of the limb bud receives the Jagged2 signal. To elucidate the requirement for the Notch pathway in limb development, we have analyzed single and compound Notch receptor mutants as well as gamma-secretase-deficient limbs. Floxed alleles were removed either from the developing limb bud ectoderm (using Msx2-Cre) or from the mesenchyme (using Prx1-Cre). Our results confirm that Jagged2 loss describes the contribution of the entire Notch pathway to the mouse limb development and revealed that both Notch1 and 2 are required in the ectoderm to receive the Jagged2 signal. Interestingly, our allelic series allowed us to determine that Notch receives this signal at an early stage in the developmental process and that memory of this event is retained by the mesenchyme, where Notch signaling appears to be dispensable. Thus, Notch signaling plays a non-autonomous role in digit septation.     

Neural crest contribution to lingual mesenchyme, epithelium and developing taste papillae and taste buds

The epithelium of mammalian tongue hosts most of the taste buds that transduce gustatory stimuli into neural signals. In the field of taste biology, taste bud cells have been described as arising from "local epithelium", in distinction from many other receptor organs that are derived from neurogenic ectoderm including neural crest (NC). In fact, contribution of NC to both epithelium and mesenchyme in the developing tongue is not fully understood. In the present study we used two independent, well-characterized mouse lines, Wnt1-Cre and P0-Cre that express Cre recombinase in a NC-specific manner, in combination with two Cre reporter mouse lines, R26R and ZEG, and demonstrate a contribution of NC-derived cells to both tongue mesenchyme and epithelium including taste papillae and taste buds. In tongue mesenchyme, distribution of NC-derived cells is in close association with taste papillae. In tongue epithelium, labeled cells are observed in an initial scattered distribution and progress to a clustered pattern between papillae, and within papillae and early taste buds. This provides evidence for a contribution of NC to lingual epithelium. Together with previous reports for the origin of taste bud cells from local epithelium in postnatal mouse, we propose that NC cells migrate into and reside in the epithelium of the tongue primordium at an early embryonic stage, acquire epithelial cell phenotypes, and undergo cell proliferation and differentiation that is involved in the development of taste papillae and taste buds. Our findings lead to a new concept about derivation of taste bud cells that include a NC origin.