Functional studies of the domains of talin.

The protein talin has two domains of approximately 200 and 47 kD, which can be cleaved apart by a variety of proteases. To examine the function of these two structural domains of talin, we have digested purified talin with a calcium-dependent protease and separated the resulting fragments chromatographically. Both fragments were radioiodinated and used to probe Western blots of whole fibroblasts and chicken gizzard extracts. The large talin fragment bound to vinculin and metavinculin. The small fragment did not demonstrate any binding in this assay. The fragments were labeled fluorescently and microinjected into fibroblasts in tissue culture. The large talin fragment incorporated quickly into focal adhesions where it remained stable for at least 14 h. The small fragment associated with focal adhesions of fibroblasts but was also distributed diffusely in the cytoplasm and the nucleus. These experiments suggest that talin has at least two sites that contribute to its localization in focal adhesions. Intact talin microinjected into Madin-Darby bovine kidney epithelial cells localized to the focal adhesions but was excluded from the zonulae adherentes, despite the localization of vinculin to both of these sites. In contrast, the large talin fragment, when microinjected into these epithelial cells, incorporated into both focal adhesions and zonulae adherentes. The difference in localization between the large talin fragment and intact talin seems to be due to the removal of the small domain. This difference in localization suggests that talin binding sites in zonulae adherentes have limited accessibility.

Fibronectin receptor exhibits high lateral mobility in embryonic locomoting cells but is immobile in focal contacts and fibrillar streaks in stationary cells.

The dynamic process of embryonic cell motility was investigated by analyzing the lateral mobility of the fibronectin receptor in various locomotory or stationary avian embryonic cells, using the technique of fluorescence recovery after photobleaching. The lateral mobility of fibronectin receptors, labeled by a monoclonal antibody, was defined by the diffusion coefficient and mobile fraction of these receptors. Even though the lateral diffusion coefficient did not vary appreciably (2 X 10(-10) cm2/S less than or equal to D less than or equal to 4 X 10(-10) cm2/S) with the locomotory state and the cell type, the mobile fraction was highly dependent on the degree of cell motility. In locomoting cells, the population of fibronectin receptors, which was uniformly distributed on the cell surface, displayed a high mobile fraction of 66 +/- 19% at 25 degrees C (82 +/- 14% at 37 degrees C). In contrast, in nonmotile cells, the population of receptors was concentrated in focal contacts and fibrillar streaks associated with microfilament bundles and, in these sites, the mobile fraction was small (16 +/- 8%). When cells were in a stage intermediate between highly motile and stationary, the population of fibronectin receptors was distributed both in focal contacts with a small mobile fraction and in a diffuse pattern with a reduced mobile fraction (33 +/- 9%) relative to the diffuse population in highly locomotory cells. The mobile fraction of the fibronectin receptor was found to be temperature dependent in locomoting but not in stationary cells. The mobile fraction could be modulated by affecting the interaction between the receptor and the substratum. The strength of this interaction could be increased by growing cells on a substratum coated with polyclonal antibodies to the receptor. This caused the mobile fraction to decrease. The interaction could be decreased by using a probe, monoclonal antibodies to the receptor known to perturb the adhesion of certain cell types which caused the mobile fraction to increase. From these results, we conclude that in locomoting embryonic cells, most fibronectin receptors can readily diffuse in the plane of the membrane. This degree of lateral mobility may be correlated to the labile adhesions to the substratum presumably required for high motility. In contrast, fibronectin receptors in stationary cells are immobilized in focal contacts and fibrillar streaks which are in close association with both extracellular and cytoskeletal structures; these stable complexes appear to provide firm anchorage to the substratum.

YY1 activates Msx2 gene independent of bone morphogenetic protein signaling.

Msx2 is a homeobox gene expressed in multiple embryonic tissues which functions as a key mediator of numerous developmental processes. YY1 is a bi-functional zinc finger protein that serves as a repressor or activator to a variety of promoters. The role of YY1 during embryogenesis remains unknown. In this study, we report that Msx2 is regulated by YY1 through protein-DNA interactions. During embryogenesis, the expression pattern of YY1 was observed to overlap in part with that of Msx2. Most notably, during first branchial arch and limb development, both YY1 and Msx2 were highly expressed, and their patterns were complementary. To test the hypothesis that YY1 regulates Msx2 gene expression, P19 embryonal cells were used in a number of expression and binding assays. We discovered that, in these cells, YY1 activated endogenous Msx2 gene expression as well as Msx2 promoter-luciferase fusion gene activity. These biological activities were dependent on both the DNA binding and activation domains of YY1. In addition, YY1 bound specifically to three YY1 binding sites on the proximal promoter of Msx2 that accounted for this transactivation. Mutations introduced to these sites reduced the level of YY1 transactivation. As bone morphogenetic protein type 4 (BMP4) regulates Msx2 expression in embryonic tissues and in P19 cells, we further tested whether YY1 is the mediator of this BMP4 activity. BMP4 did not induce the expression of YY1 in early mouse mandibular explants, nor in P19 cells, suggesting that YY1 is not a required mediator of the BMP4 pathway in these tissues at this developmental stage. Taken together, these findings suggest that YY1 functions as an activator for the Msx2 gene, and that this regulation, which is independent of the BMP4 pathway, may be required during early mouse craniofacial and limb morphogenesis.

Convergence of the BMP and EGF signaling pathways on Smad1 in the regulation of chondrogenesis.

Bone morphogenetic protein 4 (BMP4) induces, whereas epidermal growth factor (EGF) inhibits chondrogenesis. We hypothesize that BMP4 and EGF mediated intracellular signals are both coupled in the regulation of Meckel's cartilage development. Two chondrogenic experimental model systems were employed to test the hypothesis: (1) an ex vivo, serum-free, organ culture system for mouse embryonic mandibular processes, and (2) a micromass culture system for chicken embryonic mandibular processes. Chondrogenesis was assayed by alcian blue staining and expression of Sox9 and type II collagen. Exogenous EGF inhibited and BMP4 induced ectopic cartilage in a dose-dependent manner. When BMP4- and EGF-soaked beads were implanted in juxtaposition within embryonic day 10 mouse mandibular processes, the incidence and amount of ectopic cartilage, and Sox9 and type II collagen expression induced by BMP4, were significantly reduced as the concentration of EGF was increased. Similarly, in chicken serum-free micromass cultures, expression of a constitutively active BMP receptor type IB by replication competent avian retrovirus system promoted the rate and extent of chondrogenesis; however, exogenous EGF attenuated this effect. In micromass cultures, BMP signaling resulted in nuclear translocation and accumulation of the signaling molecule Smad1, whereas the addition of EGF inhibited this event. Our results suggest that BMP4 and EGF function antagonistically, yet are coupled in the regulation of initial chondrogenesis. Smad1 serves as a point of convergence for the integration of two different growth factor signaling pathways during chondrogenesis.

Subcellular compartmentalization of phosphorylated neurofilament polypeptides in neurons.

Immunocytochemistry and polyacrylamide gel electrophoresis have been used to study the distribution of phosphorylated forms of neurofilament antigens in rat brain. Immunostaining of tissue with an antisera produced against phosphatase-sensitive domains of the 200-kilodalton (kd) neurofilament polypeptide showed that phosphorylated forms of this polypeptide were present in virtually all axons and certain somata and dendrites of neurons in different brain regions. Immunoblots of whole brain homogenate or a neurofilament preparation from rat revealed that the affinity-purified anti-200-kd sera used to immunostain tissue labeled the neurofilament-associated 200-kd band in a phosphatase-sensitive manner. Fine structural analysis of this immunoreactivity in tissue showed that whenever the labeled organelle could be identified, it was a microtubule. In contrast, immunoblot analysis of twice-cycled microtubules from porcine brain revealed that microtubules in vitro did not possess the 200-kd antigen that was observed in situ. The results suggest that our antibody recognizes a phosphorylated domain on the neurofilament involved in cross-linking neurofilaments and microtubules, and that in vivo, phosphorylated epitopes of the 200-kd neurofilament polypeptide are capable of associating with microtubules.

Microinjection of antibodies against talin inhibits the spreading and migration of fibroblasts.

Talin is believed to be one of the key proteins involved in linking actin filaments to extracellular matrix receptors in focal adhesions. Our strategy for studying the function of talin has been to inactivate talin in living fibroblasts in tissue culture through the microinjection of affinity-purified, polyclonal anti-talin antibodies. The effect of the injected anti-talin antibodies on cell spreading was found to depend on how recently the cells had been plated. Cells that were in the process of spreading on a fibronectin substratum, and which had newly developed focal adhesions, were induced to round up and to disassemble many of the adhesions. However, if fibroblasts were allowed to spread completely before they were microinjected with the anti-talin antibody, focal adhesions remained intact and the flat morphology of the cells was unaffected. The percentage of cells that were able to maintain a spread morphology despite the injection of anti-talin antibodies increased during the first few hours after plating on fibronectin substrata. Fibroblasts that were allowed to spread completely before microinjection with the anti-talin antibody retained both intact focal adhesions and a flat, well-spread morphology, but failed to migrate effectively. Our experiments do not directly address the role of talin in mature focal adhesions, but they indicate that talin is essential for the spreading and migration of fibroblasts on fibronectin as well as for the development and initial maintenance of focal adhesions on this substratum.

Progress toward understanding craniofacial malformations.

Significant advances in the study of the human face have revealed the genetic and gene-environment bases of numerous common and rare craniofacial disorders. Classification of craniofacial malformations based on clinical phenotypes is sometimes quite different from the genetic findings of patients. Different mutations in a single gene can cause distinct syndromes, and mutations in different genes can cause the same syndrome. The extracellular signaling molecule SHH, fibroblast growth factor receptors, and transcription factors GLI3, MSX2, and TWIST are discussed as examples of molecules involved in interrelated signal transduction networks regulating craniofacial development. Progress in the understanding of normal and abnormal craniofacial development, through the study of morphoregulatory signaling pathways, has benefited from multifactorial approaches recommended 40 years ago at the National Institute of Dental Research-sponsored landmark Gatlinburg Conference. The utilization of biochemistry, protein structure analyses, tissue culture, and animal model systems for developmental genetics has resulted in remarkable scientific advances. The evolutionary conservation of morphoregulatory pathways has revealed the homology of genes associated with human craniofacial malformations and their counterparts that regulate the morphogenesis of fruit flies. The continued investments in basic, translational, and patient-oriented research regarding normal and abnormal craniofacial development will translate into substantial improvements in the prevention, diagnosis, and treatment of craniofacial diseases and disorders.

The Dictyostelium dual-specificity kinase splA is essential for spore differentiation.

We have studied the structure and function of the Dictyostelium kinase splA. A truncated form of the splA protein exhibited primarily tyrosine kinase activity in vitro; however, it also autophosphorylated on serine and threonine residues. The kinase domain of splA exhibits approximately 38% identity to the CTR1 kinase of Arabidopsis, which is a member of the Raf family. Outside its kinase domain, splA shares homology with the byr2 kinase of S. pombe. By aligning the sequences of splA, byr2 and STE11, a homologue of byr2 in S. cerevisiae, we have identified a conserved motif that is also found in members of the Eph family of growth factor receptor tyrosine kinases. SplA is expressed throughout development with a peak during the mound stage of morphogenesis. Strains in which the splA gene had been disrupted completed fruiting body formation; however, spore cells spontaneously lysed before completing their differentiation. Northern analysis revealed the expression of the prespore marker cotB and the prestalk markers ecmA and ecmB in the mutant strain during development. The spore differentiation marker spiA was detected in the mutant spores both by northern and immunoblotting, but these cells failed to assemble spore coats. Immunoblot analysis of the developmental pattern of tyrosine phosphorylation revealed a protein that was phosphorylated in mutants but was not phosphorylated in the wild-type cells. SplA is a novel dual specificity kinase that regulates the differentiation of spore cells.

Fibroblast growth factor signaling regulates Dach1 expression during skeletal development.

Dach1 is a mouse homologue of the Drosophila dachshund gene, which is a key regulator of cell fate determination during eye, leg, and brain development in the fly. We have investigated the expression and growth factor regulation of Dach1 during pre- and postnatal skeletal development in the mouse limb to understand better the function of Dach1. Dach1 was expressed in the distal mesenchyme of the early embryonic mouse limb bud and subsequently became restricted to the tips of digital cartilages. Dach1 protein was localized to postmitotic, prehypertrophic, and early hypertrophic chondrocytes during the initiation of ossification centers, but Dach1 was not expressed in growth plates that exhibited extensive ossification. Dach1 colocalized with Runx2/Cbfa1 in chondrocytes but not in the forming bone collar or primary spongiosa. Dach1 also colocalized with cyclin-dependent kinase inhibitors p27 (Kip1) and p57 (Kip2) in chondrocytes of the growth plate and in the epiphysis before the formation of the secondary ossification center. Because fibroblast growth factors (FGF), bone morphogenetic proteins (BMP), and hedgehog molecules (Hh) regulate skeletal patterning of the limb bud and chondrocyte maturation in developing endochondral bones, we investigated the regulation of Dach1 by these growth and differentiation factors. Expression of Dach1 in 11 days postcoitus mouse limb buds in organ culture was up-regulated by implanting beads soaked in FGF1, 2, 8, or 9 but not FGF10. BMP4-soaked beads down-regulated Dach1 expression, whereas Shh and bovine serum albumin had no effect. Furthermore, FGF4 or 8 could substitute for the apical ectodermal ridge in maintaining Dach1 expression in the limb buds. Immunolocalization of FGFR2 and FGFR3 revealed overlap with Dach1 expression during skeletal patterning and chondrocyte maturation. We conclude that Dach1 is a target gene of FGF signaling during limb skeletal development, and Dach1 may function as an intermediary in the FGF signaling pathway regulating cell proliferation or differentiation.

Positionally-dependent chondrogenesis induced by BMP4 is co-regulated by Sox9 and Msx2.

Cranial neural crest cells emigrate from the posterior midbrain and anterior hindbrain to populate the first branchial arch and eventually differentiate into multiple cell lineages in the maxilla and mandible during craniofacial morphogenesis. In the developing mouse mandibular process, the expression profiles of BMP4, Msx2, Sox9, and type II collagen demonstrate temporally and spatially restrictive localization patterns suggestive of their functions in the patterning and differentiation of cartilage. Under serumless culture conditions, beads soaked in BMP4 and implanted into embryonic day 10 (E10) mouse mandibular explants induced ectopic cartilage formation in the proximal position of the explant. However, BMP4-soaked beads implanted at the rostral position did not have an inductive effect. Ectopic chondrogenesis was associated with the up-regulation of Sox9 and Msx2 expression in the immediate vicinity of the BMP4 beads 24 hours after implantation. Control beads had no effect on cartilage induction or Msx2 and Sox9 expression. Sox9 was induced at all sites of BMP4 bead implantation. In contrast, Msx2 expression was induced more intensely at the rostral position when compared with the proximal position, and suggested that Msx2 expression was inhibitory to chondrogenesis. To test the hypothesis that over-expression of Msx2 inhibits chondrogenesis, we ectopically expressed Msx2 in the mandibular process organ culture system using adenovirus gene delivery strategy. Microinjection of the Msx2-adenovirus to the proximal position inhibited BMP4-induced chondrogenesis. Over-expression of Msx2 also resulted in the abrogation of endogenous cartilage and the down-regulation of type II collagen expression. Taken together, these results suggest that BMP4 induces chondrogenesis, the pattern of which is positively regulated by Sox9 and negatively by Msx2. Chondrogenesis only occurs at sites where Sox9 expression is high relative to that of Msx2. The combinatorial action of these transcription factors appear to establish a threshold for Sox9 function and thereby restricts the position of chondrogenesis.

Compressive force promotes sox9, type II collagen and aggrecan and inhibits IL-1beta expression resulting in chondrogenesis in mouse embryonic limb bud mesenchymal cells.

The initial modeling and subsequent development of the skeleton is controlled by complex gene-environment interactions. Biomechanical forces may be one of the major epigenetic factors that determine the form and differentiation of skeletal tissues. In order to test the hypothesis that static compressive forces are transduced into molecular signals during early chondrogenesis, we have developed a unique three-dimensional collagen gel cell culture system which is permissive for the proliferation and differentiation of chondrocytes. Mouse embryonic day 10 (E10) limb buds were microdissected and dissociated into cells which were then cultured within a collagen gel matrix and maintained for up to 10 days. Static compressive forces were exerted onto these cultures. The time course for expression pattern and level for cartilage specific markers, type II collagen and aggrecan, and regulators of chondrogenesis, Sox9 and IL-1beta, were analyzed and compared with non-compressed control cultures. Under compressive conditions, histological evaluation showed an apparent acceleration in the rate and extent of chondrogenesis. Quantitatively, there was a significant 2- to 3-fold increase in type II collagen and aggrecan expression beginning at day 5 of culture and the difference was maintained through 10 days of cultures. Compressive force also causes an elevated level of Sox9, a transcriptional activator of type II collagen. In contrast, the expression and accumulation of IL-1beta, a transcriptional repressor of type II collagen was down-regulated. We conclude that static compressive forces promote chondrogenesis in embryonic limb bud mesenchyme, and propose that the signal transduction from a biomechanical stimuli can be mediated by a combination of positive and negative effectors of cartilage specific extracellular matrix macromolecules.

Adenovirus-mediated ectopic expression of Msx2 in even-numbered rhombomeres induces apoptotic elimination of cranial neural crest cells in ovo.

Distinct cranial neural crest-derived cell types (a number of neuronal as well as non-neuronal cell lineages) are generated at characteristic times and positions in the rhombomeres of the hindbrain in developing vertebrate embryos. To examine this developmental process, we developed a novel strategy designed to test the efficacy of gain-of-function Msx2 expression within rhombomeres in ovo prior to the emigration of cranial neural crest cells (CNCC). Previous studies indicate that CNCC from odd-numbered rhombomeres (r3 and r5) undergo apoptosis in response to exogenous BMP4. We provide evidence that targeted infection in ovo using adenovirus containing Msx2 and a reporter molecule indicative of translation can induce apoptosis in either even- or odd-numbered rhombomeres. Furthermore, infected lacZ-control explants indicated that CNCC emigrated, and that 20% of these cells were double positive for crest cell markers HNK-1 and beta-gal. In contrast, there were no HNK-1 and Msx2 double positive cells emigrating from Msx2 infected explants. These results support the hypothesis that apoptotic elimination of CNCC can be induced by 'gain-of-function' Msx2 expression in even-numbered rhombomeres. These inductive interactions involve qualitative, quantitative, positional and temporal differences in TGF-beta-related signals, Msx2 expression and other transcriptional control.

Msx2 is a repressor of chondrogenic differentiation in migratory cranial neural crest cells.

During early mouse embryogenesis, cranial neural crest cells (CNCC) emigrate from the posterior midbrain and rhombomeres 1 and 2 of the anterior hindbrain into the first branchial arch-derived maxillary and mandibular processes and there provide cell lineages for several phenotypes, including cartilage, bone, and tooth. Here, we report that Sox9 and Msx2 were coexpressed in a subpopulation of CNCC during their migration. Because Sox9 is a transactivator of chondrogenesis, and Msx genes can act as transcriptional repressors, we hypothesized that Sox9 expression indicates the determination of CNCC-derived chondrogenic cell lineage and that Msx2 represses chondrogenic differentiation until CNCC migration is completed within the mandibular processes. To test whether Msx2 represses chondrogenesis, we designed experiments to inhibit Msx2 function in migratory CNCC in primary cultures through the expression of loss-of-function Msx2 mutants. We showed that infection of migratory CNCC with adenovirus Msx2 mutants accelerated the rate and extent of chondrogenesis, as indicated by the expression level of type II collagen and aggrecan, and the amount of alcian blue staining. Adenovirus infections did not apparently interfere with CNCC proliferation or migration. These findings suggest that an important early event in craniofacial morphogenesis is a transient expression of both Sox9 and Msx2 during emigration into the forming mandibular processes followed by restricted expression of Sox9 within CNCC- derived chondroprogenitor cells. We conclude that Msx2 serves as a repressor of chondrogenic differentiation during CNCC migration.

Nerve growth factor (NGF) supports tooth morphogenesis in mouse first branchial arch explants.

Posterior midbrain and anterior hindbrain neuroectoderm trans-differentiate into cranial neural crest cells (CNCC), emigrate from the neural folds, and become crest-derived ectomesenchyme within the mandibular and maxillary processes. To investigate the growth factor requirement specific for the initiation of tooth morphogenesis, we designed studies to test whether nerve growth factor (NGF) can support odontogenesis in a first branchial arch (FBA) explant culture system. FBA explants containing neural-fold tissues before CNCC emigration and the anlagen of the FBA were microdissected from embryonic day 8 (E8) mouse embryos, and cultured for 8 days in medium supplemented with 10% fetal calf serum only, or serum-containing medium further supplemented with either NGF or epidermal growth factor (EGF) at three different concentrations: 50, 100, or 200 ng/ml. Morphological, morphometric, and total protein analyses indicated that growth and development in all groups were comparable. Meckel's cartilage and tongue formation were also observed in all groups. However, odontogenesis was only detected in explants cultured in the presence of exogenous NGF. NGF-supplemented cultures were permissive for bud stage (50 ng/ml) as well as cap stage of tooth morphogenesis (100 and 200 ng/ml). Morphometric analyses of the volume of tooth organs showed a significant dose-dependent increase in tooth volume as the concentration of NGF increased. Whole-mount in situ hybridization and semiquantitative reverse transcription-polymerase chain reaction for Pax9, a molecular marker of dental mesenchyme, further supported and confirmed the morphological data of the specificity and dose dependency of NGF on odontogenesis. We conclude that (1) E8 FBA explants contain premigratory CNCC that are capable of emigration, proliferation, and differentiation in vitro; (2) serum-supplemented medium is permissive for CNCC differentiation into tongue myoblasts and chondrocytes in FBA explants; and (3) NGF controls CNCC cell fate specification and differentiation into tooth organs.

DACH: genomic characterization, evaluation as a candidate for postaxial polydactyly type A2, and developmental expression pattern of the mouse homologue.

The gene DACH is a human homologue of Drosophila melanogaster dachshund (dac), which encodes a nuclear factor essential for determining cell fates in the eye, leg, and nervous system of the fly. To investigate possible connections between DACH and inherited developmental disorders, we have characterized the human DACH genomic structure and investigated the tissue and cellular distribution of the mouse DACH1 protein during development. DACH spans 400 kb and is encoded by 12 exons. The predominant DACH transcript is 5.2 kb and encodes a 706-amino-acid protein with an observed molecular weight of 97 kDa.DACH mRNA was detected in multiple adult human tissues including kidney and heart. The mouse DACH1 protein was immunolocalized to specific cell types within the developing kidneys, eyes, cochleae, and limb buds. Data suggest genetic linkage of the limb bud patterning defect postaxial polydactyly type A (designated PAP-A2, MIM 602085) to a 28-cM interval on chromosome 13 that includes DACH. However, mutation analysis of DACH in this PAP-A2 pedigree revealed no sequence differences in the coding region, splice sites, or proximal promoter region. The data presented will allow for the analysis of DACH as a candidate for other developmental disorders affecting the limbs, kidneys, eyes, ears, and other sites of DACH expression.