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The internal and external cheek pouches found in certain rodents arise early in development by an evagination of the buccal epithelium. Differences in the epithelial eva inations that produce the internal pouches of the Syrian hamster (Mesocketus auratus) and least ckipmunk (Eutamias minimus) are consistent with the view that they evolved independently. The external cheek pouches of rodents of the superfamily Geomyoidea represent a macroevolutionary phenoty e when com ared to the internal pouches of other rodents. Externalization of an internal ouci rudiment Eund in the geom oids Di odomys and Thomomys can be explained by a simple c&nge early in its development, the erect of wiich is greatly magnified by facial growth. In this example, the traditional dichotomy between microevolutlonary and macroevolutionary theories is bridged by an understanding of developmental dynamics.

In this report we employed double-knock-out mouse embryos and fetuses (designated as Myf5-/-: MyoD-/- that completely lacked striated musculature to study bone development in the absence of mechanical stimuli from the musculature and to distinguish between the effects that static loading and weight-bearing exhibit on embryonic development of skeletal system. We concentrated on development of the mandibles (= dentary) and clavicles because their formation is characterized by intramembranous and endochondral ossification via formation of secondary cartilage that is dependent on mechanical stimuli from the adjacent musculature. We employed morphometry and morphology at different embryonic stages and compared bone development in double-mutant and control embryos and fetuses. Our findings can be summarized as follows: a) the examined mutant bones had significantly altered shape and size that we described morphometrically, b) the effects of muscle absence varied depending on the bone (clavicles being more dependent than mandibles) and even within the same bone (e.g., the mandible), and c) we further supported the notion that, from the evolutionary point of view, mammalian clavicles arise under different influences from those that initiate the furcula (wishbone) in birds. Together, our data show that the development of secondary cartilage, and in turn the development of the final shape and size of the bones, is strongly influenced by mechanical cues from the skeletal musculature.

This study provides a quantitative analysis of the active movements of the chick embryo and of the contractions of the amnion over the entire developmental period of 21 days. Four types of embryo movements are distinguished. The motor activity of the embryo shows two characteristic peaks, with maximum contraction frequencies on the 12th and on the 16th day. In contrast, the amnion activity is higher at earlier stages and decreases as the body activity increases. The amnion activity is largely independent of the body activity. Illumination has a strong influence on embryo movements. It is shown that increases of light intensity affect the patterns of activity of both the embryo and the amnion. While the effect of light on the embryo can be interpreted as being transmitted via the optic system, the mechanism of the amniotic response is unclear. The results suggest that the amnion itself may be sensitive to light. J. Exp. Zool. (Mol. Dev. Evol.) 291:186-194, 2001.

Regulation is the replacement of lost, undifferentiated embryonic cells by neighboring cells in response to environmental signals. Neural crest cells, embryonic cells unique to craniates, are good candidates for studies of regulation because they are pluripotent, and thus might be able to alter their behavior in response to environmental signals. This study investigated regulation for the loss of trunk neural crest (TNC) cells, specifically pigment derivatives, in the zebrafish, Danio rerio. The first part of the study clarifies and extends what has previously been described on normal patterns of TNC migration and differentiation. These data were then used to address the hypothesis that there is regulation for loss of TNC, and that regulation would vary with the amount removed, the position or stage of removal. Zebrafish TNC cells are large and numerous. SEM and Dil labeling revealed that TNC cells undergo several successive waves of 'sheet' and 'segmental' migration, beginning as early as the 12 somite stage. Dil-labeled TNC cells often migrated several somite lengths anteriorly and posteriorly along the trunk axis to form glial cells, ganglia, pigment, ectomesenchyme and tail reticular cells. Regulation occurred on a sliding scale, ranging from complete to incomplete. Defects in development and/or pigmentation occurred if large regions of TNC cells were removed, or if cells were removed from anterior (cardiac) and posterior (tail) extremities of the trunk. Melanophores were the cell type most visibly affected by TNC extirpations. Otherwise, pigmentation was remarkably normal. We propose that the completeness of regulation largely depends upon healing of the overlying epidermis.

Clavicles (collar bones) are variably present in mammals. Furculae (wishbones)--which may or may not be homologous with clavicles--are variably present and/or fused in birds and present in theropod dinosaurs. In this overview the development of clavicles and furculae is discussed with special attention to modes of skeletogenesis (whether intramembranous or endochondral), numbers of centres of ossification (one in chick furculae; two in murine clavicles), presence of cartilage (primary in clavicles, secondary in furculae), evidence from experimental analysis and from mutations for dependence of both clavicular and furcular growth on mechanical stimulation, and syndromes and mutations affecting clavicular development leading to both under and over development. J. Exp. Zool. 289:153-161, 2001.

We examined the temporospatial pattern of naturally occurring apoptosis in chick embryos to five days of incubation (H.H. stages 1-25; Hamburger and Hamilton, 1951) using TUNEL labeling. The initial TUNEL-positive structure was the embryonic shield at stage 1. Apoptotic cells became ubiquitously present within embryos by stage 3, which is early in gastrulation. Until stage 6, TUNEL-positive cells were restricted to the headfold region. In embryos of stages 7-8, most cell death was localized at the most anterior neural plate. TUNEL-positive neural plate, notochord and somites appeared at stage 9. Otic and optic regions became TUNEL-positive at stage 11. The aggregation of cells from which the tail bud arises contains apoptotic cells from stage 11 onwards. At stage 16, scattered TUNEL-positive cells appeared in the branchial arches. Three streams of apoptotic neural crest cells in the cranial region became most clearly visible at stage 18. The secondary neural tube from which caudal structures develop contains apoptotic cells at stage 14. Apoptotic cells are present in the branchial arches and lateral body wall for extended periods, stages 16-25 and 25 respectively. At stages 24-25, intense positive regions of cell death were confined to the caudal regions of the arches, to limb and tail buds and to the lateral body wall, the latter in relation to body wall closure. The new findings in this study are discussed along with past studies to provide the temporospatial pattern of cell death during early chick development.

Condensation is the pivotal stage in the development of skeletal and other mesenchymal tissues. It occurs when a previously dispersed population of cells gathers together to differentiate into a single cell/tissue type such as cartilage, bone, muscle, tendon, kidney, and lung and is the earliest stage during organ formation when tissue-specific genes are upregulated. We present a synopsis of our current understanding of how condensations are initiated and grown, how their boundaries and sizes are set, how condensation ceases, and how overt differentiation begins. Extracellular matrix molecules, cell surface receptors and cell adhesion molecules, such as fibronectin, tenascin, syndecan, and N-CAM, initiate condensation formation and set condensation boundaries. Hox genes (Hoxd-11-13) and other transcription factors (CFKH-1, MFH-1, osf-2), modulate the proliferation of cells within condensations. Cell adhesion is ensured indirectly through Hox genes (Hoxa-2, Hoxd-13), and directly via cell adhesion molecules (N-CAM and N-cadherin). Subsequent growth of condensations is regulated by BMPs, which activate Pax-2, Hoxa-2 and Hoxd-11 among other genes. Growth of a condensation ceases when Noggin inhibits BMP signalling, setting the stage for transition to the next stage of skeletal development, namely overt cell differentiation. BioEssays 22:138-147, 2000.

Condensation precedes chondrogenic differentiation during development of primary cartilage. While neural cell adhesion molecule (N-CAM) enhances condensation, it is unclear whether N-CAM is also required for initiation of chondrogenic differentiation. In this study, the role of N-CAM in secondary chondrogenesis from periosteal cells of the quadratojugal (QJ) from embryonic chicks was studied using several in vitro approaches. The QJ is a membrane bone and so is not preceded by cartilage formation during development. However, QJ periosteal cells can differentiate into chondrocytes to form secondary cartilage in vivo. When QJ periosteal cells were enzymatically released and plated in low density monolayer, clonal or agarose cultures, chondrogenesis was initiated in the absence of N-CAM expression. Furthermore, overexpression of the N-CAM gene in periosteal cells in monolayer culture significantly reduced the number of chondrocyte colonies, suggesting that N-CAM inhibits secondary chondrogenesis. In contrast, and consistent with expression in vivo, N-CAM is expressed during osteogenesis from QJ periosteal cells and mandibular mesenchyme in vitro. These results are discussed in relation to the role of N-CAM in osteogenesis and in primary and secondary condensation.

Patterning, cellular differentiation, and developmental sequences of dermal denticles (denticles) are described for the skate Leucoraja erinacea. Development of denticles proceeds caudo-rostrally in the tail and trunk. Once three rows of denticles form in the tail and trunk, denticles begin to appear in the region of the pelvic girdle, medio-caudal to the eyes and on the pectoral fins. Although timing of cellular differentiation of denticles differs among different locations of the body, cellular development of a denticle is identical in all locations. Thickening of the epidermis as a denticle lamina marks initiation of development. A single lamina for each denticle forms, and a small group of mesenchymal cells aggregates underneath it. The lamina then invaginates caudo-rostrally to form the inner- and outer-denticle epithelia (IDE and ODE, respectively). Before nuclei of IDE cells are polarized, enameloid matrix appears between the basement membrane of the IDE and the apical surface of the pre-odontoblasts. Pre-dentin is then laid down along with collagenous materials. Von Kossa stain visualizes initial mineralization of dentin, but not enameloid. During the growth of a denticle, dense fibrous connective tissue of the dermis forms the deep dermal tissue over the dorsal musculature. Attachment fibers and tendons anchor denticles and dorsal musculature, respectively, on deep dermal tissue. Basal tissue of the denticles develops as the denticle crown grows. If the basal tissue is bone of attachment, then the cells along the basal tissue would be osteoblasts. However, these cells could not be distinguished from odontoblasts using immunolocalization of type I pro-collagen (Col I), alkaline phosphatase (APase), and neural cell adhesion molecule (N-CAM). Well-developed dentin, (not pre-dentin), the enameloid matrix (probably when it begins to mineralize), and deep dermal tissue are Verhoeff stain-positive, suggesting that these tissues contain elastin and/or elastin-like molecules. Our study demonstrates that the cellular development of denticles resembles tooth development in elasmobranchs, but that dermal denticles differ from teeth in forming from a single denticle lamina. Whether the basal tissue of denticles is bone of attachment remains undetermined. Confirmation and function of Verhoeff-positive proteins in enameloid, dentin, and deep dermal tissue remain to be determined. We discuss these issues along with an analysis of recent findings of enamel and enameloid matrices.

Fluorescence-labeled DNA probes constructed from three whole house mouse (Mus domesticus) chromosomes were hybridized to metaphase spreads from deer mouse (Peromyscus maniculatus) to identify homologies between the species. Mus Chr 7 probe hybridized strongly to the ad-centromeric two-thirds of Peromyscus Chr 1q. Most of Mus 3 probe hybridized principally to two disjunct segments of Peromyscus Chr 3. Mus Chr 9 probe hybridized entirely to the whole Peromyscus Chr 7. Three Peromyscus linkage groups were assigned to chromosomes, based on linkage homology with Mus. The data also are useful in interpretation of chromosomal evolutionary history in myomorphic rodents.

Regulation is a significant developmental event because successful cell proliferation and migration are critical to shaping young embryos. Regulation -- the replacement of undifferentiated embryonic cells by other cells in response to signals received from the environment -- is distinct from wound healing and regeneration. Investigations on regulation of neural crest cells span all vertebrates and have revealed that regulative ability varies both among classes (even species), and spatially and temporally within individuals. In general, there is greatest regulation for cranial neural crest cells, less for trunk, and virtually none forcardiac. Regulation also appears to be more complete at early embryonic stages. Fate-mapping studies have demonstrated that large regions of neural crest cells must be removed to generate missing or morphologically reduced structures. Recent studies reveal that less extensive neural crest cell extirpations result in normal morphology of cartilaginous and neuronal elements in the head, and normal development of pigmentation in the trunk. Ablation of cardiac neural crest cells frequently generates abnormalities of the heart, great vessels and parasympathetic nerve innervation. Decreased cell death, increased division, change in fate and altered migration are possible cellular mechanisms of regulation. In mostcases, the specific mechanisms of regulation are unknown, but a major premise underlying regulation is that cell potential is greater than cell fate. This concept was born from studies which demonstrated that some cells were able to express alternative fates if transplanted to a new environment. Among the potential cellular mechanisms for regulation, cell migration has received the most attention. Following ablation of neural crest cells, replacement neural crest cells migrate into gaps, most frequently from anterior/posterior locations. Cells from surrounding epidermal and neural ectoderm may have limited regulative ability, while compensation by cells from the ventral neural tube has been demonstrated to an even lesser extent. Regulation by such non-crest cells would require their transformation into neural crest cells. The potential for regulation of neural crest by placodal cells supports a closer relationship between neural crest and placodal ectoderm than previously recognized. Decreased cell death has been discussed primarily with reference to (1) cranial ganglia that have dual contributions from neural crest and placodal cells and (2) programmed cell death in rhombomeres three and five. Increased cell division in response to neural crest ablation is likely more common than has been reported, but this mechanism is difficult to interpret without a 3-D context for viewing how patterns of division differ from normal. Lastly, changes in cell fate may be the driving factor in regulation of embryonic cells. It has been repeatedly demonstrated thatcell potential is greaterthan cell fate. Once reliable mechanisms for assessing cell potential are established, we may find that fates are commonly altered in response to environmental signals. Regulation is therefore significant both as a basic developmental mechanism and as a mechanism for evolutionary change. The more labile the fate of embryonic cells, the more potential there is for maintaining existing characters and for generating new ones. According to Ettensohn (1992, p. 50), further analysis of such systems mightshed light both on the way evolutionary processes act to modify ontogenetic programs and on the cellular and molecular mechanisms of cell interactions during development. With regard to the neural crest, studies on regulation of this vital population of cells provide insight to the origin of the neural crest, to embryonic repair, and to the source of many craniofacial malformations, heart and other embryonic defects. (ABSTRACT TRUNCATED)

We examined the relationships between litter size, embryonic growth, days of gestation, onset and duration of morphological stages and development of the first arch skeleton in three inbred strains of mice--C57BL/6, CBA/J and C3H/He. Detailed embryonic staging was based on craniofacial development between 11 and 18 days of gestation. Considerable intra- and interlitter variation of morphological stages of embryonic development exists in all three inbred strains. The relationship of morphological stages to days of gestation reveals that each stage has a different duration, being shortest at Theiler's stage 18 and longest at stage 21 in all three inbred strains. Embryos of CBA/J mice tend to reach each stage later than do embryos of the other two strains, i.e., morphological development is slowest in CBA/J. The greatest length, a measurement of embryonic growth, increases at a constant rate during gestation in all three strains. In C57BL/6 and CBA/J, more embryos tend to be implanted in the right horn of the uterus than in the left, whereas in C3H/He an even number of embryos tends to be implanted in both horns. Timing of the development of Meckel's cartilage differs between the three inbred strains: both condensation and onset of matrix deposition begin one stage earlier in C57BL/6 than in CBA/J and C3H/He. On the other hand, alkaline phosphatase, one of the earliest markers for bone development, is expressed at the same time in all three inbred strains. Differences in timing of skeletal development between the strains may be attributed in part to the genealogical closeness OF CBA/J and C3H/He mice.

Growth of chick embryonic femora, tibiotarsi and first phalanges of digit three were measured at one day intervals from day 6 through 16 of incubation. Normal controls were compared to embryos paralyzed at 5 days of incubation. Over the 10 day study period, length of the paralyzed femora, length and width of the paralyzed tibiotarsi and differences in length of the phalanges were observed. Growth in length of phalanx one of digit three was most affected by paralysis over this period. Changes in shape of these bones also occurred during growth. Normal long bones undergo changes in shape as differential growth in length and width occurs. Such changes in shape can be considered as the bone's normal growth "trajectory". Paralyzed bones displayed a different growth trajectory than normal bones. As expected, the long bones of paralyzed embryos were shorter than age-matched controls. Contrary to expectations, however, paralyzed long bones were relatively more stout than age-matched controls.

During embryonic development, neural crest derived mesenchymal (ectomesenchymal) cells in the chick mandible give rise to cartilage and membrane bone. Signaling molecules involved in the development of the mandible are less understood. To examine whether BMP-2 is involved in morphogenesis and growth of the mandible in vivo, agarose beads, loaded with BMP-2 at concentrations of 5 to 150 ng/microliter were implanted into the mandible at HH stage 22 and embryos were maintained in shell-less culture. To examine whether BMP-2 is involved in osteogenic or chondrogenic differentiation, mandibular ectomesenchyme from HH stage 22 embryos was cultured in the absence of mandibular epithelium, but in the presence of BMP-2 or BMP-2 and/or type IV collagen. Chondrogenesis and osteogenesis were examined by histological, histochemical and immunohistochemical methods. Implantation of BMP-2-containing beads in vivo retarded mandibular growth and morphogenesis in a dose-dependent manner. BMP-2 induced localized death of ectomesenchymal cells in the vicinity of the implanted bead and in proportion to the concentration of BMP-2 applied. Neither BMP-2 alone, nor BMP-2+collagen type IV, was sufficient to initiate osteogenesis in vitro in the absence of epithelium. BMP-2 inhibited chondrogenesis both in vivo and in vitro. Cartilage morphology was rod-like in the absence of BMP-2 but nodular in ectomesenchyme cultured in the presence of BMP-2. These results are discussed in relation to the stimulatory and inhibitory effects of BMPs on skeletal development.

Condensation is a multistep process, involving cell recruitment, cell-to-cell contact by cell adhesions, and concomitant changes in cell shape. Condensation of prechondrogenic cells down-regulates and/or inactivates cell proliferation and enhances the activities of cartilage-specific genes. Timing of onset and duration of condensation are thus important regulatory processes mediating cellular and molecular events during chondrogenesis. The present study was undertaken to examine timing of onset and duration of condensation and onset of matrix formation for first arch cartilages in inbred C57BL/6 mice. Because timing can only be reliably assessed in very precisely staged embryos, mice were mated for only 2 hr, pregnancy was determined by weight increase, and embryos were assigned to substages and divisions of Theiler's [1972] stages on the basis of external development of the frontonasal area, eyes, vibrissae, mandibular and hyoid auricular hillocks (pinna) and some internal structures [Miyake et al., 1996]. Condensation and matrix formation were determined using PNA lectin histochemistry, type II collagen immunohistochemistry, Mallory's trichrome, Hall and Brunt's quadruple and toluidine blue stains on serially sectioned embryos, and 3-D reconstruction. A single, continuous, first arch chondrogenic condensation was identified. It consisted of three components: 1) a rostral component for the symphysis; 2) a core component for the major portion of Meckel's cartilage; and 3) a caudal component for the caudo-lateral area of Meckel's cartilage and the two ear cartilages, the malleus and incus. The caudal component was further divided into rostral and caudal subcomponents which gave rise to the malleus and incus, respectively. Cellular arrangement differed among the three components, probably reflecting major axes of matrix growth. The condensation was only weakly recognized by peanut agglutinin lectin (PNA), unless sections were pretreated with neuraminidase, showing the masking effects of sialic acid. The core and caudal components of the condensation appeared at stage 20.12 and ended at stage 21.14 and 21.32, respectively. The symphysial component began at stage 20.2 and ended at stage 21.32. Deposition of sulfated proteoglycans began at stage 21.14 in the core component of the condensation--preceding slightly deposition of type II collagen--and at stage 21.32 in the symphysial and caudal components.

A detailed staging table of inbred C57BL/6 embryonic mice was developed to facilitate a study of the stage-by-stage cellular and molecular mechanisms underlying cranial skeletal development and elucidation of the developmental mechanisms potentially involved in evolutionary changes in cranial skeletal morphology exhibited by different inbred strains of mice. Mice were mated for only 2 hr and embryos were recovered every 2 or 4 hr between 11 and 13 days of gestation. Theiler's [1972] stages 18 through 21 were divided into substages and divisions based on the development of five external structures--the frontonasal area, eyes, auditory meatus, mandibular and hyoid auricular hillocks/pinna, and vibrissae--and three internal histological structures--eyes, tongue, and vibrissae. Each substage and division was designated with a decimal point: e.g., substage 20.1 and division 20.11. Embryos were staged using the staging table and the relationship of the substages and divisions with days of gestation was examined. The results showed considerable intra- and inter-litter variation in stages of embryos, suggesting that days of gestation are not a good indicator for staging embryos. Our staging table offers a more reliable and precise method to standardize embryonic development. Regression analyses of substages on days of gestation showed that the duration of stages increased from stages 18 to 21. Estimated durations were 3.5 hr for stages 18 and 19, 8.8 hr for stage 20, and 38.8 hr for stage 21. Our staging table also provides baseline information on development of the frontonasal area (muzzle) and vibrissae and development and the transformation of the auricular hillocks into the pinna. The developmental sequence of mystacial and labial vibrissae indicated highly regulated differentiation and morphogenesis of vibrissal development at stages 20 and 21. Three hyoid auricular hillocks transiently became four hillocks at stage 19.1 before transforming to the pinna during stage 21. The second and third hyoid auricular hillocks were the major contributor to the pinna before stage 21.2, whereas mandibular auricular hillocks contributed to the pinna from stage 21.32 onward. The staging table has already served to demonstrate stage-specific skeletogenesis of the first arch cartilages in inbred C57BL/6 mice and to reveal differences in the onset of timing of skeletogenesis among inbred C57BL/6, C3H/He and CBA/J mice.

Cell condensation is a pivotal stage in skeletal development. Although prechondrogenic condensations normally exist for some 12 h, duration can vary. Variation is seen both between condensations for different cartilages (Meckel's vs. elastic ear cartilage) and within a single condensation from which more than one skeletal element will form, as in the three components of the single first arch chondrogenic condensation. Understanding how duration of the condensation phase is established--how the condensation phase is entered and exited during cell differentiation--remains a major area for future study. During chondrogenesis, cell-specific products such as collagen types II and IX and cartilage proteoglycan appear concomitant with condensation. Therefore, during chondrogenesis, condensation precedes commitment of cells as prechondroblasts. During osteogenesis, however, differentiation of preosteoblasts precedes condensation. Therefore, during osteogenesis, condensation amplifies the number of committed osteogenic cells. Further comparative analysis of skeletogenesis should provide us with a more rigorous understanding of cell commitment, when differentiation is initiated, how commitment and differentiation are measured and the relationship of condensation to onset of differentiation. Current knowledge of molecules characteristic of condensations focused attention on extracellular matrix and cell surface components on the one hand, and on growth factors homeobox genes and transcription factors on the other. We have drawn together the molecular data for pre-chondrogenic condensations in diagrammatic form in Figure 2. Three major phases of chondrogenesis are identified: (a) epithelial-mesenchymal interactions that precede condensation, (b) condensation itself, and (c) cell differentiation. Although we label the third phase differentiation, it is important to recognize that phases a and b also constitute aspects of chondroblast cell differentiation (see Dunlop and Hall, 1995 for a discussion of this point. The pre-condensation phase is characterized by expression of Hox genes, growth factors (TGF-beta and BMP-2) and the cell surface proteoglycan receptor, syndecan-1. Expression of Msx-1 and Msx-2, growth factors and syndecan continues into the condensation phase. Other molecules, such as versican, syndecan-3 and tenascin, present in low concentrations before condensation, are up-regulated during condensation. Yet other molecules--Hox genes, transcription factors, growth factors (activin, BMP-4 and -5, GDF-5), cell adhesion molecules and proteoglycans--are only expressed during the condensation phase, while the transcription factor Pax-1, fibronectin, hyaluronan and hyaladherin are expressed both during and after condensation. During condensation mRNAs for collagen types II and IX and for the core protein of cartilage proteoglycan are up-regulated. Late in condensation and increasingly thereafter, the protein products of these genes accumulate as chondroblasts differentiate (see Fig. 2 for details). Not all the molecules present before, during of after condensation can be placed into causal sequences. Some however can. In Figure 3 we summarize the causal sequences discussed in this paper as they relate to initiation of condensation and to transit from condensation to overt differentiation during chondrogenesis. Condensations form following activation of at least three pathways: (1) Initiation of epithelial-mesenchymal interactions by tenascin, BMP-2, TGF beta-1 and Msx-1 and -2. (2) Up-regulation of N-CAM by activin. (3) Up-regulation of fibronectin by TGF-beta, further enhancing N-CAM accumulation (Fig. 3). It is by these three pathways that condensations are initiated and grow. Transition from condensation to overt cell differentiation is under both positive and negative control (Fig. 3). Syndecan blocks fibronectin and so blocks N-CAM accumulation, preventing accumulation of additional cell

We have established a method for preparing cDNA sublibraries enriched in sequences from specific chromosome regions, called selection of hybrids by affinity capture (SHAC). This procedure can be described in two stages. In the first stage, a particular chromosome region, in this study mouse chromosome 11, was microdissected, followed by PCR amplification with a universal degenerate primer. This material is referred to as the “target” DNA. In the second stage, a mouse liver cDNA library with unique linker–adapter ends, referred to as the “source” cDNA, was hybridized to the biotin-labeled target DNA prepared during the first stage. The resulting DNA duplexes were captured by streptavidin-coated magnetic beads. The cDNAs were released from their biotin-labeled target homologs by alkaline denaturation and recovered by PCR amplification. These cDNAs were referred to as the SHACcDNAs. Specificity of the SHACcDNA to chromosome 11 was verified by FISH analysis. To examine representation of the SHACcDNA, we confirmed the presence of seven genes or single-copy DNA segments known to be localized on mouse chromosome 11, using a dot blot assay. In addition, a second round of SHAC was performed to achieve even higher specificity for the resulting chromosome 11 SHACcDNA. The SHAC technology should facilitate construction of cytogenetically defined cDNA libraries and should assist in the fields of gene discovery and genome mapping.

Using degenerate primer amplification of chromosomes microdissected from banded cytogenetic preparations, we constructed both whole chromosome painting probes for mouse Chromosomes (Chrs) 1, 2, 3, and 11 and a centromere probe that strongly paints most mouse centromeres. We also amplified a Robertsonian translocation chromosome microdissected from unstained preparations to construct a painting probe for Chrs 9 and 19. The chromosomes probes uniformly painted the respective chromosomes of origin. We demonstrated the utility of the Chr 11 probe in aberration analysis by staining mutants that we had previously identified as containing a Chr 11 translocation, and in some mutant cell lines we observed chromosome rearrangements not previously detected in stained cytogenetic preparations. The technology of microdissection and amplification applies to all mouse chromosomes or to specific subchromosomal regions and will be useful in mouse genetics, in aberration analysis, and for chromosome identification.

Progenitor cells in the periosteum-perichondrium of the posterior hook of the quadratojugal (QJ, a membrane bone) in the embryonic chick are bipotential for osteogenesis and chondrogenesis. These cells switch from osteogenesis to chondrogenesis between 10 to 11 days in normal (mobile) embryos but not in paralyzed (immobile) embryos. Expression of neural cell adhesion molecule (NCAM) was studied using a monoclonal antibody in QJ hooks from normal and paralyzed chick embryos between 10 and 21 days of incubation. NCAM is expressed in osteoprogenitor cells and osteoblasts but not in chondroprogenitor cells, chondroblasts, or chondrocytes. The switch of progenitor cell differentiation from an osteogenic to a chondrogenic pathway between 10 and 11 days of incubation coincides with down-regulation of NCAM expression. Both initiation of secondary chondrogenesis and down-regulation of NCAM depend on biomechanical stimulation. In embryos paralyzed at 9 days, secondary cartilage fails to form and progenitor cells remain positive for NCAM. Furthermore, paralysis influences NCAM expression in progenitor cells before secondary chondrogenesis morphologically begins, indicating that NCAM may play a role in the initiation of secondary chondrogenesis. In 15-day normal embryos, NCAM-positive cells accumulate between the perichondrium and secondary cartilage in a position that prevents further cartilage formation in the hook. In 19-day embryos, these cells lose their NCAM expression and restart chondrogenesis in a second phase of differentiation, forming an articular cartilage. Loss of NCAM expression in this cell layer and re-commencement of chondrogenesis do not occur in embryos paralyzed at 13 days, and therefore also require biomechanical stimulation. Hence, down-regulation of NCAM expression correlates with two phases of secondary chondrogenesis in embryonic life, both of which are dependent upon embryonic movement.

Based on results of transplantation experiments, it has long been believed that trunk neural crest cells are incapable of chondrogenesis. When pigmented trunk neural crest cells of Ambystoma mexicanum are transplanted to cranial levels of albino (a/a) embryos, the graft cells ultimately produce ectopic fins, but are incapable of following the chondrogenic cranial neural crest pathways. Therefore, heterotopic transplantation does not expose these cells to the same environment experienced by cranial neural crest cells, and is neither an adequate nor a sufficient test of chondrogenic potential. However, in vitro culture of trunk neural crest cells with pharyngeal endoderm does provide a direct test of chondrogenic ability. That cartilage does not form under these conditions demonstrates conclusively that trunk neural crest cells possess no chondrogenic potential.

Meckel's cartilage in the avian mandible is a neural crest-derived permanent cartilage. To investigate whether chondrocytes that form Meckel's cartilage can be induced to undergo maturation and mineralization by manipulating the environment, we used in vitro micromass culture in which young embryonic mandibular ectomesenchymal cells were maintained at a high cell density (2 x 10(7) cells/ml) and treated with ascorbic acid (AA) or with dexamethasone and ascorbic acid (DEX + AA). Chondrogenesis and chondrocyte maturation were analyzed by histological, immunohistochemical and SDS/PAGE techniques. Chick mandibular ectomesenchymal cells from Hamburger and Hamilton (J. Morphol. 88:49-92, 1951) stage 21 (HH stage 21) chick embryos undergo chondrogenesis forming cartilage nodules when maintained under micromass culture conditions. These chondrocytes undergo maturation in response to AA but not DEX. Addition of AA to culture medium induced type X collagen expression by these chondrocytes. With prolonged culture, chondrocytes began to mineralize turning cartilage nodules into completely mineralized structures. There was no correlation between chondrocyte size and type X collagen expression. Small chondrocytes as well as large (hypertrophic) chondrocytes expressed type X collagen and then underwent mineralization. Co-treatment of cultures with DEX + AA caused reduction of chondrogenesis and inhibited chondrocyte maturation and mineralization seen with AA alone.

Neurofibromatosis type I (NFI) is a common autosomal dominant disorder with an increased risk for developing benign and malignant tumors. The NFI gene has been cloned and maps to 17q11.2, and the gene product acts as a tumor suppressor gene. Here we analyzed the role of mutations in TP53 in four malignant NFI tumors. Mutations were found in 3 out of 4 tumors. One of these mutations is a common missense mutation in codon 278 in one of the previously identified hot spots for mutations. The two other are hitherto unreported mutations, including a splice mutation of exon 3 and a nonsense mutation in exon 4. In addition, these four tumors also showed loss of heterozygosity (LOH) for markers on chromosome 17 in the region of TP53. Malignant NFI tumors are initiated by a somatic inactivation of the second NFI allele. Tumor progression, however, occurs by accumulation of additional genetic abnormalities, such as homozygous inactivation of TP53, as demonstrated in this paper.

Fracture repair offers an opportunity to study the physiology of bone formation at the fracture site. Isolation of growth factors from bone matrix has implicated growth factors as participants in bone physiology. We therefore examined the expression patterns of aFGF, IGF-I, PDGF, and TGF-beta during fracture repair. An animal model has been developed to study repair of tibial fractures. The model provides both reproducible and quantifiable results, allowing the fracture repair process to be divided into four stages (Bourque et al., Lab. Anim. Sci 42: 369-374, 1992). Fractured tibiae were examined immunohistochemically with polyclonal antibodies to four growth factors. PDGF was visualized in macrophages in close proximity to the periosteum during stage 1. aFGF was visualized in cells of the expanded cambial layer and was associated with a rapid increase in the population of fibroblast-like mesenchymal cells during stage 2. IGF-I was visualized in young chondroblasts at the edge of the cartilage mass replacing the fibrous callus during stage 3. TGF-beta was visualized in calcified matrix producing chondrocytes at the edge of ossification fronts penetrating the cartilage callus during stage 4. The immunohistochemical results suggest that these growth factors act as local simulators of the repair process.

We have devised a processing technique to embed calcified tissues, such as bone and tooth enamel, in paraffin, to preserve the delicate antigenic sites of molecules such as growth factors. The same technique, omitting the decalcification step, allows delicate tissues, such as axolotl embryos (Ambystoma mexicanum) containing large yolk masses, to be easily handled during tissue processing and to be serially sectioned. Specimens were all fixed in periodate-lysine-paraformaldehyde (PLP) fixative at 5 degrees C. Bone and teeth were decalcified in an EDTA-G solution at -4 degrees C. Maintaining a temperature of 5 degrees C, the decalcified samples were then washed (with PBS, pH 7.2, under vacuum) to remove glycerol. Both the decalcified tissues and the yolky embryos were dehydrated through an ascending series of isopropanol and embedded in low melting-point paraffin under vacuum. Acidic fibroblast growth factor (aFGF) was located in cells of the expanded cambial layer in the early fracture calluses of male CD-1 mice, demonstrating retention of antigenic sites. The results reported here have not previously been obtained with existing processing and embedding techniques.

Male CD-1 mice, 4 to 6 months of age, were used to establish a reproducible model to study the stages of fracture repair. A custom-designed fracture apparatus was constructed, and trials with it demonstrated its capacity to reliably reproduce a closed fracture of the tibia. Dietary and sleep habits in the treated mice were the same as unfractured control mice. Four stages of fracture repair were documented and the duration of each stage was quantifiable and reproducible. The last stage of fracture repair was completed by 21 days postfracture. The reproducibility of the fracture, the reproducibility of the times and stages of fracture repair, the relatively short time to complete the fracture repair process, and the minimal discomfort which allowed the mice to maintain a normal daily routine, suggest that this is an ideal animal model for studying the fracture repair process.

During neurulation, neural crest cells migrate to many regions of the body to give rise to a wide variety of cell types. Many premigratory neural crest cells are pluripotent, their potency for differentiation being gradually restricted as they migrate along definite pathways and interact with factors present in the microenvironment. Effects of growth factors on these cells have been discussed in the present review. Mediation of growth factors in differentiation varies with the cell type. Growth factors exert a direct influence on the differentiation of neural and other related neural crest-derived tissues such as endocrinal tissues but evidence for such influences on neural crest-derived mesenchymal tissues is limited. For example, NGF, BDNF, and other factors present in neural tube extracts and glioma cell conditioned medium are essential for the differentiation of sensory neurons. Similarly, NGF, insulin, IGFs and possibly other undescribed factors are necessary for the differentiation of sympathetic neurons. IGFs also enhance the proliferation of mesenchymal derivatives of both neural crest and mesodermal origin. Glucocorticoid-mediated differentiation of neural crest-derived chromaffin endocrine cells that are ontogenetically closely related to sympathetic neurons can be inhibited by NGF, and chromaffin cells can be induced to express the neuronal phenotype by NGF. Some growth factors, such as NGF, act on neural crest- and not on placodally-derived neurons, whether the former are sensory or sympathetic. Placodal sensory neurons possess NGF receptors, but only display a limited response to NGF, perhaps because of low affinity of the receptors. Other growth factors, such as BDNF, selectively act upon sensory neurons, whether neural crest- or placodally-derived. Although extracellular matrix products play a role in initiating the differentiative process, signals from growth factors are necessary for the establishment of the functionally competent phenotype of neural crest-derived neurons, a situation that does not apply for neural crest-derived mesenchymal cells. It is interactions with ECM components deposited by epithelia that govern the differentiation of mesenchymal derivatives. Growth factors do effect proliferation of mesenchymal derivatives and inhibit mesenchymal differentiation. Although direct involvement of single growth factors in transformation o f one mesenchymal phenotype to another has not been reported so far, their localization at sites of epithelial-mesenchymal interactions in palate teeth and mandible, and the ability of excess growth factors to interrupt normal development is suggestive of their possible involvement. One group of growth factors, BMPs, can influence differentiation of cartilage, including those of neural crest origin.(ABSTRACT TRUNCATED AT 400 WORDS)

SUMMARY Y alphoid primers in combination with Alu and LINEs primers generated new DNA fragments in polymerase chain reactions (PCR) on DNA from a Y-only somatic cell hybrid but not from X-only, 3-only, or 21-only hybrids. X alphoid primers used in a similar manner generated new DNA fragments from the X-only hybrid, and 1 of the primers (X2) also generated new DNA fragments on 3-only and 21-only hybrids when used in conjunction with Alu or LINEs primers. In all but one case, consensus alphoid primers generated new chromosome-specific fragments in PCR reactions with the Alu or LINEs primers. A search for cryptic Alu- or alphoid-alone PCR products as the source for one Alu-alphoid band (chosen at random) was negative. Partial sequencing of products demonstrated that alphoid and Alu sequences were indeed contiguous in some newly synthesized DNA fragments. While Alu or LINEs primers generate smears of DNA fragments on total human DNA, the alphoid-nonalphoid repeat combinations generated electrophoretically distinguishable bands of DNA when the template was total DNA. While these were distinguishable with different chromosome-specific alphoid primers, the DNA fragments were not of the same sizes as those generated with the chromosome-only hybrids.

Monoclonal antibodies with defined specifications for individual cytokeratins were used to stain the epithelia of the external auditory meatus, the middle ear and cholesteatoma. The observed staining indicated that the epithelium of the external auditory meatus has a pattern of keratin expression typical of epidermis in general and the epithelium of the middle ear resembles simple columnar epithelia. The pattern of staining of cholesteatoma closely resembled that of the skin of the external auditory meatus.

This overview discusses evolution in the context of craniofacial development and developmental processes. It begins with a discussion of the origins of the craniofacial tissues in the dentine and bone of the dermal denticles of the Ordovician jawless vertebrates, followed by a brief discussion of the mechanisms responsible for the evolution of the jaws and the origin of vertebrate dentition. Then the unique neural crest cell origin of the craniofacial skeletogenic and odontogenic tissues is discussed with emphasis on the constancy of rostrocaudal polarization of the skeletogenic cranial neural crest. Given this constancy, the variation in the craniofacial region that occurs across the vertebrates must arise because of epigenetic interactions that evoke the differentiation of craniofacial tissues. These are discussed in the context of epigenetic cascades of epithelial-mesenchymal interactions and how such epigenetic control has itself evolved. Although several mechanisms are considered, emphasis is placed on variation of the timing of development processes (heterochrony).

Osteogenesis was not initiated when Meckel's cartilages from embryonic chicks of Hamburger and Hamilton (H. H.) stages 38 and 39 were recombined with mandibular epithelia obtained from embryos of H. H. stage 22 (a stage when an epithelial-mesenchymal interaction elicits osteogenesis from mandibular mesenchyme) and grafted to the chorioallantoic membranes of host embryos for 7 to 21 days. Failure of osteogenesis was not because the cartilage inhibited or blocked the osteogenesis-initiating capabilities of mandibular epithelium for mandibular epithelia could still elicit osteogenesis when removed from Meckel's cartilages and recombined with mandibular mesenchyme. Chondrocyte hypertrophy is associated with osteogenesis in other cartilages, including Meckel's cartilage from rodent embryos. However, Meckel's cartilages from chick embryos of H. H. stages 34, 38, and 39 failed to hypertrophy when cultured in the presence of 7.5 nM thyroxine (3,3',5-triiodo-L-thyroxine), although H. H. stage 28 tibial chondrocytes cocultured with Meckel's cartilage did hypertrophy. Therefore, avian Meckelian chondrocytes fail to hypertrophy or to produce osteoprogenitor cells in response to stimuli known to evoke these events in other skeletal cells.

Mandibular epithelia and mesenchyme from chick embryos of Hamburger and Hamilton (H.H.) stage 18-25 were cultured intact, in isolation, or in recombinations in the presence or absence of 5-40 ng/ml epidermal growth factor (EGF). 3H-thymidine labelling demonstrated that mesenchyme influenced epithelial mitotic activity and vice versa. EGF can substitute for the epithelial effect. The stimulation of mesenchymal proliferation by H.H. 18 and 22 epithelia correlated with high levels of epithelial proliferation. Epithelial proliferation was low at H.H. 25 and unaffected by mesenchyme or by EGF. Epithelial stimulation of mesenchymal proliferation began earlier (H.H. 18) than did mesenchymal stimulation of epithelial proliferation (H.H. 22); i.e., within the ages tested, the epithelium initiated these reciprocal mitogenic interactions. That epithelial dependence on mesenchyme coincided with epithelial bone-evoking properties, suggested a) that mesenchyme promotes or maintains epithelial bone-promoting activity and b) that the critical differentiative influence of epithelium on mesenchyme is a mitogenic one. The temporal correlation between a sharp decline in mesenchymal proliferation and termination of the osteogenic epithelial-mesenchymal interaction at H.H. 25 further supports a connection between epithelial maintenance of mesenchymal proliferation and epithelial evocation of osteogenesis.

Standard autoradiographic techniques have been used to describe the temporal pattern in the transfer of radioactive carbon from the chlorophyte Tetraselmis suesica (Kylin) to females of the neritic copepod Temora longicornis (Muller, 1792). After 4 and 6 h exposure to labelled phytoplankton, silver grains were deposited only in the cells of the gut-wall. After 24 h exposure, grains were deposited in most tissues, with highest concentrations in cells of the gut-wall and in oocytes within the ovary. Experiments with Centropages typicus (Kroyer), Centropages hamatus (Lilljeborg), and Labidocera aestiva Wheeler demonstrated that much of the assimilated label was not turning over rapidly and that the amount of label assimilated was correlated with known food preferences of these species.

An epithelial hyperplasia is one of the reactions of skin and oral mucosa to chemical and mechanical insult. It is usually assumed that this reaction produces a more effective epithelial harrier, but there is no information as to whether a less permeable (issue results. To examine this question, hyperplasia was induced in the cheek pouches of hamsters by either chemical treatment with 0.0025% TPA in acetone or by mechanical abrasion with a rotating mop; untreated hamsters served as controls. The animals were killed and the cheek pouches were removed, mounted in diffusion chambers and the permeability to labelled water and horseradish peroxidase (IIRPO) determined. The results showed that higher values were obtained for the permeability constant of hyperplastic epithelia than for that of control tissue, suggesting that an increased epithelial thickness is not necessarily associated with an improved permeability barrier function. The presence of an inferior barrier layer in hyperplastic epithelia may be related to the increased rate of turnover of this tissue.

There is evidence for synergy between tobacco and alcohol in the etiology of oral cancer but the reason for such an effect is unclear. One possible explanation is that alcohol enhances the penetration of carcinogens through the oral lining. We measured the permeability in vitro of three regions of porcine oral mucosa to the tobacco associated carcinogen, nitrosonornicotine (NNN) alone and in the presence of 5% or 50% ethanol. 50% ethanol did not significantly alter the permeability of oral mucosa to NNN except for buccal mucosa, where it was reduced. However, there was a significant increase in the permeability of gingiva and floor of mouth mucosa (but not buccal mucosa) in the presence of 5% ethanol; this increase occurred after far shorter exposures for floor of mouth than for gingiva. These results accord well with studies showing that the floor of mouth is a "high risk area" for oral carcinoma and that there is an increased relative risk of oral cancer for heavy smokers and drinkers and, in particular, for those individuals who consume beverages with a low alcohol content.

The initiation of osteogenesis in the mandibular mesenchyme of the embryonic chick at 7 days is dependent upon an epithelial induction which occurs in the mandible up to the fourth day in ovo. In the present study, transfilter tissue recombinations were used to study this inductive mechanism. The epithelial and mesenchymal components of the mandibles were separated before the completion of the induction and recombined to form transfilter explants which were either cultured for 9 days or grafted onto the chorioallantoic membrane for host embryos for 7 days. Control experiments demonstrated that the tissue separation and recombination techniques did not interfere with the normal epithelial induction, and confirmed that mandibular mesenchyme isolated at this stage was incapable of forming bone. Bone was observed in 86% of the CAM-grafted intact mandible controls and in 80% of the cultured intact mandible controls. Bone failed to form in the mesenchyme of transfilter explants when Millipore filters with 0.45 micrometer pores were used. Bone was observed as frequently as in control explants when the mandibular mesenchyme was separated from its epithelium by 0.8 micrometer or 0.4 micrometer porosity Nuclepore filters. Only about 30% of the transfilter explants prepared with 0.1 micrometer porosity Nuclepore filters formed bone and none of the explants prepared with 0.03 micrometer porosity Nuclepore filters formed bone. SEM studies demonstrated a distinct correlation between the formation of bone in transfilter explants and the ability of the epithelium and mesenchyme to penetrate the pores of the filters. Thus, the present study provides evidence that the site of the induction is restricted to the epithelial-mesenchymal interface, and that the induction is not mediated by a diffusible substance. The nature of the inductive mechanism is discussed with respect to this and other recent studies which suggest that the induction may be mediated by a non-diffusible epithelial cell product resident in the epithelial basal lamina.

Enzymatic digestion followed by recombination of epithelia and ectomesenchyme from embryonic sclera and mandibles has been used to demonstrate that isolated scleral ectomesenchyme is only able to form scleral ossicles if in prior contact with scleral epithelium until H.H. stage 36 (10 days of incubation); that this induction by epithelial scleral papillae is a prolonged one, commencing as early as H.H. stage 30 (6.5-7 days); that scleral ectomesenchyme can respond to mandibular epithelium by forming bony ossicles and that mandibular ectomesenchyme can respond to scleral epithelium by forming bony rods, i.e. these interactions are not site, time or tissue specific. Scleral epithelia did not form scleral papillae when maintained alone in vitro or when recombined with scleral ectomesenchyme and maintained in vitro. Nor did papillae form when mandibular epithelia were cultured with scleral ectomesenchyme. These results, coupled with data from the literature, are used to argue that papillae will only form when scleral epithelia are under the tension generated by normal intraocular pressure of the growing eye.