Gestational exposure to ethanol suppresses msx2 expression in developing mouse embryos

Ethanol acts as a teratogen in developing fetuses causing abnormalities of the brain, heart, craniofacial bones, and limb skeletal elements. To assess whether some teratogenic actions of ethanol might occur via dysregulation of msx2 expression, we examined msx2 expression in developing mouse embryos exposed to ethanol on embryonic day (E) 8 of gestation and subjected to whole mount in situ hybridization on E11–11.5 using a riboprobe for mouse msx2. Control mice exhibited expression of msx2 in developing brain, the developing limb buds and apical ectodermal ridge, the lateral and nasal processes, olfactory pit, palatal shelf of the maxilla, the eye, the lens of the eye, otic vesicle, prevertebral bodies (notochord), and endocardial cushion. Embryos exposed to ethanol in utero were significantly smaller than their normal counterparts and did not exhibit expression of msx2 in any structures. Similarly, msx2 expression, as determined by reverse transcription–PCR and Northern blot hybridization, was reduced ≈40–50% in fetal mouse calvarial osteoblastic cells exposed to 1% ethanol for 48 hr while alkaline phosphatase was increased by 2-fold and bone morphogenetic protein showed essentially no change. Transcriptional activity of the msx2 promoter was specifically suppressed by alcohol in MC3T3-E1 osteoblasts. Taken together, these data demonstrate that fetal alcohol exposure decreases msx2 expression, a known regulator of osteoblast and myoblast differentiation, and suggest that one of the “putative” mechanisms for fetal alcohol syndrome is the inhibition of msx2 expression during key developmental periods leading to developmental retardation, altered craniofacial morphogenesis, and cardiac defects.

Purified hematopoietic stem cells without facilitating cells can repopulate fully allogeneic recipients across entire major histocompatibility complex transplantation barrier in mice

We report herein the successful long term engraftment of highly purified hematopoietic stem cells (HSCs) without any facilitating cells in fully allogeneic recipient mice across the entire major histocompatibility complex (MHC) transplantation barrier. This finding challenges the assumption that highly purified marrow HSCs alone cannot produce long-lived allogeneic bone marrow chimeras across the MHC barrier. In the present experiments, 1 × 105 HSCs from 5-fluorouracil (5-FU)-treated donors, without any facilitating cells, have been found to repopulate lethally irradiated fully allogeneic recipients. Low density, lineage-negative (CD4−, CD8−, B220−, Mac-1−, Gr-1−), CD71-negative, class I highly positive, FACS-sorted cells from 5-FU-treated C57BL/6 (B6) donor mice were transplanted into lethally irradiated BALB/c recipients. (BALB/c → BALB/c) → BALB/c T cell-depleted marrow cells used as compromised cells were also transplanted into the recipients to permit experiments to be pursued over a long period of time. Cells of donor origin in all recognized lineages of hematopoietic cells developed in these allogeneic chimeras. One thousand HSCs were sufficient to repopulate hemiallogeneic recipients, but 1 × 104 HSCs alone from 5-FU-treated donors failed to repopulate the fully allogeneic recipients. Transplantation of primary marrow stromal cells or bones of the donor strain into recipient, together with 1 × 104 HSCs, also failed to reconstitute fully allogeneic recipients. Suppression of resistance of recipients by thymectomy or injections of granulocyte colony-stimulating factor before stem cell transplantation enhanced the engraftment of allogeneic HSCs. Our experiments show that reconstitution of all lymphohematopoietic lineages across the entire MHC transplantation barriers may be achieved by transplanting allogeneic HSCs alone, without any facilitating cells, as long as a sufficient number of HSCs is transplanted.

Prehistoric birds from New Ireland, Papua New Guinea: Extinctions on a large Melanesian island

At least 50 species of birds are represented in 241 bird bones from five late Pleistocene and Holocene archaeological sites on New Ireland (Bismarck Archipelago, Papua New Guinea). The bones include only two of seabirds and none of migrant shorebirds or introduced species. Of the 50 species, at least 12 (petrel, hawk, megapode, quail, four rails, cockatoo, two owls, and crow) are not part of the current avifauna and have not been recorded previously from New Ireland. Larger samples of bones undoubtedly would indicate more extirpated species and refine the chronology of extinction. Humans have lived on New Ireland for ca. 35,000 years, whereas most of the identified bones are 15,000 to 6,000 years old. It is suspected that most or all of New Ireland’s avian extinction was anthropogenic, but this suspicion remains undetermined. Our data show that significant prehistoric losses of birds, which are well documented on Pacific islands more remote than New Ireland, occurred also on large, high, mostly forested islands close to New Guinea.

Fish and mammals in the economy of an ancient Peruvian kingdom

Fish and mammal bones from the coastal site of Cerro Azul, Peru shed light on economic specialization just before the Inca conquest of A.D. 1470. The site devoted itself to procuring anchovies and sardines in quantity for shipment to agricultural communities. These small fish were dried, stored, and eventually transported inland via caravans of pack llamas. Cerro Azul itself did not raise llamas but obtained charqui (or dried meat) as well as occasional whole adult animals from the caravans. Guinea pigs were locally raised. Some 20 species of larger fish were caught by using nets; the more prestigious varieties of these show up mainly in residential compounds occupied by elite families.

Diverse roles of the tumor necrosis factor family member TRANCE in skeletal physiology revealed by TRANCE deficiency and partial rescue by a lymphocyte-expressed TRANCE transgene

Tumor necrosis factor-related, activation-induced cytokine (TRANCE), a tumor necrosis factor family member, mediates survival of dendritic cells in the immune system and is required for osteoclast differentiation and activation in the skeleton. We report the skeletal phenotype of TRANCE-deficient mice and its rescue by the TRANCE transgene specifically expressed in lymphocytes. TRANCE-deficient mice showed severe osteopetrosis, with no osteoclasts, marrow spaces, or tooth eruption, and exhibited profound growth retardation at several skeletal sites, including the limbs, skull, and vertebrae. These mice had marked chondrodysplasia, with thick, irregular growth plates and a relative increase in hypertrophic chondrocytes. Transgenic overexpression of TRANCE in lymphocytes of TRANCE-deficient mice rescued osteoclast development in two locations in growing long bones: excavation of marrow cavities permitting hematopoiesis in the marrow spaces, and remodeling of osteopetrotic woven bone in the shafts of long bones into histologically normal lamellar bone. However, osteoclasts in these mice failed to appear at the chondroosseous junction and the metaphyseal periosteum of long bones, nor were they present in tooth eruption pathways. These defects resulted in sclerotic metaphyses with persistence of club-shaped long bones and unerupted teeth, and the growth plate defects were largely unimproved by the TRANCE transgene. Thus, TRANCE-mediated regulation of the skeleton is complex, and impacts chondrocyte differentiation and osteoclast formation in a manner that likely requires local delivery of TRANCE.

Soft-tissue characters in higher primate phylogenetics

Recent research has cast doubt on the reliability of bones and teeth for reconstructing phylogenetic relationships among higher primate species and genera. Herein, we investigate whether this problem is confined to hard tissues by examining the utility of higher primate soft-tissue characters for reconstructing phylogenetic relationships at low taxonomic levels. We use cladistic methods to analyze 197 soft-tissue characters for the extant hominoids and then compare the resulting phylogenetic hypotheses with the group's consensus molecular phylogeny, which is widely considered to be accurate. We show that the soft-tissue characters yield robust phylogenetic hypotheses that are compatible with the molecular phylogeny. Given the strength of the evidence for molecular phylogeny, these results indicate that, unlike craniodental hard-tissue characters, soft tissues are reliable for reconstructing phylogenetic relationships among higher primate species and genera. Thus, in higher primates at least, some types of morphological data are more useful than others for phylogeny reconstruction.

Deletion of long-range regulatory elements upstream of SOX9 causes campomelic dysplasia

Campomelic dysplasia (CD) is a rare, neonatal human chondrodysplasia characterized by bowing of the long bones and often associated with male-to-female sex-reversal. Patients present with either heterozygous mutations in the SOX9 gene or chromosome rearrangements mapping at least 50 kb upstream of SOX9. Whereas mutations in SOX9 ORF cause haploinsufficiency, the effects of translocations 5′ to SOX9 are unclear. To test whether these rearrangements also cause haploinsufficiency by altering spatial and temporal expression of SOX9, we generated mice transgenic for human SOX9-lacZ yeast artificial chromosomes containing variable amounts of DNA sequences upstream of SOX9. We show that elements necessary for SOX9 expression during skeletal development are highly conserved between mouse and human and reveal that a rearrangement upstream of SOX9, similar to those observed in CD patients, leads to a substantial reduction of SOX9 expression, particularly in chondrogenic tissues. These data demonstrate that important regulatory elements are scattered over a large region upstream of SOX9 and explain how particular aspects of the CD phenotype are caused by chromosomal rearrangements 5′ to SOX9.

Evidence of termite foraging by Swartkrans early hominids

Previous studies have suggested that modified bones from the Lower Paleolithic sites of Swartkrans and Sterkfontein in South Africa represent the oldest known bone tools and that they were used by Australopithecus robustus to dig up tubers. Macroscopic and microscopic analysis of the wear patterns on the purported bone tools, pseudo bone tools produced naturally by known taphonomic processes, and experimentally used bone tools confirm the anthropic origin of the modifications. However, our analysis suggests that these tools were used to dig into termite mounds, rather than to dig for tubers. This result indicates that early hominids from southern Africa maintained a behavioral pattern involving a bone tool material culture that may have persisted for a long period and strongly supports the role of insectivory in the early hominid diet.

Behavioral inferences from the Skhul/Qafzeh early modern human hand remains

Two groups of humans are found in the Near East ≈100,000 years ago, the late archaic Neanderthals and the early modern Skhul/Qafzeh humans. Observations that Neanderthals were more heavily muscled, had stronger upper-limb bones, and possessed unusual shapes and orientations of some upper-limb joint complexes relative to the Skhul/Qafzeh hominids, have led some researchers to conclude that significant between-group upper-limb-related behavioral differences must have been present, despite the association of the two groups with similar Middle Paleolithic archeological complexes. A three-dimensional morphometric analysis of the hand remains of the Skhul/Qafzeh hominids, Neanderthals, early and late Upper Paleolithic humans, and Holocene humans supports the dichotomy. The Skhul/Qafzeh carpometacarpal remains do not have any unique morphologies relative to the other fossil samples remains examined. However, in the functionally significant metacarpal 1 and 3 bases they resemble Upper Paleolithic humans, not Neanderthals. Furthermore, the Skhul/Qafzeh sample differs significantly from the Neanderthals in many other aspects of hand functional anatomy. Given the correlations between changes in tool technologies and functional adaptations seen in the hands of Upper Paleolithic humans, it is concluded that the Skhul/Qafzeh hand remains were adapted to Upper Paleolithic-like manipulative repertoires. These results support the inference of significant behavioral differences between Neanderthals and the Skhul/Qafzeh hominids and indicate that a significant shift in human manipulative behaviors was associated with the earliest stages of the emergence of modern humans.

Haploinsufficiency of Sox9 results in defective cartilage primordia and premature skeletal mineralization

In humans, SOX9 heterozygous mutations cause the severe skeletal dysmorphology syndrome campomelic dysplasia. Except for clinical descriptions, little is known about the pathogenesis of this disease. We have generated heterozygous Sox9 mutant mice that phenocopy most of the skeletal abnormalities of this syndrome. The Sox9+/− mice died perinatally with cleft palate, as well as hypoplasia and bending of many skeletal structures derived from cartilage precursors. In embryonic day (E)14.5 heterozygous embryos, bending of radius, ulna, and tibia cartilages was already prominent. In E12.5 heterozygotes, all skeletal elements visualized by using Alcian blue were smaller. In addition, the overall levels of Col2a1 RNA at E10.5 and E12.5 were lower than in wild-type embryos. We propose that the skeletal abnormalities observed at later embryonic stages were caused by delayed or defective precartilaginous condensations. Furthermore, in E18.5 embryos and in newborn heterozygotes, premature mineralization occurred in many bones, including vertebrae and some craniofacial bones. Because Sox9 is not expressed in the mineralized portion of the growth plate, this premature mineralization is very likely the consequence of allele insufficiency existing in cells of the growth plate that express Sox9. Because the hypertrophic zone of the heterozygous Sox9 mutants was larger than that of wild-type mice, we propose that Sox9 also has a role in regulating the transition to hypertrophic chondrocytes in the growth plate. Despite the severe hypoplasia of cartilages, the overall organization and cellular composition of the growth plate were otherwise normal. Our results suggest the hypothesis that two critical steps of the chondrocyte differentiation pathway are sensitive to Sox9 dosage. First, an early step presumably at the stage of mesenchymal condensation of cartilage primordia, and second, a later step preceding the transition of chondrocytes into hypertrophic chondrocytes.

Targeted overexpression of parathyroid hormone-related peptide in chondrocytes causes chondrodysplasia and delayed endochondral bone formation.

Parathyroid hormone-related peptide (PTHrP) was initially identified as a product of malignant tumors that mediates paraneoplastic hypercalcemia. It is now known that the parathyroid hormone (PTH) and PTHrP genes are evolutionarily related and that the products of these two genes share a common receptor, the PTH/PTHrP receptor. PTHrP and the PTH/PTHrP receptor are widely expressed in both adult and fetal tissues, and recent gene-targeting and disruption experiments have implicated PTHrP as a developmental regulatory molecule. Apparent PTHrP functions include the regulation of endochondral bone development, of hair follicle formation, and of branching morphogenesis in the breast. Herein, we report that overexpression of PTHrP in chondrocytes using the mouse type II collagen promoter induces a novel form of chondrodysplasia characterized by short-limbed dwarfism and a delay in endochondral ossification. This features a delay in chondrocyte differentiation and in bone collar formation and is sufficiently marked that the mice are born with a cartilaginous endochondral skeleton. In addition to the delay, chondrocytes in the transgenic mice initially become hypertrophic at the periphery of the developing long bones rather than in the middle, leading to a seeming reversal in the pattern of chondrocyte differentiation and ossification. By 7 weeks, the delays in chondrocyte differentiation and ossification have largely corrected, leaving foreshortened and misshapen but histologically near-normal bones. These findings confirm a role for PTHrP as an inhibitor of the program of chondrocyte differentiation. PTHrP may function in this regard to maintain the stepwise differentiation of chondrocytes that initiates endochondral ossification in the midsection of endochondral bones early in development and that also permits linear growth at the growth plate later in development.

Targeted disruption of the arylsulfatase B gene results in mice resembling the phenotype of mucopolysaccharidosis VI.

Mucopolysaccharidosis VI (MPS VI) is a lysosomal storage disease with autosomal recessive inheritance caused by a deficiency of the enzyme arylsulfatase B (ASB), which is involved in degradation of dermatan sulfate and chondroitin 4-sulfate. A MPS VI mouse model was generated by targeted disruption of the ASB gene. Homozygous mutant animals exhibit ASB enzyme deficiency and elevated urinary secretion of dermatan sulfate. They develop progressive symptoms resembling those of MPS VI in humans. Around 4 weeks of age facial dysmorphia becomes overt, long bones are shortened, and pelvic and costal abnormalities are observed. Major alterations in bone formation with perturbed cartilaginous tissues in newborns and widened, perturbed, and persisting growth plates in adult animals are seen. All major parenchymal organs show storage of glycosaminoglycans preferentially in interstitial cells and macrophages. Affected mice are fertile and mortality is not elevated up to 15 months of age. This mouse model will be a valuable tool for studying pathogenesis of MPS VI and may help to evaluate therapeutical approaches for lysosomal storage diseases.

Premature suture closure and ectopic cranial bone in mice expressing Msx2 transgenes in the developing skull.

The coordinate growth of the brain and skull is achieved through a series of interactions between the developing brain, the growing bones of the skull, and the fibrous joints, or sutures, that unite the bones. These interactions couple the expansion of the brain to the growth of the bony plates at the sutures. Craniosynostosis, the premature fusion of the bones of the skull, is a common birth defect (1 in 3000 live births) that disrupts coordinate growth and often results in profoundly abnormal skull shape. Individuals affected with Boston-type craniosynostosis, an autosomal dominant disorder, bear a mutated copy of MSX2, a homeobox gene thought to function in tissue interactions. Here we show that expression of the mouse counterpart of this mutant gene in the developing skulls of transgenic mice causes craniosynostosis and ectopic cranial bone. These mice provide a transgenic model of craniosynostosis as well as a point of entry into the molecular mechanisms that coordinate the growth of the brain and skull.