Maternally controlled (beta)-catenin-mediated signaling is required for organizer formation in the zebrafish.

We have identified and characterized a zebrafish recessive maternal effect mutant, ichabod, that results in severe anterior and dorsal defects during early development. The ichabod mutation is almost completely penetrant, but exhibits variable expressivity. All mutant embryos fail to form a normal embryonic shield; most fail to form a head and notochord and have excessive development of ventral tail fin tissue and blood. Abnormal dorsal patterning can first be observed at 3.5 hpf by the lack of nuclear accumulation of (beta)-catenin in the dorsal yolk syncytial layer, which also fails to express bozozok/dharma/nieuwkoid and znr2/ndr1/squint. At the onset of gastrulation, deficiencies in expression of dorsal markers and expansion of expression of markers of ventral tissues indicate a dramatic alteration of dorsoventral identity. Injection of (beta)-catenin RNA markedly dorsalized ichabod embryos and often completely rescued the phenotype, but no measurable dorsalization was obtained with RNAs encoding upstream Wnt pathway components. In contrast, dorsalization was obtained when RNAs encoding either Bozozok/Dharma/Nieuwkoid or Znr2/Ndr1/Squint were injected. Moreover, injection of (beta)-catenin RNA into ichabod embryos resulted in activation of expression of these two genes, which could also activate each other. RNA injection experiments strongly suggest that the component affected by the ichabod mutation acts on a step affecting (beta)-catenin nuclear localization that is independent of regulation of (beta)-catenin stability. This work demonstrates that a maternal gene controlling localization of (beta)-catenin in dorsal nuclei is necessary for dorsal yolk syncytial layer gene activity and formation of the organizer in the zebrafish.

Identification of a mouse germ cell-less homologue with conserved activity in Drosophila.

Drosophila Germ cell-less (Gcl) has previously been shown to be important in early events during the formation of pole cells, which are the germ cell precursors in the fly. We have isolated a 524 amino acid mouse gene with 32% identity and 49% similarity to Drosophila gcl, termed mgcl-1. Like Drosophila Gcl, mGcl-1 localizes to the nuclear envelope. Ectopic expression of mgcl-1 in Drosophila rescues the gcl-null phenotype, indicating that mGcl-1 is a functional homologue of Gcl. mgcl-1 maps to chromosome 6 at 47.3 cM, and is expressed at low levels at all embryonic stages examined from 8.5 to 18.5 d.p.c. as well as in many adult tissues. Different from Drosophila gcl, mgcl-1 is not highly expressed at the time the primordial germ cells appear in the mouse, but high mgcl-1 expression is found in selected mouse adult male germ cells. The differences in these expression patterns in light of conserved activity between the two genes is discussed.

germ cell-less is required only during the establishment of the germ cell lineage of Drosophila and has activities which are dependent and independent of its localization to the nuclear envelope.

The germ cell precursors of Drosophila (pole cells) are specified by maternally supplied germ plasm localized to the posterior pole of the egg. One component of the germ plasm, germ cell-less (gcl) mRNA, encodes a novel protein which specifically localizes to the nuclear envelope of the pole cell nuclei. In addition to its maternal expression, gcl is zygotically expressed through embryonic development. In this report, we have characterized a null allele of germ cell-less to determine its absolute requirement during development. We have found that gcl activity is required only for the establishment of the germ cell lineage. Most embryos lacking maternal gcl activity fail to establish a germline. No other developmental defects were detected. Examination of germline development in these mutant embryos revealed that gcl activity is required for proper pole bud formation, pole cell formation, and pole cell survival. Using this null mutant we have also assayed the activity of forms of Gcl protein with altered subcellular distribution and found that localization to the nuclear envelope is crucial for promoting pole cell formation, but not necessary to initiate and form proper pole buds. These results indicate that gcl acts in at least two different ways during the establishment of the germ cell lineage.

Early chick limb cartilaginous elements possess polarizing activity and express hedgehog-related morphogenetic factors.

Skeletal patterning and morphogenesis in the developing limb are thought to be regulated by instructive factors and cues from the zone of polarizing activity (ZPA), the apical ectodermal ridge (AER), and the dorsal ectoderm. However, the activities of the ZPA and AER dwindle early in embryogenesis and soon after ceases, when in fact the proximal skeletal elements are still rudimentary in structure and the more distal ones are yet to become recognizable. Thus, we asked whether the chondrocytes emerging within each mesenchymal condensation may themselves start expressing properties similar to those of ZPA and/or AER and, in so doing, may bring skeletal development to completion. Indeed, we found that the cartilaginous, but not precartilaginous, tissues in early chick limbs possess ZPA-like properties. They expressed an endogenous factor related to Sonic hedgehog (Shh), most likely Indian hedgehog (Ihh), and when fragments were grafted to the anterior margin of host stage 16-20 chick wing buds, they induced supernumerary skeletal elements (polarizing activity). The acquisition of polarizing activity by the cartilaginous structures followed clear proximo-to-distal and posterior-to-anterior routes. Thus, (1) stage 25 cartilaginous humerus had polarizing activity while stage 25 prospective radius did not, (2) posteriorly-located stage 29 ulna had stronger activity than anteriorly-located stage 29 radius, and (3) ulna's diaphysis had stronger activity at stage 29 than 31 while radius's diaphysis was stronger at stage 31 than 29. Prior to inducing extra digit formation, the cartilaginous grafts induced Hoxd-12 and Hoxd-13 gene expression in adjacent competent mesenchymal tissue. Strikingly, the cartilaginous grafts activity also expression of Shh and polarizing activity in adjacent mesenchyme, which ZPA grafts cannot do; thus, the cartilaginous structures displayed activities "upstream" of those of the ZPA. The results support our hypothesis that chondrocytes may themselves direct skeletal morphogenesis. In so doing and as a result of their inductive activities, the cells may also have an important role in the completion of limb patterning and morphogenesis.

Syndecan-3 and the control of chondrocyte proliferation during endochondral ossification.

During endochondral ossification, chondrocytes progress through several stages of maturation before they are replaced by bone cells. Chondrocyte proliferation, the first step in this complex multistage process, is strictly controlled both spatially and temporally but its underlying mechanisms of regulation remain unclear. In this study we asked whether chondrocytes produce syndecan-3, a cell surface receptor for growth factors such as fibroblast growth factor 2 (FGF-2), and whether syndecan-3 may play a role in proliferation during chondrocyte maturation. We found that proliferating immature cartilage from chick embryo tibia and sternum contained significant amounts of syndecan-3 mRNA, whereas mature hypertrophic cartilage contained markedly lower transcript levels. Immunohistochemical analyses on sections of Day 18 chick embryo tibia revealed that syndecan-3 was spatially restricted and indeed detectable only in immature proliferating chondrocytes in the top zone of growth plate. These syndecan-3-rich proliferating chondrocytes lay beneath developing articular chondrocytes rich in their typical matrix protein tenascin-C, resulting in a striking boundary between these two populations of chondrocytes. Immature proliferating chondrocyte populations reared in growth-promoting culture conditions displayed strong continuous syndecan-3 gene expression; upon induction of maturation by vitamin C treatment, syndecan-3 gene expression was markedly down-regulated. Treatment with FGF-2 for 24 h stimulated both syndecan-3 gene expression and chondrocyte proliferation; this growth stimulation was counteracted by cotreatment with heparinase I or III. The results of the study indicate that syndecan-3 participates in the maturation of chondrocytes during endochondral ossification and represents a regulator of the proliferative phase of this multistage process.

Expression of syndecan-3 and tenascin-C: possible involvement in periosteum development.

The development of cartilaginous elements of long bone during embryogenesis and postnatal bone repair processes is a complex process that involves skeletal cells and surrounding mesenchymal periosteal cells. Relatively little is known of the mechanisms underlying these processes. Previous studies from this and other laboratories have suggested that the extracellular matrix protein tenascin-C is involved in skeletogenesis. Using in situ hybridization and immunofluorescence, we extended those studies by comparing the expression of tenascin-C with that of syndecan-3, which belongs to a family of cell surface receptors with which tenascins are known to interact. We found that syndecan-3 transcripts at first were very abundant in the presumptive periosteum surrounding the diaphysis of early chondrocytic skeletal elements in chick limb. As the elements developed further, syndecan-3 gene expression decreased in the diaphyseal periosteum, whereas it became stronger around the early epiphysis and within the forming articular cells. However, as the diaphyseal periosteum initiated osteogenesis and gave rise to the intramembranous bone collar, syndecan-3 gene expression increased again. At early stages of skeletogenesis: the tenascin-C gene exhibited patterns of expression that were similar to and temporally followed, those of the syndecan-3 gene. At later stages, however, tenascin-C gene expression was markedly reduced during intramembranous osteogenesis around the diaphysis. In addition, although syndecan-3 gene expression was low in osteoblasts and osteocytes located deep into trabecular bone, tenascin-C gene expression remained strong. Thus, tenascin-C and syndecan-3 display distinct temporal and spatial patterns of expression in periosteum and during the development of long bone. Given their multidomain structure and specific patterns of expression, these macromolecules may regulate site-specific skeletal processes, including interactions between developing periosteum and chondrocytes and delineation of the early cartilaginous skeletal elements.

Polarizing activity, Sonic hedgehog, and tooth development in embryonic and postnatal mouse.

Tooth development involves reciprocal epithelial-mesenchymal interactions, polarized growth, mesenchyme condensation, and complex morphogenetic events. Because these processes bear similarities to those occurring in the developing limb, we asked whether morphogenetic signals found in the limb also occur in the developing tooth. We grafted mouse embryo tooth germs to the anterior margin of host chick embryo wing buds and determined whether the dental tissues had polarizing activity. Indeed, the grafts induced supernumerary digits. Activity of both molar and incisor tooth germs increased from bud to cap stages and was maximal at late bell stage in newborn. With further development the polarizing activity began to decrease, became undetectable in adult molar mesenchyme but persisted in incisor mesenchyme, correlating with the fact that incisors grow throughout postnatal life while molars do not. When different portions of neonatal incisors were assayed, a clear proximo-distal gradient of activity was apparent, with maximal activity restricted to the most proximal portion where undifferentiated mesenchyme and enamel organ reside. In situ hybridizations demonstrated that prior to induction of supernumerary digits, the tooth germ grafts induced expression in host tissue of Hoxd-12 and Hoxd-13. In addition, whole-mount in situ hybridizations and immunohistochemistry showed that developing tooth germs express Sonic hedgehog (Shh). Shh expression was first detected in bud stage tooth germs; at later stages Shh transcripts were prominent in enamel knot and differentiating ameloblasts at the cuspal region. We concluded that tooth germs possess polarizing activity and produce polarizing factors such as Shh. As in the limb, these factor(s) and activity probably play key roles in establishing polarity and regulating morphogenesis during early tooth development. Given its subsequent association with differentiating ameloblasts, Shh probably participates also in cytogenetic events during odontogenesis.

Syndecan-3, tenascin-C, and the development of cartilaginous skeletal elements and joints in chick limbs.

The mechanisms by which the early limb cell condensations and interzone mesenchyme give rise to skeletal elements and joints are poorly understood. Previous work from this laboratory has shown that the extracellular matrix protein tenascin-C is associated with articular cartilage and joint tissue development; others have shown that tenascin-C may exert its biological activities via interactions with cell surface receptors, such as syndecans. To further analyze the roles of tenascin-C and its putative receptors in skeletal development, we carried out a detailed in situ hybridization analysis of tenascin-C and syndecan-3 gene expression during development of chick limb skeletal elements and joints. We found that as the early mesenchymal condensations chondrify around day 5 (E5) of development, they become surrounded by a thick syndecan-3 rich perichondrium while tenascin-C transcripts are much fewer and restricted to diaphyseal perichondrium and developing interzones. Similar patterns were observed as distal carpal and digit condensations formed in older embryos. As the cartilaginous long bone models elongated proximo-distally and joint formation proceeded with age, we observed that syndecan-3 transcripts decrease significantly along the diaphysis and remain very abundant along the metaphysis and in the epiphyseal articular cap and interzone. Conversely, tenascin-C RNAs remain abundant along the diaphysis and begin to increase at the epiphysis and in interzone-derived tissues, such as menisci and joint capsule. By E10, the skeletal elements have well-defined morphologies, endochondral ossification has initiated in their diaphysis, and diaphyseal perichondrium has become periosteum. These developmental changes were accompanied by equally marked changes in gene expression; these included a marked increase in tenascin-C gene expression in articular cap, fragmentation of tenascin-C gene expression along the periosteum, reinitiation of syndecan-3 gene expression in periosteum, and differential gene expression in osteoprogenitor cells. The sheer complexity of the gene expression patterns documented in this study attests to the complexity of processes that bring about normal skelatogenesis. Clearly, tenascin-C and syndecan-3 appear to be closely associated with several of these processes, particularly in establishing tissue boundaries (perichondrium and periosteum) between condensations and surrounding mesenchymal cells, in regulating perichondral cell differentiation and incorporation into the growing skeletal elements, and in the genesis of epiphyseal chondrocytes and associated joint tissues.

Tenascin is associated with articular cartilage development.

The roles of tenascin in cartilage development and function remain unclear. Based on the observation that tenascin is particularly abundant at the epiphyseal extremities of developing cartilaginous models of long bones in chick and mouse embryo, we tested the hypothesis that tenascin is involved in articular cartilage development. Immunofluorescence analysis revealed that tenascin was first localized in the cell condensation region of Day 4 chick embryo limb buds, where the cartilaginous models form. With further development, tenascin gene expression became indeed restricted to the articular cap of the models. Tenascin persisted in the articular cartilage of postnatal chickens but appeared to decrease with age. The protein was also abundant in embryonic and adult tracheal cartilage rings which, like articular cartilage, persist throughout postnatal life. Similar patterns of tenascin expression were seen in mouse. Using monoclonal antibodies to avian tenascin variants, we found that the bulk of articular cartilage contained the shortest tenascin variant (Tn190), whereas the largest variant (Tn230) was present in tissues associated or interacting with articular cartilage (ligaments and meniscus). The protein and its mRNA, however, were undetectable in growth plate cartilage undergoing maturation and endochondral ossification. This inverse correlation between chondrocyte maturation and tenascin production was corroborated by the finding that tenascin gene expression decreased markedly during maturation of chondrocytes in culture and during formation of a secondary ossification center within the articular cap in vivo. Thus, tenascin is intimately associated with the development of articular cartilage and other permanent cartilages whereas absence or reduced amounts of this matrix protein characterize transient cartilages which undergo maturation and are replaced by bone.

Age-dependent influence of hypoxia on methionine-enkephalin concentration within rabbit brainstem nuclei.

We examined the effect of hypoxia (FIO2 = 0.10) on methionine-enkephalin concentrations in brainstem nuclei involved in the integration of cardiopulmonary control in 3- and 21-day-old rabbits. Rabbit pups were confined in environmental chambers for 6 h and exposed to one of four conditions. Control, 21% O2 for 6 h; intermittent hypoxia, 12 cycles of 20 min 21% O2 followed by 10 min of 10% O2; acute hypoxia, 4 h of 21% O2 followed by 2 h of 10% O2; recovery, 2 h of 10% O2 followed by 4 h of 21% O2. Methionine-enkephalin was measured by radioimmunoassay in the nucleus tractus solitarius, nucleus ambiguus, nucleus parabrachialismedialis, and nucleus reticulogigantocellularis. In 3-day-old rabbits, exposure to 10% O2 did not affect methionine-enkephalin concentrations in any brainstem nuclei studied. In contrast, 21-day-old pups demonstrated a decrease in methionine-enkephalin concentration in three of the four nuclei studied when exposed to intermittent hypoxia, as well as an apparent ability to recover from an acute hypoxic exposure (p less than 0.05). These data support an age-, nucleus-, and stimulus-specific effect of hypoxia on methionine-enkephalin concentration within specific brainstem nuclei and suggest a possible mechanism for the newborn's increased cardiopulmonary instability under hypoxia.

Effect of hypoxia on brainstem concentration of biogenic amines in postnatal rabbits.

The effects of hypoxia FiO2 = 0.10 on concentration of biogenic amines in specific brainstem nuclear groups were investigated in 3-and 21-day-old rabbits. The rabbit pups were confined to temperature-controlled water-jacketed chambers and exposed to 6h of 21% O2 or to one of 3 combinations of 21% O2 and 10% O2. These 3 combinations were either intermittent hypoxia, or 4 h of normoxia followed by 2 h constant hypoxia, or 2 h of hypoxia followed by recovery for 4 h in normoxia. Radioenzymatic assays were used to determine the concentration of dopamine, norepinephrine and serotonin in the following brainstem nuclei: substantia nigra, locus coeruleus, dorsal raphe and the nucleus reticularis pontis oralis. Compared with control, hypoxia did not affect dopamine levels at either age. The concentration of norepinephrine was inconsistently affected by hypoxia at either age. In contrast, in the 3-day-old rabbits serotonin was consistently reduced in each of the nuclei. In the 21-day-old rabbits, serotonin was either unchanged or increased following hypoxia. Our results show that hypoxia alters the concentration of serotonin in an age-specific manner. This change in serotonin concentration may reflect altered serotonin metabolism and suggests a possible mechanism by which hypoxia disrupts physiologic homeostasis in newborns.

Development of methionine-enkephalin in microdissected areas of the rabbit brain.

Microdissected areas of the rabbit brain were isolated at prenatal day E-29, postnatal days P-3, 7, 14, 21, 2 months and adults. Methionine-enkephalin (ME) was assayed by RIA and ME concentration [ME] was expressed relative to the protein content of the extracted brain tissues. In brain nuclei with important roles in respiratory control [ME] was higher in prenatal and early postnatal life than in adults. In contrast, the prenatal and early postnatal [ME] levels in other nuclei were lower than or equal to adult values. These data suggest an important and changing role for ME in respiratory control throughout development. Early high [ME] levels within brainstem respiratory control nuclei may contribute to the newborn's increased susceptibility to respiratory depression.

Effects of albumin and/or furosemide therapy on pulmonary edema induced by hydrochloric acid aspiration in rabbits.

Aspiration of hydrochloric acid in rabbits resulted in an increased P(A-a)O2 together with increases in both lung water volume and lung extravascular albumin. This finding suggests lung damage following acid aspiration is related to changes in capillary permeability, with pulmonary edema resulting from the movement of albumin and water into the interstitial space. Therapy with albumin and furosemide together reduced the lung water and albumin accumulation and decreased P(A-a)O2. Treatment with albumin or furosemide alone was ineffective. Caution should be exercised in administering albumin alone for therapy of pulmonary edema when plasma protein is not clearly decreased, or when increased pulmonary capillary permeability is suspected.