Relationships among msx gene structure and function in zebrafish and other vertebrates.

The zebrafish genome contains at least five msx homeobox genes, msxA, msxB, msxC, msxD, and the newly isolated msxE. Although these genes share structural features common to all Msx genes, phylogenetic analyses of protein sequences indicate that the msx genes from zebrafish are not orthologous to the Msx1 and Msx2 genes of mammals, birds, and amphibians. The zebrafish msxB and msxC are more closely related to each other and to the mouse Msx3. Similarly, although the combinatorial expression of the zebrafish msx genes in the embryonic dorsal neuroectoderm, visceral arches, fins, and sensory organs suggests functional similarities with the Msx genes of other vertebrates, differences in the expression patterns preclude precise assignment of orthological relationships. Distinct duplication events may have given rise to the msx genes of modern fish and other vertebrate lineages whereas many aspects of msx gene functions during embryonic development have been preserved.

Specific craniofacial cartilage dysmorphogenesis coincides with a loss of dlx gene expression in retinoic acid-treated zebrafish embryos.

Treatments of zebrafish embryos with retinoic acid (RA), a substance known to cause abnormal craniofacial cartilage development in other vertebrates, result in dose- and stage-dependent losses of dlx homeobox gene expression in several regions of the embryo. Dlx expression in neural crest cells migrating from the hindbrain and in the visceral arch primordia is particularly sensitive to RA treatment. The strongest effects are observed when RA is administered prior to or during crest cell migration but effects can also be observed if RA is applied when the cells have entered the primordia of the arches. Losses of dlx expression correlate either with the loss of cartilage elements originating from hindbrain neural crest cells or with abnormal morphology of these elements. Cartilage elements that originate from midbrain neural crest cells, which do not express dlx genes, are less affected. Taken together with the observation that the normal patterns of visceral arch dlx expression just prior to cartilage condensation resemble the morphology of the cartilage elements that are about to differentiate, our results suggest that dlx genes are an important part of a multi-step process in the development of a subset of craniofacial cartilage elements.

Osteogenic protein-1 is required for mammalian eye development.

Osteogenic Protein-1 (OP-1/BMP-7) is a bone morphogenetic protein in the transforming growth factor-beta superfamily and has been shown to be expressed temporally and spatially during epithelial-mesenchymal interactions mediating tissue morphogenesis in early embryogenesis. In order to identify the primary role(s) for OP-1 in development, we carried out whole rat embryo cultures, over a 72-h period from primitive streak stages to early limb bud stages, in rat sera containing either OP-1 blocking antibodies (10 micrograms/ml) or nonreactive IgG. Rat embryos cultured with control antibodies developed normally, while those cultured with anti-OP-1 antibodies consistently exhibited over-all reduced size and absence of eyes. Histological sections revealed a greater reduction in neural retina development in the embryos treated with anti-OP-1 blocking antibodies. In situ hybridization and immunolocalization analyses indicate that OP-1 is expressed in the neuroepithelium of the optic vesicle at E11.5, is limited to the presumptive neural retina and developing lens placode, and is subsequently expressed in the neural retina, lens and developing cornea at E12.5-E13.5. Our results indicate that OP-1 mediates the inductive signals involved in mammalian eye development.

In vitro analysis of the spatial organization of chondrogenic regions of avian mandibular mesenchyme.

The mechanism(s) which control patterning in the face remain elusive, due in large part to the absence of morphologically identifiable controlling regions such as the AER of the limb bud. In order to identify the controlling region(s) and timing of patterning in the face, an investigation was launched to determine the spatial organization of tissues within this region, beginning with the chondrogenic zones of the avian (chick and quail) mandible. The mandibles from HH stage 23/24 chick and equivalent stage quail embryos were initially bisected in three planes giving rostral or caudal, proximal or distal, and medial or lateral halves. The mesenchyme from these various regions was isolated, plated out in high density micromass cultures, and grown for 4 days. Additionally, further cultures were grown, consisting of mandibular mesenchyme subdivided into quarters along the long axis of the mandible (e.g., rostro-proximal quarter) as well as the bisecting of medial or lateral halves (e.g., medial-rostral quarter). Nodule number and area were determined by morphometric analysis for each culture as well as whole mandible controls. The demarcation between chondrogenic and non-chondrogenic regions was dramatic. Of the bisected halves, proximal and lateral were the most chondrogenic with the lateral subdivision displaying much more cartilage than whole mandible. The nodules of the lateral cultures fused into a sheet of cartilage. In contrast mesenchyme from the medial half was virtually non-chondrogenic. When ranked by the amount of chondrogenesis, the order was, lateral > proximal = whole = core > distal > caudal > rostral > periphery >> medial. Interestingly, when subdivided further an altered pattern appeared.(ABSTRACT TRUNCATED AT 250 WORDS)

Chondrogenic differentiation in cultures of embryonic rat mesenchyme.

The morphology and fine structure of day 12 rat embryonic mesenchyme from forelimb bud, mandibular arches, and frontonasal prominence is described as the cells undergo chondrogenesis in high density, micromass culture. The cultures began as a multilayered "pavement" of flattened mesenchymal cells, 3-4 deep, with moderate intercellular space but little identifiable electron-dense extracellular matrix. Pre-cartilage condensations, which consisted of aggregates of cells which had rounded up, displaying little or no intercellular space, formed within the first 24 h in limb mesenchyme and after an additional 24 h in mandibular and frontonasal cultures. Gap junctions occur between these cells, indicating a phase of direct cell-cell communication. Chondrogenesis within these aggregates began within the next 24 h in limb cultures but was delayed an additional 24-48 h in the frontonasal and especially in mandibular cultures. The aggregates in both limb and mandibular mesenchyme form discrete nodules bordered by a perichondrium consisting of 2-3 layers of flattened cells. Evidence from late stage mandibular cultures suggests that chondroblasts are added to the nodules from the perichondrium, as occurs in vivo. By contrast, the frontonasal cartilage is initially unbordered and forms anastomosing trabecular arrays. Some of these arrays fuse into larger structures with time, but others become surrounded by a single, flattened perichondrium, resulting in the stacking of these trabeculae as chondrification proceeds. The sequence of cartilage matrix production, as revealed in long-term facial cultures, appears to occur in three stages, an early phase in which the extracellular matrix consists primarily of proteoglycans, followed by a phase of homogeneous collagen-proteoglycan matrix and a mature, territorial matrix. In all three cultures the cartilage ultimately produced resembles mature rat hyaline cartilage with chondroblasts surrounded by a territorial matrix of type II collagen and proteoglycan granules.

Stable, position-related responses to retinoic acid by chick limb-bud mesenchymal cells in serum-free cultures.

Retinoic acid (RA) has dramatic effects on limb-skeletal patterning in vivo and may well play a pivotal role in normal limb morphogenesis. RA's effects on the expression of pattern-related genes in the developing limb are probably mediated by cytoplasmic RA-binding proteins and nuclear RA-receptors. Little is known, however, about how RA modifies specific cellular behaviors required for skeletal morphogenesis. Earlier studies supported a role for regional differences in RA concentration in generating the region-specific cell behaviors that lead to pattern formation. The present study explores the possibility that position-related, cell-autonomous differences in the way limb mesenchymal cells respond to RA might have a role in generating pattern-related cell behavior. Mesenchymal cells from different proximodistal regions of stage 21-22 and 23-24 chick wing-buds were grown in chemically defined medium and exposed to 5 or 50 ng/ml of RA for 4 days in high-density microtiter cultures. The effects of RA on chondrogenesis in these cultures clearly differed depending on the limb region from which the cells were isolated. Regional differences in RA's effects on growth over 4 days in these cultures were less striking. The region-dependent responses of these cells to RA proved relatively stable in culture despite ongoing cytodifferentiation. This serum-free culture model will be useful in exploring the mechanisms underlying the region-dependent responsiveness of these cells to RA.

Differential and stage dependent effects of retinoic acid on chondrogenesis and synthesis of extracellular matrix macromolecules in chick craniofacial mesenchyme in vitro.

Retinoic acid (RA) is well known to be a potent teratogen and induces a variety of facial defects in vivo, but at concentration levels lower than those that cause facial defects, RA seems to play an important role in normal facial development. In a previous study, we demonstrated the ability of RA to stimulate chondrogenesis in vitro in HH stage 23/24 chick mandibular (MND) but not frontonasal (FNP) mesenchyme cultured in a serum-free medium. The present study furthers these results by examining the effects of RA on chondrogenesis of chick facial mesenchyme at earlier embryonic stages and the effects on cell proliferation and synthesis of specific extracellular matrix macromolecules at stage 23/24. MND and FNP cells were cultured as micromasses for 4 days in defined media. As described previously, chondrogenesis in stage 23/24 MND cells was significantly enhanced by concentrations of RA of 0.1-1 ng/ml; however, at all earlier stages examined (18 to 22) RA at these concentrations had no significant effect. Higher concentrations of the retinoid inhibited chondrogenesis in MND cultures from all stages tested. Cells of the FNP from all stages displayed no significant change in chondrogenesis below 1 ng/ml RA and a dose dependent inhibition at higher concentrations. Thus RA's promotional effects in the face are not only tissue specific (MND), but also stage-dependent (HH 23/24). The specific effects of RA on matrix production and cell proliferation of stage 23/24 MND and FNP cells was examined by analysis of 35S sulfate, 3H thymidine and 3H proline incorporation. Analysis of 35S sulfate incorporation into sulfated proteoglycans confirmed that concentrations of RA of 0.1-1 ng/ml stimulated cartilage matrix production in MND but not FNP cultures. Above this level of RA, 35S sulfate incorporation was reduced in both. Likewise, 3H proline incorporation into collagenous protein, and to a lesser extent non-collagenous proteins, was stimulated by low levels of RA in MND, but not FNP cultures. Higher concentrations of the retinoid in either MND or FNP cultures did not lower collagen production, undoubtedly due to stimulation of non-chondrogenic cells within the population. This indicates that levels of RA as high as 100 ng/ml cause phenotypic change rather than cell death. This last point is corroborated by the analysis of 3H thymidine uptake in the cultures which was only transiently modified in most. The data indicate that cell proliferation occurred even in the presence of high RA levels.

Myogenic potential of chick limb bud mesenchyme in micromass culture.

The myogenic potential of chick limb mesenchyme from stages 18-25 was assessed by micromass culture under conditions conductive to myogenesis, and was measured as the proportion of differentiated (muscle myosin-positive) mononucleated cells detected. It was found that similar myogenic potentials existed in mesenchyme from whole limbs between stages 18 and 19, but this potential was halved by stage 20. At stage 21, proximal mesenchyme showed significantly more myogenesis than distal mesenchyme, but this difference was abolished by stage 22. Thereafter, myogenesis was increasingly restricted from the distal mesenchyme, whilst the potential in more proximal regions did not significantly increase after stage 23. When the ratio between total limb myoblasts which differentiated on days 1 and 4 of culture was analysed, it was found that two distinct peaks existed at stages 20 and 23. The significance of these ratio peaks is unclear, but may be related to different proliferative potentials of the pre-myoblasts at these stages.

Formation of chondrous and osseous tissues in micromass cultures of rat frontonasal and mandibular ectomesenchyme.

Rat frontonasal and mandibular mesenchyme was isolated from day-12 1/2 (stage-22) rat embryos and cultured at high density for up to 12 days. The stage chosen was based on the observation that mandibular mesenchyme at this stage became independent of its epithelium with respect to the production of both cartilage and bone. Frontonasal cultures developed aggregates of anastomosing columns of cells within 2 days. These grew as the cells enlarged, laying down an Alcian-blue-positive matrix by day 3 of culture. Significant mineral was detected by von Kossa staining by day 5 at which time the aggregates covered a large portion of the culture, eventually covering the entire micromass by day 10-12. Mandibular cultures developed centrally located nodular aggregates by 3 days of culture. These nodules increased in number, spreading outwards as the cells enlarged, laying down an Alcian-blue-positive matrix by day 4 and mineral by days 6-7. At this time the nodules began to elongate and coalesce, but never covered the entire culture over the 12-day period. Antibody staining revealed that in both cultures the cells were initially positive for type I collagen. Subsequently, the aggregates began expressing type II collagen, followed by type X, which coincided with the onset of mineralization. At this time some cells were negative for these cartilage markers, but positive for osteoblast markers, bone sialoprotein II, osteocalcin and type I collagen. In addition osteonectin and alkaline phosphatase were demonstrable in all of the aggregate cells late in the culture period. This provided clear evidence that chondroblast and osteoblast differentiation was proceeding within these cultures. The culture of rat facial mesenchyme should prove very useful, not only for the analysis of bone and cartilage induction and lineage relationships, but also in furthering our knowledge of craniofacial differentiation, growth and pattern formation by extending our analysis to a mammalian system.

Developmental processes, developmental sequences and early vertebrate phylogeny.

(1) We have put forth the position that evolutionary sequences can be deduced by an analysis of fundamental developmental sequences. Such sequences are highly conserved within a group and 'contain steps which are necessary to achieve a developmental fate'. The premise of our work then, is that such fundamental sequences do not arise de novo time and time again but can be traced back through their evolutionary history in organisms which contain portions of the sequence. (2) These highly conserved developmental sequences are in fact developmental constraints to evolution in as much as natural selection has not been able to discard them, but rather has utilized them in achieving evolutionary change. (3) We have demonstrated the ability to use developmental data by producing an evolutionary sequence for the origin of the vertebrates using the processes of neuralization and cephalization, the latter due primarily to the influences of the neural crest and epidermal placodes. The evolutionary sequence created, while not novel in structure, is distinct in that it was created solely by following a developmental sequence that is highly conserved among the vertebrates. The sequence is: (a) Chordamesoderm differentiates from the surrounding mesoderm and induces an overlying neural tube. (b) Through the influence of neuralizing morphogens, the neural tube differentiates into anterior (fore-, mid- and hindbrain) and posterior (spinal cord) parts. Cephalization has begun. (c) Cephalization proceeds via the development of two new populations of embryonic cells, the neural crest, a derivative of the neural epithelium and the epidermal placodes, derivatives of the ectoderm immediately adjacent to the neural tube. These two populations contribute significantly to the subsequent development of the vertebrate head including the skeleton, connective tissues, cranial nerve and sensory organs. Sequence (a) occurs in the most primitive protochordates and is one of the differences between the chordates and deuterostome invertebrates. Sequence (b) occurred next leading to a protochordate with a differentiated central nervous system, but lacking most vertebrate head structures. Sequence (c) signalled the beginning of the true vertebrates or branchiates (after the branchial arches which all 'vertebrates' share) since the production of a neurocranium, viscerocranium, cephalic armour, teeth and cranial peripheral ganglia was only possible with the acquisition of this developmental step.(ABSTRACT TRUNCATED AT 400 WORDS)

Differential effects of physiological concentrations of retinoic acid in vitro on chondrogenesis and myogenesis in chick craniofacial mesenchyme.

Recent studies have shown that in the developing limb bud retinoic acid is a skeletal morphogen at physiological levels, but a potent teratogen at higher levels. Retinoic acid has also been shown to be teratogenic during facial development, but very low levels may have an as yet unspecified role in normal development. In the present study the effects of retinoic acid on chondrogenesis and myogenesis by craniofacial cells grown in micromass cell culture were investigated. Retinoic acid, at concentrations of 0.01-100 ng/ml, was supplied to cells derived from day-4 (H.H stage 23/24) chick embryo mandibular, maxillary and frontonasal processes, grown in micromass cultures for 4 days in both serum-containing and defined media. Based on Alcian-blue-staining, concentrations of retinoic acid of 0.1-1 ng/ml were found to enhance chondrogenesis by mandibular cells grown in defined medium, while greater concentrations up to 100 ng/ml inhibited chondrogenesis. By contrast, chondrogenesis was generally retarded by all concentrations of retinoic acid applied to frontonasal cells grown in defined medium and when applied to both mandibular and frontonasal cells when grown in serum-containing medium. Cells from stage-23/24 maxillae did not display any significant chondrogenic activity in either medium under these culture conditions. Unlike chondrogenesis, myogenesis in mandibular, frontonasal and maxillary cultures was greater in defined than serum-containing medium, based on the appearance of immunologically detectable muscle myosin, and was reduced considerably less in defined medium by all concentrations of retinoic acid tested. In the presence of serum however, myogenesis was retarded with increasing concentrations of retinoic acid beyond 1 ng/ml in micromass cultures from all three facial regions.(ABSTRACT TRUNCATED AT 250 WORDS)

Selective stimulation of in vitro limb-bud chondrogenesis by retinoic acid.

Embryonic exposure to pharmacologic doses of vitamin A analogs (retinoids) is a well-known cause of limb-skeletal deletions, limb truncation and other skeletal malformations. The exclusively inhibitory effect of retinoic acid (RA) on chondrogenesis in standard serum-containing cultures of limb-bud mesenchymal cells is equally well known and has provided a means to explore the cellular basis for RA-mediated skeletal teratogenesis. Recent studies showing that lower RA concentrations can cause skeletal duplication when applied directly to the anterior border of a developing limb, suggest that RA may have a role in normal limb development as a diffusible morphogen capable of regulating skeletal pattern. While RA treatment causes both, skeletal deletions and duplications are clearly different (if not opposing) effects, the latter of which is difficult to reconcile with RA's heretofore exclusively inhibitory effect on in vitro chondrogenesis. In the present study. RA's effects on chondrogenesis and myogenesis were examined in serum-free cultures of chick limb-bud mesenchymal cells and compared with its effects on similar cultures grown in serum-containing medium. When added to serum-free medium, concentrations of RA known to cause skeletal duplication in vivo dramatically enhanced in vitro chondrogenesis (to over 200% of control values) as judged by both Alcian-blue staining and [35S]sulfate incorporation, while having little effect on myogenesis. Higher concentrations inhibited both chondrogenesis and myogenesis. The results indicate that at physiological concentrations. RA can selectively modulate chondrogenic expression and suggest that at higher concentrations, RA's inhibitory effects are less specific.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of the neural crest in development of the cartilaginous cranial and visceral skeleton of the medaka, Oryzias latipes (Teleostei).

Neural crestectomies were performed on neurula stage medaka embryos to remove neural crest with tungsten needles from one of five anteriorly located zones. The embryos were allowed to develop to stage 35 (immediately posthatching) larvae, then cleared and stained for cartilage. An analysis of changes to the head skeletons indicated that most of the anterior neurocranium and the entire viscerocranium received neural crest contributions during development. The elements involved included; the lamina orbitonasalis of the nasal capsule, the trabeculae, Meckels' cartilage and the quadrate of the lower jaw, the pterygoid process, the orbital cartilages and the epiphyseals of the neurocranial roof, as well as all the elements of the hyoid and branchial arches. By further analysis of only those neural crest ablations which produced alterations to the head skeleton, the neural crest cells which contributed to the development of each element were mapped. They originated principally, from one of three regions; the mesencephalon (second most anterior zone removed, number II), the preotic rhombencephalon (zone III), or the postotic rhombencephalon (zone IV). Neural crest from the level of the prosencephalon (zone I) was not chondrogenic nor was neural crest from the fifth region (zone V) which extended beyond the 5th to about the 8th or 10th somite and marked the anterior end of trunk neural crest. The data are discussed and are found to be consistent with the results from other vertebrates and support the central role of the neural crest in the development and evolution of the vertebrate head skeleton.

The organ culture and grafting of lamprey cartilage and teeth.

Cartilage from larval (ammocoetes) and adult (prespawning upstream migrant) lamprey was successfully maintained both when cultured in vitro or grafted in vivo (on the chorioallantoic membrane (CAM) of host chick embryos). In addition teeth from adult lamprey were successfully cultured in vitro. Cartilages were cultured in supplemented Lebovitz's l15 medium at 15 and 20 degrees C for periods of up to 56 d and in supplemented BGJb medium at 37 degrees C for periods of up to 14 d. Cartilages were also grafted onto the CAM for up to 16 d. Both the cultured and grafted cartilages retained their structural and cellular integrity as verified histologically. The viability of the cartilage, even after extended culture periods, was demonstrated ultrastructurally by the presence of chondrocytes displaying abundant rough endoplasmic reticulum, mitochondria, and Golgi apparatii with associated vesicles. In addition the cartilages were shown to be metabolically active in vitro by the incorporation of radioactive sulfur into the matrix. Some cell outgrowth from other tissues, such as connective tissue, muscle, and gill when left adjacent to the cartilage, occurred over time in cultures. No cell outgrowth was observed in CAM-grafted tissue nor was there any invasion of the agnathan tissue by chorioallantoic blood vessels. Teeth cultured in L15-supplemented media for up to 14 d at either 15 or 20 degrees C retained their structural and cellular integrity as observed histologically, with no apparent cell outgrowth. With the successful culture of these tissues, their development, biochemistry, and physiology, potentially of great importance in understanding early vertebrate evolution, can be better understood.