Somite chondrogenesis in vitro: 2. Changes in the hyaluronic acid synthesis

The role of hyaluronic acid in limb morphogenesis (chondrogenesis) has been well defined. In the present study, we found that hyaluronic acid synthesis in somite explants steadily increased until day 6, then decreased, and inclusion of notochord did not accelerate the rate of synthesis. Analysis of hyaluronidase activity in the somite explants indicated an increase in the enzyme level in day-6 cultures. Again, inclusion of notochord did not alter this pattern. The decrease in hyaluronic acid after day 6 and the increase in sulfated proteoglycan synthesis from day 6 resemble the pattern described during limb development. Subsequent studies showed that, with time, the size of the hyaluronic acid synthesized by somites increased and, again, inclusion of notochord did not influence this pattern. The results indicate that unstimulated somites are capable of synthesizing cartilage-specific proteoglycans in a relatively restricted manner, and the inclusion of notochord resulted in accelerated synthesis of stable proteoglycan aggregates typical of differentiated chondrocytes. Metabolic events in somites related to hyaluronic acid are not influenced by the notochord.     

Somite chondrogenesis: alterations in cyclic AMP levels and proteoglycan synthesis

Cyclic AMP (cAMP) levels have been shown to have a positive influence on chondrogenesis in limb buds and pelvic cartilage. In the present study the level of cAMP was measured during somite chondrogenesis in vitro and found to decrease from 1.38 pmol/micrograms DNA on day 0 to 0.9 pmol/micrograms DNA on day 6. Inclusion of notochord with somites caused a marked reduction, with levels decreasing from 1.41 pmol/micrograms DNA on day 0 to 0.36 pmol/micrograms DNA on day 6. Concurrently, the incorporation of radioactive sulfate into sulfated glycosaminoglycans increased from day 3 to day 6 by 38% in somite and 77% in somite-notochord explants. The aggregation of proteoglycans was analyzed by gel chromatography and found to increase with a corresponding decrease in cAMP levels. The results indicate that a decrease in cAMP levels may be necessary for chondrogenic expression in somites.     

Notochordal stimulation of in vitro somite chondrogenesis before and after enzymatic removal of perinotochordal materials.

In the present investigation, evidence is presented directly implicating proteoglycans produced by the embryonic notochord in the control of somite chondrogenesis. It has been demonstrated by several histochemical techniques that during the period of its interaction with somites, the notochord synthesizes perinotochordal proteoglycans, and these proteoglycans have been shown to contain chondroitin 4-sulfate (40%), chondroitin 6-sulfate (40%), and heparan sulfate (20%). Dissection of notochords from embryos with the aid of a brief treatment with trypsin results in the removal of perinotochordal extracellular matrix materials including proteoglycans, while dissection of notochords without the aid of enzyme treatment or with a low concentration of collagenase results in their retention. There is a considerable increase in the rate and amount of cartilage formation and a corresponding 2 to 3-fold increase in the amount of sulfated glycosaminoglycan accumulated by somites cultured in association with notochords dissected under conditions in which perinotochordal materials are retained. Treatment of collagenase-dissected or freely dissected notochords with highly purified enzymes (chondroitinase ABC, AC, and testicular hyaluronidase) which specifically degrade proteoglycans causes a loss of histochemically detectable perinotochordal proteoglycans. These notochords are considerably impaired in their ability to support in vitro somite chondrogenesis. In addition, when trypsin-treated notochords are cultured (“precultured”) for 24 hr on nutrient agar (in the absence of somites), perinotochordal material reaccumulates. Somites cultured in association with such “precultured” notochords exhibit considerable increase in the amount of cartilage formed and a 2- to 3-fold increase in the amount of sulfated glycosaminoglycan accumulated as compared to somites cultured in association with trypsin-treated notochords which have not been “precultured.” This observation indicates that trypsin-treated notochords reacquire their ability to maximally stimulate in vitro somite chondrogenesis by resynthesizing and accumulating perinotochordal material. Finally, “precultured” notochords treated with chondroitinase to remove perinotochordal proteoglycans are considerably impaired in their ability to support in vitro somite chondrogenesis. These observations are consonant with the concept that proteoglycans produced by the embryonic notochord play an important role in somite chondrogenesis.     

The influence of axial structures on chick somite formation

The influence of the axial structures on somite formation was investigated by culturing, on a nutritive agar substrate, segmental plates from chick embryos having 8 to 20 pairs of somites. In the first set of experiments, segmental plate was explanted together with adjacent notochord and approximately the lateral halves of the neural tube and node region. These explants formed 18 to 20 somites within 30 hr. In a second series of experiments, the notochord and neural tube were included as before, but further regression movements in the explants were prevented by removing the node region. These explants formed only 11.9 ± 1.1 somites. Finally, explants of segmental plate that included no neural tube, notochord, or node region were made. These explants had formed 10.7 ± 1.1 somites 14 to 17 hr later. When such explants were cultured for periods longer than 17 hr, there was a marked tendency for the more posterior somites to disperse and for all of the somites to develop a peculiar “hollow” morphology. It was concluded from these results that during the period of development when chick embryos possess 8 to 20 pairs of somites, the segmental plate mesoderm (1) represents about 12 prospective somites, (2) may segment into its full complement of somites without further contact with the axial structures, but (3) requires continued intimate contact with the axial structures for normal somite morphologic differentiation and stability.     

Elevation of maternal glucocorticoid hormones alters neurotransmitter phenotypic expression in embryos

The tissue interaction between the notochord and the somites of the vertebrate embryo establishes the proper shape and constitution of the vertebral cartilage. Soon after somite formation, the somite differentiates into a cartilage-forming part, the sclerotome, and a muscle and skin-forming part, the dermamyotome. These components of the somite were dissected from $\text{3}\text{1}\text{2}\text{-}\text{day-old}$ chick embryos (stage $\text{18}\text{1}\text{2}\text{–19}$ and cultured in vitro in the presence or absence of notochord. It was found that the sclerotome cells respond to the notochord by an increased incidence of hyaline cartilage nodules, greater accumulation of sulfated glycosaminoglycans, synthesis of larger aggregates of proteoglycans, increased DNA accumulation, and accelerated DNA synthesis. The dermamyotome did not show these changes. These results indicate that the notochord enhances cartilage differentiation in the sclerotome. Under these conditions, the notochord did not elicit cartilage formation in the dermamyotome.     

VACUOLATION OF EXPLANTED AND IMPLANTED NOTOCHORDS OF AMPHIBIA

Processes of vacuolation were investigated with the explanted and implanted rudiments of amphibian notochord. Explanted notochords generally underwent vacuolation when they were surrounded by other differentiating tissues. In the absence of these tissues, they were not only unable to vacuolate but also unable to survive. When the notochords were implanted into the ventral mass of yolk granules of neurula, they showed no vacuolation and died within a few days if they were alone and not surrounded by other differentiating tissues. These facts suggest that the presence of other differentiating tissues surrounding the notochord is a requisite for the notochord to vacuolate. Further, the fact that in explanted notochords, vacuolation was frequently interrupted and was left unfinished when the surrounding tissues were scarce, suggests that the quantity of the surrounding tissues is important in promoting vacuolation. Interruption of the vacuolation occurs in the explanted notochord at any stage from the beginning to the end of the process. Therefore, almost all the stages of vacuolation were observed in these notochords. Very frequently, different parts of a single notochord presented different stages of vacuolation. All these stages were essentially the same as those found in the normal notochord. From these results, it emerges that although vacuolation of the explanted and implanted notochords is carried out with the same process as in the normal intact one, it is accomplished only when they are surrounded by a sufficient quantity of other differentiating tissues.     

Somite Chondrogenesis in vitro: Differential Induction by Modified Matrix- a Biochemical and Morphological Study*

The stimulation of somite chondrogenesis by extracellular matrix components was studied by monitering the synthesis of cartilage-specific large proteoglycan aggregates. Chick embryonic sternal proteoglycans were separated into various components: monomers, hyaluronic acid, link protein and glycosaminoglycan side chains. The effects of these components, either individually or in various combinations, on somite chondrogenesis was examined. Proteoglycan monomers, alone or in a mixture with other components, induced chondrogenesis. The other components did not have any stimulating effect of their own. The results of these induction studies were also observed on a Sepharose CL–2B column and correlated using electron microscopy. Stimulation of somites resulted in an increase in the amount of proteoglycan aggregation (material excluded from the column) and was in agreement with the morphological appearance of the matrix in that there was increased accumulation of large proteoglycan granules. A matrix mixture of collagen and proteoglycans showed significant stimulation. When the matrix environment of the somites was altered to be unfavorable to the explants (medium containing hyaluronic acid) there was altered synthesis of cartilage-specific molecules. The results presented in this report strongly suggest that the composition of the extracellular matrix material is critical for somite chondrogenesis.     

Articular cartilage cultured with interleukin 1. Increased release of link protein, hyaluronate-binding region and other proteoglycan fragments.

Pig articular cartilage was maintained in culture for 3 days with and without porcine interleukin 1. The proteoglycans remaining in the cartilage and those released into the medium were analysed by using radioimmunoassays for the hyaluronate-binding region, link protein and keratan sulphate. In interleukin 1-treated cultures after 3 days there was 38% release of total glycosaminoglycans into the medium, 18% release of binding region, 14% release of link protein and 20% release of keratan sulphate epitope, whereas in control cultures the proportions released were much less (16, 9, 10 and 7% respectively). Characterization of the proteoglycans in the media after 1.5 days and 3 days of culture showed that interleukin 1 promoted the release of proteoglycan of large average size and also the release of link protein and of low-Mr binding region which was unattached to proteoglycan. Both the link protein and binding region released were able to bind to exogenously added hyaluronate, whereas the proteoglycan in the medium was not. The proteoglycans extracted from cultured cartilage were similar to those from fresh cartilage: they contained a high proportion of aggregating proteoglycans and some low-Mr binding region. The proportion of this binding region extracted from the interleukin 1-treated cartilage was increased. The presence of interleukin 1 in the cultures therefore appeared to increase the rate of proteolytic degradation of proteoglycan in the matrix and to lead to a more rapid loss of intact binding region, of link protein and of large proteoglycan fragments into the medium.     

Determination of somite cells: independence of cell differentiation and morphogenesis

Somites are mesodermal structures which appear transiently in vertebrates in the course of their development. Cells situated ventromedially in a somite differentiate into the sclerotome, which gives rise to cartilage, while the other part of the somite differentiates into dermomyotome which gives rise to muscle and dermis. The sclerotome is further divided into a rostral half, where neural crest cells settle and motor nerves grow, and a caudal half. To find out when these axes are determined and how they rule later development, especially the morphogenesis of cartilage derived from the somites, we transplanted the newly formed three caudal somites of 2.5-day-old quail embryos into chick embryos of about the same age, with reversal of some axes. The results were summarized as follows. (1) When transplantation reversed only the dorsoventral axis, one day after the operation the two caudal somites gave rise to normal dermomyotomes and sclerotomes, while the most rostral somite gave rise to a sclerotome abnormally situated just beneath ectoderm. These results suggest that the dorsoventral axis was not determined when the somites were formed, but began to be determined about three hours after their formation. (2) When the transplantation reversed only the rostrocaudal axis, two days after the operation the rudiments of dorsal root ganglia were formed at the caudal (originally rostral) halves of the transplanted sclerotomes. The rostrocaudal axis of the somites had therefore been determined when the somites were formed. (3) When the transplantation reversed both the dorsoventral and the rostrocaudal axes, two days after the operation, sclerotomes derived from the prospective dermomyotomal region of the somites were shown to keep their original rostrocaudal axis, judging from the position of the rudiments of ganglia. Combined with results 1 and 2, this suggested that the fate of the sclerotomal cells along the rostrocaudal axis was determined previously and independently of the determination of somite cell differentiation into dermomyotome and sclerotome. (4) In the 9.5-day-old chimeric embryos with rostrocaudally reversed somites, the morphology of vertebrae and ribs derived from the explanted somites were reversed along the rostrocaudal axis. The morphology of cartilage derived from the somites was shown to be determined intrinsically in the somites by the time these were formed from the segmental plate. The rostrocaudal pattern of the vertebral column is therefore controlled by factors intrinsic to the somitic mesoderm, and not by interactions between this mesoderm and the notochord and/or neural tube, arising after segmentation.     

Mediolateral patterning of somites: multiple axial signals, including Sonic hedgehog, regulate Nkx-3.1 expression

The axial structures, the notochord and the neural tube, play an essential role in the dorsoventral patterning of somites and in the differentiation of their many cell lineages. Here, we investigated the role of the axial structures in the mediolateral patterning of the somite by using a newly identified murine homeobox gene, Nkx-3.1, as a medial somitic marker in explant in vitro assays. Nkx-3.1 is dynamically expressed during somitogenesis only in the youngest, most newly-formed somites at the caudal end of the embryo. We found that the expression of Nkx-3.1 in pre-somitic tissue explants is induced by the notochord and maintained in newly-differentiated somites by the notochord and both ventral and dorsal parts of the neural tube. We showed that Sonic hedgehog (Shh) is one of the signaling molecules that can reproduce the effect of the axial structures by exposing explants to either COS cells transfected with a Shh expression construct or to recombinant SHH. Shh could induce and maintain Nkx-3.1 expression in pre-somitic mesoderm and young somites but not in more mature, differentiated ones. The effects of Shh on Nkx-3.1 expression were antagonized by a forskolin-induced increase in the activity of cyclic AMP-dependent protein kinase A. Additionally, we confirmed that the expression of the earliest expressed murine myogenic marker, myf 5, is also regulated by the axial strucutres but that Shh by itself is not capable of inducing or maintaining it. We suggest that the establishment of somitic medial and lateral compartments and the early events in myogenesis are governed by a combination of positive and inhibitory signals derived from the neighboring structures, as has previously been proposed for the dorsoventral patterning of somites.     

Neuronal induction and regional identity by co-culture of adherent human embryonic stem cells with chicken notochords and somites

The role of somites and notochords in neuroectoderm differentiation from the embryonic ectoderm and its subsequent patterning into regional compartments along rostro-caudal and dorso-ventral axes, especially in humans, remains elusive. Here, we demonstrate the co-culture effect of somites and notochords isolated from chicken embryos on the neuronal differentiation and regional identity of an adherent culture of human embryonic stem cells (hESCs). Notochord increased the efficiency and speed of neuronal induction, whereas somites had a weak neuronal inducing effect on hESCs. However, a synergistic effect was not observed when notochords and somites were used together. Moreover, in somite and notochord co-culture groups, hESCs-derived neuronal cells expressed HOXB4, OTX2, IRX3 and PAX6, indicative of dorsal hindbrain and ventral anterior identities, respectively. Our results reveal the influence of embryonic notochord and somite co-culture in providing neuronal induction as well as rostro-caudal and dorso-ventral regional identity of hESCs-derived neuronal cells. This study provides a model through which in vivo neuronal induction events may be imitated.     

Apoptosis regulates notochord development in Xenopus

The notochord is the defining characteristic of the chordate embryo and plays critical roles as a signaling center and as the primitive skeleton. In this study we show that early notochord development in Xenopus embryos is regulated by apoptosis. We find apoptotic cells in the notochord beginning at the neural groove stage and increasing in number as the embryo develops. These dying cells are distributed in an anterior to posterior pattern that is correlated with notochord extension through vacuolization. In axial mesoderm explants, inhibition of this apoptosis causes the length of the notochord to approximately double compared to controls. In embryos, however, inhibition of apoptosis decreases the length of the notochord and it is severely kinked. This kinking also spreads from the anterior with developmental stage such that, by the tadpole stage, the notochord lacks any recognizable structure, although notochord markers are expressed in a normal temporal pattern. Extension of the somites and neural plate mirrors that of the notochord in these embryos, and the somites are severely disorganized. These data indicate that apoptosis is required for normal notochord development during the formation of the anterior-posterior axis, and its role in this process is discussed.     

Temporal and spatial expression pattern of the myostatin gene during larval and juvenile stages of the Chilean flounder (Paralichthys adspersus)

The full length cDNA sequence of the myostatin gene was cloned from a teleostean fish, the Chilean flounder (Paralichthys adspersus) through RT-PCR amplification coupled with the RACE approach to complete the 5'- and 3'-region. The deduced amino acid sequence encodes a protein of 377 amino acid residues, including the structural domains responsible for its biological activity. Amino acid sequence comparison revealed high sequence conservation, and confirmed that the isolated sequence corresponds to the MSTN1 gene. Gene expression analysis showed that cfMSTN mRNA is present in a wide variety of tissues in juvenile fish. In addition, we assessed the spatial expression pattern of the MSTN mRNA during embryos and larval stages through whole mount in situ hybridization. No expression was observed in embryos, whereas in larvae of 8 and 9 days post fertilization, the notochord, somites, intestine and some discrete territories in the head, such as brain and eye, were positive for MSTN mRNA. Our results contribute to the knowledge of the MSTN system in larval and juvenile stages; in particular the strong expression observed in the notochord suggests that MSTN, in synchronization with positive growth signals, may play an important role in the control of the development of larvae somites.     

Analysis of perinotochordal materials. I. Studies on proteoglycans synthesis

Perinotochordal proteoglycans have been shown to influence somite chondrogenic differentiation. However, information concerning the composition of the proteoglycan molecules synthesized by the notochord, or the exact type of molecule necessary for the induction of somite chondrogenesis is not known. The results of the present study indicate that the proteoglycan extracted from the 8 day old notochord culture consists of predominantly small proteoglycans, while the large aggregates form less than 30% of the total. The chondroitin sulfate composition also shows a cartilage type of proteoglycan molecules synthesized by the notochord.     

In vitro chondrogenesis and cell viability

Anterior somites cultured with (NSA) or without (SA) notochord, and posterior somites cultured with (NSP) or without notochord (SP) were compared with respect to changes in their DNA content, their potential to synthesize the active sulfate principle phosphoadenosine phosphosulfate (PAPS), and their ability to accumulate ³⁵S-sulfate.Chondrogenesis was observed in the NSA, NSP, and SP explants, but was rarely noted in the SA explants. A decrease in DNA content during the initial 48 hr of culture was common to all explants. After this initial decrease, DNA content increased most in those explants forming cartilage. The synthesis of PAPS by cell-free extracts of each type of somite explant also decreased during the initial period of culture. Only extracts of those explants undergoing chondrogenesis showed increases in PAPS synthesis with continued culture. Each type of somite explant accumulated ³⁵S-sulfate into chondroitin sulfate during the first hours of culture. The non-chondrogenic SA explants accumulated little ³⁵S-sulfate during the period of culture. At varying times after 24 hr the chondrifying explants (NSA, SP, and NSP) initiated an increased rate of accumulation of ³⁵S-sulfate.Cartilage nodules, increases in DNA content, PAPS synthesis and ³⁵S-sulfate accumulation occurred within the same 24 hr period, during the 2nd day in NSP explants, the 3rd day in NSA explants, and between the 3rd and 4th day for SP explants. A hypothesis of in vitro somite chondrogenesis based on differential cell viability is presented.     

Variation in development of rat embryos at the presomite period.

Rat embryos at a single gestational time in the presomite period were studied for their variation in development and their fate after culture. They were explanted at 8 A.M. on day 9 of gestation from timed-pregnant Sprague-Dawley rats which were obtained by mating between 8 and 10 A.M. (plug day = day 0). In the first experiment, a total of 203 embryos from 20 litters were examined for their variation in development. Several dimensions of embryo/egg cylinder were measured and development of various embryonic/extraembryonic structures were assessed using a scoring system that we developed for the present study. Embryos were then divided into different stages of development based on their scores using the staging system that we developed previously. A large variation in developmental stage was demonstrated; the youngest embryo was at the early primitive streak stage with no signs of amniotic folds and the oldest one was at the late neural plate stage with a foregut pocket but without visible somites. No strong correlation was demonstrated between developmental stage and size of embryo/egg cylinder, nor between developmental stage and development of the proamniotic tube, ectoplacental cavity, or allantois. In the second experiment, embryos were explanted at the same time and those at different stages were cultured separately in rotating bottles and their outcomes were compared after 49 hours. The difference in mean somites number of embryos cultured from the mid primitive streak and late neural plate stages was 6.1. This difference corresponds to approximately 10 hours based on the known linear increase of somites number on day 11 of approximately 0.6 somites per hour. These results indicate a large variation in development of presomite period embryos supposedly of the same gestational age and suggest the importance of careful staging at the time of explantation if precision is needed for whole embryo culture experiments.     

Dorsoventral Differences of Morphogenetic Potencies of the Loach Blastoderm in Experiments with Alteration of Mass of Explanted Fragments

We studied the influence of doubling the mass of explanted fragments of the dorsal and ventral loach blastoderm at the early gastrula stage on their capacity for differentiation of axial structures. The dorsoventral differences are as follows: the differentiation of somites correlates, according to the results of factor analysis, with the shape complication only in double dorsal explants, while the notochord is more differentiated in the ventral fragments, if it is present, than in the dorsal ones. Doubling of the mass of dorsal fragments of the blastoderm enhances their morphogenetic potencies and shifts differentiation towards the formation of trunk axial structures. The increased mass of ventral fragments does not affect their differentiation and morphogenesis, but disturbs the correlation of these processes.     

Dorsoventral differences of morphogenetic potencies of the loach blastoderm in experiments with alteration of the mass of explanted fragments

We studied the influence of doubling the mass of explanted fragments of the dorsal and ventral loach blastoderm at the early gastrula stage on their capacity for differentiation of axial structures. The dorsoventral differences are as follows: the differentiation of somites correlates, according to the results of factor analysis, with the shape complication only in double dorsal explants, while the notochord is more differentiated in the ventral fragments, if it is present, than in the dorsal ones. Doubling of the mass of dorsal fragments of the blastoderm enhances their morphogenetic potencies and shifts differentiation towards the formation of trunk axial structures. The increased mass of ventral fragments does not affect their differentiation and morphogenesis, but disturbs the correlation of these processes.     

Formation of germ layers and roles of the dorsal lip of the blastopore in normally developing embryos of the newt Cynops pyrrhogaster

Three-dimensional relationships between tissues during the formation of germ layers were studied in sections of normally developing embryos of the newt, Cynops pyrrhogaster. In gastrulae, the inner postinvolution layer was not in direct contact with the outer preinvolution layer as a result of the presence of an intervening layer of cells. Only after the formation of the yolk plug, a narrow strip of primitive notochord, which consisted of columnar cells, established a close contact with the central part of the overlaying presumptive neural plate. The primitive notochord was also linked to endoderm at its right and left margins, facing the archenteron. Mesodermal cells other than notochord cells were mesenchymal until the neurula stage, when primitive somites appeared on both sides of the notochord. From a comparison of the relative locations of tissues in embryos at different stages of development, it was shown that the notochord elongates by a remodeling of the mass of the primitive notochord, and that, as the anteriorly directed translocation of the neural area and the invagination of endoderm occur, these processes keep pace with the elongation of the notochord. These observations suggest organizing or guiding roles for the notochord in the formation of germ layers. A role for the dorsal lip of the blastopore as the organizer is discussed in relation to the origin of the notochord.