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The time of appearance and location of three distinct collagen gene transcripts termed 1 alpha, 2 alpha, and 3 alpha, were monitored in the developing S. purpuratus embryo by in situ hybridization. The 1 alpha and 2 alpha transcripts of fibrillar collagens were detected simultaneously in the primary (PMC) and secondary (SMC) mesenchyme cells of the late gastrula stage and subsequently expressed in the spicules and gut associated cells of the pluteus stage. The 3 alpha transcripts of the basement membrane collagen appeared earlier than 1 alpha and 2 alpha, and were first detected in the presumptive PMC at the vegetal plate of the late blastula stage. The PMC exhibited high expression of 3 alpha at the mesenchyme blastula stage, but during gastrulation the level of expression was reduced differentially among the PMC. In the late gastrula and pluteus stages, both PMC and SMC expressed 3 alpha mRNA, and thus at these stages all three collagen genes displayed an identical expression pattern by coincidence. This study thus provides the first survey of onset and localization of multiple collagen transcripts in a single sea urchin species.

The preotic neural tube has a variable ability for regeneration of neural crest depending on the neuraxial level. There is robust regeneration of neural crest in the caudal midbrain/rostral hindbrain. In contrast, removal of the cardiac neural crest results in cardiovascular abnormalities suggesting the lack of regeneration in this area, although the regenerative capacity of the cardiac crest region has never been tested directly. Premigratory cardiac neural crest was ablated bilaterally using laser irradiation or extirpation by tungsten needle, and the remaining ventral neural tube was labeled with DiI to examine any neural crest regeneration from the neural tube. The results indicate that there is very little regeneration of crest cells from the cardiac region of the neural tube if the ablation is done prior to the 5-somite stage and no regeneration after the 6-somite stage with either ablation procedure. Furthermore no compensatory response occurs from the adjacent regions of the neural crest. By contrast, we were able to confirm that regeneration of neural crest occurs in the preotic rhombencephalic neural tube even after laser irradiation. An analysis in the trunk region suggests that the trunk neural tube is similar to the cardiac region in that it does not regenerate crest cells in the ventral migratory pathway after ablation. However, melanocytes generated cranial and caudal to the ablated region migrate radially and fill in the ablated region so that there is no interruption of the normal pigment pattern. This study indicates that even though there is a variable capacity for crest regeneration in the preotic neural tube, the postotic neural tube does not have such regenerative ability.

By utilizing myosin immunostaining, we were able to identify early rat myocardium as a thin epithelial sheet and realized that its cohesive movement toward the midline leads to the straight heart tube formation. Localization study of fibronectin mRNA and protein was, therefore, carried out to investigate its tissue origin and possible roles in facilitating mesoderm migration and heart formation. Fibronectin mRNAs were first detected throughout the mesoderm during the early primitive streak stage, suggesting that the mesoderm is the source of fibronectin. By pre-head fold (pre-somite) and head fold (early somite) stages, the mesoderm became largely down-regulated for fibronectin mRNAs, while it was also at these stages when myosin-positive myocardium formed itself into the epithelium and was subsequently folding toward the midline. Thus, there appears to be little fibronectin synthesis during and directly relevant to early heart tube formation. Later, during the early straight heart tube stage (5 somite and older), endocardium became highly positive for fibronectin mRNAs, suggesting that the endocardium is the major source of fibronectin for the cardiac jelly. Based on the results, we present a map for the early mammalian heart in which the heart is a single crescentic band lying in front of the prechordal plate. We also suggest a process for heart tube formation based on the cohesive movement of the myocardial epithelium. During heart tube formation, fibronectin protein had been deposited previously by the mesoderm and was found uniformly in the ECM and not newly produced by any adjacent tissue. The data contradict the endodermal guidance of heart migration by fibronectin gradient and suggest, instead, a permissive role for the fibronectin substrate.

Correct assembly of fibrillar collagen networks plays a critical role in animal morphogenesis. Very little is known about the contribution of the so-called minor fibrillar collagens (types V and XI) to fibrillogenesis. Here we examined the developmental expression of the mouse pro-alpha 2(V) collagen gene (col5a2) after the cloning and sequencing of cDNAs that cover the entire length of the message. Transcripts of col5a2, detectable as early as 9 days of gestation, localize with distinct patterns in the tissues of day 12.5 and day 16.5 fetuses. The earlier developmental stage is characterized by low and diffuse col5a2 expression in the peritoneal membranes and intestinal and craniofacial mesenchymes. The later stage exhibits higher and more restricted col5a2 mRNA accumulation in primary ossified regions, perichondrium, joints, tendon, atrioventricular valve of the heart, and selected portions of the head. A parallel analysis using a cartilage-specific pro-alpha 1(II) collagen (col2a1) probe confirmed that these two collagen genes are transcribed in a mutually exclusive manner during mouse embryogenesis. On the other hand, the developmental pattern of col5a2 expression closely resembles that of the type I collagen, thus further substantiating the notion that these macromolecules cooperate in the formation of fibrillar networks in non-cartilaginous matrices.

The HstI gene is a transforming gene, coding for a protein of the fibroblast growth factor family (Sakamoto et al., 1986). Previous RNA hybridization studies with the mouse homolog demonstrated the presence of a 3.0-kb transcript in Day 11 and 14 mouse embryos. Here we detect a 3.0-kb transcript in the limb and body of the dissected Day 11 mouse embryo. PCR amplification using HstI-specific primers also showed comparable results. In order to localize the HstI transcripts during development, corresponding HstI cDNA was isolated, and an HstI-specific region was used as a probe for in situ hybridization analysis. Serial sections of embryos from Day 8 (early-somite stages) through Days 9, 10, 11, and 12 of gestation were examined. With the antisense probe, a signal was detected in the Day 11 and 12 embryo, where it was localized to the apical ectodermal ridge (AER) of the limb bud. This structure is well known for its role in promoting the distal outgrowth of the developing limb bud. Signal was detected in both fore- and hindlimbs during the period of rapid distal growth. This restricted localization suggests a role for HstI in normal embryogenesis, including outgrowth of the limb bud.

Here we describe the isolation and characterization of a new chicken homeobox-containing gene, G-Hox 7, which is related to Drosophila msh. The deduced amino acid sequence of the cDNA shows greater than 96% homology to the homeo domain of other vertebrate msh-like genes. As for other species, the amino and carboxy termini of the protein are, however, greatly divergent when compared phylogenetically. In situ hybridization studies revealed the early and wide-spread expression of G-Hox 7 during chick development. This includes its expression in the primitive streak and extraembryonic cells undergoing epiboly, and its expression along the neural axis, including the forebrain. Expression was also observed in the neural crest, neural crest-derived facial and branchial structures, the otocyst, limb, and heart valves. This widespread and recurrent expression of the transcript suggests that the gene may play an essential role at multiple sites during the initiation of new developmental pathways.

The pattern of type II collagen expression during Xenopus laevis embryogenesis has been established after isolating specific cDNA and genomic clones. Evidence is presented suggesting that in X. laevis there are two transcriptionally active copies of the type II procollagen gene. Both genes are activated at the beginning of neurula stage and steady-state mRNA levels progressively increase thereafter. Initially, the transcripts are localized to notochord, somites, and the dorsal region of the lateral plate mesoderm. At later stages of development and parallel to increased mRNA accumulation, collagen expression becomes progressively more confined to chondrogenic regions of the tadpole. During the early period of mRNA accumulation, there is also a transient pattern of expression in localized sites that will later not undergo chondrogenesis, such as the floor plate in the ventral neural tube. At later times and coincident with the appearance of chondrogenic tissues in the developing embryo, expression of the procollagen genes is characterized by the production of an additional, alternatively spliced transcript. The alternatively spliced sequences encode the cysteine-rich globular domain in the NH2-propeptide of the type II procollagen chain. Immunohistochemical analyses with a type II collagen monoclonal antibody documented the deposition of the protein in the extracellular matrix of the developing embryo. Type II collagen expression is therefore temporally regulated by tissue-specific transcription and splicing factors directing the synthesis of distinct molecular forms of the precursor protein in the developing Xenopus embryo.

We have isolated cDNAs encoding a novel Xenopus homeodomain-containing protein homologous to the mouse Hox-7.1 and the Drosophila muscle segment homebox (msh). Northern blot and RNAase protection experiments established that transcripts of the frog gene, termed Xhox-7.1, first appear at about the beginning of gastrulation. After a rapid increase, mRNA levels plateau between the neurula and middle-tailbud stages, and decrease steadily thereafter. In situ hybridization localized the Xhox-7.1 message to the dorsal mesodermal mantle of gastrula stage embryos. Comparison of the hybridization patterns of progressively more anterior cross-section of tailbud stage embryos localized the signal to the dorsal neural tube and neural crest, to specific regions of the lateral plate mesoderm, and to the cardiogenic region. By the tadpole stage, the Xhox-7.1 message appears only at specific sites in the central nervous system, such as in the dorsal hindbrain. Thus, during embryonic development levels of Xhox-7.1 expression decrease as the transcript becomes more progressively localized. Finally, evidence is presented of a distinct msh-like transcript (provisionally termed Xhox-7.1') which begins to accumulate at early-gastrula stage, as well.

We have cloned and characterized several overlapping cDNAs that specify a large portion of a Paracentrotus lividus fibrillar collagen molecule. Our conclusions are based on sequencing data, which showed that the clones code for a 786-amino acid collagenous domain composed of an uninterrupted series of Gly-X-Y repeats and for a 265-amino acid carboxyl-terminal globular extension. The latter domain exhibits features highly reminiscent of those of the vertebrate counterparts, notably a putative carboxyl-peptidase cleavage site, a series of similarly arranged cysteinyl residues, and an N-linked glycosylation attachment site. In situ and Northern blot hybridizations have established the size, time of appearance, and tissue localization of the collagen mRNA during sea urchin development. The collagen transcript, 9 kilobases in length, is first detected in the primary and, more predominantly, in the secondary mesenchyme cells of late gastrulae where it progressively accumulates thereafter. This and other work (D'Alessio, M., Ramirez, F., Suzuki, H.R., Solursh, M., and Gambino, R. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 9303-9307) provide evidence of a genetic heterogeneity of fibrillar collagens in the sea urchin embryo and suggest that the two genes are activated in the same cell lineages at distinct developmental stages.

We have isolated and characterized cDNA and genomic clones that specify a Paracentrotus lividus procollagen chain. The cDNAs code for 160 uninterrupted Gly-Xaa-Yaa triplets and a 252-amino acid carboxyl propeptide. Analysis of the deduced amino acid sequences indicated that the sea urchin polypeptide exhibits structural features that are characteristic of the fibril-forming class of collagen molecules. Partial characterization of two genomic recombinants revealed that the 3' end of the echinoid gene displays a complex organization that closely resembles that of a prototypical vertebrate fibrillar collagen gene. In situ and Northern (RNA) blot hybridizations established the size, time of appearance, and tissue distribution of the collagen transcripts in the developing sea urchin embryo. Collagen mRNA, approximately equal to 6 kilobases in size, is first detected in the forming primary mesenchyme cells of late blastulae where it progressively accumulates until the free swimming/feeding pluteus larval stage. Interestingly, collagen transcripts are also detected in the forming secondary mesenchyme cells of late gastrulae, and by the prism stage, their derivatives appear to be the most intensively labeled cells.

When the leg buds from chick embryos at stages 17-23 were cultured as organ cultures in F12 medium, which contained chick serum and an extract of chick embryos, leg cartilage structures developed that included pelvic girdle, femur, tibiofibula, and tarsometatarsus. The proportion of explants with definite distal elements increased when the leg buds from later embryos were used. Supplementation with chick serum was essential. These results suggest that the pattern of formation of limbs in chick embryos can be studied in organ culture.

We have previously succeeded in culturing whole leg bud from stage 21-23 chick embryos and observed a leg structure with typical cartilage pattern in vitro. In the present study, we have attempted the organ culture of the fragmented leg bud and investigated its capacity to form cartilage. Leg buds from stages 17-21 chick embryos were dissected into four pieces in the anteroposterior sequence (named 1, 2, 3, and 4, respectively) and cultured on a membrane filter in a medium consisting of Ham's F-12, chick serum, and chick embryo extract. After 6 days in culture, two central fragments (2 and 3) developed into large cartilaginous masses, while anterior (1) and posterior (4) fragments formed few or small cartilaginous masses. In addition, when these less chondrogenic fragments were combined, pinned together, and cultured, large cartilaginous masses were formed from 1 + 4 combinations but not from 1 + 1 or 4 + 4 combinations. These observations were analyzed quantitatively by measurement of 35SO4 incorporation into the sulfated glycosaminoglycan (S-GAG) and of final DNA content per explant, and by histological reconstruction of the chick-quail chimera explant. The results showed that (a) the 1 + 4 combination resulted in higher S-GAG synthesis and final DNA content than the 1 + 1 or 4 + 4 combinations in stage 18 and 21 leg buds (P less than 5%); (b) removal of ectoderm from the leg bud inhibited the increase observed for the 1 + 4 combination; c) in chick-quail chimera explants the cartilage formed from the 1 + 4 combination was largely of fragment 1 origin. These results demonstrate, first, the presence of a difference in chondrogenic capacity along the anteroposterior axis in the leg bud and, second, the occurrence of an interaction between anterior and posterior fragments which mimics the effects of grafting a zone of polarizing activity (ZPA). The mechanism of ZPA function is still unknown but the ectoderm may play some role. Some roles for ectoderm in ZPA function and differences in mesodermal responsiveness to ZPA factor(s) are suggested.