Developmental structure of the vertebral column,fins, scutes and scales in bester sturgeon,a hybrid of beluga Huso huso and sterlet Acipenser ruthenus

In the larval bester, a hybrid sturgeon of beluga Huso huso and sterlet Acipenser ruthenus, development of cartilage around the notochord began 7 days post hatch (dph) (14·0 mm, total length, L_T). The vertebral cartilage develops in the following sequence: basidorsals and basiventrals, neural canals, neural spines and ribs. The development of ribs remained incomplete in the largest specimen (181 dph, 179 mm L_T) that was examined. Endoskeletal development of the fins began 4 dph for the dorsal and anal fins, 6 dph for the pectoral fin and 10 dph for the caudal and pelvic fins. Complete elements of all fins were observed by 91 dph and complete ossification of fin rays was observed by 122 dph in the double‐stained specimens. Observation of the histological sections, however, suggested that ossification occurred soon after the formation of the organic matrix in the fin rays. Dorsal scutes were first visible by 25 dph, followed by the lateral and ventral scutes, which were visible by 37 and 44 dph, respectively. The number of scutes was fixed at 44, 59 and 91 dph and ossification was complete by 59 (dorsal) and 91 dph (lateral and ventral scutes) in the double‐stained specimens. Ossification occurred soon after the formation of the scute organic matrix in the histological sections. Four types of scales were observed in the H. huso×A. ruthenus hybrid. Median predorsal, preanal and small scales on the anterior section of the head were visible by 59 dph. Scales on the caudal fin were visible by 91 dph and a variable assemblage of scales anterior to the anal fin was visible by 122 dph. Both the scutes and scales developed in a process that is similar to that of intramembranous ossification.     

Immunological analysis of chick notochord and cartilage matrix development with antisera to cartilage matrix macromolecules

Transverse frozen sections from the postcephalic region of stage 9-16 chick embryos and from the wing bud region of stage 17-31 embryos were stained with antibodies to the major extracellular matrix components of cartilage. These probes included unfractionated A1 and A2 antisera to the major cartilage proteoglycan, affinity-purified purified antibodies to the proteoglycan core protein and to Type II collagen, and a monoclonal antibody to keratan sulfate. In embryos as early as stage 10, notochord stained specifically with the keratan sulfate monoclonal antibody. At this stage the notochord, as well as surrounding tissues, were negative to cartilage proteoglycan and collagen antibodies. Positive staining with the latter probes was coordinately acquired by notochord cells and their accompanying sheath around stage 15, while surrounding tissues remained negative. At this stage, the ventral region of the perispinal cord sheath exhibited light staining with the proteoglycan and keratan sulfate antibodies though failing to react to Type II collagen antibodies. Positive staining of notochord and ventral spinal cord persisted through later developmental stages. As revealed by immunofluorescence, definitive vertebral chondroblasts first emerged at approximately stage 23 and definitive limb chondroblasts at stage 25. The results are discussed in terms of the possible multiple roles of notochord in early embryogenesis.     

Conditional ablation of osteoblasts in medaka

Different from tetrapods, teleost vertebral centra form without prior establishment of a cartilaginous scaffold, in two steps: First, mineralization of the notochord sheath establishes the vertebral centra. Second, sclerotome derived mesenchymal cells migrate around the notochord sheath. These cells differentiate into osteoblasts and deposit bone onto the mineralized notochord sheath in a process of intramembranous bone formation. In contrast, most skeletal elements of the cranial skeleton arise by chondral bone formation, with remarkably similar mechanisms in fish and tetrapods. To further investigate the role of osteoblasts during formation of the cranial and axial skeleton, we generated a transgenic osx:CFP-NTR medaka line which enables conditional ablation of osterix expressing osteoblasts. By expressing a bacterial nitroreductase (NTR) fused to Cyan Fluorescent Protein (CFP) under control of the osterix promoter these cells become sensitive towards Metronidazole (Mtz). Mtz treatment of stable osx:CFP-NTR transgenic medaka for several consecutive days led to significant loss of osteoblasts by apoptosis. Live staining of mineralized bone matrix revealed reduced ossification in head skeletal elements such as cleithrum and operculum, as well as in the vertebral arches. Interestingly in Mtz treated larvae, intervertebral spaces were missing and the notochord sheath was often continuously mineralized resulting in the fusion of centra. We therefore propose a dual role for osx-positive osteoblasts in fish. Besides a role in bone deposition, we suggest an additional border function during mineralization of the chordal centra. After termination of Mtz treatment, osteoblasts gradually reappeared, indicating regenerative properties in this cell lineage. Taken together, the osx:CFP-NTR medaka line represents a valuable tool to study osteoblast function and regeneration at different stages of development in whole vertebrate specimens in vivo.     

Notochordal and elastic components of the axial skeleton of fishes and their functions in locomotion

Structural details of the notochord and elastic longitudinal ligaments (dorsal and ventral) of fish are presented with discussions on their possible contribution to the speed, power and modes of swimming by conferring an automatic spring-like resilience to the vertebral axis as a whole. The notochord is also believed to function as a series of ring-like hinges placed intervertebrally which dictates that the centra must be biconcave (amphicoelous) to support and house them. Examination of about 100 species shows that, whilst the dorsal ligament is always present, the ventral is found in primitive teleosts only. The phylogenetic significance of this in relation to the different efferent branchial systems will be submitted (with diagrammatic recording of dissections) for publication in the near future. The dorsal and ventral ligaments are suitably situated to assist the circulation of lymph and blood respectively in the small lateral vessels associated with the main longitudinal one of the appropriate system. Experimental work is required to test the hypotheses presented.     

Vertebral column and associated elements in dipnoans and comparison with other fishes: development and homology.

A vertebral column consisting of a persistent notochord and ossified arcocentra is the primitive condition for Gnathostomata; it still persists in primitive actinopterygians and sarcopterygians. Advanced actinopterygians and sarcopterygians develop numerous types of centra that include, among others, the presence of holocentrum, chordacentrum, and autocentrum. The chordacentrum, a mineralization or calcification of the fibrous sheath of the notochord, is only found in actinopterygians, whereas an autocentrum is a synapomorphy of teleosts above Leptolepis coryphaenoides. The chordacentrum, formed by migration of cartilaginous cells from the arches into the fibrous sheath of the notochord and usually covered by a thin calcification, is a unique feature of chondrichthyans. The actinopterygian chordacentrum and the chondrichthyan chordacentrum are not homologous. The postcaudal cartilaginous centrum is only known in postcaudal vertebrae of living dipnoans. The holocentrum is present in certain fossil dipnoans and actinopterygians, where it has been independently acquired. It is formed by proliferation of cartilage cells around the elastica externa of the notochord. These cells later ossify, forming a compact centrum. A vertebral column formed by a persistent notochord without vertebral centra is the primitive pattern for all vertebrates. The formation of centra, which is not homologous among vertebrate groups, is acquired independently in some lineages of placoderms, most advanced actinopterygians, and some dipnoans and rhipidistians. Several series of structures are associated with the vertebral column such as the supraneurals, interhaemals, radials, and ribs. In living dipnoans median neural spine, "supraneural," and dorsal radial result from growth and distal differentiation of one median cartilage into two or three median bones during ontogeny. The median neural spine articulates with the neural arch and fuses with it in the caudal vertebrae early in ontogeny. Two bones differentiate in the anterior abdominal vertebrae, i.e., the proximal neural spine and the distal "supraneural." Three bones differentiate in front of the dorsal fin, i.e., the proximal neural spine, the middle "supraneural", and the distal radial; the same pattern is observed in front of the anal fin (the proximal haemal spine, the middle interhaemal, and the distal radial). Considering that the three dorsal (and also the three ventral) bones originate from growth of only one cartilage, they cannot be serial homologs of the neural spines, or "supraneural." They are linear homologs of the median neural cartilage in living dipnoans. The development of these elements differs within osteichthyans from sarcopterygians to actinopterygians, in which the neural spine originates as a continuation of the basidorsal arcualia and in which the supraneural and radial originate from independent cartilages that appear at different times during early ontogeny. The ribs of living dipnoans are unique in that they are not articulated with parapophyses, like in primitive fossil dipnoans, but a remnant of the ventral arcuale surrounded by a small arcocentrum remains at its base. A true caudal fin is absent in living dipnoans. The postcaudal cartilages extend to the caudal tip of the body separating dorsal and ventral rays (or the camptotrichia). Actinotrichia are present in young dipnoans. They are also known in extant actinistians and actinopterygians. They probably represent the primitive state for teleostomes. In contrast, the camptotrichia are unique for extant dipnoans (and probably Carboniferous and younger dipnoans). Lepidotrichia apparently developed many times among osteichthyans.     

Morphological development of the swim bladder in hatchery-reared striped trumpeter Latris lineata

This study examined swim bladder morphogenesis in three cohorts of striped trumpeter (Latris lineata), a euphysoclist species with physostomous larvae. The swim bladder was first discernible 1–2 days after hatching as an evagination on the dorsal surface of the incipient digestive tract. It comprised a cluster of mesenchymal cells surrounding an inner primordium of epithelial cells. At mouth opening in larvae of 5.3 mm standard length (SL), the swim bladder was noticeably enlarged. Histologically, the swim bladder lumen was dilated and liquid filled. The pneumatic duct was first seen during the dilation stage and the rete mirabile began forming among the connective tissue surrounding the swim bladder. Initial swim bladder inflation occurred on day 11 post‐hatching in Cohort 1, at 14°C, and day 9 post‐hatching, in Cohorts 2 and 3, at 16°C. Histologically, the lumens of inflated swim bladders were ellipsoid and the epithelium was squamous, except for cuboidal gas gland cells at the anterio‐ventral and anterio‐lateral regions of the swim bladder. During the initial inflation interval the pneumatic duct was dilated in larvae both with and without swim bladder inflation. The pneumatic duct began to regress in some larvae over 7.5 mm SL. The swim bladder of striped trumpeter was similar to larvae of other altricial perciform marine fish in respect to organ derivation, tissue differentiation, luminal dilation and initial gaseous inflation. However, variations, particularly the delay in initial swim bladder inflation until after the start of feeding, were observed that could be fundamental to problems encountered during larval rearing.     

Dorsal Blastomeres in the Equatorial Region of the 32-Cell Xenopus Embryo Autonomously Produce Progeny Committed to the Organizer

For testing the autonomic differentiation abilities of dorsal equatorial blastomeres of 32-cell Xenopus embryos, their roles in head formation in normal development and the organizer-inducing capabilities of the dorsal-most vegetal cells, interspecific transplantations were made using Xenopus borealis and X. laevis . When transplanted into the ventral region, the dorsal blastomeres produced descendants that differentiated into prechordal mesoderm, notochord and somites in the recipient according to their fates. They induced formation of the secondary embryo with the head and tail. The prechordal mesoderm and notochord in the secondary structure consisted of progeny of the graft, whereas somites and the CNS were chimeric and the pronephros was composed of host cells. Replacement of the dorsal blastomeres by ventral equatorial cells caused complete arrest of head formation in the recipient. Without exception, the notochord was completely absent or very thin. These results confirm the assumption that the presumptive head organizer in the Xenopus embryo is localized in the dorsal equatorial region at the 32-cell stage and comes into existence not under the inductive influence of the dorsal-most vegetal cells, but owing to allocation of morphogenetic determinants residing in the fertilized egg to the dorsal equatorial blastomeres of the 32-cell embryo.     

Dorsal and ventral cells of cleavage-stage Xenopus embryos show the same ability to induce notochord and somite formation

Cells in the dorsal marginal zone of the amphibian embryo acquire the potential for mesoderm formation during the first few hours following fertilization. An examination of those early cell interactions may therefore provide insight on the mechanisms important for organization of axial structures. The formation of mesoderm (notochord, somites, and pronephros) was studied by combining blastomeres from the animal pole region of Xenopus embryos (32- to 512-cell stages) with blastomeres from different regions of the vegetal hemisphere. The frequency of notochord and somite development was similar in combinations made with dorsal or ventral blastomeres, or with both. Our results show that during early cleavage stages the ventral half of the vegetal hemisphere has the potential to organize axial structures, a property previously believed to be limited to the dorsal region.     

Induction of notochord by the organizer inXenopus

Summary One important step in understanding early development is to define the cell interactions involved in establishing tissue types. In amphibian embryos, one such interaction is the induction by the organizer region after the late blastula stage of lateral and ventral regions of the marginal zone (MZ) to form dorsal tissue types such as muscle. It is not known whether the organizer can also induce lateral MZ to form notochord after the late blastula stage. We find that this induction occurs under experimental conditions and plays a role in normalXenopus development. The ability to induce notochord is strongest at the center of the organizer along the dorsal midline and weaker at the lateral edges of the organizer. Organizer tissue along the dorsal midline, which would differentiate as notochord in normal development, can exhibit organizer functions such as the induction of the dorsolateral MZ to form notochord without later differentiating as notochord itself. Thus organizer activity can be dissociated from subsequent notochord formation.     

Structure and Development of the Notochord ‘Elastica Externa’ and Nearby Components of the Elastic Fibre System of Agnathans

Between 15 days and 3 months in age, the ‘elastica externa’ of the notochord sheath of larval lampreys develops from patches of moderately dense and amorphous material into a thick, continuous and electron-dense layer. In both lampreys and hagfish, this layer stains strongly with Verhoeff's elastic stain and aldehyde fuchsin and is penetrated by collagen fibrils on both its outer and inner boundaries. Peroxidase labelling using an antibody raised against human elastin specifically labels both the notochord ‘elastica externa’ and the elastic fibre system of lampreys. The diameters of the microfibrils (10–13 nm) of the oxytalan, elaunin and elastic fibres of lampreys and hagfish are the same as those of higher vertebrates. The connective tissue immediately dorsal and ventral to the notochord of lampreys contains mainly oxytalan fibres in very young ammocoetes, a combination of oxytalan, elaunin and elastic fibres in older ammocoetes, and predominantly elastic fibres in adult lampreys. While the region of the endomeninx at the base of the spinal cord contains almost exclusively oxytalan fibres in young ammocoetes, it also possesses numerous elastic fibres in adult lampreys. These findings indicate that, as in higher vertebrates, the elastic fibres of lampreys develop from oxytalan fibres via elaunin fibres.     

Tissue interactions affecting the migration and differentiation of neural crest cells in the chick embryo.

A series of microsurgical operations was performed in chick embryos to study the factors that control the polarity, position and differentiation of the sympathetic and dorsal root ganglion cells developing from the neural crest. The neural tube, with or without the notochord, was rotated by 180 degrees dorsoventrally to cause the neural crest cells to emerge ventrally. In some embryos, the notochord was ablated, and in others a second notochord was implanted. Sympathetic differentiation was assessed by catecholamine fluorescence after aldehyde fixation. Neural crest cells emerging from an inverted neural tube migrate in a ventral-to-dorsal direction through the sclerotome, where they become segmented by being restricted to the rostral half of each sclerotome. Both motor axons and neural crest cells avoid the notochord and the extracellular matrix that surrounds it, but motor axons appear also to be attracted to the notochord until they reach its immediate vicinity. The dorsal root ganglia always form adjacent to the neural tube and their dorsoventral orientation follows the direction of migration of the neural crest cells. Differentiation of catecholaminergic cells only occurs near the aorta/mesonephros and in addition requires the proximity of either the ventral neural tube (floor plate/ventral root region) or the notochord. Prior migration of presumptive catecholaminergic cells through the sclerotome, however, is neither required nor sufficient for their adrenergic differentiation.     

Regulation of the Dorsal Axial Structures in Cell-Deficient Embryos of Xenopus laevis

To study the regulation of the dorsal axial structures, we removed the right animal dorsal and the right vegetal dorsal cells from an 8-cell embryo of Xenopus laevis .
Most of the right dorsal cell-deficient embryos developed to normally proportioned tailbud embryos. No detectable delay was observed in their development. Examinations of serial sections revealed that they had restored bilateral symmetry. The cell numbers of the somite and the notochord had recovered to more than 90% and 70%, respectively, those of controls. Since the right dorsal cell-deficient embryo retained roughly three-quarters of the prospective region for the somites and half of that for the notochord, respectively, the cell number was more than that expected from the remaining prospective regions. Cell lineage analyses showed that progeny of the right ventral cells had formed almost all of the right dorsal axial structures, which are normally formed by the progeny of the right dorsal cells. However, almost all the notochord cells had been derived from the remaining left dorsal cells.
These results indicate that some quantitative aspects of regulation as expressed in terms of the cell number were different between the two tissues examined.     

BMP antagonism by Noggin is required in presumptive notochord cells for mammalian foregut morphogenesis

Esophageal atresia with tracheoesophageal fistula (EA/TEF) is a serious human birth defect, in which the esophagus ends before reaching the stomach, and is aberrantly connected with the trachea. Several mouse models of EA/TEF have recently demonstrated that proper dorsal/ventral (D/V) patterning of the primitive anterior foregut endoderm is essential for correct compartmentalization of the trachea and esophagus. Here we elucidate the pathogenic mechanisms underlying the EA/TEF that occurs in mice lacking the BMP antagonist Noggin, which display correct dorsal/ventral patterning. To clarify the mechanism of this malformation, we use spatiotemporal manipulation of Noggin and BMP receptor 1A conditional alleles during foregut development. Surprisingly, we find that the expression of Noggin in the compartmentalizing endoderm is not required to generate distinct tracheal and esophageal tubes. Instead, we show that Noggin and BMP signaling attenuation are required in the early notochord to correctly resolve notochord cells from the dorsal foregut endoderm, which in turn, appears to be a prerequisite for foregut compartmentalization. Collectively, our findings support an emerging model for a mechanism underlying EA/TEF in which impaired notochord resolution from the early endoderm causes the foregut to be hypo-cellular just prior to the critical period of compartmentalization. Our further characterizations suggest that Noggin may regulate a cell rearrangement process that involves reciprocal E-cadherin and Zeb1 expression in the resolving notochord cells.     

Embryonic origin of skeletal muscle cells in the iris of the duck and quail

Summary The origin of skeletal muscle cells in avian iris muscle was investigated by quantitative analysis of heterochromatin profiles at the electron-microscopic level in irides of six types of quail-duck chimeras. Each of the following tissues was transplanted into the head region from quail to duck between stages 9 and 10: cranial neural crest; trunk neural crest; midbrain and adjacent mesoderm; forebrain; forebrain without neural crest; and forebrain without neural crest and mesoderm. The average ratio of heterochromatin profile to nucleus profile in iris skeletal muscle cells was high (quail type) in the dorsal iris, but low (duck type) in the ventral iris of the chimeras resulting from isotopic transplantation of cranial neural crest. Heterotopic transplantation of trunk neural crest to cranial position resulted in failure of development of skeletal muscle cells in the dorsal iris, but not in the appearance of skeletal muscle cells in the ventral iris. The average ratio of heterochromatin profile to nucleus profile in iris skeletal muscle cells was high in the chimeras resulting from transplantation of midbrain region and the chimeras resulting from transplantation of forebrain region, intermediate in the chimeras resulting from transplantation of forebrain region without neural crest, and low in the chimeras resulting from transplantation of forebrain region without neural crest and mesoderm. These results indicate that the skeletal muscle cells in the dorsal iris are of cranial neural crest origin while those in the ventral iris are not, and could possibly arise from cranial mesoderm.     

Nerves and nerve plexuses of the human vertebral column

The origin, distribution, and termination pattern of nerves supplying the vertebral column and its associated structures have been studied in the human fetus by means of an acetylcholinesterase whole-mount method. The vertebral column is surrounded by ventral and dorsal nerve plexuses which are interconnected. The ventral nerve plexus consists of the nerve plexus associated with the anterior longitudinal ligament. This longitudinally oriented nerve plexus has a bilateral supply from many small branches of the sympathetic trunk, rami communicantes, and perivascular nerve plexuses of segmental arteries. In the thoracic region, the ventral nerve plexus also is connected to the nerve plexuses of costovertebral joints. The dorsal nerve plexus is made up of the nerve plexus associated with the posterior longitudinal ligament. This nerve plexus is more irregular and receives contributions only from the sinu-vertebral nerves. The sinu-vertebral nerves originate from the rami communicantes and, in the cervical region, also from the nerve plexus of the vertebral artery. Thick and thin sinu-vertebral nerves are found. Most frequently three types of thick sinu-vertebral nerves are observed, i.e., ascending, descending, or dichotomizing ones. Finally, the distribution of the branches of the ventral and dorsal nerve plexuses and of the sinu-vertebral nerves is described.     

The murine Bapx1 homeobox gene plays a critical role in embryonic development of the axial skeleton and spleen

Our previous studies in both mouse and human identified the Bapx1 homeobox gene, a member of the NK gene family, as one of the earliest markers for prechondrogenic cells that will subsequently undergo mesenchymal condensation, cartilage production and, finally, endochondral bone formation. In addition, Bapx1 is an early developmental marker for splanchnic mesoderm, consistent with a role in visceral mesoderm specification, a function performed by its homologue bagpipe, in Drosophila. The human homologue of Bapx1 has been identified and mapped to 4p16.1, a region containing loci for several skeletal diseases. Bapx1 null mice are affected by a perinatal lethal skeletal dysplasia and asplenia, with severe malformation or absence of specific bones of the vertebral column and cranial bones of mesodermal origin, with the most severely affected skeletal elements corresponding to ventral structures associated with the notochord. We provide evidence that the failure of the formation of skeletal elements in Bapx1 null embryos is a consequence of a failure of cartilage development, as demonstrated by downregulation of several molecular markers required for normal chondroblast differentiation (&agr; 1(II) collagen, Fgfr3, Osf2, Indian hedgehog, Sox9), as well as a chondrocyte-specific alpha1 (II) collagen-lacZ transgene. The cartilage defects are correlated with failed differentiation of the sclerotome at the time when these cells are normally initiating chondrogenesis. Loss of Bapx1 is accompanied by an increase in apoptotic cell death in affected tissues, although cell cycling rates are unaltered.