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Degeneration of axotomized GABAergic septohippocampal neurones has been shown to be enhanced in ciliary neurotrophic factor (CNTF)-deficient mice following fimbria-fornix transection (FFT), indicating a neuroprotective function of endogenous CNTF. Paradoxically, however, the cholinergic population of septohippocampal neurones was more resistant to axotomy in these mutants. As leukaemia inhibitory factor (LIF) has been identified as a potential neuroprotective factor for the cholinergic medial septum (MS) neurones, FFT-induced responses were compared in CNTF(-/-), LIF(-/-) and CNTF/LIF double knockout mice. In CNTF(-/-) mice, FFT-induced cholinergic degeneration was confirmed to be attenuated as compared with wildtype mice. The expression of both LIF and LIF receptor beta was increased in the MS providing a possible explanation for the enhanced neuronal resistance to FFT in these animals. However, ablation of the LIF gene also produced paradoxical effects; following FFT in LIF(-/-) mice no loss of GABAergic or cholinergic MS neurones was detectable during the first postlesional week, suggesting that other efficient neuroprotective mechanisms are activated in these animals. In fact, enhanced activation of astrocytes, a source of neurotrophic proteins, was indicated by increased up-regulation of glial fibrillary acidic protein and vimentin expression. In addition, mRNA levels for neurotrophin signalling components (e.g. nerve growth factor, p75(NTR)) were differentially regulated. The positive effect on axotomized cholinergic neurones seen in CNTF(-/-) and LIF(-/-) mice as well as the increased up-regulation of astrogliose markers was abolished in CNTF/LIF double knockout animals. Our results indicate that endogenous CNTF and LIF are involved in the regulation of neuronal survival following central nervous system lesion and are integrated into a network of neurotrophic signals that mutually influence their expression and function.

Summary Glucocorticoids have been shown to influence trophic processes in the nervous system. In particular, they seem to be important for the development of cholinergic neurons in various brain regions. Here, we applied a genetic approach to investigate the role of the glucocorticoid receptor (GR) on the maturation and maintenance of cholinergic medial septal neurons between P15 and one year of age by using a mouse model carrying a CNS-specific conditional inactivation of the GR gene (GR NesCre ). The number of choline acetyltransferase and p75 NTR immuno-positive neurons in the medial septum (MS) was analyzed by stereology in controls versus mutants. In addition, cholinergic fiber density, acetylcholine release and cholinergic key enzyme activity of these neurons were determined in the hippocampus. We found that in GR NesCre animals the number of medial septal cholinergic neurons was significantly reduced during development. In addition, cholinergic cell number further decreased with aging in these mutants. The functional GR gene is therefore required for the proper maturation and maintenance of medial septal cholinergic neurons. However, the loss of cholinergic neurons in the medial septum is not accompanied by a loss of functional cholinergic parameters of these neurons in their target region, the hippocampus. This pinpoints to plasticity of the septohippocampal system, that seems to compensate for the septal cell loss by sprouting of the remaining neurons.

It is an open question to what extent neuroprotective mechanisms involving neurotrophic proteins are activated after central nervous system (CNS) lesions. Results from previous studies have indicated that ciliary neurotrophic factor (CNTF) and other members of the family of gp130-associated cytokines have neuroprotective effects on septohippocampal projection neurons axotomized by fimbria-fornix transection (FFT). Here we demonstrate that the transcription factor STAT3, a component of the primary cytokine signal transduction pathway, is transiently activated after FFT in the medial septum and in the lateral septum deafferented by the lesion. Immunocytochemical double-labeling showed nuclear signals for phosphorylated STAT3 in both types (GABAergic and cholinergic) of axotomized medial septal neurons around day 4 postlesion. This response temporally coincided with the cell-type-specific upregulation of the cytokine receptor components CNTF receptor alpha and LIF receptor beta in the same neurons. However, neuronal STAT3-activation was not abolished in CNTF- or LIF-deficient mouse mutants. Furthermore, lesion-induced STAT3 signaling was also found in reactive GFAP-positive astrocytes of the medial and lateral septum. Interestingly, basal GFAP expression was reduced but postlesional upregulation was markedly enhanced in CNTF(-/-) animals. These results demonstrate transient activation of cytokine signaling after CNS lesion and suggest that gp130-associated cytokines have multiple functions in the lesioned CNS by directly acting on axotomized neurons and on reactive astrocytes.

Application of neurotrophic proteins including ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF), members of the family of gp130-associated cytokines, can rescue CNS neurons from injury-induced degeneration. However, it is not clear so far if these effects reflect a physiological function of the endogenous cytokines. Using fimbria-fornix transection as a model, we examined whether responses of GABAergic and cholinergic septohippocampal neurons to axotomy are altered in mice lacking CNTF. In addition, we studied the cellular expression of CNTF, LIF and related cytokine receptor components in the septal complex following lesion. Degeneration of septohippocampal GABAergic neurons in the medial septum as indicated by the loss of parvalbumin-immunoreactive neurons was accelerated and permanently enhanced in CNTF(-/-) mice as compared to wild-type animals. Unexpectedly, the number of axotomized cholinergic MS neurons was significantly higher in CNTF-deficient mice during the first 2 weeks postlesion. Both in wild-type and in CNTF(-/-) mutants, expression of mRNA for the CNTF-specific alpha-subunit of the cytokine receptor complex was specifically upregulated in axotomized GABAergic septal neurons, whereas enhanced expression of the LIF-binding beta-subunit was specifically observed in axotomized cholinergic neurons. Following lesion, CNTF expression in wild-type mice was induced in activated astrocytes surrounding the axotomized neurons and at the lesion site. Expression of LIF mRNA was localized in the GABAergic and cholinergic septohippocampal neurons. These results strongly indicate that endogenous CNTF, supplied by reactive glia cells, acts as a neuroprotective factor for axotomized CNS neurons. In the septum, endogenous CNTF specifically supports lesioned GABAergic projection neurons, whereas LIF may play a similar role for the cholinergic counterparts.

Somites contribute myogenic and endothelial precursor cells to the limb bud. Transplantations of single somites have shown the pattern of muscle cell distribution from individual somites to individual limb muscles. However, the pattern of the endothelial cell distribution from individual somites to the limb has not been characterized. We have mapped quail muscle and endothelial cell distribution in the distal part of the chick limb after single somite transplantation to determine if there is a spatial relationship between muscle and endothelial cells originating from the same somite. Single brachial somites from quail donor embryos were transplanted into chick embryos, and, following incubation, serial sections were stained with a quail-endothelial cell-specific monoclonal antibody (QH-1), an anti-quail antibody (QCPN) and an anti-desmin antibody to distinguish the quail endothelial and muscle cells from chick cells. Our results show that transplants of somite 16-21 each gave rise to quail endothelial cells in the wing. The anterioposterior position of the blood vessels formed by somitic endothelial cells corresponded to the craniocaudal position of the somite from which they have originated. Endothelial cells were located not only in the peri- and endomysium but also in the subcutaneous, intermuscular, perineural and periost tissues. There was no strict correlation between the distribution of muscle and endothelial cell from a single transplanted somite. Blood vessels formed by grafted quail endothelial cells could invade the muscle that did not contain any quail muscle cells, and conversely a muscle composed of numerous quail muscle cells was lacking any endothelial cells of quail origin. Furthermore, a chimeric limb with very little quail muscle cells was found to contain numerous quail endothelial cells and vice versa. These results suggest that muscle and endothelial cells derived from the same somite migrate on different routes in the developing limb bud.

Controversy has surrounded the process of resegmentation of cervico-occipital somites. We have reinvestigated this topic by grafting single somites of quail embryos homotopically into chick embryos. Somites one to five contribute to the skull. Somites one and two contribute to the parasphenoid, which develops by direct ossification in a non-segmental fashion. All cartilaginous derivatives of the somites are segmental. Somite two forms a stripe of cells in the basioccipital, exoccipital and supraoccipital. Somites three to five give rise to the subsequent caudal parts of the basioccipital and exoccipital. Somite five forms the first motion segment including the occipital condyle, the cranial part of the atlas and the tip of the dens axis. Therefore, the border between head and neck is in the centre of somite five, and corresponds to the expression boundary of Choxb-3. Somite six forms the caudal part of the atlas and the cranial part of the axis. Somites two to eight all contribute to the cranio-cervical muscles with the exception of the Mm. rectus capitis dorsalis and ventralis and the M. biventer cervicis, which do not receive contributions from somite two. In contrast, the M. cucullaris capitis is exclusively formed by myogenic cells from somite two, which parallels its exclusive innervation by the accessory nerve. Our data confirm the segmental nature of the occiput, and show that resegmentation is a very regular process involving all except the four cranialmost somites. Except for somites one and two, all of the somites contribute to the muscles located at the appropriate levels.

Bones of the postcranial skeleton of higher vertebrates originate from either somitic mesoderm or somatopleural layer of the lateral plate mesoderm. Controversy surrounds the origin of the scapula, a major component of the shoulder girdle, with both somitic and lateral plate origins being proposed. Abnormal scapular development has been described in the naturally occurring undulated series of mouse mutants, which has implicated Pax1 in the formation of this bone. Here we addressed the development of the scapula, firstly, by analysing the relationship between Pax1 expression and chondrogenesis and, secondly, by determining the developmental origin of the scapula using chick quail chimeric analysis. We show the following. (1) The scapula develops in a rostral-to-caudal direction and overt chondrification is preceded by an accumulation of Pax1-expressing cells. (2) The scapular head and neck are of lateral plate mesodermal origin. (3) In contrast, the scapular blade is composed of somitic cells. (4) Unlike the Pax1-positive cells of the vertebral column, which are of sclerotomal origin, the Pax1-positive cells of the scapular blade originate from the dermomyotome. (5) Finally, we show that cells of the scapular blade are organised into spatially restricted domains along its rostrocaudal axis in the same order as the somites from which they originated. Our results imply that the scapular blade is an ossifying muscular insertion rather than an original skeletal element, and that the scapular head and neck are homologous to the ‘true coracoid’ of higher vertebrates.

According to the concept of resegmentation, the boundaries of vertebrae are shifted one half a segment compared with somite boundaries. This theory has been experimentally confirmed by interspecific transplantations of single somites. Due to the difficulty of exactly orientating individual somites in the host embryo, the outcome and interpretations of these experiments have occasionally been questioned. This is especially true for the formation of neural arches, their processes, and the ribs. We reinvestigated the formation of vertebrae in the avian embryo by grafting one and one half somites from quail to chick embryos. This method eliminates the possibility of a wrong somite orientation in the host embryo. Results show that the vertebral body, the neural arch and its processes are made up of material of two adjacent somites. This is also true for the rib, with the exception of the costal head, which is formed by only one somite. Whereas in the proximal part of the costal body the chick and quail cell regions border on each other in the middle of the rib, in its distal part quail cells gradually begin to mix with chick cells. The intersegmental muscles and their skeletal attachments sites are formed from the same somite. These results support and complete the data of previous studies and confirm the resegmentation concept.

The somites of vertebrate embryos give rise to sclerotomes and dermomyotomes. The sclerotomes form the axial skeleton, whereas the dermomyotomes give rise to all trunk muscles and the dermis of the back. The ribs were thought to be ventral processes of the axial skeleton and therefore to be derived from the sclerotomes; however, recently a dermomyotomal origin of the distal rib (the costal shaft) was suggested, with only the proximal parts (head and neck of the rib) being of sclerotomal origin. We have re-investigated the development of the ribs in quail-chick chimeras and carried out three experimental series. (1) Single dermomyotomes and (2) single sclerotomes were grafted homotopically, and (3) the ectoderm overlying the unsegmented paraxial mesoderm was removed in the prospective thoracic region. We found that the cells of the dermomyotome gave rise to epaxial and hypaxial trunk muscles, dermis of the back and endothelial cells, but not to ribs. Cells of the sclerotome formed the axial skeleton and all parts of the ribs. Ablation of the ectoderm, which affects dermomyotome development, results in severe malformations of the ribs, probably due to disturbed interactions between dermomyotome and sclerotome. Our results strongly confirm the traditional view of the sclerotomal origin of the ribs.

The musculature of the vertebrate tongue is composed of cells recruited from the somites. In this paper we have investigated the migration and organisation of the muscle cells that give rise to the tongue muscle during chick embryogenesis. At the molecular level, our data suggests that a population of Tbx-3 expressing cells migrate away from the occipital somites prior to the migration of muscle precursors that express Pax-3. Both populations take the same pathway and form the hypoglossal cord. The first signs of muscle cell differentiation were not detected until cells had migrated some distance from the somites. We have determined the contribution of single somites to the musculature of the tongue and show in contrast to previous data that somites 2-6 take part in the formation of all glossal and infrahyoid muscles to the same extent but do not contribute to suprahyoid muscle. This is particularly interesting since glossal and infrahyoid muscle differ from the suprahyoid muscles not only in their morphology, but also in their developmental origin. Furthermore we show that myocytes cross the midline and contribute to the contralateral glossal and infrahyoid muscles. This is supported from our molecular data, which showed that the migratory precursor population was maintained primarily at the rostral tip of the developing hypoglossal cord.

Glucocorticoids have been shown to influence trophic processes in the nervous system. In particular, they seem to be important for the development of cholinergic neurons in various brain regions. Here, we applied a genetic approach to investigate the role of the glucocorticoid receptor (GR) on the maturation and maintenance of cholinergic medial septal neurons between P15 and one year of age by using a mouse model carrying a CNS-specific conditional inactivation of the GR gene (GRNesCre). The number of choline acetyltransferase and p75NTR immuno-positive neurons in the medial septum (MS) was analyzed by stereology in controls versus mutants. In addition, cholinergic fiber density, acetylcholine release and cholinergic key enzyme activity of these neurons were determined in the hippocampus. We found that in GRNesCre animals the number of medial septal cholinergic neurons was significantly reduced during development. In addition, cholinergic cell number further decreased with aging in these mutants. The functional GR gene is therefore required for the proper maturation and maintenance of medial septal cholinergic neurons. However, the loss of cholinergic neurons in the medial septum is not accompanied by a loss of functional cholinergic parameters of these neurons in their target region, the hippocampus. This pinpoints to plasticity of the septo-hippocampal system, that seems to compensate for the septal cell loss by sprouting of the remaining neurons.

We have studied the derivatives of the first somite using the quail-chick marking technique. After transplantation of the somite, the chick embryos were reincubated for periods ranging from 4 h to 11 days. Coronal and sagittal sections of the embryos were prepared for parallel staining with Feulgen-reaction, anti-quail antibody, anti-desmin antibody and QH-1 antibody. The first somite consists of an epithelial envelope surrounding somitocoele cells. Like other somites, it forms sclerotome, dermatome and myotome. Cells contribute to the occipital and parasphenoid bone, the meninges, the dermis in the occipital region and the pharyngeal connective tissue. The contribution of the first somite to bones, meninges, dermis and pharyngeal connective tissue is characterised by sharp anterior and posterior boundaries. In contrast, other derivatives such as connective tissue surrounding the vagus nerve, the carotid artery, and jugular vein exceed 10 to 18 segments. This is also true for myogenic cells participating in the formation of the cucullaris capitis muscle that extends from the temporal bone to the shoulder. In one third of the embryos, myocytes of the intrinsic laryngeal muscles are derived from the grafted first somite. Moreover, endothelial cells originate from this somite and migrate into the head (hind-brain, meninges, dermis), neck (pharynx, connective tissue surrounding the vagus nerve, carotid artery and jugular vein) and thorax. With respect to differentiation and derivatives the first somite is similar to other somites.

We have studied the angiogenic potential of the unsegmented paraxial mesoderm and epithelial somites of the trunk with homotopical grafts between quail and chick embryos. Quail endothelial cells of the grafts were stained with the QH-1 antibody after 1-6 days of reincubation. The unsegmented paraxial mesoderm and all parts of the epithelial somite were found to contain angioblasts which develop into QH-1 positive endothelial cells. These cells are incorporated into the lining of the host's blood vessels such as the perineural vascular plexus and the dorsal branches of the aorta. There is a certain preference as concerns the location of endothelial cells derived from different parts of the somites. Angioblasts from ventral somite halves are mainly found in ventrolateral blood vessels. Those from dorsomedial quadrants form vessels in the dermis of the back, and those from dorsolateral quadrants can be found in the ventrolateral body wall and the wing. With the exception of the dorsal perineural vascular plexus, angioblasts do not cross the median plane of the body. This shows that, although angioblasts migrate extensively, there is bilaterality of the vascular system in the trunk. It remains to be studied whether the notochord plays a role in the establishment of this bilaterality.

The early somite of avian embryos is made up of an epithelial wall and mesenchymal cells located within the somitocoele. We have studied the fate of somitocoele cells for a period of up to 6 days, using the quail-chick marker technique. We also applied the QH-1 antibody, which specifically stains hemangiopoietic cells of quail origin, and studied the proliferative activity of epithelial somites with the BrdU anti-BrdU method. Our results show that somitocoele cells mainly give rise to the ribs and peripheral parts of the intervertebral discs. After 1 and 2 days of reincubation, the grafted somitocoele cells were located in the lateral part of the sclerotome, and only a few cells migrated axially towards the notochord. In frontal sections, the cells were located in a triangular area within the cranial part of the caudal sclerotome half. After 3 days of reincubation, some of the cells had migrated cranially along the myotome. After longer reincubation periods, cells grafted into one somite could be found in two adjacent ribs. The studies with the QH-1 antibody show that a subpopulation of somitocoele cells has angiogenic potency. Endothelial cells originating from the mesenchyme of the somitocoele migrated actively and even invaded the ipsilateral half of the neural tube. In the epithelial wall of the somite, BrdU-labelled nuclei were found basally, whereas more apically the nuclei were not stained, but mitotic figures were frequently present. The somitocoele cells also showed a high proliferative activity with about 26% of nuclei labelled with BrdU.