Selective agenesis of the dorsal pancreas in mice lacking homeobox gene Hlxb9.

The initial stages of pancreatic development occur early during mammalian embryogenesis, but the genes governing this process remain largely unknown. The homeodomain protein Pdx1 is expressed in the developing pancreatic anlagen from the approximately 10-somite stage, and mutations in the gene Pdx1 prevent the development of the pancreas. The initial stages of pancreatic development, however, still occur in Pdx1-deficient mice. Hlxb9 (encoding Hb9; ref. 6) is a homeobox gene that in humans has been linked to dominant inherited sacral agenesis and we show here that Hb9 is expressed at early stages of mouse pancreatic development and later in differentiated beta-cells. Hlxb9 has an essential function in the initial stages of pancreatic development. In absence of Hlxb9 expression, the dorsal region of the gut epithelium fails to initiate a pancreatic differentiation program. In contrast, the ventral pancreatic endoderm develops but exhibits a later and more subtle perturbation in beta-cell differentiation and in islet cell organization. Thus, dorsally Hlxb9 is required for specifying the gut epithelium to a pancreatic fate and ventrally for ensuring proper endocrine cell differentiation.     

Sensory transmitters regulate intracellular calcium in dorsal horn neurons

Primary afferent terminals in the dorsal horn of the spinal cord release excitatory amino acid and peptide transmitters that initiate the central processing of nociceptive information. The postsynaptic actions of amino acid transmitters on spinal neurons have been well characterized, but the cellular basis of peptide actions remains unclear. Substance P is the best characterized of the peptides present in sensory neurons and has been shown to depolarize dorsal horn neurons and to facilitate nociceptive reflexes. To determine the mechanisms by which substance P contributes to afferent synaptic transmission, we have monitored the levels of intracellular calcium in single isolated rat dorsal horn neurons and report that substance P can produce a prolonged elevation in calcium concentration by mobilizing its release from intracellular stores. This elevation may contribute to the long-term changes in the excitable properties of dorsal horn neurons that occur following afferent fibre stimulation. We have also found that L-glutamate elevates intracellular calcium in substance P-sensitive dorsal horn neurons by increasing calcium influx. These results provide a direct demonstration of intracellular calcium changes in response to neuropeptides in mammalian central neurons. They also indicate that there is convergent regulation of intracellular calcium in dorsal horn neurons by two different classes of sensory transmitters that are co-released from the same afferent terminals.     

Chemotropic guidance of developing axons in the mammalian central nervous system

In the developing nervous system, axons project considerable distances along stereotyped pathways to reach their targets. Axon guidance depends partly on the recognition of cell-surface and extracellular matrix cues derived from cells along the pathways. It has also been proposed that neuronal growth cones are guided by gradients of chemoattractant molecules emanating from their intermediate or final cellular targets. Although there is evidence that the axons of some peripheral neurons in vertebrates are guided by chemotropism and the directed growth of some central axons to their targets is consistent with such a mechanism, it remains to be determined whether chemotropism operates in the central nervous system. During development of the spinal cord, commissural axons are deflected towards a specialized set of midline neural epithelial cells, termed the floor plate, which could reflect guidance by substrate cues or by diffusible chemoattractant molecules. Here we provide evidence in support of chemotropic guidance by demonstrating that the rat floor-plate cells secrete a diffusible factor(s) that influences the pattern and orientation of commissural axon growth in vitro without affecting other embryonic spinal cord axons. These findings support the hypothesis that chemotropic mechanisms guide developing axons to their intermediate targets in the vertebrate CNS.     

Genetic ablation reveals that the roof plate is essential for dorsal interneuron specification

During neural development in vertebrates, a spatially ordered array of neurons is generated in response to inductive signals derived from localized organizing centres. One organizing centre that has been proposed to have a role in the control of neural patterning is the roof plate. To define the contribution of signals derived from the roof plate to the specification of neuronal cell types in the dorsal neural tube, we devised a genetic strategy to ablate the roof plate selectively in mouse embryos. Embryos without a roof plate lack all the interneuron subtypes that are normally generated in the dorsal third of the neural tube. Using a genetically based lineage analysis and in vitro assays, we show that the loss of these neurons results from the elimination of non-autonomous signals provided by the roof plate. These results reveal that the roof plate is essential for specifying multiple classes of neurons in the mammalian central nervous system.     

ATP excites a subpopulation of rat dorsal horn neurones

The peripheral receptive properties and central projections of different classes of dorsal root ganglion neurones are well characterized. Much less is known about the transmitters used by these neurones. Excitatory amino acids have been proposed as sensory transmitters but the sensitivity of virtually all central neurones to those compounds has made it difficult to assess their precise role in sensory transmission. Several neuropeptides have been localized within discrete subclasses of primary sensory neurones that project to the superficial dorsal horn of the spinal cord and may be afferent transmitters. However, only about one-third of spinal sensory neurones have been shown to contain neuropeptides. We have recently described the presence of a 5'-nucleotide hydrolysing acid phosphatase in a separate subpopulation of dorsal root ganglion neurones that project to the superficial dorsal horn. This enzyme also appears in certain autonomic and endocrine cells that contain high concentrations of releasable nucleotides in their storage granules. It is possible that the presence of this enzyme in sensory neurones is also associated with a releasable pool of nucleotides. Holton and Holton have provided evidence that ATP is released from the peripheral terminals of unmyelinated sensory fibres and have suggested that release of ATP might also occur from central sensory terminals. To investigate the possibility that nucleotides act as central sensory transmitters we have examined their actions on rat dorsal horn and dorsal root ganglion neurones maintained in dissociated cell culture. We report here a selective and potent excitation of subpopulations of both neuronal types by ATP.     

A monoclonal antibody defining antigenic determinants on subpopulations of mammalian neurones and Trypanosoma cruzi parasites

An IgM lambda class monoclonal antibody raised against membranes from rat dorsal root ganglia defines a novel antigenic determinant expressed by subpopulations of mammalian central and peripheral neurones. In the presence of complement the antibody is cytotoxic to mammalian neurones in vitro. The same antibody labels Trypanosoma cruzi, the protozoan responsible for Chagas' disease. Classes of mammalian neurones and cardiac muscle that are labelled by the antibody are known to degenerate in Chagas' disease. The common neuronal and trypanosomal antigens recognized by the antibody may therefore be important in pathogenic events underlying this disorder.