Neurohypophysial dysmorphogenesis in mice lacking the homeobox gene Uncx4.1.

A number of transcription factors have been implicated in the development of the hypothalamo-neurohypophysial system (HNS). Null mutations for these factors caused severe defects in proliferation, migration and survival during early embryogenesis. While they have informed about early events of HNS developments no insights in mechanisms of late development and maturation of this major peptidergic system have been obtained as yet. In a screen for adult-expressed homeobox genes we identified Uncx4.1 as a gene expressed in adult and embryonic magnocellular neurons of the (HNS). Null mutation of Uncx4.1 left these neurons viable and able to express neuropeptides. However, the connectivity of magnocellular neurons with posterior pituitary elements was compromised. As a consequence neuronal fibres traversed to the adenohypophysis. The penetrance of this phenotype was about 50%. The data show a selective role of Uncx4.1 in controlling the development of connections of hypothalamic neurons to pituitary elements, allowing central neurons to reach the peripheral blood circulation and to deliver hormones for control of peripheral functions.

Survey for paired-like homeodomain gene expression in the hypothalamus: restricted expression patterns of Rx, Alx4 and goosecoid.

Homeobox genes are important regulators of cellular identity. Several homeobox genes are known to be specifically expressed in subsets of neurons in the forebrain, exclusively, or in distinct combinations. In this study, we explored the expression of homeobox genes in the forebrain of the adult rat by a degenerate polymerase chain reaction cloning strategy. We identified the expression of 12 homeobox genes, several of which display a remarkable restricted expression pattern in the adult brain. We demonstrated the expression of goosecoid in a very small set of neurons in the hypothalamus. By using Otp as a marker, these goosecoid-positive cells were found to constitute a small area just beside the paraventricular nucleus. Furthermore, we found expression of Rx in the pineal gland, along with Alx4. Rx was additionally found in the posterior pituitary and in cells aligning the bottom of the third ventricle. These findings form a starting point to reveal functions of the described homeobox genes in the forebrain.

The homeobox genes Lhx7 and Gbx1 are expressed in the basal forebrain cholinergic system.

The specific combination of homeobox genes is proposed to be decisive in the terminal differentiation of neuronal systems. In order to identify combined expression of homeobox genes in the ventral forebrain, a reverse transcriptase-polymerase chain reaction strategy using degenerated primers was employed. We identified, amongst others, Lhx7 and Gbx1, displaying a marked overlapping expression in septal and pallidal areas. Gbx1 and Lhx7 were both expressed in those adult brain nuclei that collectively form the basal forebrain cholinergic system, a prime target of neurodegeneration in Alzheimer's disease. Indeed, we detected Lhx7 within cholinergic neurons, whereas the related Lhx6 gene was found in adjacent neurons. From these data we suggest that combined expression of Lhx7 and Gbx1 plays a role in the development of the cholinergic system of the basal forebrain. It is speculated that both genes remain participating in molecular processes in the adult cholinergic neurons, and can be employed to study regulation and survival of these neurons under normal and pathological conditions.

A second independent pathway for development of mesencephalic dopaminergic neurons requires Lmx1b.

We identified the LIM homeodomain transcription factor Lmx1b in the mesencephalic dopamine (mesDA) systems of embryos and adults. Analysis of spatiotemporal expression in Lmx1b null mutants and wild-type mice implicated a cascade involving Lmx1b in the early development of mesDA neurons. Although disruption of this cascade did not block induction of tyrosine hydroxylase (TH), a key enzyme in DA synthesis, or Nurr1, a nuclear hormone receptor, Lmx1b knockout mice failed to induce the mesDA-specific homeodomain gene Ptx3 in TH-positive neurons. Eventually, this small set of TH-positive neurons was lost during embryonic maturation. The data suggest that at least two molecular cascades operate during the specification of the mesDA system, one specifying neurotransmitter phenotype and another essential for other aspects of mesDA neuron differentiation.

Co-expression in Xenopus neurons and neuroendocrine cells of messenger RNA homologues of exocytosis proteins DOC2 and munc18-1.

The proteins munc18-1 and DOC2 are assumed to play a role in docking of synaptic vesicles in neurotransmitter exocytosis at the presynaptic junction. As the proteins are known to interact, they should co-exist within neurons. We have tested this hypothesis for exocytosis of both classical and peptidergic messengers, by investigating the distribution of the messenger RNAs of munc 18-1 and DOC2 homologues in the brain and pituitary gland of the clawed toad Xenopus laevis, using in situ hybridization. For this purpose we cloned a partial complementary DNA encoding Xenopus unc18 (xunc18) and used a corresponding RNA probe, together with an RNA probe for Xenopus DOC2. At the messenger RNA level DOC2 and xunc18 were found to be expressed throughout the Xenopus brain. All brain nuclei expressing DOC2-messenger RNA showed xunc18-messenger RNA expression as well. Co-expression was shown at the individual cell level in consecutive sections of large-sized neurons. A strong expression was demonstrated in the suprachiasmatic and magnocellular nuclei and in peptidergic endocrine cells in the intermediate and anterior lobes of the pituitary gland, suggesting roles of DOC2 and xunc18 in messenger release from peptidergic secretory systems. Combined in situ hybridization and immunocytochemical analyses show that neuropeptide Y-containing cells in the suprachiasmatic nucleus also express DOC2 and xunc18 messenger RNAs. Since these cells have a high secretory activity, controlling the activity of the pituitary pars intermedia, the levels of expression of DOC2 and xunc18 may be indicators for neuronal secretory activity. The present data represent the first evidence for the co-existence of DOC2 and munc18-1 and suggest co-ordinate action of these proteins at the level of brain nuclei, individual neurons and endocrine cells.

Hypothalamic transcription factors and the regulation of the hypothalamo-neurohypophysial system.

The transcription factors that confer high level expression and regulate the genes encoding neurohypophysial hormones are largely unknown. A number of different approaches have been taken to identify these factors and to elucidate molecular mechanisms of physiological gene regulation. In this chapter two transcription factor families are considered: homeodomain proteins and nuclear receptors. Their identification in the hypothalamus and actions on the OT gene are addressed here.