Donna M. Simmons

A tissue-specific transcription factor containing a homeodomain specifies a pituitary phenotype

Multiple related cis-active elements required for cell-specific activation of the rat prolactin gene appear to bind a pituitary-specific positive transcription factor(s), referred to as Pit-1. DNA complementary to Pit-1 mRNA, cloned on the basis of specific binding to AT-rich cell-specific elements in the rat prolactin and growth hormone genes, encodes a 33 kd protein with significant similarity at its carboxyl terminus to the homeodomains encoded by Drosophila developmental genes. Pit-1 mRNA is expressed exclusively in the anterior pituitary gland in both somatotroph and lactotroph cell types, which produce growth hormone and prolactin, respectively. Pit-1 expression in heterologous cells (HeLa) selectively activates prolactin and growth hormone fusion gene expression, suggesting that Pit-1 is sufficient to confer a characteristic pituitary phenotype. The structure of Pit-1 and its recognition elements suggests that metazoan tissue phenotype is controlled by a family of transcription factors that bind to related cis-active elements and contain several highly conserved domains.

Immunohistochemical localization of neuronal nicotinic receptors in the rodent central nervous system

The distribution of nicotinic acetylcholine receptors (AChR) in the rat and mouse central nervous system has been mapped in detail using monoclonal antibodies to receptors purified from chicken and rat brain. Initial studies in the chicken brain indicate that different neuronal AChRs are contained in axonal projections to the optic lobe in the midbrain from neurons in the lateral spiriform nucleus and from retinal ganglion cells. Monoclonal antibodies to the chicken and rat brain AChRs also label apparently identical regions in all major subdivisions of the central nervous system of rats and mice, and this pattern is very similar to previous reports of 3H-nicotine binding, but quite different from that of alpha-bungarotoxin binding. In several instances, the immunohistochemical evidence has strongly indicated that neuronal AChR undergoes axonal transport. The clearest example of this has been in the visual system, where labeling was observed in the retina, the optic nerve and tract, and in all of the major terminal fields of the optic nerve except the ventral suprachiasmatic nucleus. This was confirmed in unilateral enucleation experiments in the rat, where labeling was greatly reduced in the contralateral optic tract, ventral lateral geniculate nucleus, pretectal nuclei receiving direct visual input, superficial layers of the superior colliculus, and medical terminal nucleus, and was significantly reduced in the dorsal lateral geniculate nucleus. Clear neuronal labeling was also observed in dorsal root ganglion cells and in cranial nerve nuclei containing motoneurons that innervate branchial arch-derived muscles, although the possibility that neuronal AChR undergoes axonal transport in the latter cells was not tested experimentally.(ABSTRACT TRUNCATED AT 250 WORDS)

Basic Fibroblast Growth Factor (FGF) in the Central Nervous System: Identification of Specific Loci of Basic FGF Expression in the Rat Brain

The expression of basic FGF mRNA, while virtually absent in peripheral tissues, appears to be constitutively expressed in the central nervous system. As such, while it is difficult to detect any mRNA encoding basic FGF in the heart, lung, kidneys, ovaries, liver, or pituitary of rats, the levels are easily detected in brain. A regional analysis of the expression of basic FGF mRNA in brain reveals that it is widely distributed in the cortex (frontal, parietal, and occipital), the hippocampus, hypothalamus, and pons. Only a few loci of basic FGF synthesis are detected by in situ hybridization and include layers 2 and 6 of the medial (cingulate) cortex, the indusium griseum, fasciola cinereum, and field CA2 of the hippocampus. The identification of specific cell populations in the brain, and particularly in the hippocampus, that synthesize basic FGF supports the notion that this potent neurotrophic factor is involved in normal CNS function and that the presence (or absence) of its expression may be linked to the pathogenesis of the neurogenerative diseases characterizing these various loci. The significance of these findings with respect to the regulation of basic FGF expression in peripheral tissue and the central nervous system is discussed.

Regulation of intracellular pH by a neuronal homolog of the erythrocyte anion exchanger

We have isolated AE3, a novel gene expressed primarily in brain neurons and in heart. The predicted AE3 polypeptide shares a high degree of identity with the anion exchange and cytoskeletal binding domains of the erythrocyte band 3 protein. Expression of AE3 cDNA in COS cells leads to chronic cytoplasmic acidification and to chloride- and bicarbonate-dependent changes in intracellular pH, confirming that this gene product is an anion exchanger. Characterization of an AE3 mutant lacking the NH2-terminal 645 amino acids demonstrates that the COOH-terminal half of the polypeptide is both necessary and sufficient for correct insertion into the plasma membrane and for anion exchange activity. The NH2-terminal domain may play a role in regulating the activity of the exchanger and may be involved in the structural organization of the cytoskeleton in neurons.

Brain 4: a novel mammalian POU domain transcription factor exhibiting restricted brain-specific expression.

The POU domain gene family of transcription factors share a conserved bipartite DNA binding domain, and exhibit distinct temporal and spatial patterns of expression during development, particularly in the forebrain. A cDNA encoding a new member of the POU‐III class of the POU domain gene family, referred to as Brn‐4, was isolated from a rat hypothalamic cDNA library. Like other mammalian POU‐III genes previously characterized (Brn‐1, Brn‐2, Tst‐1), Brn‐4 transcripts are initially widely expressed at all levels of the developing neural tube, but in contrast to other previously described POU‐III genes, are subsequently restricted to only a few regions of the adult forebrain, including the supraoptic and paraventricular nuclei of the hypothalamus. Brn‐4 was shown to bind to DNA sequences containing the octamer motif and to trans‐activate promoters containing this DNA binding motif, based on the actions of a unique N‐terminal information. This ontogenic pattern of Brn‐4 expression in concert with that of Oct‐2 and Pit‐1, indicates that certain POU domain genes potentially exert their primary functions widely during early neural development, and in a very limited set of neurons in the mature brain.

Comparison of the spatial distribution of seven types of neuroendocrine neurons in the rat paraventricular nucleus: Toward a global 3D model

The paraventricular nucleus of the hypothalamus (PVH) coordinates neuroendocrine, autonomic, and behavioral responses to help maintain energy and body water balance. The rat paraventricular nucleus has three major divisions: descending with axonal projections to somatomotor‐behavioral and autonomic circuitry, magnocellular neuroendocrine with projections directly to the posterior pituitary, and parvicellular neuroendocrine with projections to the median eminence for controlling anterior pituitary hormone secretion. The present work was undertaken to provide high‐resolution mapping of spatial relationships among the two magnocellular neuroendocrine and five parvicellular neuroendocrine neuron types throughout the nucleus. Double immunohistochemical labeling for two neuron types combined with retrograde labeling to identify neuroendocrine neurons positively was used in individual sections spaced 45 μm apart, along with a grid transfer method for reducing plane of section artifacts when comparing staining pattern data between animals. The results indicate that whereas each neuroendocrine neuron phenotype displays a unique distribution pattern, there is extensive partial overlap in a complex pattern between small “hot spots” with a relatively high density of a particular neuron type and few if any other phenotypes. In addition, the distribution of non‐neuroendocrine neurons staining with each of the markers (but not retrogradely labeled) was mapped and compared with each other and with the neuroendocrine neuron populations. This spatial organization raises important questions about the differential functional regulation of individual—and perhaps sets of—neuroendocrine motor neuron populations in the PVH by synaptic mechanisms and by less traditional mechanisms like dendritic neurotransmitter release and gap junctions within and between neuron types. J. Comp. Neurol. 516:423–441, 2009.

Neuronal expression of chimeric genes in transgenic mice.

Gene expression may occur in unexpected ectopic sites when diverse genetic elements are juxtaposed as chimeric genes in transgenic mice. To determine the specific contribution of the promoter and reporter gene in ectopic expression, we have analyzed the expression of 14 different fusion genes in transgenic mice. Chimeric genes containing the mouse metallothionein-I promoter linked to either the rat or human growth hormone gene or the calcitonin/CGRP gene are expressed in a very similar pattern of neuronal regions. This ectopic expression is not a unique feature of the metallothionein promoter, since transferring the human growth hormone gene to four other heterologous promoters resulted in varying degrees of ectopic expression in overlapping subsets of cortical and hypothalamic neurons. The novel pattern of ectopic expression suggests that these otherwise unrelated neurons share a common developmental regulatory machinery for activation of gene transcription.

The Distribution of Progesterone Receptor Immunoreactivity and mRNA in the Preoptic Area and Hypothalamus of the Ewe: Upregulation of Progesterone Receptor mRNA in the Mediobasal Hypothalamus by Oestrogen

The distribution of progesterone receptors (PR) was mapped in the hypothalamus of the ewe using immunocytochemistry. These results were confirmed using in situ hybridization with a sheep‐specific 35S‐labelled riboprobe. In addition, the effect of oestrogen on the level of PR mRNA in the hypothalamus was examined in ovariectomized (OVX) ewes following treatment with an oestrogen implant or without treatment. PR immunoreactive (‐ir) cells were readily detected in OVX animals. Labelled cells were observed in four main hypothalamic regions: the preoptic area (POA), including the organum vasculosum of the lamina terminalis, periventricular nucleus (PeVN), ventromedial nucleus (VMN) and the arcuate nucleus (ARC) (including the region ventral to the mamillary recess). In addition, lightly stained PR‐ir cells were observed in the supraoptic nucleus and a few PR‐ir cells were also found in the diagonal band of Broca. No PR‐ir cells were found in the brainstem. PR mRNA‐containing cells were found in the same hypothalamic regions as the PR‐ir cells. Image analysis of emulsion‐dipped slides following in situ hybridization indicated that oestrogen treatment increased (P<0.01) the mean number of silver grains/cell and the density of labelled cells in the VMN and ARC but had no effect on the level of PR mRNA expression in the POA or PeN. The distribution of PR‐containing cells in the hypothalamus is similar to that described in other species and all cells were located in nuclei that contain large populations of oestrogen receptor‐containing cells. These include regions implicated in the regulation of reproductive neuroendocrine function, and reproductive behaviour. Oestrogen and progesterone synergize to inhibit GnRH secretion and the present results suggest that these functions may involve cells of the VMN and ARC, with oestrogen acting to upregulate PR.

Enkephalin mRNA production by cochlear and vestibular efferent neurons in the gerbil brainstem

SummaryPreproenkephalin mRNA production by efferent neurons projecting to the gerbil inner ear was assessed using combined in situ hybridization and retrograde labeling with florescent tracers. Virtually all vestibular efferent neurons were positive for preproenkephalin mRNA. Of the cochlear efferents, one-half of the medial olivocochlear neurons were positive for enkephalin. All lateral olivocochlear neurons were negative for enkephalin. The results suggest that there are two, biochemically distinct subpopulations of medial olivocochlear efferents in the gerbil.

RNA levels of neuronal nicotinic acetylcholine receptor subunits are differentially regulated in axotomized facial motoneurons: an in situ hybridization study

In situ hybridization histochemistry using complementary RNA probes revealed that alpha 3 and beta 2 neuronal nicotinic acetylcholine receptor subunit mRNAs were expressed in 12% and 40% of facial motoneurons of the rat, respectively. The alpha 3 subunit mRNA signals disappeared in response to axotomy, whereas the beta 2 subunit mRNA signal was remarkably enhanced, suggesting that mRNA levels of receptor subunits are differentially regulated in axotomized motoneurons.