Adeno-associated virus serotypes 9 and rh10 mediate strong neuronal transduction of the dog brain.

Canine models have many advantages for evaluating therapy of human central nervous system (CNS) diseases. In contrast to nonhuman primate models, naturally occurring canine CNS diseases are common. In contrast to murine models, the dog's lifespan is long, its brain is large and the diseases affecting it commonly have the same molecular, pathological and clinical phenotype as the human diseases. We compared the ability of four intracerebrally injected adeno-associated virus vector (AAV) serotypes to transduce the dog brain with green fluorescent protein as the first step in using these vectors to evaluate both delivery and efficacy in naturally occurring canine homologs of human diseases. Quantitative measures of transduction, maximum diameter and area, identified both AAV2/9 and AAV2/rh10 as significantly more efficient than either AAV2/1 or AAV2/5 at transducing cerebral cortex, caudate nucleus, thalamus and internal capsule. Fluorescence co-labeling with cell-type-specific antibodies demonstrated that AAV2/9 and AAV2/rh10 were capable of primarily transducing neurons, although glial transduction was also identified and found to be more efficient with the AAV2/9 vector. These data are a prerequisite to evaluating the efficacy of recombinant AAV vectors carrying disease-modifying transgenes to treat naturally occurring canine models in preclinical studies of human CNS disease therapy.

Clusterin gene transcription is activated by caudal-related homeobox genes in intestinal epithelium.

Caudal-related homeobox (Cdx) proteins play an important role in development and differentiation of the intestinal epithelium. Using cDNA differential display, we identified clusterin as a prominently induced gene in a Cdx2-regulated cellular model of intestinal differentiation. Transfection experiments and DNA-protein interaction assays showed that clusterin is an immediate downstream target gene for Cdx proteins. The distribution of clusterin protein in the intestine was assessed during development and in the adult epithelium using immunohistochemistry. In the adult mouse epithelium, clusterin protein was localized in both crypt and villus compartments but not in interstitial cells of the intestinal mucosa. Together, these data suggest that clusterin is a direct target gene for Cdx homeobox proteins, and the pattern of clusterin protein expression suggests that it is associated with the differentiated state in the intestinal epithelium.

Cdx1 and cdx2 expression during intestinal development.

BACKGROUND & AIMS: The intestine-specific transcription factors Cdx1 and Cdx2 are candidate genes for directing intestinal development, differentiation, and maintenance of the intestinal phenotype. This study focused on the complex patterns of expression of Cdx1 and Cdx2 during mouse gastrointestinal development. METHODS: Embryonic and postnatal mouse tissues were analyzed by immunohistochemistry to determine protein expression of Cdx1 and Cdx2 in the developing intestinal tract. RESULTS: Cdx2 protein expression was observed at 9. 5 postcoitum (pc), whereas weak expression of Cdx1 protein was first seen at 12.5 pc in the distal developing intestine (hindgut). Expression of Cdx1 increased from 13.5 to 14.5 pc during the endoderm/epithelial transition with predominately distal expression. In contrast to Cdx1, there was intense expression of Cdx2 in all but the distal portions of the developing intestine. Cdx2 expression remained low in the distal colon throughout postnatal development. A gradient of expression formed in the crypt-villus axis, with Cdx1 primarily in the crypt and Cdx2 primarily in the villus. CONCLUSIONS: Direct comparison of the patterns of Cdx1 and Cdx2 protein expression during development as performed in this study provides new insights into their potential functional roles. The relative expression of Cdx1 to Cdx2 protein may be important in the anterior to posterior patterning of the intestinal epithelium and in defining patterns of proliferation and differentiation along the crypt-villus axis.

PRL-1 PTPase expression is developmentally regulated with tissue-specific patterns in epithelial tissues.

The mechanisms controlling tyrosine phosphorylation of cellular proteins are important in the regulation of many cellular processes, including development and differentiation. Protein tyrosine phosphatases (PTPases) may be as important as protein tyrosine kinases (PTKs) in these processes. PRL-1 is a distinct PTPase originally identified as an immediate-early gene in liver regeneration whose expression is associated with growth in some tissues but with differentiation in others. We now demonstrate that the PRL-1 protein is expressed during development in a number of digestive epithelial tissues. It is expressed at variable time points in the developing intestine, but its expression is limited to the developing villus enterocytes. In the gastric epithelium, PRL-1 expression in the adult is restricted to zymogen cells. PRL-1 is also expressed in the developing liver and esophagus and in the epithelia of the kidney and lung. In each of these contexts, the expression of PRL-1 is associated with terminal differentiation, suggesting that it may play a role in this important developmental process.

Recovery of neurofilament expression selectively in regenerating reticulospinal neurons.

During regeneration of lamprey spinal axons, growth cones lack filopodia and lamellipodia, contain little actin, and elongate much more slowly than do typical growth cones of embryonic neurons. Moreover, these regenerating growth cones are densely packed with neurofilaments (NFs). Therefore, after spinal hemisection the time course of changes in NF mRNA expression was correlated with the probability of regeneration for each of 18 identified pairs of reticulospinal neurons and 12 cytoarchitectonic groups of spinal projecting neurons. During the first 4 weeks after operation, NF message levels were reduced dramatically in all axotomized reticulospinal neurons, on the basis of semiquantitative in situ hybridization for the single lamprey NF subunit (NF-180). Thereafter, NF expression returned toward normal in neurons whose axons normally regenerate beyond the transection but remained depressed in poorly regenerating neurons. The recovery of NF expression in good regenerators was independent of axon growth across the lesion, because excision of a segment of spinal cord caudal to the transection site blocked regeneration but did not prevent the return of NF-180 mRNA. The early decrease in NF mRNA expression was not accompanied by a reduction in NF protein content. Thus the axotomy-induced loss of most of the axonal volume resulted in a reduced demand for NF rather than a reduction in volume-specific NF synthesis. We conclude that the secondary upregulation of NF message during axonal regeneration in the lamprey CNS may be part of an intrinsic growth program executed only in neurons with a strong propensity for regeneration.

Developmental increases in expression of neurofilament mRNA selectively in projection neurons of the lamprey CNS.

Neurofilaments of the sea lamprey are unique in being homopolymers of a single subunit (NF-180). Digoxigenin-labeled RNA probes complementary to NF-180 were used to determine the distribution and timing of expression of neurofilament message in the brain and spinal cord of the lamprey. In the brainstem, detection of NF-180 mRNA was restricted to neurons with axons projecting to the spinal cord or the periphery. The majority of brainstem neurons, whose axons project locally, did not express NF-180 within the detection limits of this technique. NF-180-positive neurons included cells with a wide range of axon diameters, suggesting neurofilament mRNA expression was linked to axon length rather than caliber. To further evaluate this hypothesis, expression was studied in animals of different developmental stages between larvae and adults. In younger (shorter) larvae, the large Mauthner and rhombencephalic Müller cells did not express NF-180 mRNA, even though their axons are among the largest caliber in the animal and extend the entire length of the spinal cord. In contrast, many other reticulospinal neurons, whose axons are smaller in diameter than those of the Müller and Mauthner cells, expressed NF-180 message throughout larval development. Furthermore, neurons of the cranial motor nuclei did not express NF-180 until later developmental stages and the extraocular motor neurons did not label until metamorphosis. Therefore, while detectable neurofilament mRNA expression in the lamprey is restricted to neurons with long axons, its expression in this population of neurons appears to be developmentally regulated by factors still not determined. It is postulated that need for NF message is determined by a balance between the volume of axon to be filled and the rate of turnover of NF in that axon.

Metamorphosis of spinal-projecting neurons in the brain of the sea lamprey during transformation of the larva to adult: normal anatomy and response to axotomy.

The spinal projecting system of the sea lamprey (Petromyzon marinus) has been used extensively in studies of axonal regeneration in both larvae and adults. However, little is known about the changes that are undergone by this system during metamorphosis. In order to determine the developmental changes in the size of the descending spinal projection and in the morphology of its neurons, larval, transforming, and adult lamprey brains were labeled by retrograde transport of horseradish peroxidase (HRP) injected into the spinal cord at 25% of body length. Examination of brain wholemount preparations revealed that the total number of labeled neurons doubled during metamorphosis. Most of this increase could be explained by elongation of reticulospinal axons from the rostralmost segments of the spinal cord to locations caudal to the injection site. There were no additions or deletions of either identified reticulospinal neurons or of reticulospinal nuclear groups between the larval and the adult stages. The proportions of Müller and Mauthner cells that were labeled reached a maximum of 93% during the early stages of metamorphosis. Axons of these neurons are known to project almost the entire length of the cord, even in larvae. Therefore, the efficiency of retrograde transport appears to be greater during metamorphosis than during larval or adult stages. While changes in efficiency of retrograde transport could account for some of the apparent increase in reticulospinal neuron numbers between larvae and animals undergoing metamorphosis, this could not contribute to the further increase in the apparent size of the reticulospinal system in the adult, since efficiency of retrograde labeling in these animals was lower than that at earlier stages. With retrograde labeling, a significant increase was seen in the profusion of dendritic arborization of some Müller and Mauthner cells during the early stages of metamorphosis. This correlated with an increase in the incidence of extreme axonal die-back, as indicated by the presence of retraction bulbs within the brainstem. However, intracellular injection of Neurobiotin in untransected animals showed similar degrees of dendritic arborization at all examined stages of development. Therefore, the dendritic profusion did not reflect developmental changes in neuronal morphology but rather reflected an increased sensitivity to axotomy during metamorphosis. We conclude that, during the transformation of the lamprey from the large larval to the adult form, there is little change in either the size or the dendritic morphology of the identified giant reticulospinal neurons. With respect to the smaller reticulospinal neurons, the distance of projection of many of their axons increases during metamorphosis, but there is very little increase in the number of reticulospinal neurons.

A method for in situ hybridization in wholemounted lamprey brain: neurofilament expression in larvae and adults.

Nonisotopic in situ hybridization (NISH) using both cDNA and cRNA probes is rapidly gaining favor over autoradiographic methods. Typically, either biotinylated or digoxigenin-labeled probes are used to detect mRNAs in sectioned tissue or in cultured cells. With a few exceptions, most applications of NISH in wholemount preparations have been limited to Drosophila embryos. A protocol developed for NISH in whole adult Drosophila CNS was extended to wholemounted larval and adult lamprey brain preparations. Digoxigenin-labeled RNA probes were transcribed from cloned fragments of a lamprey neurofilament (NF180) cDNA. Hybridization with these probes, and comparisons with Nissl-stained wholemounts and wholemounts retrogradely labeled by injections of tracer into the spinal cord, demonstrated that NF180 mRNA was expressed in only a subset of neurons in the lamprey CNS. These included primarily neurons with long axons that project out of the brainstem, e.g., reticulospinal neurons and cranial motor neurons. Metamorphosis from the larval to the adult form was accompanied by an increase in the number of neurons expressing NF180 and in the apparent level of NF expression as judged by the intensity of labeling. For example, in the oculomotor and trochlear nuclei, expression of NF180 was seen in postmetamorphic young adult lampreys but not in larvae. In the trigeminal motor nucleus, both the number of neurons expressing NF180 and the intensity of the hybridization labeling increased with metamorphosis. The ability to do NISH in lamprey brain wholemounts eliminates the need for serial reconstructions and thus facilitates the study of selected gene expression during metamorphosis and regeneration.

Cytoarchitecture of spinal-projecting neurons in the brain of the larval sea lamprey.

The descending spinal projecting system of the lamprey is of interest because it includes axons that activate swimming pattern generators and because regeneration of this system is involved in the behavioral recovery of lampreys following spinal transection. However, little is known about the true size of this projection and of the distribution of its terminations along the spinal cord. Brain neurons with spinal projections were studied in larval sea lampreys by using wholemount preparations labeled retrogradely with horseradish peroxidase (HRP) from spinal injections at 10%, 15%, 25%, 50%, 70%, and 75% of body length from the anterior end. Neurons projecting to different levels of the spinal cord were mapped. A large number of descending axons terminated within nine segments caudal to the last gill. The spinal projection system was divided into 10 bilateral groups based on cytoarchitectural landmarks. All of the lateral nuclear groups had contralateral spinal projections. In addition to the 12 pairs of Müller cells, the pair of Mauthner cells, and the pair of auxiliary Mauthner cells described by previous authors, the study revealed four pairs of smaller neurons that were individually identifiable.

Passover: a gene required for synaptic connectivity in the giant fiber system of Drosophila.

Passover (Pas) flies fail to jump in response to a light-off stimulus. The mutation disrupts specific synapses of the giant fibers (GFs), command neurons for this response. Pas was cloned from a P element-induced allele. The cDNA encodes a putative membrane protein of 361 amino acids. Null, hypomorphic, and dominant alleles were sequenced. In the adult central nervous system, and in the pupa during GF synapse formation, Pas is consistently expressed in the GF and in a large thoracic cell in the location of its postsynaptic targets. Pas establishes a new gene family. The Drosophila ogre protein, required for postembryonic neuroblast development, is 47% identical; the C. elegans Unc-7 protein, which when mutated alters the connectivity of a few neurons, is 33% identical.

A deficiency chromosome in Drosophila alters neuritic projections in an identified motoneuron.

The tergotrochanteral muscles (TTM) in the second thoracic segment of the fruitfly, Drosophila melanogaster, power the jump-escape response. The cell bodies of the motoneurons innervating these muscles, located in the thoracico-abdominal ganglion, have prominent posterior and medial neurites. While in wildtype flies of the Canton-S (C-S) and Oregon-R (O-R) strains, the medial neurite of a TTM motoneuron rarely crossed the midline (C-S: 0/17; O-R: 2/8), in heterozygous flies with a deficiency at the base of the X-chromosome, Df(1)16-3-22, the medial neurite frequently crossed the midline (Df(1)16-3-22/C-S: 7/12; Df(1)16-3-22/O-R: 18/22).