Microvascular Casting of the Lung: Vascular Lavage

Corrosion casting is an important tool for the study of microvascular structure, but unlike tissue preparation for light and electron microscopy, few controlled studies of the methods for preparation of microvascular casts for scanning electron microscopy have been reported. Most vascular casting begins with rinsing cells from the lumen of the blood vessels, but the necessity of this has not been shown. Because of a report of successful casting without rinsing and light microscopic evidence of edema with complete rinsing prior to casting, this preparation procedure was examined. Casts of lung vasculature that were thoroughly rinsed were compared to those that were not. Of the six rats that were not lavaged, only one gave a completely filled cast, but of the six animals that were well rinsed, only one did not fill. Except for the filling and mild periarterial edema, no significant difference in cast quality or features was detected. Rinsing improves the frequency of obtaining completely filled casts. The edema does not greatly interfere with the capillary image and its location gives insight into this process in the lung

Zfh1, a somatic motor neuron transcription factor, regulates axon exit from the CNS

AbstractMotor neurons are defined by their axon projections, which exit the CNS to innervate somatic or visceral musculature, yet remarkably little is known about how motor axons are programmed to exit the CNS. Here, we describe the role of the Drosophila Zfh1 transcription factor in promoting axon exit from the CNS. Zfh1 is detected in all embryonic somatic motor neurons, glia associated with the CNS surface and motor axons, and one identified interneuron. In zfh1 mutants, ventral projecting motor axons often stall at the edge of the CNS, failing to enter the muscle field, despite having normal motor neuron identity. Conversely, ectopic Zfh1 induces a subset of interneurons—all normally expressing two or more “ventral motor neuron transcription factors” (e.g. Islet, Hb9, Nkx6, Lim3)—to project laterally and exit the CNS. We conclude that Zfh1 is required for ventral motor axon exit from the CNS

Combinatorial RNAi: a method for evaluating the functions of gene families in Drosophila

Individual members of gene families often have partially redundant functions during nervous system development, making conventional genetic analysis problematic. Here we review experiments showing that several genes can be silenced together by injection of double-stranded RNAs into wild-type Drosophila embryos. By dye-labeling single neuroblasts in injected embryos, the effects of multigene silencing on individual CNS axon pathways can now be examined

Complex Genetic Interactions among Four Receptor Tyrosine Phosphatases Regulate Axon Guidance in Drosophila

Four receptor-linked protein tyrosine phosphatases are selectively expressed on central nervous system axons in the Drosophila embryo. Published data show that three of these (DLAR, DPTP69D, DPTP99A) regulate motor axon guidance decisions during embryonic development. Here we examine the role of the fourth neural phosphatase, DPTP10D, by analyzing double-, triple-, and quadruple-mutant embryos lacking all possible combinations of the phosphatases. This analysis shows that all four phosphatases participate in guidance of interneuronal axons within the longitudinal tracts of the central nervous system. In the neuromuscular system, DPTP10D works together with the other three phosphatases to facilitate outgrowth and bifurcation of the SNa nerve, but acts in opposition to the others in regulating extension of ISN motor axons past intermediate targets. Our results provide evidence for three kinds of genetic interactions among the neural tyrosine phosphatases: partial redundancy, competition, and collaboration

An Optimized Method for Histological Detection of Dopaminergic Neurons in Drosophila melanogaster

Parkinson's disease (PD) affects >1 million Americans and is marked by the loss of dopaminergic neurons in the substantia nigra. PD has been linked to two causative factors: genetic risks (hereditary PD) and environmental toxins (idiopathic PD). In recent years, considerable effort has been devoted to the development of a Drosophila model of human PD that might be useful for examining the cellular mechanisms of PD pathology by genetic screening. In 2000, Feany and Bender reported a Drosophila model of PD in which transgenic flies expressing human mutant α-synuclein exhibited shortened life spans, dopaminergic losses, Parkinsonian behaviors, and Lewy bodies in surviving dopaminergic neurons. Since then, a number of studies have been published that validate the model or build on it; conversely, a number report an inability to replicate the results and suggest that most protocols for dopaminergic histology underreport the actual numbers of dopaminergic neurons in the insect brain. Here we report the optimization of dopaminergic histology in Drosophila and identification of new dopaminergic neurons, show the remarkable dendritic complexity of these neurons, and provide an updated count of these neurons in adult brains. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials. (J Histochem Cytochem 56:1049–1063, 2008