Pat Miethke

Bird-like sex chromosomes of platypus imply recent origin of mammal sex chromosomes

In therian mammals (placentals and marsupials), sex is determined by an XX female: XY male system, in which a gene (SRY) on the Y affects male determination. There is no equivalent in other amniotes, although some taxa (notably birds and snakes) have differentiated sex chromosomes. Birds have a ZW female: ZZ male system with no homology with mammal sex chromosomes, in which dosage of a Z-borne gene (possibly DMRT1) affects male determination. As the most basal mammal group, the egg-laying monotremes are ideal for determining how the therian XY system evolved. The platypus has an extraordinary sex chromosome complex, in which five X and five Y chromosomes pair in a translocation chain of alternating X and Y chromosomes. We used physical mapping to identify genes on the pairing regions between adjacent X and Y chromosomes. Most significantly, comparative mapping shows that, contrary to earlier reports, there is no homology between the platypus and therian X chromosomes. Orthologs of genes in the conserved region of the human X (including SOX3, the gene from which SRY evolved) all map to platypus chromosome 6, which therefore represents the ancestral autosome from which the therian X and Y pair derived. Rather, the platypus X chromosomes have substantial homology with the bird Z chromosome (including DMRT1) and to segments syntenic with this region in the human genome. Thus, platypus sex chromosomes have strong homology with bird, but not to therian sex chromosomes, implying that the therian X and Y chromosomes (and the SRY gene) evolved from an autosomal pair after the divergence of monotremes only 166 million years ago. Therefore, the therian X and Y are more than 145 million years younger than previously thought.

Cholinergic amacrine cells are not required for the progression and atropine-mediated suppression of form-deprivation myopia

Muscarinic cholinergic pathways have been implicated in the visual control of ocular growth. However, the source(s) of acetylcholine and the tissue(s) which regulate ocular growth via muscarinic acetylcholine receptors (mAChRs) remain unknown. We sought to determine whether retinal sources of acetylcholine and mAChRs contribute to visually guided ocular growth in the chick. Cholinergic amacrine cells were ablated by intraocular injections of either ethylcholine mustard aziridinium ion (ECMA; a selective cholinotoxin) or quisqualic acid (QA; an excitotoxin that destroys many amacrine cells, including those that release acetylcholine). Disruption of cholinergic pathways was assessed immunocytochemically with antibodies to the acetylcholine-synthesizing enzyme choline acetyltransferase (ChAT) and three different isoforms of mAChR, and by biochemical assay for ChAT activity. ECMA (25 nmol) destroyed two of the four subtypes of cholinergic amacrine cells and attenuated retinal ChAT activity, but left retinal mAChR-immunoreactivity intact. QA (200 nmol) destroyed the majority of all four subtypes of cholinergic amacrine cells, and ablated most mAChR-immunoreactivity and ChAT activity in the retina. ECMA and QA had no apparent effect on mAChRs or cholinergic fibres in the choroid, only marginally reduced choroidal ChAT activity, and had little effect on ChAT activity in the anterior segment. Toxin-treated eyes remained emmetropic and responded to form-deprivation by growing excessively and becoming myopic. Furthermore, daily intravitreal injection of 40 microg atropine for 6 days into form-deprived toxin-treated eyes completely prevented ocular elongation and myopia. We conclude that neither cholinergic amacrine cells nor mAChRs in the retina are required for visual regulation of ocular growth, and that atropine may exert its growth-suppressing influence by acting upon extraretinal mAChRs, possibly in the choroid, retinal pigmented epithelium, or sclera.

Characterizing the chromosomes of the Australian model marsupial Macropus eugenii (tammar wallaby)

Marsupials occupy a phylogenetic middle ground that is very valuable in genome comparisons of mammal and other vertebrate species. For this reason, whole genome sequencing is being undertaken for two distantly related marsupial species, including the model kangaroo species Macropus eugenii (the tammar wallaby). As a first step towards the molecular characterization of the tammar genome, we present a detailed description of the tammar karyotype, report the development of a set of molecular anchor markers and summarize the comparative mapping data for this species.

Is nitric oxide a transmitter of the centrifugal projection to the avian retina

The chicken retina contains a population of prominent elements in the inner nuclear layer, which stain for NADPH-diaphorase. In distribution and morphology, these elements resemble the terminals of the centrifugal projection from the isthmo-optic nucleus. This identification was confirmed by showing that the NADPH-diaphorase-positive elements in the retina degenerated after destruction of the isthmo-optic nucleus or tract. These results indicate that the centrifugal projection to the retina in birds uses nitric oxide as a messenger or transmitter, in addition to a more conventional but as yet unidentified transmitter.

Parallel suppression of retinal and pineal melatonin synthesis by retinally mediated light.

WE have recently shown that light, over a narrow range of low intensities suppresses the activity of the enkepha-lin-immunoreactive amacrine cells of the chicken retina. In this paper, we show that over the same range of low light intensities the rate of melatonin synthesis in both the retina and the pineal of the chicken is suppressed. We further show that the effects of light on the pineal at these low intensities are mediated by the retina and not by direct actions on the pineal. Combined with our evidence that dopaminergic pathways within the retina are involved in controlling the state of activity of the pineal, these results suggest, but do not prove, that the change in state of a microcircuit within the retina involving the photoreceptors, dopaminergic amacrine cells and enkephalin-immunoreactive amacrine cells may be causally related to changes in the state of the pineal.

Pineal activity is under the control of retinal D1-dopaminergic pathways

The role of dopaminergic pathways in the retina in controlling the functional state of the pineal was investigated. Dopaminergic agents were injected into the eyes of dark-adapted chickens which were maintained in the dark. Changes in the activity of N-acetyltransferase (NAT) in the retina and pineal were then monitored. Injection of the non-specific dopamine agonist 6,7-ADTN depressed retinal and pineal NAT. The D1-specific agonist SKF38393 did not affect retinal NAT but depressed pineal NAT. In contrast, quinpirole, a D2-specific agonist, depressed retinal NAT, but did not depress pineal NAT. Thus, D1- rather than D2-dopaminergic pathways in the retina are involved in the retinal circuit which control pineal function.

Characterizing the chromosomes of the platypus (Ornithorhynchus anatinus)

Like the unique platypus itself, the platypus genome is extraordinary because of its complex sex chromosome system, and is controversial because of difficulties in identification of small autosomes and sex chromosomes. A 6-fold shotgun sequence of the platypus genome is now available and is being assembled with the help of physical mapping. It is therefore essential to characterize the chromosomes and resolve the ambiguities and inconsistencies in identifying autosomes and sex chromosomes. We have used chromosome paints and DAPI banding to identify and classify pairs of autosomes and sex chromosomes. We have established an agreed nomenclature and identified anchor BAC clones for each chromosome that will ensure unambiguous gene localizations.

Neural barriers affect the action of nitric oxide synthase inhibitors in the intact chicken retina.

Nitric oxide synthase (NOS) activity, as measured by the formation of L-[3H]citrulline from L-[3H]arginine, was blocked by micromolar concentrations of NOS inhibitors in retinal homogenates, but concentrations approximately 20-3000 times higher were needed in intact retina. The higher concentrations could be related to transport of the NOS inhibitors into neuronal cells and/or their sequestration within glial cells. NG-monomethyl-L-arginine and N-iminoethyl-L-ornithine significantly inhibited L-[3H]arginine uptake, whereas N omega-nitro-L-arginine methyl ester and N omega-nitro-L-arginine had little or no effect on L-[3H]arginine uptake. The high concentrations of the inhibitors needed to inhibit nitric oxide production in intact tissue and their different interactions with arginine uptake systems may explain some of the conflicting results on the activity of NOS inhibitors on a range of physiological parameters in vivo.