Genetic ablation of root cap cells in Arabidopsis

The root cap is increasingly appreciated as a complex and dynamic plant organ. Root caps sense and transmit environmental signals, synthesize and secrete small molecules and macromolecules, and in some species shed metabolically active cells. However, it is not known whether root caps are essential for normal shoot and root development. We report the identification of a root cap-specific promoter and describe its use to genetically ablate root caps by directing root cap-specific expression of a diphtheria toxin A-chain gene. Transgenic toxin-expressing plants are viable and have normal aerial parts but agravitropic roots, implying loss of root cap function. Several cell layers are missing from the transgenic root caps, and the remaining cells are abnormal. Although the radial organization of the roots is normal in toxin-expressing plants, the root tips have fewer cytoplasmically dense cells than do wild-type root tips, suggesting that root meristematic activity is lower in transgenic than in wild-type plants. The roots of transgenic plants have more lateral roots and these are, in turn, more highly branched than those of wild-type plants. Thus, root cap ablation alters root architecture both by inhibiting root meristematic activity and by stimulating lateral root initiation. These observations imply that the root caps contain essential components of the signaling system that determines root architecture.

Targeted ablation of the vitamin D receptor: An animal model of vitamin D-dependent rickets type II with alopecia

Vitamin D, the major steroid hormone that controls mineral ion homeostasis, exerts its actions through the vitamin D receptor (VDR). The VDR is expressed in many tissues, including several tissues not thought to play a role in mineral metabolism. Studies in kindreds with VDR mutations (vitamin D-dependent rickets type II, VDDR II) have demonstrated hypocalcemia, hyperparathyroidism, rickets, and osteomalacia. Alopecia, which is not a feature of vitamin D deficiency, is seen in some kindreds. We have generated a mouse model of VDDR II by targeted ablation of the second zinc finger of the VDR DNA-binding domain. Despite known expression of the VDR in fetal life, homozygous mice are phenotypically normal at birth and demonstrate normal survival at least until 6 months. They become hypocalcemic at 21 days of age, at which time their parathyroid hormone (PTH) levels begin to rise. Hyperparathyroidism is accompanied by an increase in the size of the parathyroid gland as well as an increase in PTH mRNA levels. Rickets and osteomalacia are seen by day 35; however, as early as day 15, there is an expansion in the zone of hypertrophic chondrocytes in the growth plate. In contrast to animals made vitamin D deficient by dietary means, and like some patients with VDDR II, these mice develop progressive alopecia from the age of 4 weeks.

Subplate neuron ablation alters neurotrophin expression and ocular dominance column formation

Ocular dominance column formation in visual cortex depends on both the presence of subplate neurons and the endogenous expression of neurotrophins. Here we show that deletion of subplate neurons, which supply glutamatergic inputs to visual cortex, leads to a paradoxical increase in brain-derived neurotrophic factor mRNA in the same region of visual cortex in which ocular dominance columns are absent. Subplate neuron ablation also increases glutamic acid decarboxylase-67 levels, indicating an alteration in cortical inhibition. These observations imply a role for this special class of neurons in modulating activity-dependent competition by regulating levels of neurotrophins and excitability within a developing cortical circuit.

Ablation of P/Q-type Ca2+ channel currents, altered synaptic transmission, and progressive ataxia in mice lacking the α1A-subunit

The Ca2+ channel α1A-subunit is a voltage-gated, pore-forming membrane protein positioned at the intersection of two important lines of research: one exploring the diversity of Ca2+ channels and their physiological roles, and the other pursuing mechanisms of ataxia, dystonia, epilepsy, and migraine. α1A-Subunits are thought to support both P- and Q-type Ca2+ channel currents, but the most direct test, a null mutant, has not been described, nor is it known which changes in neurotransmission might arise from elimination of the predominant Ca2+ delivery system at excitatory nerve terminals. We generated α1A-deficient mice (α1A−/−) and found that they developed a rapidly progressive neurological deficit with specific characteristics of ataxia and dystonia before dying ≈3–4 weeks after birth. P-type currents in Purkinje neurons and P- and Q-type currents in cerebellar granule cells were eliminated completely whereas other Ca2+ channel types, including those involved in triggering transmitter release, also underwent concomitant changes in density. Synaptic transmission in α1A−/− hippocampal slices persisted despite the lack of P/Q-type channels but showed enhanced reliance on N-type and R-type Ca2+ entry. The α1A−/− mice provide a starting point for unraveling neuropathological mechanisms of human diseases generated by mutations in α1A.

Bax ablation prevents dopaminergic neurodegeneration in the 1-methyl- 4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) damages dopaminergic neurons in the substantia nigra pars compacta (SNpc) as seen in Parkinson's disease. Here, we show that the pro-apoptotic protein Bax is highly expressed in the SNpc and that its ablation attenuates SNpc developmental neuronal apoptosis. In adult mice, there is an up-regulation of Bax in the SNpc after MPTP administration and a decrease in Bcl-2. These changes parallel MPTP-induced dopaminergic neurodegeneration. We also show that mutant mice lacking Bax are significantly more resistant to MPTP than their wild-type littermates. This study demonstrates that Bax plays a critical role in the MPTP neurotoxic process and suggests that targeting Bax may provide protective benefit in the treatment of Parkinson's disease.

Selective ablation of retinoid X receptor α in hepatocytes impairs their lifespan and regenerative capacity

Retinoid X receptors (RXRs) are involved in a number of signaling pathways as heterodimeric partners of numerous nuclear receptors. Hepatocytes express high levels of the RXRα isotype, as well as several of its putative heterodimeric partners. Germ-line disruption (knockout) of RXRα has been shown to be lethal in utero, thus precluding analysis of its function at later life stages. Hepatocyte-specific disruption of RXRα during liver organogenesis has recently revealed that the presence of hepatocytes is not mandatory for the mouse, at least under normal mouse facility conditions, even though a number of metabolic events are impaired [Wan, Y.-J., et al. (2000) Mol. Cell. Biol. 20, 4436–4444]. However, it is unknown whether RXRα plays a role in the control of hepatocyte proliferation and lifespan. Here, we report a detailed analysis of the liver of mice in which RXRα was selectively ablated in adult hepatocytes by using the tamoxifen-inducible chimeric Cre recombinase system. Our results show that the lifespan of adult hepatocytes lacking RXRα is shorter than that of their wild-type counterparts, whereas proliferative hepatocytes of regenerating liver exhibit an even shorter lifespan. These lifespan shortenings are accompanied by increased polyploidy and multinuclearity. We conclude that RXRα plays important cell-autonomous function(s) in the mechanism(s) involved in the lifespan of hepatocytes and liver regeneration.

Perilipin ablation results in a lean mouse with aberrant adipocyte lipolysis, enhanced leptin production, and resistance to diet-induced obesity

Perilipin coats the lipid droplets of adipocytes and is thought to have a role in regulating triacylglycerol hydrolysis. To study the role of perilipin in vivo, we have created a perilipin knockout mouse. Perilipin null (peri−/−) and wild-type (peri+/+) mice consume equal amounts of food, but the adipose tissue mass in the null animals is reduced to ≈30% of that in wild-type animals. Isolated adipocytes of perilipin null mice exhibit elevated basal lipolysis because of the loss of the protective function of perilipin. They also exhibit dramatically attenuated stimulated lipolytic activity, indicating that perilipin is required for maximal lipolytic activity. Plasma leptin concentrations in null animals were greater than expected for the reduced adipose mass. The peri−/− animals have a greater lean body mass and increased metabolic rate but they also show an increased tendency to develop glucose intolerance and peripheral insulin resistance. When fed a high-fat diet, the perilipin null animals are resistant to diet-induced obesity but not to glucose intolerance. The data reveal a major role for perilipin in adipose lipid metabolism and suggest perilipin as a potential target for attacking problems associated with obesity.

Targeted ablation of the 25-hydroxyvitamin D 1α-hydroxylase enzyme: Evidence for skeletal, reproductive, and immune dysfunction

The active form of vitamin D, 1α,25-dihydroxyvitamin D [1α,25(OH)2D], is synthesized from its precursor 25 hydroxyvitamin D [25(OH)D] via the catalytic action of the 25(OH)D-1α-hydroxylase [1α(OH)ase] enzyme. Many roles in cell growth and differentiation have been attributed to 1,25(OH)2D, including a central role in calcium homeostasis and skeletal metabolism. To investigate the in vivo functions of 1,25(OH)2D and the molecular basis of its actions, we developed a mouse model deficient in 1α(OH)ase by targeted ablation of the hormone-binding and heme-binding domains of the 1α(OH)ase gene. After weaning, mice developed hypocalcemia, secondary hyperparathyroidism, retarded growth, and the skeletal abnormalities characteristic of rickets. These abnormalities are similar to those described in humans with the genetic disorder vitamin D dependent rickets type I [VDDR-I; also known as pseudovitamin D-deficiency rickets (PDDR)]. Altered non-collagenous matrix protein expression and reduced numbers of osteoclasts were also observed in bone. Female mutant mice were infertile and exhibited uterine hypoplasia and absent corpora lutea. Furthermore, histologically enlarged lymph nodes in the vicinity of the thyroid gland and a reduction in CD4- and CD8-positive peripheral T lymphocytes were observed. Alopecia, reported in vitamin D receptor (VDR)-deficient mice and in humans with VDDR-II, was not seen. The findings establish a critical role for the 1α(OH)ase enzyme in mineral and skeletal homeostasis as well as in female reproduction and also point to an important role in regulating immune function.

How αβ T cells deal with induced TCRα ablation

On deletion of the gene encoding the constant region of the T cell antigen receptor (TCR)α chain in mature T cells by induced Cre-mediated recombination, the cells lose most of their TCR from the cell surface within 7–10 days, but minute amounts of surface-bound TCRβ chains are retained for long periods of time. In a situation in which cellular influx from the thymus is blocked, TCR-deficient naïve T cells decay over time, the decay rates being faster for CD8+ cells (t1/2 ≈ 16 days) than for CD4+ cells (t1/2 ≈ 46 days). TCR+ naïve cells are either maintained (CD8+) or decay more slowly (CD4+; t1/2 ≈ 78 days.) Numbers of TCR-deficient memory T cells decline very slowly (CD8+ cells; t1/2 ≈ 52 days) or not at all (CD4+ cells), but at the population level, these cells fail to expand as their TCR+ counterparts do. Together with earlier data on T cell maintenance in environments lacking appropriate major histocompatibility complex antigens, these data argue against the possibility that spontaneous ligand-independent signaling by the αβTCR contributes significantly to T-cell homeostasis.

Ablation of suprachiasmatic nucleus alters timing of hibernation in ground squirrels.

Hibernation patterns were monitored continuously for 2.5 years in female squirrels that were neurologically intact or in which the hypothalamic suprachiasmatic nucleus (SCN) was completely ablated (SCNx). The number of hibernation bouts in SCNx squirrels increased by 159%, total hibernation time increased by 58%, and periodic arousals from hibernation were 47% longer in SCNx than in control squirrels; the duration of individual torpor bouts was 2 days shorter and far more variable in SCNx than in control animals. Some SCNx squirrels cycled through bouts of torpor continuously for nearly 2 years. The SCN appears to be part of the mechanism that controls the duration of the hibernation season and the temporal structure of individual torpor bouts.

Localization of the central rhythm generator involved in spontaneous consummatory licking in rats: functional ablation and electrical brain stimulation studies.

Localization of the central rhythm generator (CRG) of spontaneous consummatory licking was studied in freely moving rats by microinjection of tetrodotoxin (TTX) into the pontine reticular formation. Maximum suppression of spontaneous water consumption was elicited by TTX (1 ng) blockade of the oral part of the nucleus reticularis gigantocellularis (NRG), whereas TTX injections into more caudal or rostral locations caused significantly weaker disruption of drinking. To verify the assumption that TTX blocked the proper CRG of licking rather than some relay in its output, spontaneously drinking thirsty rats were intracranially stimulated via electrodes chronically implanted into the oral part of the NRG. Lick-synchronized stimulation (a 100-ms train of 0.1-ms-wide rectangular pulses at 100 Hz and 25-150 microA) applied during continuous licking (after eight regular consecutive licks) caused a phase shift of licks emitted after stimulus delivery. The results suggest that the stimulation has reset the CRG of licking without changing its frequency. The reset-inducing threshold current was lowest during the tongue retraction and highest during the tongue protrusion period of the lick cycle. It is concluded that the CRG of licking is located in the oral part of NRG.

Genetic ablation of parietal cells in transgenic mice: a new model for analyzing cell lineage relationships in the gastric mucosa.

The gastric mucosa of mammalian stomach contains several differentiated cell types specialized for the secretion of acid, digestive enzymes, mucus, and hormones. Understanding whether each of these cell lineages is derived from a common stem cell has been a challenging problem. We have used a genetic approach to analyze the ontogeny of progenitor cells within mouse stomach. Herpes simplex virus 1 thymidine kinase was targeted to parietal cells within the gastric mucosa of transgenic mice, and parietal cells were ablated by treatment of animals with the antiherpetic drug ganciclovir. Ganciclovir treatment produced complete ablation of parietal cells, dissolution of gastric glands, and loss of chief and mucus-producing cells. Termination of drug treatment led to the reemergence of all major gastric epithelial cell types and restoration of glandular architecture. Our results imply the existence of a pluripotent stem cell for the gastric mucosa. Parietal cell ablation should provide a model for analyzing cell lineage relationships within the stomach as well as mechanisms underlying gastric injury and repair.