Activity, non-selective attention and emotionality in dopamine D2/D3 receptor knock-out mice.

In order to assess the role of dopamine (DA) D2 and D3 receptors in the modulation of behaviour, we analysed exploration in a spatial novelty in mouse model systems. Genetically engineered mice mutants have been used that carry normal, partial or no expression of D2R, D3R, or both D2R/D3R (double mutants) DA receptor subtypes. Adult male mice were exposed for 30 min to a Làte-maze. The behaviour was analysed for indices of activity, orienting (rearing frequency), scanning times (rearing duration) and defecation score (emotionality). D2R - / - and + / - as well as the D2R/D3R double homozygous mutants were less active than wild-type (WT) controls in travelled distance. In contrast D3R + / - were more active than WT mice in the first part of the test. As to orienting frequency, the D2R - / - were less active than WT during the entire test-period, whereas the D2 + / - mutants were less active than WT only in the second part of the test. Moreover, the D3R - / - and + / - mutants showed less and more rearing frequency than WT, respectively, during the entire test. Finally, the D2/D3R - / - double mutants were also less active than WT during the entire test period. As to scanning times, D2R + / - and - / - mutants were higher than WT during the entire test or only in the second part, respectively. The D3R + / - and - / - were not different from WT, whereas the D2/D3R - / - double mutants showed shorter scanning times only in the first part of the test. As to emotionality index, the defecation score, was lower only in D3R + / - mutants. Thus, the dopamine D2 and D3 receptor subtypes appear to be differentially involved in the modulation of activity, orienting and scanning phases of attention. Lastly double mutation experiments reveal an interaction between D2R and D3R with the former prevailing on the latter.

Dickkopf1 is required for embryonic head induction and limb morphogenesis in the mouse.

Dickkopf1 (Dkk1) is a secreted protein that acts as a Wnt inhibitor and, together with BMP inhibitors, is able to induce the formation of ectopic heads in Xenopus. Here, we show that Dkk1 null mutant embryos lack head structures anterior of the midbrain. Analysis of chimeric embryos implicates the requirement of Dkk1 in anterior axial mesendoderm but not in anterior visceral endoderm for head induction. In addition, mutant embryos show duplications and fusions of limb digits. Characterization of the limb phenotype strongly suggests a role for Dkk1 both in cell proliferation and in programmed cell death. Our data provide direct genetic evidence for the requirement of secreted Wnt antagonists during embryonic patterning and implicate Dkk1 as an essential inducer during anterior specification as well as a regulator during distal limb patterning.

Expression screening for Lhx3 downstream genes identifies Thg-1pit as a novel mouse gene involved in pituitary development.

Thg-1pit, a novel mouse gene, was detected in a screen for genes that are differentially expressed in the developing pituitary of wild-type and Lhx3 null mutant embryos. The predicted translation product of the Thg-1pit gene contains a C-terminal TSC-box adjacent to a leucine zipper motif. These features are characteristic for the TSC-22/DIP/bun family of proteins. The onset of prominent Thg-1pit expression coincides with Lhx3 activation at early stages of pituitary development. Expression is further enhanced as cells begin to differentiate within the developing pituitary gland. No expression is observed in the pituitary rudiment of mutants that lack Lhx3 function. A possible role is thus suggested for Lhx3 activities in the regulation of Thg-1pit function during early steps of pituitary organogenesis.

The consequences of the Odra flood (summer 1997) for the Odra lagoon and the beaches of Usedom: what can be expected under extreme conditions?

The exceptional flood of the river Odra in July/August 1997 caused severe damage, especially on the Polish side of the Odra valley. An additional 5 km3 of water were discharged during the flood. This represents about a third of the normal annual Odra discharge of 17 km3. Large agricultural and industrial areas were submerged, as well as towns and villages. However, as regards the Odra lagoon and the beaches of the Isle of Usedom, the substances transported, such as nutrients and pollutants, did not cause much damage, due to strong dilution. Hygienic investigations (human pathogenic bacteria and viruses) showed that the water had bathing quality during the whole flood.

Learning impairments and motor dysfunctions in adult Lhx5-deficient mice displaying hippocampal disorganization.

Lhx5 is a member of the LIM homeobox gene family that regulates development of the nervous system. Adult mice generated with a mutation in Lhx5 were found to display absent or disorganized hippocampal neuroanatomy. The pyramidal cell layer in Ammon's horn and the granule cell layer in the dentate gyrus were absent or poorly defined in the hippocampus of adult Lhx5 knockout mice. Behavioral phenotyping of Lhx5 null mutants detected deficits on learning and memory tasks, including the Barnes maze spatial learning task, spontaneous alternation recognition memory, and contextual and cued fear conditioning. General health, neurological reflexes, and sensory abilities appeared to be normal in Lhx5 knockout mice. Motor tests showed impaired performance on some measures of motor activity, coordination, balance, and gait. These results reveal functional outcomes of Lhx5 gene deletion on the integrity of hippocampal neuroanatomy and behavior in the adult mouse.

A large upstream region is not necessary for gene expression or hypersensitive site formation at the mouse beta -globin locus.

Developmental expression at the beta-globin locus is regulated in part by the locus control region, a region upstream of the genes containing at least five major DNase I hypersensitive sites (HSs) in mammalian erythrocytes. Sequences farther 5' of these HSs are conserved in mouse and human, and both loci are embedded within a cluster of functional odorant receptor genes. In humans, distant upstream sequences have been implicated in regulation of the beta-globin genes. In this study, the role of the 5'-most HSs and their adjacent sequence was investigated by deletion of an 11-kb region from the mouse locus, including 5'HS 4.2, 5'HS 5, 5'HS 6, and the 5'beta1 odorant receptor gene. Mice that were homozygous for this deletion were fully viable, and no significant effect on adult beta-globin gene expression was seen. 5'HSs 1-4, which are located downstream of the deletion, were still present in the mutant mice. In addition, two new upstream HSs, HS -60.7 and HS -62.5, were found in erythroid tissue of both wild-type and mutant mice. Therefore, although the possibility of a minor role still exists, neither the HSs nor the other regions deleted in this study are essential for beta-globin gene expression, and it is unlikely that chromatin structure is affected either upstream or downstream of the deletion. This is the largest deletion at the mouse locus control region to show no apparent phenotype, and focuses attention on the possible contribution of sequences even farther upstream.

Distinct but overlapping roles of histone acetylase PCAF and of the closely related PCAF-B/GCN5 in mouse embryogenesis.

PCAF plays a role in transcriptional activation, cell-cycle arrest, and cell differentiation in cultured cells. PCAF contributes to transcriptional activation by acetylating chromatin and transcription factors through its intrinsic histone acetylase activity. In this report, we present evidence for the in vivo function of PCAF and the closely related PCAF-B/GCN5. Mice lacking PCAF are developmentally normal without a distinct phenotype. In PCAF null-zygous mice, protein levels of PCAF-B/GCN5 are drastically elevated in lung and liver, where PCAF is abundantly expressed in wild-type mice, suggesting that PCAF-B/GCN5 functionally compensates for PCAF. In contrast, animals lacking PCAF-B/GCN5 die between days 9.5 and 11.5 of gestation. Normally, PCAF-B/GCN5 mRNA is expressed at high levels already by day 8, whereas PCAF mRNA is first detected on day 12.5, which may explain, in part, the distinct knockout phenotypes. These results provide evidence that PCAF and PCAF-B/GCN5 play distinct but functionally overlapping roles in embryogenesis.

Involvement of neurogranin in the modulation of calcium/calmodulin-dependent protein kinase II, synaptic plasticity, and spatial learning: a study with knockout mice.

Neurogranin/RC3 is a neural-specific Ca(2+)-sensitive calmodulin (CaM)-binding protein whose CaM-binding affinity is modulated by phosphorylation and oxidation. Here we show that deletion of the Ng gene in mice did not result in obvious developmental or neuroanatomical abnormalities but caused an impairment of spatial learning and changes in hippocampal short- and long-term plasticity (paired-pulse depression, synaptic fatigue, long-term potentiation induction). These deficits were accompanied by a decreased basal level of the activated Ca(2+)/CaM-dependent kinase II (CaMKII) ( approximately 60% of wild type). Furthermore, hippocampal slices of the mutant mice displayed a reduced ability to generate activated CaMKII after stimulation of protein phosphorylation and oxidation by treatments with okadaic acid and sodium nitroprusside, respectively. These results indicate a central role of Ng in the regulation of CaMKII activity with decisive influences on synaptic plasticity and spatial learning.

Positive and negative interactions of GDNF, NTN and ART in developing sensory neuron subpopulations, and their collaboration with neurotrophins.

Glial cell line-derived neurotrophic factor (GDNF), neurturin (NTN) and neublastin/artemin (ART) are distant members of the transforming growth factor beta family, and have been shown to elicit neurotrophic effects upon several classes of peripheral and central neurons. Limited information from in vitro and expression studies has also substantiated a role for GDNF family ligands in mammalian somatosensory neuron development. Here, we show that although dorsal root ganglion (DRG) sensory neurons express GDNF family receptors embryonically, they do not survive in response to their ligands. The regulation of survival emerges postnatally for all GDNF family ligands. GDNF and NTN support distinct subpopulations that can be separated with respect to their expression of GDNF family receptors, whereas ART supports neurons in populations that are also responsive to GDNF or NTN. Sensory neurons that coexpress GDNF family receptors are medium sized, whereas small-caliber nociceptive cells preferentially express a single receptor. In contrast to brain-derived neurotrophic factor (BDNF)-dependent neurons, embryonic nerve growth factor (NGF)-dependent nociceptive neurons switch dependency to GDNF, NTN and ART postnatally. Neurons that survive in the presence of neurotrophin 3 (NT3) or neurotrophin 4 (NT4), including proprioceptive afferents, Merkel end organs and D-hair afferents, are also supported by GDNF family ligands neonatally, although at postnatal stages they lose their dependency on GDNF and NTN. At late postnatal stages, ART prevents survival elicited by GDNF and NTN. These data provide new insights on the roles of GDNF family ligands in sensory neuron development.

Role for GDNF in biochemical and behavioral adaptations to drugs of abuse.

The present study examined a role for GDNF in adaptations to drugs of abuse. Infusion of GDNF into the ventral tegmental area (VTA), a dopaminergic brain region important for addiction, blocks certain biochemical adaptations to chronic cocaine or morphine as well as the rewarding effects of cocaine. Conversely, responses to cocaine are enhanced in rats by intra-VTA infusion of an anti-GDNF antibody and in mice heterozygous for a null mutation in the GDNF gene. Chronic morphine or cocaine exposure decreases levels of phosphoRet, the protein kinase that mediates GDNF signaling, in the VTA. Together, these results suggest a feedback loop, whereby drugs of abuse decrease signaling through endogenous GDNF pathways in the VTA, which then increases the behavioral sensitivity to subsequent drug exposure.

Glial cell line-derived neurotrophic factor is essential for postnatal survival of midbrain dopamine neurons.

Glial cell line-derived neurotrophic factor (GDNF) is one of the most potent trophic factors that have been identified for midbrain dopamine (DA) neurons. Null mutations for trophic factor genes have been used frequently for studies of the role of these important proteins in brain development. One problem with these studies has been that often only prenatal development can be studied because many of the knockout strains, such as those with GDNF null mutations, will die shortly after birth. In this study, we looked at the continued fate of specific neuronal phenotypes from trophic factor knockout mice beyond the time that these animals die. By transplanting fetal neural tissues from GDNF -/-, GDNF +/-, and wild-type (WT) mice into the brain of adult wild-type mice, we demonstrate that the continued postnatal development of ventral midbrain dopamine neurons is severely disturbed as a result of the GDNF null mutation. Ventral midbrain grafts from -/- fetuses have markedly reduced DA neuron numbers and fiber outgrowth. Moreover, DA neurons in such transplants can be "rescued" by immersion in GDNF before grafting. These findings suggest that postnatal survival and/or phenotypic expression of ventral mesencephalic DA neurons is dependent on GDNF. In addition, we present here a strategy for studies of maturation and even aging of tissues from trophic factor and other knockout animals that do not survive past birth.

The LIM homeobox gene Lhx9 is essential for mouse gonad formation.

During mammalian embryonic development, the ovaries and testes develop from somatic cells of the urogenital ridges as indifferent gonads, harbouring primordial germ cells that have migrated there. After sex determination of the gonads, the testes produce testosterone and anti-Mullerian hormone which mediate male sexual differentiation, and the female developmental pathway ensues in their absence. Here we show that transcripts of the LIM homeobox gene Lhx9 are present in urogenital ridges of mice at embryonic day 9.5; later they localize to the interstitial region as morphological differentiation occurs. In mice lacking Lhx9 function, germ cells migrate normally, but somatic cells of the genital ridge fail to proliferate and a discrete gonad fails to form. In the absence of testosterone and anti-Mullerian hormone, genetically male mice are phenotypically female. The expression of steroidogenic factor 1 (Sf1), a nuclear receptor essential for gonadogenesis, is reduced to minimal levels in the Lhx9-deficient genital ridge, indicating that Lhx9 may lie upstream of Sf1 in a developmental cascade. Unlike mice lacking other genes that mediate early stages of gonadogenesis, Lhx9 mutants do not exhibit additional major developmental defects. Thus, LHX9 mutations may underlie certain forms of isolated gonadal agenesis in humans.

A dynamic regulation of GDNF-family receptors correlates with a specific trophic dependency of cranial motor neuron subpopulations during development.

Glial cell line-derived neurotrophic factor (GDNF) family ligands promote the survival of developing motor neurons in vivo and in vitro. However, not all neurons survive with any single ligand in culture and GDNF null mutant mice display only a partial motor neuron loss. An interesting possibility is that subpopulations of motor neurons based on their function and/or their myotopic organization require distinct members of GDNF family ligands. Because responsiveness to the different ligands depends on the expression of their cognate ligand-binding receptor we have herein addressed this issue by examining the expression of GDNF-family receptors (gfr) during development and in the adult in cranial motor nuclei subpopulations. We have furthermore examined the in vivo role of GDNF for cranial motor neuron subpopulations. The shared ret receptor was expressed in all somatic, branchial and visceral cranial embryonic motor nuclei examined, showing that they are all competent to respond to GDNF family ligands during development. At early stages of development both the GDNF receptor, gfralpha1, and the neurturin (NTN) receptor, gfralpha2, were expressed in the oculomotor, facial and spinal accessory, and only gfralpha1 in the trochlear, superior salivatory, trigeminal, hypoglossal and weakly in the dorsal motor nucleus of the vagus and the ambiguous nucleus. The abducens nucleus was negative for both gfralpha1 and gfralpha2. The artemin (ART) receptor, gfralpha3, was expressed only in the superior salivatory nucleus. A motor neuron subnuclei-specific expression of gfralpha1 and gfralpha2 was seen in the facial and trigeminal nuclei which corresponded to their dependence on GDNF in null mutant mice. We found that the expression was dynamic in these nuclei, which may reflect developmental changes in their trophic factor dependency. Analysis of GDNF null mutant mice revealed that the dynamic receptor expression is regulated by the ligand in vivo, indicating that the attainment of changes in dependency could be ligand induced. Our results indicate that specific GDNF family ligands support selective muscle-motor neuron circuits during development.

Regulation of cell fate decision of undifferentiated spermatogonia by GDNF.

The molecular control of self-renewal and differentiation of stem cells has remained enigmatic. Transgenic loss-of-function and overexpression models now show that the dosage of glial cell line-derived neurotrophic factor (GDNF), produced by Sertoli cells, regulates cell fate decisions of undifferentiated spermatogonial cells that include the stem cells for spermatogenesis. Gene-targeted mice with one GDNF-null allele show depletion of stem cell reserves, whereas mice overexpressing GDNF show accumulation of undifferentiated spermatogonia. They are unable to respond properly to differentiation signals and undergo apoptosis upon retinoic acid treatment. Nonmetastatic testicular tumors are regularly formed in older GDNF-overexpressing mice. Thus, GDNF contributes to paracrine regulation of spermatogonial self-renewal and differentiation.

Glial cell line-derived neurotrophic factor receptor alpha1 availability regulates glial cell line-derived neurotrophic factor signaling: evidence from mice carrying one or two mutated alleles.

Glial cell line-derived neurotrophic factor receptor alpha1 (GFRalpha1, also known as GDNFR-alpha) is a glycolipid-anchored membrane protein of the GFRalpha family, which binds glial cell line-derived neurotrophic factor [Jing S. et al. (1996) Cell 85, 1113-1124; Treanor J. J. et al. (1996) Nature 382, 80-83], a survival factor for several populations of central and peripheral neurons, including midbrain dopamine neurons [Lin L. F. et al. (1993) Science 260, 1130-1132], and mediates its ligand-induced cell response via a tyrosine kinase receptor called Ret [Takahashi M. et al. (1988) Oncogene 3, 571-578; Takahashi M. and Cooper G. M. (1987) Molec. Cell Biol. 7, 1378-1385]. In this paper, we show that mice with a null mutation of the GFRalpha1 gene manifest epithelial-mesenchymal interaction deficits in kidney and severe disturbances of intestinal tract development similar to those seen with glial cell line-derived neurotrophic factor or Ret null mutations. There is a marked renal dysgenesis or agenesis and the intrinsic enteric nervous system fails completely to develop. We also show that newborn GFRalpha1-deficient mice display no or minimal changes in dorsal root and sympathetic ganglia. This is in contrast to the deficits reported in these neuronal populations in glial cell line-derived neurotrophic factor and Ret null mutations. Mesencephalic dopaminergic neurons in the substantia nigra and ventral tegmental area appear intact at the time of birth of the mutated mice. Mice homozygous for the GFRalpha1 null mutation die within 24 h of birth because of uremia. Heterozygous animals, however, live to adulthood. There is a significantly reduced neuroprotective effect of glial cell line-derived neurotrophic factor in such heterozygous animals, compared with wild-type littermates, after cerebral ischemia. Taken together with previous data on glial cell line-derived neurotrophic factor and Ret, our results strongly suggest that GFRalpha1 is the essential GFRalpha receptor for signaling in the glial cell line-derived neurotrophic factor-Ret pathway in the kidney and enteric nervous system development, and that GFRalpha2 or GFRalpha3 cannot substitute for the absence of GFRalpha1. Moreover, neuroprotective actions of exogenous glial cell line-derived neurotrophic factor also require full GFRalpha1 receptor expression.

Functions of LIM-homeobox genes.

Homeobox genes play fundamental roles in development. They can be subdivided into several subfamilies, one of which is the LIM-homeobox subfamily. The primary structure of LIM-homeobox genes has been remarkably conserved through evolution. Have their functions similarly been conserved? A host of new data has been derived from mutational analysis in diverse organisms, such as nematodes, flies and vertebrates. These studies have revealed a prominent involvement of LIM-homeodomain proteins in tissue patterning and differentiation, and their function in neural patterning is evident in all organisms studied to date. Here, we summarize the recent findings on LIM-homeobox gene function, compare the function of these genes from different organisms and describe specific co-factor requirements.

Insight into the physiological function(s) of uteroglobin by gene-knockout and antisense-transgenic approaches.

To determine the physiological function(s) of uteroglobin (UG), a steroid-inducible, homodimeric, secreted protein, we have generated transgenic mice that either are completely UG-deficient due to UG gene-knockout (UG-KO) or are partially UG-deficient due to the expression of UG antisense RNA (UG-AS). Both the UG-KO and UG-AS mice develop immunoglobulin A (IgA) nephropathy (IgAN), characterized by microhematuria, albuminuria, and renal glomerular deposition of IgA, fibronectin (Fn), collagen, and C3 complement. This phenotype of both UG-KO and UG-AS mice is virtually identical to that of human IgAN, the most common primary glomerulopathy worldwide. The molecular mechanism by which UG prevents this disease in mice appears to center around UG's interaction with Fn. Since Fn, IgA, and UG are present in circulation and high plasma levels of IgA-Fn complex have been reported in human IgAN, we sought to determine whether UG interacts with Fn and prevents Fn-Fn and/or IgA-Fn interactions, essential for abnormal tissue deposition of Fn and IgA. Our coimmunoprecipitation studies uncovered the formation of Fn-UG heteromers in vitro and these heteromers are detectable in the plasma of normal mice, but not UG-KO mice. Further, high plasma levels of IgA-Fn complex, a characteristic of human IgAN patients, were also found in UG-KO mice. Finally, coadministration of UG + Fn or UG + IgA to UG-KO mice prevented glomerular deposition of Fn and IgA, respectively. Our results define a possible molecular mechanism of IgAN and provide insight into at least one important physiological function of UG in maintaining normal renal function in mice.

Genomic structure, chromosomal localization and expression of the human LIM-homeobox gene LHX5.

The LIM-homeobox gene Lhx5 plays an essential role in the regulation of neuronal differentiation and migration during development of the central nervous system. Mice lacking Lhx5 function show severely disorganized brain morphology and are impaired in cognition and motor coordination. In this study, we characterized the cDNA and genomic organization of the human LHX5 gene and analyzed its expression and chromosomal location. The human gene was found to contain five exons encoding a protein composed of 402 amino acids that is 98.8% identical to mouse Lhx5. By reverse transcriptase polymerase chain reaction, LHX5 transcripts were detected in fetal brain and in various regions of the adult central nervous system including the spinal cord, the thalamus, and the cerebellum. Fluorescence in situ hybridization mapped the LHX5 gene to chromosome 12, position 12q24.31-24.32. These results provide a framework for future analysis of possible association of human hereditary disorders with mutations in LHX5.

Isolated cleft palate in mice with a targeted mutation of the LIM homeobox gene lhx8.

Formation of the mammalian secondary palate is a highly regulated and complex process whose impairment often results in cleft palate, a common birth defect in both humans and animals. Loss-of-function analysis has linked a growing number of genes to this process. Here we report that Lhx8, a recently identified LIM homeobox gene, is expressed in the mesenchyme of the mouse palatal structures throughout their development. To test the function of Lhx8 in vivo, we generated a mutant mouse with a targeted deletion of the Lhx8 gene. Our analysis of the mutant animals revealed a crucial role for Lhx8 in palatogenesis. In Lhx8 homozygous mutant embryos, the bilateral primordial palatal shelves formed and elevated normally, but they often failed to make contact and to fuse properly, resulting in a cleft secondary palate. Because development of other craniofacial structures appeared normal, the impaired palatal formation in Lhx8-mutant mice was most likely caused by an intrinsic primary defect in the mesenchyme of the palatal shelves. The cleft palate phenotype observed in Lhx8-mutant mice suggests that Lhx8 is a candidate gene for the isolated nonsyndromic form of cleft palate in humans.