Evelyne Souil

Localization of dopamine D3 receptor mRNA in the rat brain using in situ hybridization histochemistry: comparison with dopamine D2 receptor mRNA

The messenger RNA (mRNA) of the recently characterized D3 dopamine receptor was visualized on rat brain sections using in situ hybridization with a 32P-labeled ribonucleic acid probe corresponding to a major part of the third cytoplasmic loop, a domain in which D2 and D3 dopamine receptors display little homology. For the purpose of comparison, D2 receptor mRNA was also specifically visualized on adjacent sections. The areas that expressed D2 and/or D3 receptors were also compared with those previously detected using [125I]iodosulpride, a ligand that binds to both D2 and D3 receptors with a similar affinity. The localization of D3 receptor mRNa markedly differs from that of D2 receptor mRNA. Whereas D2 receptor mRNA is expressed in all major brain areas receiving dopaminergic projections, particularly in the whole striatal complex, D3 receptor mRNA is expressed in a more restricted manner. It is mainly detected in telencephalic areas receiving dopaminergic inputs from the A10 cell group, e.g. accumbens nucleus, islands of Calleja, bed nucleus of the stria terminalis and other limbic areas such as the hippocampus and the mammillary nuclei. D2 and D3 receptor mRNAs were also detected at the level of the substantia nigra, suggesting that these receptors function as both autoreceptor and postsynaptic receptors. In several dopaminergic projection areas, e.g. ventral straitum, septal or mammillary nuclei, the distributions of D2 and D3 receptor mRNAs appeared complementary without overlap. The distribution of [125I]iodosulpride binding sites generally overlapped that of D2 or D3 receptor mRNAs, the latter being most abundant in dopaminergic areas known to be associated with cognitive and emotional functions.

Six1 and Six4 homeoproteins are required for Pax3 and Mrf expression during myogenesis in the mouse embryo

In mammals, Six5, Six4 and Six1 genes are co-expressed during mouse myogenesis. Six4 and Six5 single knockout (KO) mice have no developmental defects, while Six1 KO mice die at birth and show multiple organ developmental defects. We have generated Six1Six4 double KO mice and show an aggravation of the phenotype previously reported for the single Six1 KO. Six1Six4 double KO mice are characterized by severe craniofacial and rib defects, and general muscle hypoplasia. At the limb bud level, Six1 and Six4 homeogenes control early steps of myogenic cell delamination and migration from the somite through the control of Pax3 gene expression. Impaired in their migratory pathway, cells of the somitic ventrolateral dermomyotome are rerouted, lose their identity and die by apoptosis. At the interlimb level, epaxial Met expression is abolished, while it is preserved in Pax3-deficient embryos. Within the myotome, absence of Six1 and Six4 impairs the expression of the myogenic regulatory factors myogenin and Myod1, and Mrf4 expression becomes undetectable. Myf5 expression is correctly initiated but becomes restricted to the caudal region of each somite. Early syndetomal expression of scleraxis is reduced in the Six1Six4 embryo, while the myotomal expression of Fgfr4 and Fgf8 but not Fgf4 and Fgf6 is maintained. These results highlight the different roles played by Six proteins during skeletal myogenesis.

Altered myogenesis in Six1-deficient mice.

Six homeoproteins are expressed in several tissues, including muscle, during vertebrate embryogenesis, suggesting that they may be involved in diverse differentiation processes. To determine the functions of the Six1 gene during myogenesis, we constructed Six1-deficient mice by replacing its first exon with the lacZ gene. Mice lacking Six1 die at birth because of severe rib malformations and show extensive muscle hypoplasia affecting most of the body muscles in particular certain hypaxial muscles. Six1–/– embryos have impaired primary myogenesis, characterized, at E13.5, by a severe reduction and disorganisation of primary myofibers in most body muscles. While Myf5, MyoD and myogenin are correctly expressed in the somitic compartment in early Six1–/– embryos, by E11.5 MyoD and myogenin gene activation is reduced and delayed in limb buds. However, this is not the consequence of a reduced ability of myogenic precursor cells to migrate into the limb buds or of an abnormal apoptosis of myoblasts lacking Six1. It appears therefore that Six1 plays a specific role in hypaxial muscle differentiation, distinct from those of other hypaxial determinants such as Pax3, cMet, Lbx1 or Mox2.

Thymus, kidney and craniofacial abnormalities in Six1 deficient mice

Six genes are widely expressed during vertebrate embryogenesis, suggesting that they are implicated in diverse differentiation processes. To determine the functions of the Six1 gene, we constructed Six1-deficient mice by replacing its first exon by the beta-galactosidase gene. We have previously shown that mice lacking Six1 die at birth due to thoracic skeletal defects and severe muscle hypoplasia affecting most of the body muscles. Here, we report that Six1(-/-) neonates also lack a kidney and thymus, as well as displaying a strong disorganisation of craniofacial structures, namely the inner ear, the nasal cavity, the craniofacial skeleton, and the lacrimal and parotid glands. These organ defects can be correlated with Six1 expression in the embryonic primordium structures as revealed by X-Gal staining at different stages of embryogenesis. Thus, the fetal abnormalities of Six1(-/-) mice appear to result from the absence of the Six 1 homeoprotein during early stages of organogenesis. Interestingly, these Six1 defects are very similar to phenotypes caused by mutations of Eya 1, which are responsible for the BOR syndrome in humans. Close comparison of Six1 and Eya 1 deficient mice strongly suggests a functional link between these two factors. Pax gene mutations also lead to comparable phenotypes, suggesting that a regulatory network including the Pax, Six and Eya genes is required for several types of organogenesis in mammals.

Coexpression of dopamine D1 and D3 receptors in islands of Calleja and shell of nucleus accumbens of the rat: opposite and synergistic functional interactions

Using double in situ hybridization, we found extensive coexpression of dopamine D1 and D3 receptor (D1R and D3R) mRNAs in neurons of the island of Calleja major (ICjM) and ventromedial shell of nucleus accumbens (ShV), respectively. Thus, at least 79 and 63% of D3R mRNA‐expressing neurons in ICjM and ShV also expressed the D1R mRNA. Coexpression of D1R and D3R mRNAs was found to occur in substance P (SP) mRNA‐expressing neurons in both areas, suggesting SP mRNA as a marker of the activity of coexpressing neurons. Administration of SKF 38393, a D1R receptor agonist, increased c‐fos mRNA in ICjM, whereas administration of quinpirole, a D2R/D3R agonist, decreased it; SCH 23390, a D1R antagonist and nafadotride, a preferential D3R antagonist, given alone, had effects opposite to those of the corresponding agonists. These data indicate that basal c‐fos expression in ICjM is maintained by endogenous dopamine acting tonically upon two receptor subtypes subserving opposite effects on the same cell. However, in ShV, whereas SKF 38393 also increased c‐fos mRNA, quinpirole had no effect, a difference presumably reflecting the lower fraction of neurons coexpressing D1R and D3R in this area. In contrast, in ShV from reserpine‐treated rats, SKF 38393 increased SP mRNA and quinpirole potentiated this effect. These contrasting interactions of D1R‐ and D3R‐mediated signalling events, i.e. in either opposite or synergistic directions, most likely occurring at the single cell level, may serve to increase the dopamine response threshold of the target cells in ICjM and to maintain a strong tonic activity of ShV neurons.

Gain-of-function mutant of angiotensin II receptor, type 1A, causes hypertension and cardiovascular fibrosis in mice

The role of the renin-angiotensin system has been investigated by overexpression or inactivation of its different genes in animals. However, there is no data concerning the effect of the constitutive activation of any component of the system. A knockin mouse model has been constructed with a gain-of-function mutant of the Ang II receptor, type 1A (AT(1A)), associating a constitutively activating mutation (N111S) with a C-terminal deletion, which impairs receptor internalization and desensitization. In vivo consequences of this mutant receptor expression in homozygous mice recapitulate its in vitro characteristics: the pressor response is more sensitive to Ang II and longer lasting. These mice present with a moderate (~20 mmHg) and stable increase in BP. They also develop early and progressive renal fibrosis and cardiac fibrosis and diastolic dysfunction. However, there was no overt cardiac hypertrophy. The hormonal parameters (low-renin and inappropriately normal aldosterone productions) mimic those of low-renin human hypertension. This new model reveals that a constitutive activation of AT(1A) leads to cardiac and renal fibrosis in spite of a modest effect on BP and will be useful for investigating the role of Ang II in target organs in a model similar to some forms of human hypertension.

Functional Type I PTH/PTHrP Receptor in Freshly Isolated Newborn Rat Keratinocytes: Identification by RT‐PCR and Immunohistochemistry

The presence of identical or distinct type I parathyroid hormone (PTH)/parathyroid hormone‐related peptide (PTHrP) receptors in keratinocytes is still a matter of debate. We studied the expression and functionality of PTHrP receptors in freshly isolated keratinocytes from newborn rat skin. Four overlapping primers, amplifying different regions in the rat PTH receptor, were used for reverse transcriptase‐polymerase chain reaction (RT‐PCR). The first region corresponded to the N‐terminal extracellular region and the first transmembrane domain (S/M1), the second region amplified the connecting intracellular and extracellular loops transmembrane domain (E2/M5), the third spanned the range from the transmembrane to the intracellular domain (M4/T), and the fourth region amplified the C‐terminal tail (M6/7/T). The PCR products from the keratinocyte RNA were identical to those from kidney RNA of the same rats. The cloned four transcripts showed 100% of homologies with the cDNA sequence from bone ROS cells. Keratinocytes, freshly isolated or present in situ in the epidermis, recognized an anti‐PTH receptor antibody (PTH‐II) directed against the receptor extracellular domain. Western blotting showed the same protein patterns in keratinocytes, kidney, and ROS cell extracts. Low doses of PTHrP(1–34) (10−12–10−9 M) increased the cell number studied by [3H]thymidine incorporation and DNA content. Treatment with the PTH/PTHrP receptor antagonist [Asn10, Leu11, D Trp12] PTHrP(7–34) or two different PTH receptor antibodies inhibited the increase in cell proliferation induced by PTHrP(1–34). All these findings indicate that newborn rat epidermis and keratinocytes express functional PTHrP receptors, which are identical to type I PTH/PTHrP receptor and are recognized by PTHrP(1–34).

The Third Dopamine Receptor (D3): New Perspectives in Therapeutics

Until recently it was widely accepted that dopamine affects its target cells in brain and endocrine tissues via interaction with only two receptor subtypes, termed D1 and D2, differing from each other by their pharmacological specificity and their opposite effect on adenylate cyclase (Kebabian and Calne 1979). It was also generally admitted that the therapeutic efficacy of antipsychotics derived from their high affinity binding to D2 receptors.