Loss of Gq/11 Family G Proteins in the Nervous System Causes Pituitary Somatotroph Hypoplasia and Dwarfism in Mice

Heterotrimeric G proteins of the G_q/11 family transduce signalsfrom a variety of neurotransmitter and hormone receptors andhave therefore been implicated in various functions of the nervoussystem. Using the Cre/loxP system, we generated mice which lackthe genes coding for the ${alpha}$ subunits of the two main members ofthe G_q/11 family, gnaq and gna11, selectively in neuronal andglial precursor cells. Mice with defective gnaq and gna11 geneswere morphologically normal, but they died shortly after birth.Mice carrying a single gna11 allele survived the early postnatalperiod but died within 3 to 6 weeks as anorectic dwarfs. Inthese mice, postnatal proliferation of pituitary somatotrophcells was strongly impaired, and plasma growth hormone (GH)levels were reduced to 15%. Hypothalamic levels of GH-releasinghormone (GHRH), an important stimulator of somatotroph proliferation,were strongly decreased, and exogenous administration of GHRHrestored normal proliferation. The hypothalamic effects of ghrelin,a regulator of GHRH production and food intake, were reducedin these mice, suggesting that an impairment of ghrelin receptorsignaling might contribute to GHRH deficiency and abnormal eatingbehavior. Taken together, our findings show that G_q/11 signalingis required for normal hypothalamic function and that impairmentof this signaling pathway causes somatotroph hypoplasia, dwarfism,and anorexia.

INTRODUCTION

Top

Introduction

The G_q/11 family of heterotrimeric G proteins mediates the cellulareffects of numerous neurotransmitter receptors, e.g., the metabotropicglutamate receptor subtypes 1 and 5, the M₁ muscarinic-acetylcholinereceptor, the 5-HT₂ serotonin receptors, or the ${alpha}$ ₁ adrenergicreceptor. G_q/11-coupled receptors are also involved in the signaltransduction of several hypothalamic peptide hormone receptors,such as the receptors for thyrotropin-releasing hormone (39),gonadotropin-releasing hormone (13), and prolactin-releasinghormones (3). There is an increasing amount of evidence thatthe release of hypothalamic hormones themselves is also controlledby G_q/11-coupled receptors. Kisspeptins, for example, have beensuggested to control the release of gonadotropin-releasing hormonevia the G_q/11-coupled receptor GPR54 (14, 20), and the gastrointestinalpeptide hormone ghrelin regulates the production of growth hormone-releasinghormone (GHRH) via the hypothalamic growth hormone secretagoguereceptor (GHS-R) (12, 22). G_q/11 family G proteins mediate theactivation of ß isoforms of phospholipase C, resultingin the activation of protein kinase C and intracellular calciummobilization (2). The G_q/11 family consists of four members,two of which, G_q and G₁₁, are expressed almost ubiquitouslyin the central nervous system (26). Genetic inactivation ofthe gnaq gene, which codes for the ${alpha}$ subunit of G_q (G ${alpha}$ _q), leadsto a defect in primary hemostasis (16) and cerebellar ataxia(15). In contrast, mice homozygous for a null allele of thegene coding for G ${alpha}$ ₁₁, gna11, did not show any phenotypic abnormalities(17). These defects were relatively mild when one considersthe number of potential transmitter systems affected, and thisfact is probably due to the high functional redundancy of G ${alpha}$ _qand G ${alpha}$ ₁₁, which share 88% amino acid sequence identity (24).Indeed, mice lacking both G ${alpha}$ _q and G ${alpha}$ ₁₁ die at day 10.5 of embryonicdevelopment (17). To circumvent this embryonic lethality, weused the Cre/loxP system (18) to generate a mouse line whichallows conditional, tissue-specific inactivation of gnaq inconstitutively G ${alpha}$ ₁₁-deficient mice (36). Postnatal inactivationof G_q/11-mediated signaling in the forebrain by use of the Camkcre4mouse line was shown to inhibit maternal behavior (35). To investigatethe role of G ${alpha}$ _q and G ${alpha}$ ₁₁ in pre- and postnatal development andthe function of the whole nervous system, we used in this studythe NestinCre mouse line (31), which causes Cre-mediated recombinationin neuronal and glial precursor cells starting at embryonicday 9.5 (40). Interestingly, the G ${alpha}$ _q/G ${alpha}$ ₁₁ deficiency in descendantsof neuronal and glial precursors did not cause gross morphologicalabnormalities of the developing nervous system but was incompatiblewith postnatal survival. In addition, we provide evidence thatG_q/11-mediated signaling is critically involved in the regulationof hypothalamic GHRH production and consecutive control of somatotrophproliferation.

MATERIALS AND METHODS

Top

Materials and Methods

Generation of mice lacking G ${alpha}$ _q and G ${alpha}$ ₁₁ in neuronal precursor cells. Mice in which the gene coding for G ${alpha}$ _q, gnaq, is flanked by loxPsites (gnaq^flox) (36) were crossed to the constitutively G ${alpha}$ ₁₁-deficientmouse line (17) and to mice which express the recombinase Creunder the control of the Nestin promoter (31, 40). This actionled to the generation of mice which lacked one, two, three,or all of the four gnaq/gna11 alleles in derivatives of neuronaland glial precursor cells. Genotyping for the gnaq^flox allele,for wild-type and knockout gna11 alleles, and for the Cre transgenewas performed as described previously (36).

Hormone levels. Plasma samples were obtained from 15-day-old mice at the beginningof the dark period. Levels of insulin-like growth factor I (IGF-I)were determined with an OCTEIA rat and mouse IGF-I kit (IDSInc., Boldon, United Kingdom), ghrelin levels were determinedwith a rat and mouse ghrelin radioimmunoassay (RIA) kit (Phoenix,Belmont, Calif.), thyroxine levels were determined with a Microenzyme immunoassay kit (Leinco, St. Louis, Mo.), and adrenocorticotropichormone (ACTH) levels were determined with an immunometric assaykit (Nichols Institute Diagnostics, San Clemente, Calif.). Corticosteronewas measured by RIA in the Steroid Laboratory of the PharmacologyInstitute, Heidelberg, Germany. GH and thyroid-stimulating hormone(TSH) were measured by RIA in the laboratory of the NationalHormone and Pituitary Program, Torrance, Calif.

Histology. Mice were deeply anesthetized with pentobarbital (100 mg/kgof body weight) intraperitoneally (i.p.) and perfused with 4%paraformaldehyde (PFA) via the left cardiac ventricle. Brainswere postfixed in 4% PFA overnight and then stored in 0.5% PFAat 4°C. For immunohistochemistry, 50-µm-thick vibratomesections were cut and incubated at 4°C with the followingantibodies: rabbit anti-c-fos antibody (sc-52; Santa Cruz Biotechnology,Santa Cruz, Calif.) at a 1:20,000 dilution for 3 days, rabbitanti-G ${alpha}$ _q/11 antibody (sc-392; Santa Cruz Biotechnology) at a1:1,000 dilution for 16 h, rabbit anti-GHRH antibody (obtainedfrom F. Talamantes, University of California, Santa Cruz, SantaCruz, Calif.) at a 1:20,000 dilution for 16 h, and mouse anti-NeuNantibody (Chemicon, Hofheim, Germany) at a dilution of 1:2,000for 16 h. For staining, we used the Vectastain Elite ABC kit(Vector Laboratories, Burlingame, Calif.). Internal organs andbrains were weighed after preparation, and hematoxylin-eosinstaining was performed on 5-µm-thick paraffin sectionsaccording to standard protocols. For immunofluorescence stainingof pituitary somatotroph cells, 5-µm-thick paraffin sectionswere incubated for 16 h with a rabbit anti-GH antibody (dilution,1:20,000; obtained from A. F. Parlow) and then with fluoresceinisothiocyanate (FITC)-labeled goat anti-rabbit antibody (dilution,1:200; Dianova, Hamburg, Germany) for 2 h. For BrdU labeling,5-day-old mice were injected intraperitoneally on three consecutivedays with 10 µg of GHRH (Phoenix)/kg or with a comparablevolume of saline. In the afternoon of the third day, mice wereintraperitoneally injected with 10 µg of BrdU/kg of bodyweight and sacrificed after 2 h. Paraffin sections of the pituitarywere stained with anti-BrdU antibody (dilution, 1:100; BD Biosciences,San Diego, Calif.) and FITC-labeled goat anti-rabbit antibody(dilution, 1:200; Jackson ImmunoResearch) for 2 h. For ghrelinimmunohistochemistry, 16-µm-thick cryotome sections ofthe stomach were stained with goat anti-ghrelin antibody (dilution,1:300; Santa Cruz Biotechnology) and by using a Vectastain EliteABC kit (Vector Labs). The numbers of c-fos-, NeuN-, ghrelin-,or BrdU-positive cells, as well as the area of GHRH immunoreactivity,were determined with the CellExplorer 2003 program (BioSciTec,Frankfurt, Germany).

Food uptake. Food uptake during the dark phase was determined in 3-to 4-week-oldmice by weighing standard chow before and after the test period.Water was accessible ad libitum. To determine the effect ofghrelin on food intake, mice were injected intraperitoneallywith 1 mg of rat ghrelin (Bachem, Heidelberg, Germany) or saline/kgof body weight, and food uptake during the following 3 h wasdetermined every 30 min. In some cases, mice were sacrificed1 h after ghrelin injection for c-fos immunohistochemistry oftheir arcuate nuclei (arcN).

Statistics. Values were expressed as means ± standard errors of themeans. Differences between two groups were statistically analyzedby using the unpaired Student's t test. Statistical significancewas accepted at a P value of <0.05.

RESULTS

Top

Results

We used the NestinCre mouse line (31) to generate mice whichlacked the G protein ${alpha}$ subunit G ${alpha}$ _q selectively in neuronal andglial precursor cells, and we then mated these mice with theconstitutively G ${alpha}$ ₁₁-deficient mouse line (17). This procedureresulted in the generation of mice which lacked one, two, three,or all four gnaq/gna11 alleles in neuronal and glial precursorcells. To prove that Cre expression leads to the recombinationof the gnaq^flox allele and thereby to a loss of G ${alpha}$ _q protein,we performed Western blot experiments with extracts from thebrains of newborn mice by using an antibody directed againstG ${alpha}$ _q/11 (Fig. 1a). In the brains of NestinCre^+/^– gnaq^flox/floxgna11^–^/^– mice (i.e., no gnaq or gna11 alleles wereleft), neither G ${alpha}$ _q nor G ${alpha}$ ₁₁ protein was detectable, whereas NestinCre^+/^–gnaq^flox/wt gna11^–^/^– (i.e., one gnaq allele is intact)or NestinCre^+/^– gnaq^flox/flox gna11^+/^– mouse brains(i.e., one gna11 allele is intact) lacked only G ${alpha}$ ₁₁ or G ${alpha}$ _q, respectively.These results show that the NestinCre-mediated recombinationof gnaq^flox alleles indeed leads to a loss of G ${alpha}$ _q protein inthe nervous system.

View larger version (88K):
[in this window]
[in a new window]
FIG. 1. The survival of Nestin-G ${alpha}$ _q/11-deficient mice depends on the number of remaining gnaq/gna11 alleles. (a) Western blot for G ${alpha}$ _q/11 proteins in brain extracts (10 µg per lane) from newborn mice with different genotypes. +, wild-type allele; fl, recombined floxed allele; –, null allele. (b) Reduced body length of a mouse with only one remaining gna11 allele (bottom) compared to a control littermate (top) at postnatal day 20. (c and d) Survival rate (c) and postnatal weight gain (d) of NestinCre^+/^– gnaq^flox/flox gna11^–^/^– mice (asterisks), NestinCre^+/^– gnaq^flox/flox gna11^+/^– mice (circles), NestinCre^+/^– gnaq^flox/wt gna11^–^/^– mice (triangles), or control mice (squares) (24 to 30 mice per group). p, postnatal day. (e) Representative hematoxylin-eosin stainings of frontal brain sections at embryonic day 18.5 from a control animal (left) and a NestinCre^+/^– gnaq^flox/flox gna11^–^/^– mouse (right) (magnification, x2.5). (f) Tenfold magnification of the mouse sensory cortices shown in panel e.

Matings between triple-heterozygous mice (NestinCre^+/^–gnaq^flox/wt gna11^+/^–) showed that mice of all genotypiccombinations were born at expected frequencies (data not shown).However, the postnatal survival of a newborn strongly dependedon the number of intact gnaq/gna11 alleles (Fig. 1c). Normalnumbers of newborns in which all four gnaq/gna11 alleles wereinactivated were born without obvious malformations, but theydid not take up rhythmic breathing after delivery. Accordingly,the lungs of these animals were only partially inflated (datanot shown). Neither histological nor basic immunohistochemicalanalysis of the brains of these animals revealed major structuralabnormalities (Fig. 1e and f and data not shown). The peripheralnervous system, including the phrenic nerve, was histologicallynormal in each mouse. Initial reflexes towards tactile stimuliand basal cardiovascular function seemed to be normal, suggestingthat inefficient respiration caused the deaths of these newborns.

Mice with at least one intact gnaq allele or two intact gna11alleles were viable and fertile. Of note, mice lacking bothgnaq alleles showed an ataxic gait comparable to the one seenin constitutively G ${alpha}$ _q-deficient mice (16), whereas animals witha single intact gnaq allele were normal in this respect. Incontrast, mice with only one gna11 allele were strongly retardedin their somatic growth (Fig. 1b and d) and died as underweightdwarfs between the third and sixth postnatal weeks (Fig. 1c).

Growth retardation in mice with a single gna11 allele was firstdetectable between postnatal days 5 and 9 (Fig. 1d). Duringthe following weeks, the body weights increased only slowly,peaked between the second and third weeks, and finally decreasedagain (Fig. 1d), usually leading to the deaths of the animalsbetween the third and sixth weeks (Fig. 1c). Except for theirsmall size and slightly retarded motor development, these animalsappeared to be normal, i.e., they did not show obvious behavioralabnormalities or signs of illness. Neither the removal of normallittermates to decrease competition for the dam's milk nor thepresence of easily accessible extra food changed the final outcome.To determine whether the growth retardation and premature deathsof mice with only one intact gna11 allele could be attributedto the failure of a specific organ system, we performed macroscopicand microscopic analyses of their central nervous systems andinternal organs. Brains, hearts, lungs, kidneys, livers, thyroids,and adrenal glands did not show major morphological abnormalities(data not shown). Analyses of organ weight/body weight ratiosshowed that kidneys and hearts were of normal sizes relativeto the body weights of the mice (Fig. 2a and data not shown),whereas the relative brain weights were increased (Fig. 2b).In contrast, the relative weights of the stomach and of retrorenalfat deposits were decreased (Fig. 2c and d), and this was probablydue to reduced food uptake (Fig. 2e). Most strikingly, the pituitarieswere much too small in relation to body weights, especiallyat older ages, and this circumstance was due mainly to the reducedsize of the anterior pituitary in each mouse, since the posteriorpituitary was of normal size (Fig. 2f and g).

View larger version (55K):
[in this window]
[in a new window]
FIG. 2. Organ weights and food uptake of wild-type mice and mice with only one intact gna11 allele (NestinCre^+/– gnaq^fl^ox^/fl^ox gna11^+/^–). (a to d, f) Organ weights as percentages of body weight (%bw) for kidneys (a), brains (b), stomachs (c), retrorenal fat deposits (d), and pituitaries (f) at postnatal days 15 (p15) and 25 (p25) (five to six mice per group). (e) Food uptake in 3-week-old mice (five to six mice per group). *, P < 0.05; **, P < 0.01; ***, P < 0.005. (g) Pituitaries from a control mouse (left) and an animal with only one intact gna11 allele (right) at postnatal day 15. Borders of the posterior pituitary are indicated by hatched ovals.

As somatic growth is regulated mainly through secretion of GHfrom somatotroph cells of the anterior pituitary, we reasonedthat the observed dwarfism might be due to impaired somatotrophfunction. Immunofluorescence staining showed that the densitiesof GH-positive cells were reduced to about 50% of that of thecontrol cells in pituitaries from 2-week-old mice (Fig. 3a and b).This reduced density of GH-positive cells, together withthe massive reduction of anterior pituitary volume (Fig. 2f and g),leads to GH deficiency, with plasma GH levels at 15%of the normal level (Fig. 3c). Consistent with this, IGF-I levelswere reduced to 30% of normal levels in 2-week-old animals (Fig.3d). Interestingly, other anterior pituitary functions, as determinedby levels of TSH, thyroxine, ACTH, or the mouse glucocorticoidanalogue corticosterone in plasma, were normal on postnatalday 15 (Fig. 3e through h). These findings show that mice withonly one intact gna11 allele in the nervous system suffer fromselective somatotroph hypoplasia, leading to strongly reducedplasma GH and IGF-I levels and, consecutively, proportionatedwarfism.

View larger version (70K):
[in this window]
[in a new window]
FIG. 3. Reduced numbers of somatotrophs in pituitaries of mice with only one intact gna11 allele (NestinCre^+/– gnaq^fl^ox^/fl^ox gna11^+/^–). (a) Immunofluorescence staining for GH in the anterior pituitary of 15-day-old control mice (left) and mice with one remaining gna11 allele (right). (b) Statistical evaluation of immunofluorescence staining (two mice per group, three sections each). (c to h) Levels in plasma, at postnatal day 15, of GH (15 mice per group) (c), IGF-I (6 to 8 mice per group) (d), ACTH (8 mice per group) (e), mouse cortisol analogue corticosterone (8 mice per group) (f), TSH (g), and thyroxine, T4 (h) (6 to 8 mice per group). ***, P < 0.001.

To test whether hypoplasia of the anterior pituitary was dueto a defect in prenatal development, we histologically analyzedpituitaries from mice at embryonic day 18.5. At this age, pituitarysizes and development were normal for mice of all genotypes(Fig. 4a), indicating that somatotroph hypoplasia is due toa defect in postnatal proliferation. To test this concept, weperformed BrdU labeling of pituitaries in the first postnatalweek and found that the number of proliferating cells was reducedin mice with only one intact gna11 allele to 38% of that ofthe control littermates (Fig. 4b and c). An important stimulatorof somatotroph proliferation is GHRH (4), which is producedin neurons of the hypothalamic arcN and which is released intothe pituitary portal system at the median eminence. To testwhether pituitaries from mice with only one intact gna11 allelewould be responsive to exogenously administered GHRH, we treatedpups of different genotypes with daily intraperitoneal injectionsof saline or GHRH from postnatal days 5 through 7. This treatmentstrongly enhanced anterior pituitary proliferation in mice withonly one intact gna11 allele, which almost reached the proliferationlevels of saline-treated control littermates (Fig. 4b and c).These findings suggest that somatotroph cells are responsiveto GHRH in these mice and that somatotroph hypoplasia is notdue to a defect of the pituitary itself but rather to inappropriateGHRH release from the hypothalamus. Consistent with this, ß-galactosidasestaining of mice carrying both the NestinCre transgene and aRosa26-lacZ reporter construct (23) did not show any recombinationin the anterior pituitary, whereas the posterior pituitary andhypothalamus were recombined (Fig. 4d and data not shown).

View larger version (99K):
[in this window]
[in a new window]
FIG. 4. Impaired postnatal proliferation of somatotroph cells can be rescued by administration of GHRH. (a) Hematoxylin-eosin staining of coronal head sections from a control animal (left) and a mouse with only one intact gna11 allele (right) at embryonic day 18.5. (b) BrdU-labeled cells in the anterior pituitary of 7-day-old control animals (left) and mice with only one intact gna11 allele (right) after 3 days of intraperitoneal administration of saline (top) or GHRH (bottom). (c) Statistical analysis of BrdU-positive (pos.) cells (two mice per group, three sections each). Black bars, control mice; white bars, mice with one intact gna11 allele; ***, P < 0.001. (d) ß-Galactosidase staining of a pituitary from a mouse carrying both the Rosa26-lacZ reporter construct and the NestinCre transgene. (a and d) ap, anterior pituitary; ht, hypothalamus; il, intermediate lobe; pp, posterior pituitary; sb, sphenoid bone.

Immunohistochemical analyses of the hypothalamic arcN and medianeminence, in which GHRH-producing neurons are located, revealeda considerable immunoreactivity for G ${alpha}$ _q/G ${alpha}$ ₁₁ in wild-type controls,which was strongly reduced in mice with only one intact gna11allele (Fig. 5a). To quantify G ${alpha}$ _q/G ${alpha}$ ₁₁ protein expression in thehypothalami of mice with only one intact G ${alpha}$ _q or G ${alpha}$ ₁₁ allele, weperformed Western blotting experiments using extracts from hypothalamiof wild-type or G ${alpha}$ _q/G ${alpha}$ ₁₁-deficient mice. We found that in wild-typemouse hypothalami, G ${alpha}$ _q levels were severalfold higher than G ${alpha}$ ₁₁levels and that the reduction of G ${alpha}$ _q/G ${alpha}$ ₁₁ immunoreactivity wasmuch stronger in hypothalami from animals with one intact G ${alpha}$ ₁₁allele than in hypothalami from mice with one intact G ${alpha}$ _q allele(Fig. 5b).

View larger version (82K):
[in this window]
[in a new window]
FIG. 5. Immunohistochemical changes in the hypothalami of mice with only one gna11 allele. (a) Staining with antibodies against G ${alpha}$ _q/11 in the arcN of a control mouse (left) and a mouse with one intact gna11 allele (right). (b) Western blot of extracts from wild-type and mutant mouse hypothalami (10 µg per lane) with antibodies against G ${alpha}$ _q/11 and total extracellular signal-regulated kinase (ERK) as the loading control. +, wild-type allele; fl, recombined floxed allele; –, null allele. (c) Staining with antibodies against GHRH in the arcN of a control mouse (left) and a mouse with one intact gna11 allele (right). (d) Statistical evaluation of GHRH immunoreactivity (GHRH-IR) in the median eminence (two mice per group, three sections each). Black bars, control mice; white bars, mice with only one intact gna11 allele. ***, P < 0.001.

Immunohistochemistry using an antibody directed against GHRHrevealed that the number of GHRH-immunoreactive terminals wasmarkedly decreased in mice with only one intact G ${alpha}$ ₁₁ allele (Fig.5c and d) but that the total number of arcN neurons, as determinedby NeuN immunohistochemistry, was not changed (not shown). Thesefindings suggest that the loss of G ${alpha}$ _q/G ${alpha}$ ₁₁-mediated signalingin the arcN leads to impaired GHRH production, which in turncauses impaired somatotroph proliferation and, consecutively,dwarfism.

The peptide hormone ghrelin, in addition to having a regulatoryfunction in food uptake and energy homeostasis, is a potentinducer of hypothalamic GHRH release, and the ghrelin receptorGHS-R was reported to couple to G_q/11 family G proteins (12,22). We therefore tested whether impaired GHS-R signaling mightcontribute to reduced GHRH release and weight loss in mice withonly one intact gna11 allele. Since ghrelin administration isknown to cause c-fos expression in the arcN of rodents (8, 30,33), we performed c-fos immunohistochemistry after intraperitonealghrelin application and found that the number of activated neuronsin these mice was reduced to 65% of the number in controls (Fig.6a and b). In contrast to its effect on wild-type animals, ghrelinadministration did not increase plasma IGF-I levels in micewith only one gna11 allele (Fig. 6c), indicating that ghrelin-inducedGH release is inhibited in these mice. We then tested whetherimpaired GHS-R signaling led to a compensatory up-regulationof ghrelin production and found both the number of ghrelin-immunoreactivecells in the stomach (Fig. 6d and e) and the plasma ghrelinlevels (Fig. 6f) to be increased. These findings suggest thatdefective signaling via the ghrelin receptor GHS-R contributesto deregulation of somatic growth and food uptake in mice withonly one intact gna11 allele.

View larger version (63K):
[in this window]
[in a new window]
FIG. 6. Impaired ghrelin actions in mice with only one intact gna11 allele (NestinCre^+/– gnaq^fl^ox^/fl^ox gna11^+/^–). (a) c-fos immunohistochemistry of the arcN in control mice (left) or mice with one intact gna11 allele (right) after the i.p. administration of saline (top) or ghrelin (bottom). (b) Statistical evaluation of c-fos immunohistochemistry of mouse arcN (two mice per group, three sections each). (c) Relative increase in plasma IGF-I levels after i.p. administration of ghrelin (six mice per group). (d) Immunohistochemistry for ghrelin-positive cells in the stomachs of control mice (left) and mice with only one intact gna11 allele (right). (e) Statistical evaluation of immunochemistry for ghrelin-positive cells (two mice per group, three sections each). (f) Plasma ghrelin levels on postnatal day 20 (eight mice per group). (b, c, e, and f) **, P < 0.005; ***, P < 0.001.

DISCUSSION

Top

Discussion

We show in this study that conditional inactivation of G_q/11-mediatedsignaling in the nervous system leads to different degrees ofimpairment, depending on the number of intact gnaq/gna11 alleles.Newborn mice which lacked all four alleles did not show anygross morphological abnormalities but did not take up rhythmicbreathing activity. This outcome suggests that G ${alpha}$ _q/G ${alpha}$ ₁₁-mediatedsignaling is crucial for the postnatal function of the nervoussystem but not for its prenatal development. Pups carrying oneintact gna11 allele survived the early postnatal period butdied as underweight dwarfs between the third and sixth postnatalweeks. In contrast, mice which inherited one intact gnaq allelewere viable and fertile. gnaq and gna11 are obviously equallyable to guarantee vital functions in neonates, but normal postnataldevelopment and weight gain depend on the presence of an intactgnaq allele. Two factors might account for these differencesbetween G ${alpha}$ _q and G ${alpha}$ ₁₁: functional differences and differentialexpression patterns of the two proteins. Potential functionaldifferences between G ${alpha}$ _q and G ${alpha}$ ₁₁ have been intensively studied,but up to now, neither differential receptor coupling (34, 38)nor differences with regard to effector preferences (7, 29,38) have been described. Our data show that total G ${alpha}$ _q/G ${alpha}$ ₁₁ immunoreactivityis much lower in hypothalami from mice with only one intactG ${alpha}$ ₁₁ allele than in hypothalami from mice with one intact G ${alpha}$ _qallele. Though both G ${alpha}$ _q and G ${alpha}$ ₁₁ are basically ubiquitously expressedin the central nervous system (26), differences in expressionlevels have also been described for other brain regions, e.g.,in the cerebellum or the hippocampus, in which expression levelsof G ${alpha}$ ₁₁ are also lower than those of G ${alpha}$ _q (6, 11, 15). Thus, asingle gna11 allele, but not a single gnaq allele, might beunable to compensate for the loss of the other alleles. Thisfinding is in line with observations for constitutively gnaq-and/or gna11-deficient mice, in which the presence of eithertwo gna11 alleles or one gnaq allele ensures normal cardiomyocytedevelopment (17), craniofacial development (10, 17), and melanoblastmigration and proliferation (32).

We showed that growth retardation in mice with only one intactgna11 allele is due to a massive hypoplasia of anterior pituitarysomatotroph cells, resulting in GH deficiency. The anteriorpituitary is a derivative of the oral ectoderm and is not ofneuronal origin; hence, it does not undergo NestinCre-mediatedrecombination (Fig. 4d) (31). The defect leading to somatotrophhypoplasia is therefore unlikely to reside in the pituitarybut rather involves brain regions which regulate somatotrophproliferation, e.g., the hypothalamus. In rats, somatotrophcells are first detected at embryonic day 18.5 and rapidly increasein number during the first 10 days after birth (27). An importantstimulator of somatotroph proliferation is GHRH, and the overexpressionof GHRH leads to somatotroph hyperplasia, increased growth,and tumorigenesis, whereas inactivating mutations cause somatotrophhypoplasia and dwarfism (4). Our data show that impaired somatotrophproliferation in mice with only one gna11 allele is accompaniedby decreased production of GHRH in the hypothalamus and thatexogenous application of GHRH can restore normal proliferation.

The synthesis and release of GHRH from arcN neurons are modulatedby a variety of neuropeptides and neurotransmitters, e.g., somatostatin,galanin, neuropeptide Y, and the peptide hormone ghrelin (1).Ghrelin enhances GHRH release via the activation of the GHS-R,which was shown to activate protein kinase C and to releasecalcium from intracellular stores via the pathway mediated byphospholipase C-ß and IP₃ (12). However, several othereffectors seem to contribute to GHS-R function, like adenylylcyclase-mediated cyclic AMP production or L-type Ca²⁺ channels(12, 22). We showed that impaired hypothalamic G_q/11 signalingled to a significant reduction of ghrelin-mediated c-fos activationin arcN neurons, suggesting that G_q/11 proteins are indeed crucialfor the signal transduction of the GHS-R. This notion is corroboratedby the finding that ghrelin-induced IGF-I surges are inhibitedin mice with only one intact gna11 allele. The latter findingalso suggests that the pituitary GHS-R (5, 9), which shouldsignal normally because gnaq is not inactivated in the anteriorpituitary of these mice, does not play a major role in ghrelin-mediatedGH release. This finding is in line with the observation thatGHRH antiserum inhibits the GH response to ghrelin in rats (28)and that patients with hypothalamopituitary disconnection donot respond with GH release to synthetic GHS-R ligands (19).

Impaired proliferation of the somatotroph cell line explainsvery well the dwarf phenotype but not the premature deaths ofthese mice. Dwarfism itself is not expected to be lethal, butit might handicap the pup in competing for the dam's milk orin reaching the food tray. However, even in very small littersand when food and water were put directly into the cage, thesurvival of dwarf mice was not improved. In addition to beingof short stature, these mice are also impaired with regard tomotor development, i.e., they develop an ataxia resembling theone seen in constitutively G ${alpha}$ _q-deficient mice (15). However,constitutively G ${alpha}$ _q-deficient mice have, except for an increasedperinatal mortality due to defective primary hemostasis (16),a normal life expectancy (15), and therefore, ataxia cannotaccount for the premature deaths of mice with only one intactgna11 allele. We hypothesized that additional defects must existin these mice, but neither levels of other pituitary hormonesin plasma (Fig. 3) nor histological analyses of internal organsrevealed any abnormalities. However, stomach weight, size ofretrorenal fat deposits, and food uptake were reduced in micewith only one intact gna11 allele (Fig. 2), suggesting thatthey suffer from a deregulation of energy homeostasis and/orappetite control. Ghrelin, besides having a role as a growth-hormonesecretagogue, is a well-known regulator of food intake and energyexpenditure (12), and impaired hypothalamic ghrelin signalingis expected to result in dwarfism and reduced food intake. Surprisingly,genetic inactivation of the ghrelin gene did not affect growthor food intake in mice (25, 37). On the other hand, a smallinterfering RNA-based knockdown of the GHS-R in rats impairedsomatic growth, weight gain, and food uptake (21). These studiessuggest that the GHS-R, but not ghrelin itself, is indispensablefor normal regulation of growth and energy homeostasis. It isquite possible that parallel hormonal systems, known or unknown,are involved in the regulation of these vital processes, andour findings strongly suggest that they converge on G ${alpha}$ _q/G ${alpha}$ ₁₁-coupledreceptors, including the GHS-R.

Taken together, our data show that the complete loss of G ${alpha}$ _q/G ${alpha}$ ₁₁-mediatedsignaling in the developing nervous system does not cause obviousdevelopmental abnormalities but leads to functional defectsincompatible with extrauterine life. The presence of a singlegnaq allele or a single gna11 allele is sufficient to maintainvital functions after birth, but only the presence of a gnaqallele, not of a gna11 allele, allows normal postnatal proliferationof the somatotroph cell line. In addition to showing somatotrophhypoplasia and consecutive dwarfism, mice with only one intactgna11 allele show abnormal eating behavior, and our findingssuggest that both defects involve impaired signaling via theGHS-R.

ACKNOWLEDGMENTS

We thank Rüdiger Klein for providing the NestinCre lineand Anke Rogatzki and Marianne Hillesheim for expert technicalassistance.

This work was funded by Collaborative Research Center 488 ofthe Deutsche Forschungsgemeinschaft.

FOOTNOTES

* Corresponding author. Mailing address: Institute of Pharmacology, University of Heidelberg, Im Neuenheimer Feld 366, 69120 Heidelberg, Germany. Phone: 49-6221-548255. Fax: 49-6221-548549. E-mail: Nina.Wettschureck{at}urz.uni-heidelberg.de.

REFERENCES

Top