<p>A solid-phase sequencing technique was applied to quantify the mitochondrial A3243G mutation in three families with maternally inherited diabetes and deafness. A correlation between the level of heteroplasmy and age at onset was found. The fluctuation of the heteroplasmy levels of the A3243G mutation was monitored from 4 to 18 years, in three female patients. Using the minisequencing method, the level of heteroplasmy was found to decrease over time in endothelial cell samples from all three patients. </p><p> With a similar strategy, the heteroplasmy levels of two neutral polymorphisms in the non-coding region of the mtDNA in healthy individuals were monitored. It has recently been suggested that heteroplasmy occurs frequently at neutral nucleotide positions in the control region of mtDNA and that the heteroplasmy level changes with age. The level of heteroplasmy of the neutral polymorphisms was found to remain unchanged over a time period of up to 25 years in four individuals. </p><p> Wilson disease (WD) is caused by mutations in the ATP7B gene that encodes a mitochondrial copper-transporting ATPase. The worldwide prevalence of WD has been estimated to 1 in 30 000. Based on the number of diagnosed patients the estimated prevalence in the Swedish population would be 1 in 300 000. The prevalence of WD in Sweden was estimated indirectly by quantitative minisequencing analysis of two WD-causing mutations in pooled DNA samples. In addition, the population frequencies of eight SNPs in the ATP7B gene were determined. Our results confirmed that WD is truly more rare in Sweden than in other populations.</p><p> A previously nondetectable diversity of alleles at the KIT locus, determining the coat color of pigs, was found by using three quantitative methods, minisequencing, pyrosequencing and the "TaqMan" 5' exonuclease assay. A splice-site mutation and a duplication of the KIT gene, encoding the mast/stem cell growth factor receptor causes the allelic diversity. Despite of a strong selection for white color dating from the medevial era, the desired phenotype has not been fixed. This study provides tools for genotyping the complicated KIT locus in pigs, which may be used for the purpose of breeding true for white color.</p>

Neuroblastoma is a malignant childhood cancer, originating from sympathetic neuroblasts of the peripheral nervous system. Neuroblastoma is a heterogenous group of tumours, while some are highly malignant others can spontaneosly mature into a more benign form or regress. Less than half of the patients survive and this statistics has improved only modestly over the past 20 years. SH-SY5Y is a human neuroblastoma cell line established from a highly malignant tumour. The cells have retained a capacity to differentiate in vitro in response to low concentrations of the phorbolester 12-O-tetradecanoylphorbol-13-acetate (TPA) in the presence of serum or defined growth factors. Differentiated cells are characterised by neurite formation and upregulation of neuronal marker genes. SH-SY5Y are unresponsive to nerve growth factor (NGF), but when transfected to express the NGF-receptor TrkA, they differentiate in response to NGF. Protein kinase C (PKC) is pivotal for the differentiation response to take place. We have investigated the role of signaling through the Ras-MAPK pathway in differentiating SH-SY5Y, with respect to neurite formation, expression of neuronal marker genes and growth control. Our results show that differentiation-promoting treatment induced a sustained activation and nuclear accumulation of the MAPK ERK in SH-SY5Y. The nuclear accumulation of ERK was PKC-dependent. However, nuclear accumulation of ERK was not sufficient for a differentiation response to take place in these cells, but ERK activity was needed for the characteristic upregulation of NPY and GAP-43 induced by TPA. ERK activity did not induce neurite formation, neither was it necessary for TPA-induced neurite formation. Instead, stimulation of a pathway distinct from MEK/ERK, but downstream of Ras, was needed for morphological differentiation. We could also show that differentiated cells still entered S-phase and that there was no correlation between expression of the CKI p21cip1 (an ERK target), BrdU-incorporation or neurite formation.

Glioblastoma multiforme is a malignant brain tumor characterized by heterogeneity.Interactions between heterogeneous tumor cells are supposed to affect the behavior of awhole tumor cell population. In this thesis an in vitro model system of clonal glioma celllines originating from one glioblastoma tumor was used, and the behavior of cells incocultures was studied and compared the behavior of cells grown separately. The resultsindicate the presence of two types of interactions. In one, paracrine signals acted via extra-cellular media. This was associated with increased growth of the whole co-culture followedby a selective force driving one clone to dominance. In the other type, the cell clones grewside by side without signs of paracrine signalling, in a balance resulting in an increasedterminal cell density. Further investigations focused on mechanisms of interactions in thiscombination. Two cell clones were chosen, a GFAP+ and a GFAP-, for further experiments. Withdifferential display PCR it was possible to investigate their specific gene expressionpatterns. Seventeen cDNA fragments were differentially expressed, among them twocorresponded to known transcription factors, ATF3 and prox-1, one to a cytoskeletal protein,α-tropomyosin. The collection also contained eight ESTs (Expressed Sequence Tags) wherethe corresponding genes are unknown at present. Expression of the isolated sequences werealso analyzed in a panel of 12 different glioma cell lines and the results illustrate thecomplexity of gene expression and of tumor heterogeneity. Genes, the expression levels ofwhich were modulated in co-cultures and/or were cell density dependent, were alsoidentified. PDGF B is suggested to play a role in sarcomas. The gene codes for an mRNA transcriptwith long UTRs, parts of which are deleted in the homologous oncogene v-sis. The UTRs ofPDGF B mRNAs in human sarcomas were investigated for deletions similar to v-sis thatmight result in increased protein levels. A new transcript variant was identified, lacking a149 base region in the 3'UTR, but its presence was not associated with increased levels ofprotein. Alterations in the 5'UTR were found more likely to be associated with increasedprotein levels.

Medulloblastoma is a highly malignant cerebellar childhood tumor. As in many other brain tumors, expression of platelet-derived growth factor (PDGF) and its receptors has been shown in medulloblastoma. To reveal the importance of this growth factor in cerebellar development and tumorigenesis, analyses were performed on human medulloblastoma cell lines and on tissue from normal mouse brain at different stages of development. The in vivo effect of a forced expression of PDGF-B in the cerebellar primordium was examined in transgenic mice. In the normal mouse embryo, we found PDGF receptor-α-positive cells in the early neuroepithelium and on neuronal precursors. In the postnatal cerebellum, cells in the external germinal layer and Purkinje cells expressed the receptor. In the medulloblastoma cells, expression of all the three PDGF isoforms and PDGF receptors was seen and correlated to neuronal differentiation. Endogenously activated, i.e. tyrosine phosphorylated, PDGF receptors were identified. To reveal the role of PDGF in normal cerebellar development, we established transgenic mice where a PDGF-B cDNA was introduced via homologous recombination into the engrailed-1 gene. Engrailed-1 is specifically expressed at the mid-/hindbrain boundary of the early neural tube, i.e. in an area from which the cerebellar primordium develops. The ectopic expression of PDGF-B caused a disturbance of cerebellar development. Midline fusion of the cerebellar primordium did not occur properly, which resulted in cerebellar dysplasia in the adult mouse. In a parallel study, the expression pattern of a glial fibrillary acidic protein (GFAP)-lacZ transgene was followed in the embryonic mouse central nervous system. It was shown that the human GFAP promoter was already active by embryonic day 9.5 and as development proceeded, expression occured in different, independent cell populations. Among these cell populations were the radial glial cells in the neocortex.

Myelomatose (benmargskreft) er en blodsyk dom som rammer ca 200 nordmenn årlig. Sykdommen kan ikke kureres og karakteriseres av symptomer som benmargssvikt og infeksjonstendenns, men kanskje først og fremst av sykelig nedbrytning av skjelettet. Pasientene rammes i høy utstrekning av benbrudd, hvirvelsammenfall og skjelettsmerter. Mekanismene for bennedbrytning og vekstkontroll står sentralt i avhandlingsarbeidet som består av fem artikler om cytokiners rolle i myelomatose. Cytokiner er signalsubstanser som benyttes i celle-celle-kommunikasjon. Det er sannsynligvis ubalanse av cytokiner som forårsaker den sykelige nedbrytningen av bensubstansen. Det første delarbeidet omhandler funnet av hepatocyttvekstfaktor (HGF) som er uttrykt hos nesten alle pasienter med myelomatose Dette påvises med forskjellige teknikker og det benyttes bl a en separasjonsmetode for myelomceller basert på Ugelstadkuler som ble utviklet ved IKM i 1993. Videre påvises forhøyede nivåer av HGF i serum fra pasienter. Et interessant funn er at HGF reseptor også er uttrykt i pasientprøver, hvilket kan tale for at myelomceller kan ha en selvstimulerende (autokrin) funksjon. I det andre delarbeidet vises en dyremodell for myelomatose i immundefekte mus. Et hovedpoeng er at det lar seg gjøre å få vekst av myelomceller i musebenmarg med påvisbare tegn til patologisk bennedbrytning på røntgen og ved histologisk undersøkelse. Musene har forhøyede nivåer av HGF i serum. Benlesjonene ble karakterisert ved hjelp av histomorfometri. Denne undersøkelse viste 99% reduksjon av de bendannende cellene (osteoblaster) og 33% reduksjon av bennedbrytende celler (osteklaster). I tredje delarbeidet viser man at HGF induserer interleukin (IL)-11-produksjon i osteoblaster. IL-11 er en kjent påskynder av benresorpsjon og osteoklastaktivator. Et interessant fenomen er at HGF ser ut til å være bundet til heparansulfat på cellemembranen og at slikt membranbundet HGF virker bedre enn løselig HGF. Effekten av HGF potensieres av cytokinene TGF-beta og IL-1. En styrke ved arbeidet er at såvel ferskisolerte pasientceller som cellelinjer viser identiske mønstre. Arbeidet angir en mulig måte som HGF kan befremme bennedbrytning. I fjerde delarbeid vises at cytokinet IL-15 forhindrer programmert celledød (apoptose) i myelomcellelinjen OH-2. Det var fra før kjent at myelomceller relativt hyppig lar seg stimulere av cytokinet IL-6, som fortsatt er den mest anerkjente myelomvekstfaktoren. IL-15 var tilnærmet like potent antiapoptotisk som IL-6, og befremmet også kortvarig proliferasjon. IL-15s effekt kunne potensieres av TNF-alfa I femte delarbeid påvises at cytokinet benmorfogent protein (BMP)-4 hemmer vekst av myelomceller. BMP-4 befremmer bendannelse. Effekten av BMP-4 kom fram i IL-6-stimulerte cellelinjer og pasientprøver. Effekten skyldtes såvel induksjon av apoptose som stopp i cellesyklus G1-fase. Dette er et mulig viktig funn siden man kan tenke seg at pasienter med myelomatose kunne behandles med BMP-4 eller lignende substanser. På slik måte ville såvel skjelettnedbrytningen som myelomcellevekst kunne påvirkes gunstig. Arbeidet bidrar til forståelse av molekylære mekanismer for bendestruksjon og myelomcellevekst og ble veiledet av profesor dr. med. Anders Waage. Henrik Hjorth-Hansen har vært stipendiat i Den norske kreftforening, og undersøkelsen ble dessuten støttet av Kreftfondet ved RiT og Blix’ legat.

Seasonal variations of mood, behavior and physiology have been of increasing interest. At least two different seasonal rhythms seem to exist: Descriptions of Seasonal Affective Disorder (SAD) with increased weight, increased sleep and fatigue during winter have attracted attention in academic psychiatry and in the general public the last two decades. In addition to such a difference in mood, weight and sleep between summer and winter, many studies describe a spring and fall increase in frequency of suicides and of admissions to hospital for mood disorders. In searching for a possible etiology for these seasonal changes, the main focus has been on variations in length of day. The objective of this thesis was to study the existence and pattern of seasonal variation in some forms of behavior and of psychiatric illness among children and adults in Norway. Possible statistical connections between seasonal variations of behavior and changes in length of day and the influences of latitude, sex and age were also studied. The monthly numbers of incidents in different groups were studied: All suicides in Norway 1969-96 (N=14.503), admissions to hospital for depression and mania in some hospitals 1992-96 (N=4.341), all violent episodes recorded by the police in Norway 1991-97 (N=82.537), all patient-staff incidents in a psychiatric department 1990-97 (N=502), all telephone calls to the Red Cross help-line for children and adolescents in Norway 1996-98 (N=691.787calls, 220.602 conversations) and in Trondheim, Norway 1991-97 (N=80.983 calls, 22.698 conversations) were included in the thesis. The monthly frequencies of these incidents were compared to an expected equal daily frequency of incidents through the year. Changes with increasing age and increasing latitude were examined. Correlations between the monthly frequencies of incidents and the length of day, with maximum impact at midsummer, and correlations between the monthly frequencies of incidents and the speed of change in length of day, with maximum impact at the equinoxes, were also studied. In this thesis, an increased activity in April-June and in October-November is described for all the groups that were studied. In summer and winter there is less activity than in the rest of the year. Among children calling the help-line, a steady diminishing seasonal variation in number of calls with increasing age from 7 to 17 years of age and an increasing seasonal variation in number of calls with increasing latitude were found. Also the seasonal variation of violence increases with increasing latitude in Norway. Among men there is a correlation between the monthly number of suicides and the monthly number of admissions for mania and a correlation between the monthly number of suicides and the monthly number of admissions for depression. Among women there is a diminishing seasonal variation of admissions for depressions with increasing age. The monthly frequency of violence in Norway and the monthly frequency of calls to the Red Cross help-Line for children and adolescents correlated with a delay of 1-2 months with the monthly change in length of day with maximum impact at the equinoxes. The results in the thesis correspond with earlier studies describing an increase in the frequency of suicides and an increase in admissions for depressions in spring and fall. A corresponding rhythm for other forms of human behavior is described in the present thesis, indicating that the seasonal rhythm of psychiatric illness reflects a seasonal rhythm of behavior in greater parts of the population. The seasonal variation in behavior seems to increase with increasing latitude, to be more dramatic in the northern than in the southern parts of Norway. In this thesis results supporting a hypothesis of human behavior being influenced by changes in length of day are given. Changes in length of day may induce changes in sleep and other disturbances in the daily rhythm that could change mood and other emotional qualities in vulnerable individuals. The demands on our capability to adapt to changes in length of day are largest at the equinoxes. / <b>Årstidsvariasjon av sinnstemning og adferd.</b> Det er økende interesse for årstidsvariasjon av adferd og av forekomsten av psykiske lidelser. Det synes å foreligge minst to ulike årstidsrytmer i befolkningen; Størst oppmerksomhet har oppdagelsen av vinterdepresjon karakterisert ved tristhet, tretthet, økt vekt og forlenget søvn vakt. I tillegg til en slik forskjell i humør, vekt og søvn mellom sommer og vinter, er det en rekke beskrivelser av overhyppighet av selvmord og av innleggelser i sykehus for depresjoner vår og høst. Årsakene til disse to ulike årstidsrytmene er ikke kjent, men man har antatt at variasjon i dagslengde gjennom året spiller en rolle. Hensikten med denne avhandlingen har vært å undersøke om det er årstidsvariasjon i forekomsten av ulike former for adferd og av psykiske lidelser hos barn og voksne i Norge. Videre er eventuelle statistiske sammenhenger mellom adferd og dagslengde gjennom året undersøkt. Til sist er forskjeller i årstidsrytme knyttet til breddegrad, alder og kjønn undersøkt. Antallet hendelser pr måned i ulike grupper ble studert; Alle selvmord i Norge 1969-96 (N=14.503), innleggelser for depresjon og mani i en del sykehus 1992-96 (N=4.341), alle registrerte voldsepisoder i Norge 1991-97 (N= 82.537), personalskader i et psykiatrisk sykehus 1991-97 (N=502), alle telefoner til Røde Kors Kontakttelefon for barn og unge i Norge 1996–98 (N=691.787 oppringninger, 220.602 samtaler) og i Trondheim 1991-97 (N=80.983 oppringninger, 22.698 samtaler) ble inkludert i arbeidet. Hyppigheten av alle disse hendelsene i hver måned ble sammenlignet med en forventet lik fordeling av hendelsene året igjennom. Endringer med økende alder og med økende breddegrad ble undersøkt. Videre ble det gjort sammenligninger med dagslengde som er lengst ved sommersolverv og kortest ved vintersolverv, og sammenligninger med endringer av dagslengde som er hurtig ved vår og høstjamndøgn og sakte ved solvervene. I alle disse materialene er det en økt aktivitet april – juni og oktober – november, videre er det stille perioder om vinteren og om sommeren. Blant barn som ringer kontakttelefonen er det gradvis avtagende årstidsvariasjon av henvendelser med økende alder fra 7 til 17 år og økende årstidsvariasjon i antallet henvendelser jo lenger nord man kommer i Norge. Også årstidsvariasjonen av vold i Norge endrer seg jo lengre nord man kommer i landet. Blant menn er der en korrelasjon mellom det månedlige antallet av selvmord og av innleggelser for mani og mellom antallet selvmord og innleggelser for depresjon. Blant kvinner er det en avtagende årstidsvariasjon av innleggelser for depresjon med økende alder. Den månedlige endring av dagslengde som er raskest ved jamndøgnene korrelerer med en viss forsinkelse med forekomsten av vold i Norge og med antallet oppringninger til Barn og Unges kontakttelefon. Funnene i avhandlingen er i samsvar med tidligere beskrivelser av en markert økning av suicid og av innleggelser for depresjoner om våren og til dels om høsten. I avhandlingen er en tilsvarende rytme funnet for annen adferd. Dette tyder på at årstidsrytmen av psykiatrisk sykelighet avspeiler en årstidsrytme av adferd i store deler av befolkningen. Videre ser det ut til at forskjellene i adferd gjennom året blir mer markerte jo lengre nord man kommer i landet. I avhandlingen er det funn som støtter en hypotese om at endringer i dagslengde påvirker mennesket, det er mulig at dette skjer gjennom endret søvn og andre forstyrrelser i døgnrytmen. Vår døgnrytme er utsatt for størst krav til å tilpasse seg hurtige endringer i lysforhold rundt jamndøgnene.

The aims of this dissertation were to investigate alcohol-related seizures in clinical neurological practice. We wanted to assess the extent of this problem, to classify the seizures, and to investigate methods to improve the clinical diagnosis of such seizures. We propose an arbitrary but simple and reproducible way of diagnosing alcohol-related seizures and alcohol withdrawal seizures. Papers I and II relate to seizure classification and the extent of the problem in relation to the level and weekly pattern of alcohol use. Paper III investigates the performance of various biological markers as aids in the diagnosis of alcohol-related seizures. Paper IV explores pitfalls in the result interpretation for two methods for detection of CDT in patients with neurological disorders. Paper V investigates the utility of standard EEG for the identification of alcohol-related seizures. Even though the general alcohol consumption in our region is low, every third patient with an epileptic seizure leading to hospitalisation had hazardous alcohol consumption. Evidence of focal lesions or focal seizure start was found in a high proportion of alcohol-related seizures. All such seizures were secondarily generalized and thus, we challenge the establishment impression that the vast majority of alcohol-related seizures are primarily generalized. Binge drinking (more than six drinks for men or four drinks for women, in a single drinking occasion) was common, but had little influence on seizure susceptibility or timing of seizures. In contrast to prior knowledge, we found that in some patients there was no time lag from cessation of drinking to the occurrence of a seizure, but falling intake levels prior to withdrawal seizures were demonstrated. This indicates that a state of relative withdrawal while still drinking may be sufficient to induce a seizure. Carbohydrate-deficient transferring (CDT) is the most accurate biomarker for alcohol use and good adjunct to the diagnosis of alcohol-related seizures, but its accuracy does not compete with a good clinical investigation. Generally poor accuracy should be expected for fertile women. Women on enzyme-inducing antiepileptic drugs who drink no or little alcohol seem to be at risk of having false positive CDT. Other variables associated with increased CDT were low body mass index, or having total transferring levels outside normal range. A definitely abnormal EEG suggests epilepsy or symptomatic seizures unrelated to alcohol use. The predictive value of a normal EEG is limited, but the typical post-ictal finding in alcohol-related seizures is nevertheless a normal low-amplitude EEG record. The best method for identification of alcohol-related seizures is a clinical work-up based on a thorough medical history. The Alcohol Use Disorders Identification Test (AUDIT) provides a reliable measure of drinking habits. CDT is a good supplement to the clinical diagnosis when there is doubt, if factors associated with false-positive values are appreciated. The diagnostic value of EEG is limited.

A solid-phase sequencing technique was applied to quantify the mitochondrial A3243G mutation in three families with maternally inherited diabetes and deafness. A correlation between the level of heteroplasmy and age at onset was found. The fluctuation of the heteroplasmy levels of the A3243G mutation was monitored from 4 to 18 years, in three female patients. Using the minisequencing method, the level of heteroplasmy was found to decrease over time in endothelial cell samples from all three patients. With a similar strategy, the heteroplasmy levels of two neutral polymorphisms in the non-coding region of the mtDNA in healthy individuals were monitored. It has recently been suggested that heteroplasmy occurs frequently at neutral nucleotide positions in the control region of mtDNA and that the heteroplasmy level changes with age. The level of heteroplasmy of the neutral polymorphisms was found to remain unchanged over a time period of up to 25 years in four individuals. Wilson disease (WD) is caused by mutations in the ATP7B gene that encodes a mitochondrial copper-transporting ATPase. The worldwide prevalence of WD has been estimated to 1 in 30 000. Based on the number of diagnosed patients the estimated prevalence in the Swedish population would be 1 in 300 000. The prevalence of WD in Sweden was estimated indirectly by quantitative minisequencing analysis of two WD-causing mutations in pooled DNA samples. In addition, the population frequencies of eight SNPs in the ATP7B gene were determined. Our results confirmed that WD is truly more rare in Sweden than in other populations. A previously nondetectable diversity of alleles at the KIT locus, determining the coat color of pigs, was found by using three quantitative methods, minisequencing, pyrosequencing and the "TaqMan" 5' exonuclease assay. A splice-site mutation and a duplication of the KIT gene, encoding the mast/stem cell growth factor receptor causes the allelic diversity. Despite of a strong selection for white color dating from the medevial era, the desired phenotype has not been fixed. This study provides tools for genotyping the complicated KIT locus in pigs, which may be used for the purpose of breeding true for white color.

Adenovirus infected cells provide a good model system for studying nuclear organization during RNA production and transport. This thesis is focused on the dynamic organization of splicing factors during the late phase of Adenovirus infection in HeLa cells, the nuclear localization of viral RNA, and the pathway used for viral RNA transport to the cytoplasm. Splicing factors are relocalized from interchromatin granule clusters to sites of transcription in Adenovirus infected cells at intermediate times of infection. Later, splicing factors and viral RNA accumulate posttranscriptionally in interchromatin granule clusters. The release of the splicing factors from transcription sites was energy dependent or preceded by energy requiring mechanisms. Our data indicated that phosphorylation events inhibited by staurosporine, and 3' cleavage of the transcript are two possible mechanisms involved prior to the release of the RNP complex from transcription sites. A viral protein derived from orf6 of early region 4, 34K, is important for the nuclear stability and transport of late viral mRNA derived from the major late transcription unit. A viral mutant lacking this region is defective for posttranscriptional accumulation of viral mRNA in interchromatin granule clusters, and for the accumulation of viral RNA in the cytoplasm. These results suggest that posttranscriptional accumulation of viral RNA in interchromatin granule clusters may contribute to the maturation of the RNP complex or sorting of RNAs and proteins, to prepare the final RNP complex for transport to the cytoplasm. A previous model suggested that adenoviral late mRNA is transported to the cytoplasm by utilizing the CRM-1 pathway. This pathway can be blocked by the drug leptomycin B. The data presented in paper IV suggests that this model might not be applicable, since leptomycin B did not inhibit adenoviral late gene expression.

Platelet-derived growth factor receptor α (PDGFRα) plays an important role during embryogenesis. After implantation, the patterns of expression of Pdgfrα and its ligand Pdgf-A undergo an "autocrine-paracrine transition", in that Pdgf-A becomes expressed in the ectoderm and epithelia, while Pdgfrα is expressed in the adjacent mesenchymal tissue. In human tumors, such as malignant glioma, both PDGF and PDGFRα are overexpressed within the same tissue, indicating that an autocrine PDGF loop is generated in the tumors. This thesis is focused on the in vivo functionality of the PDGFRα gene (PDGFRA) promoter, arid on the effect of autocrine PDGF-A stimulation in transgenic n-iice during embryogenesis. To test the in vivo promoter function of a human PDGFRA 2.2 kb 5' flanking fragment, we generated transgenic mouse lines and found that the 2.2 kb fragment was able to promote lacZ reporter gene expression in most of the endogenous Pdgfra expressing tissues. Absence of expression and "ectopic" expression of the transgenic lacZ were also observed. To investigate the autocrine PDGF effect, we produced autocrine PDGF-As (A short-chain) transient transgenic embryos. These transgenic embryos carried a 6 kb mouse Pdgfra 5' flanking sequence linked to a human PDGF-As cDNA. The pattern of expression of the PDGF-As transgene mRNA was similar to that of lacZ. Some of the transgenic embryos exhibited severe abnormal phenotypes, such as midline fusion defects in the cephalic and craniofacial region and small body size, and these embryos die at mid-gestation stage. These findings indicate that a paracrine pattern of expression and the dosage of PDGF are important for sustaining normal embryo development, especially with regard to the middline fusion in craniofacial regions. The possible signaling pathways that may be involved in regulating Pdgfra activity were also studied by comparison of patterns of mRNA expression of Gli, Ptc, and Paxl with that of Pdgfra. The results pointed to the possibility that the Shh signaling pathway may be involved in the regulation of Pdgfra expression for example during early bone and foregut development. The specific regulatory mechanisms may vary for different tissues.