Jennifer V. Schmidt

Dlk1 expression marks developing endothelium and sites of branching morphogenesis in the mouse embryo and placenta

The protein product of the Delta‐like 1 (Dlk1) gene belongs to the Delta‐Notch family of signaling molecules, proteins involved in cell fate determination in many tissues during development. The DLK1 protein is believed to function as a growth factor, maintaining the proliferative state of undifferentiated cells, and is usually down‐regulated as immature cells differentiate. The expression pattern of the DLK1 protein has been described in certain human tissues; however, Dlk1 expression is not well understood in the mouse, the most tractable mammalian genetic model system. To better understand the role of Dlk1 in embryonic development, the tissue‐specific expression pattern of Dlk1 mRNA during mouse embryogenesis was analyzed by in situ hybridization. In embryonic day 12.5 (e12.5) embryos, high levels of Dlk1 were found in the developing pituitary, pancreas, lung, adrenal, and many mesodermally derived tissues. Strikingly, Dlk1 expression also marks the growing branches of organs that develop through the process of branching morphogenesis. At e16.5, Dlk1 expression is down‐regulated in most tissues but remains in the pituitary, the adrenal gland, and in skeletal muscle. In the placenta, expression of Dlk1 is detected in endothelial cells lining the fetal blood vessels of the labyrinth. This pattern is distinct from that seen in the human placenta and suggests a role for Dlk1 in regulating maternal–fetal interactions. Developmental Dynamics 235:1115–1123, 2006.

Dlk1 is necessary for proper skeletal muscle development and regeneration.

Delta-like 1homolog (Dlk1) is an imprinted gene encoding a transmembrane protein whose increased expression has been associated with muscle hypertrophy in animal models. However, the mechanisms by which Dlk1 regulates skeletal muscle plasticity remain unknown. Here we combine conditional gene knockout and over-expression analyses to investigate the role of Dlk1 in mouse muscle development, regeneration and myogenic stem cells (satellite cells). Genetic ablation of Dlk1 in the myogenic lineage resulted in reduced body weight and skeletal muscle mass due to reductions in myofiber numbers and myosin heavy chain IIB gene expression. In addition, muscle-specific Dlk1 ablation led to postnatal growth retardation and impaired muscle regeneration, associated with augmented myogenic inhibitory signaling mediated by NF-κB and inflammatory cytokines. To examine the role of Dlk1 in satellite cells, we analyzed the proliferation, self-renewal and differentiation of satellite cells cultured on their native host myofibers. We showed that ablation of Dlk1 inhibits the expression of the myogenic regulatory transcription factor MyoD, and facilitated the self-renewal of activated satellite cells. Conversely, Dlk1 over-expression inhibited the proliferation and enhanced differentiation of cultured myoblasts. As Dlk1 is expressed at low levels in satellite cells but its expression rapidly increases upon myogenic differentiation in vitro and in regenerating muscles in vivo, our results suggest a model in which Dlk1 expressed by nascent or regenerating myofibers non-cell autonomously promotes the differentiation of their neighbor satellite cells and therefore leads to muscle hypertrophy.

Analysis of candidate imprinted genes linked to Dlk1-Gtl2 using a congenic mouse line.

The study of genomic imprinting requires the use of DNA sequence polymorphisms between interfertile mouse species or strains. Most commonly, crosses between Mus musculus domesticus and Mus musculus castaneus or Mus spretus animals are used. Difficulties arise in the maintenance of these wild-derived mice in conventional animal facilities, however, and can be overcome by the use of a congenic strain for the region under study. We describe here the generation of a new mouse line, congenic for a region on distal Chromosome (Chr) 12 that encompasses the Dlk1–Gtl2 imprinted domain. We have taken a first step towards demonstrating the utility of these animals by assaying known genes located within the congenic interval for imprinted expression. We show that the two genes located immediately proximal to Dlk1, the Yy1 and Wars genes, are expressed in a biallelic manner. In addition, we have analyzed the Dio3 gene, located distal to Gtl2. This gene displays preferential expression of the paternal allele, with approximately 75% of the total message level originating from the paternal allele and 25% originating from the maternal allele. These data delineate the position of the Wars gene as the proximal boundary of the Dlk1–Gtl2 imprinted domain, and identify Dio3 as another potentially imprinted gene within this domain.

Conditional deletions refine the embryonic requirement for Dlk1

Numerous studies have implicated Delta-like 1 (DLK1), a transmembrane protein that shares homology with Notch ligands, in embryonic growth and differentiation. Dlk1 expression is widespread, though not ubiquitous, during early development, but is confined to a few specific cell types in adults. Adult Dlk1-expressing tissues include the Insulin-producing β-cells of the pancreas and the Growth hormone-producing somatotrophs of the pituitary gland. Previously generated Dlk1 null mice (Dlk1(Sul-pat)), display a partially penetrant neonatal lethality and a complex pattern of developmental and adult phenotypes. Here we describe the generation of a conditional Dlk1 mouse line (Dlk1(flox)) to facilitate cell type-specific deletion of the Dlk1 gene, providing a powerful system to explore each aspect of the Dlk1 null phenotype. Four tissue-specific Cre mouse lines were used to produce individual Dlk1 deletions in pancreatic β-cells, pituitary somatotrophs and the endothelial cells of the embryo and placenta, key candidates for the Dlk1 phenotype. Contrary to expectations, all of these conditional mice were fully viable, and none recapitulated any aspect of the Dlk1(Sul-pat) null mice. Dlk1 expression is therefore not essential for the normal development of β-cells, somatotrophs and endothelial cells, and the tissues responsible for the Dlk1 null phenotype remain to be identified. Dlk1(flox) mice will continue to provide an important tool for further research into the function of Dlk1.

Dlk1 Promotes a Fast Motor Neuron Biophysical Signature Required for Peak Force Execution

Quick, Quick, Slow The slow muscles of postural stability and the fast muscles of running and jumping are driven by motor neurons that are differentiated by fast and slow biophysical properties. By retrograde labeling of mouse and chick muscle fibers, Müller et al. (p. 1264) characterized the developmental distinctions between fast and slow motor neurons. A transmembrane protein, when over- or underexpressed, was discovered to drive specification of the motor neurons and a downstream effector specified some, but not all, of the biophysical attributes. The fast versus slow profile of motor neurons is controlled by expression of a membrane protein. Motor neurons, which relay neural commands to drive skeletal muscle movements, encompass types ranging from “slow” to “fast,” whose biophysical properties govern the timing, gradation, and amplitude of muscle force. Here we identify the noncanonical Notch ligand Delta-like homolog 1 (Dlk1) as a determinant of motor neuron functional diversification. Dlk1, expressed by ~30% of motor neurons, is necessary and sufficient to promote a fast biophysical signature in the mouse and chick. Dlk1 suppresses Notch signaling and activates expression of the K+ channel subunit Kcng4 to modulate delayed-rectifier currents. Dlk1 inactivation comprehensively shifts motor neurons toward slow biophysical and transcriptome signatures, while abolishing peak force outputs. Our findings provide insights into the development of motor neuron functional diversity and its contribution to the execution of movements.

Oxytocin selectively increases ERα mRNA in the neonatal hypothalamus and hippocampus of female prairie voles

During neonatal development exogenous oxytocin increases ERalpha immunoreactivity in the hypothalamus of female prairie voles. The purpose of this study was to determine if the increase in ERalpha is associated with an increase in ERalpha mRNA expression and to determine if the effect is specific to ER subtype or if oxytocin also influences ERbeta mRNA expression. On the day of birth female prairie vole pups were treated with oxytocin, an oxytocin antagonist, or saline. Brains were collected and RT-PCR was used to determine the effect of treatment on ER mRNA production in the hypothalamus, hippocampus, and cortex. Within 2h of treatment oxytocin significantly increased ERalpha mRNA expression in the hypothalamus and hippocampus, but not the cortex, while inhibiting the effects of endogenous oxytocin reduced the expression of ERalpha mRNA in the hippocampus. Neonatal treatment did not affect the expression of ERbetamRNA. The results demonstrate that the effects of oxytocin treatment are region and ER subtype specific and that during the neonatal period oxytocin can affect the expression of ERalpha by altering message production. The regional specific changes in ERalpha mRNA expression in females are consistent with studies examining the behavioral and physiological effects of neonatal manipulation of oxytocin in females.

Identification of imprinting regulators at the Meg3 differentially methylated region

Genomic imprinting at the Delta-like 1 (Dlk1)-Maternally expressed gene 3 (Meg3) locus is regulated by the Meg3 differentially methylated region (DMR), but the mechanism by which this DMR acts is unknown. The goal of this study was to analyze the Meg3 DMR during imprinting establishment and maintenance for the presence of histone modifications and trans-acting DNA binding proteins using chromatin immunoprecipitation. In embryonic stem (ES) cells, where Meg3 is biallelically expressed, the DMR showed variable DNA methylation, with biallelic methylation at one region but paternal allele-specific methylation at another. All histone modifications detected at the Meg3 DMR of ES cells were biallelic. In embryonic day 12.5 (e12.5) embryos, where Meg3 is maternally expressed, the paternal Meg3 DMR was methylated, and activating histone modifications were specific to the maternal DMR. DNA-binding proteins that represent potential regulatory factors were identified in both ES cells and embryos.

Novel imprinted transcripts from the Dlk1-Gtl2 intergenic region, Mico1 and Mico1os, show circadian oscillations

Most of the known imprinted genes are assembled into clusters that share common imprinting control regions (ICRs). Non-coding transcripts are often associated with ICRs and implicated in imprinting regulation. We undertook a systematic search for transcripts originating from the Dlk1-Gtl2 intergenic region that contains the ICR for the chromosome 12 imprinted cluster and identified two overlapping transcripts expressed from opposite strands exclusively from the maternal chromosome. These novel imprinted transcripts most likely represent non-coding RNAs and are located telomeric to the IG DMR, extending the proximal boundary of the region of maternal-specific transcription. Their expression is tissue-specific and shows diurnal and circadian oscillations. Therefore, we named these novel transcripts maternal intergenic circadian oscillating 1 (Mico1) and Mico1, opposite strand (Mico1os).

Parental regulation of central patterns of estrogen receptor α

Reduced levels of estrogen receptor alpha (ERalpha) in the medial amygdala (MeA) and bed nucleus of stria terminalis (BST) have been hypothesized to play a significant role in the expression of male behaviors associated with monogamy. Therefore, the regulation of ERalpha could be a critical factor in determining male behavior and the evolution of monogamy. Central expression of ERalpha immunoreactivity was compared in hybrid offspring from crosses between two phenotypically distinct populations of prairie voles (Microtus ochrogaster). Illinois voles (IL) are socially monogamous and display low levels of ERalpha, while Kansas voles (KN) display some characteristics associated with polygyny and have higher levels of ERalpha. In offspring from hybrid crosses, the pattern of ERalpha expression was dependent upon parentage; the two types of hybrid crosses did not produce the same ERalpha pattern in the offspring. In the BST and MeA, hybrid males expressed ERalpha patterns consistent with those of males from their mothers population, while hybrid females had ERalpha patterns typical of females belonging to their fathers population. The parental-specific patterns of ERalpha expression are suggestive of genomic imprinting, therefore, the vole ERalpha (Esr1) gene was cloned and sequenced, and examined for allele-specific expression. Results from this study indicate that while maternal factors may play a major role the expression of ERalpha in their male offspring, genomic imprinting is unlikely to be involved, suggesting another mechanism is responsible.

Localizing transcriptional regulatory elements at the mouse Dlk1 locus.

Much effort has focused recently on determining the mechanisms that control the allele-specific expression of genes subject to genomic imprinting, yet imprinting regulation is only one aspect of configuring appropriate expression of these genes. Imprinting control mechanisms must interact with those regulating the tissue-specific expression pattern of each imprinted gene in a cluster. Proper expression of the imprinted Delta-like 1 (Dlk1) - Maternally expressed gene 3 (Meg3) gene pair is required for normal fetal development in mammals, yet the mechanisms that control tissue-specific expression of these genes are unknown. We have used a combination of in vivo and in vitro expression assays to localize cis-regulatory elements that may regulate Dlk1 expression in the mouse embryo. A bacterial artificial chromosome transgene encompassing the Dlk1 gene and 77 kb of flanking sequence conferred expression in most endogenous Dlk1-expressing tissues. In combination with previous transgenic data, these experiments localize the majority of Dlk1 cis-regulatory elements to a 41 kb region upstream of the gene. Cross-species sequence conservation was used to further define potential regulatory elements, several of which functioned as enhancers in a luciferase expression assay. Two of these elements were able to drive expression of a lacZ reporter transgene in Dlk1-expressing tissues in the mouse embryo. The sequence proximal to Dlk1 therefore contains at least two discrete regions that may regulate tissue-specificity of Dlk1 expression.