A gene network switch enhances the oxidative capacity of ovine skeletal muscle during late fetal development

Abstract

Background: The developmental transition between the late fetus and a newborn animal is associated with profound changes in skeletal muscle function as it adapts to the new physiological demands of locomotion and postural support against gravity. The mechanisms underpinning this adaption process are unclear but are likely to be initiated by changes in hormone levels. We tested the hypothesis that this developmental transition is associated with large coordinated changes in the transcription of skeletal muscle genes.

Results: Using an ovine model, transcriptional profiling was performed on Longissimus dorsi skeletal muscle taken at three fetal developmental time points (80, 100 and 120 d of fetal development) and two postnatal time points, one approximately 3 days postpartum and a second at 3 months of age. The developmental time course was dominated by large changes in expression of 2,471 genes during the interval between late fetal development (120 d fetal development) and 1-3 days postpartum. Analysis of the functions of genes that were uniquely up-regulated in this interval showed strong enrichment for oxidative metabolism and the tricarboxylic acid cycle indicating enhanced mitochondrial activity. Histological examination of tissues from these developmental time points directly confirmed a marked increase in mitochondrial activity between the late fetal and early postnatal samples. The promoters of genes that were up-regulated during this fetal to neonatal transition were enriched for estrogen receptor 1 and estrogen related receptor alpha cis-regulatory motifs. The genes down-regulated during this interval highlighted de-emphasis of an array of functions including Wnt signaling, cell adhesion and differentiation. There were also changes in gene expression prior to this late fetal--postnatal transition and between the two postnatal time points. The former genes were enriched for functions involving the extracellular matrix and immune response while the latter principally involved functions associated with transcriptional regulation of metabolic processes.

Conclusions: It is concluded that during late skeletal muscle development there are substantial and coordinated changes in the transcription of a large number of genes many of which are probably triggered by increased estrogen levels. These changes probably underpin the adaption of muscle to new physiological demands in the postnatal environment.

Figure 1

Experimental design. LD skeletal muscle samples were taken from three animals at each of five developmental stages i.e. 80, 100, and 120 days of fetal development, 150 days of development (i.e. 1-3 days post- partum) and 230 days of development (i.e. 83 days post- partum). Birth was at approximately 147 days of development.

Figure 2

Visual representation of the changes in global gene expression in ovine skeletal muscle during late fetal and postnatal development. Affymetrix MAS5 data were globally scaled to 200 on each microarray and normalized across all microarrays to enable visualization of the expression of all genes. Each analysis time point consisted of triplicate samples, and the mean for each developmental stage is presented. The genes are differentially colored according to their normalized expression intensity relative to the global gene intensity at 80 days of fetal development. Genes with mean intensities less than the global average at 80 days are colored green, while those with mean intensities greater than the global average at 80 days are colored red. Gene intensities that were not different from the 80 day fetal global mean are colored black. Genes represented by unsaturated colors were of low confidence according to a defined set of criteria. The ordinate shows the logarithm of the MAS5 intensity data while the abscissa corresponds to the five developmental stages i.e. 80, 100 and 120 days of fetal development, 150 days of development (i.e. 1-3 days after birth) and 230 days of development (young lamb).

Figure 3

qRT-PCR expression patterns for selected genes from the microarray gene expression clusters. The mRNA expression patterns of selected genes from the major gene expression clusters were also measured by qRT-PCR. The expression levels are expressed as mean normalised expression (MNE) values relative to the reference gene, 18S RNA. The corresponding gene expression cluster derived from microarray data is shown in brackets. Fetal samples at 80, 100 and 120 days of development and postnatal samples at 150 d and 230 d of development are shown. The result for each gene shows the mean (n = 3) while the error bar denotes 1 S.D.

Figure 4

qRT-PCR expression patterns for selected genes not represented or not reporting on the microarray. The mRNA expression patterns of genes that were not represented or not reporting on the microarray were measured by qRT-PCR. The expression levels were expressed as mean normalised expression (MNE) values relative to 18S RNA. Fetal samples at 80, 100 and 120 days of development and postnatal samples at 150 d and 230 d of development are shown. The result for each gene shows the mean (n = 3), while the error bar denotes 1 S.D.

Figure 5

Muscle fibre type histochemistry showing the frequency of oxidative fibres in samples across the developmental period. NADH-Tr histochemistry was used to identify oxidative fibres in sections of LD muscles from animals sampled at 80, 100, 120, 150 and 230 days of development (n = 3 at each developmental stage). (a) Examples of histochemical staining patterns in a 120 d fetal sample (left panel) and a sample taken at 230 d of development (young immature lamb) (right panel). (b) A series of random grey-scale images was collected from each section and converted to pixel grayscale intensity values. The average frequency across the images from each sample was calculated and the mean for the three animals at each developmental time was then graphed. Greater pixel intensity corresponded with increased staining and therefore higher oxidative activity. The developmental times are differentially colored: 80 d (black); 120 d (yellow); 100 d (red); 150 d (blue), and; 230 d (purple).