Dynamics of muscle fibre growth during postnatal mouse development

Background  

Postnatal growth in mouse is rapid, with total skeletal muscle mass increasing several-fold in the first few weeks. Muscle growth can be achieved by either an increase in muscle fibre number or an increase in the size of individual myofibres, or a combination of both. Where myofibre hypertrophy during growth requires the addition of new myonuclei, these are supplied by muscle satellite cells, the resident stem cells of skeletal muscle.     

Properties of mouse retinal ganglion cell dendritic growth during postnatal development

The property of dendritic growth dynamics during development is a subject of intense interest. Here, we investigated the dendritic motility of retinal ganglion cells (RGCs) during different developmental stages, using ex vivo mouse retina explant culture, Semliki Forest Virus transfection and time-lapse observations. The results illustrated that during development, the dendritic motility underwent a change from rapid growth to a relatively stable state, i.e., at P0 (day of birth), RGC dendrites were in a highly active state, whereas at postnatal 13 (P13) they were more stable, and at P3 and P8, the RGCs were in an intermediate state. At any given developmental stage, RGCs of different types displayed the same dendritic growth rate and extent. Since the mouse is the most popular mammalian model for genetic manipulation, this study provided a methodological foundation for further exploring the regulatory mechanisms of dendritic development.     

Roles of growth hormone and insulin-like growth factor 1 in mouse postnatal growth

To examine the relationship between growth hormone (GH) and insulin-like growth factor 1 (IGF1) in controlling postnatal growth, we performed a comparative analysis of dwarfing phenotypes manifested in mouse mutants lacking GH receptor, IGF1, or both. This genetic study has provided conclusive evidence demonstrating that GH and IGF1 promote postnatal growth by both independent and common functions, as the growth retardation of double Ghr/Igf1 nullizygotes is more severe than that observed with either class of single mutant. In fact, the body weight of these double-mutant mice is only approximately 17% of normal and, in absolute magnitude ( approximately 5 g), only twice that of the smallest known mammal. Thus, the growth control pathway in which the components of the GH/IGF1 signaling systems participate constitutes the major determinant of body size. To complement this conclusion mainly based on extensive growth curve analyses, we also present details concerning the involvement of the GH/IGF1 axis in linear growth derived by a developmental study of long bone ossification in the mutants.     

Expression of epidermal growth factor in the kidney and submandibular gland during mouse postnatal development

Earlier work has demonstrated that the salivary glands and kidneys are the major sites of epidermal growth factor (EGF) synthesis in adult mice. The precise timing of the onset of endogenous EGF synthesis in these tissues is not yet clear. In the present study we assessed the ontogenesis of EGF expression in the Swiss-Webster mouse. Paraformaldehyde-fixed frozen sections of neonatal kidneys and salivary glands were probed with proEGF cRNA labelled with 35S for in situ hybridization and with rabbit antisera to mouse EGF for immunocytochemistry. Both EGF mRNA and immunoreactivity were first detected in the developing distal nephron between days 3 and 5 postpartum. Juxtamedullary nephrons underlying the superficial nephrogenic zone were the first to express EGF. During the 2nd week after birth, EGF-expressing tubules became more abundant and distributed to medullary as well as cortical regions, corresponding to the thick ascending limb of Henle and distal convoluted tubule. Initial EGF mRNA and immunoreactivity in the submandibular gland were first detected between days 18 and 20 postpartum and increased notably during the following weeks.     

Axial mechanical properties of fresh human cerebral blood vessels

Human cerebral blood vessels are frequently damaged in head impact, whether accidental or deliberate, resulting in intracranial bleeding. Additionally, the vasculature constitutes the support structure for the brain and, hence, plays a key role in the cranial load response. Quantification of its mechanical behavior, including limiting loads, is thus required for a proper understanding and modeling of traumatic brain injury--as well as providing substantial assistance in the development and application of preventive measures. It is believed that axial stretching is the dominant loading mode for the blood vessels, regardless of the nature of the insult. Eighteen arteries and fourteen veins were obtained from the cortical surface of the cerebral temporal lobe of patients undergoing surgery. These vessels were stretched to failure in the longitudinal direction, either quasi-statically or dynamically. The significance of specimen and experiment parameters was determined using multivariate analysis of variance (MANOVA) testing. Results demonstrate that the arteries were considerably stiffer than the veins, carrying approximately twice as much stress at failure but withstanding only half as much stretch. No significant rate dependence was measured over a strain rate range of more than four orders of magnitude (0.01 to 500 s -1).     

Lead acetate delays rapid postnatal mouse brain and body growth

Starting at parturition and continuing until weaning, mothers of five mouse litters received tap water while five others had 10 mg PbAc/ml in their drinking water. The offspring receiving lead from the mothers had significantly lower body weights after the first days of receiving lead; their slowed body growth led to a 2-day delay of onset (usually at 16-18 days) of their last rapid body growth stage. They also had significantly smaller brain weights between age 14 days and weaning (23 days). The onset of rapid brain growth was delayed from its usual onset at 16-18 days to about 22-23 days before rising to about the same value as the control mice at 26 days. Thus, the initial effect on brain growth is decidedly greater than on body growth, though brain weight later reaches close to the control value.     

Immunohistochemical localization of transforming growth factor β1 and β2 in mouse testes during postnatal development

We examined age-related changes in the expression of transforming growth factor-β₁ (TGF-β₁) and transforming growth factor-β₂ in mouse testes. The mice were assigned to three age groups: 35, 50, and 75 days old. Paraffin embedded testis sections were processed for the standard streptavidin biotin peroxidase complex immunohistochemistry method. TGF-β₁ expression increased in aging round spermatids over the time studied. There was no expression in 35-day-old Leydig cells, whereas strong expression of TGF-β₁ was observed in 50-day-old Leydig cells. Expression decreased in 75-day-old Leydig cells. TGF-β₂ expression was weak in 35- and 50-day-old mouse spermatids, but expression was greater in 75-day-old elongated spermatids. In Leydig cells, TGF-β₂ expression was strong in both 35- and 50-day-old mice, whereas the expression of TGF-β₂ was less in 75-day-old Leydig cells. Our results suggest that TGF-β₁ and TGF-β₂ may play significant roles in testicular functions and germ cell development in mice.     

Effects of organophosphate insecticides on mechanical properties of rat aorta

The present study was carried out to search whether organophosphate pesticides affect the mechanical properties of the thoracic aorta. Wistar female rats (aged 6-8 weeks) were assigned randomly to a control group and groups treated with either dichlorvos or chlorpyriphos for 90 days at a dose of 5 mg/kg/day. After that period, animals were killed and thoracic aorta strips in longitudinal direction were isolated. The stress, strain and elastic modulus were obtained from the strips. Our results showed that chronic administration of chlorpyriphos and dichlorvos caused downward shift of the stress-strain relations compared to the control curve. The elastic modulus-stress curve revealed distinct characteristics in the low and high stress regions. A power function was used to simulate the low stress region while a line was fit to the high stress region. Curve fitting procedure illustrated that both pesticides influenced mainly the high stress region, but they had diverse effects at the low stress region. The results also imply that chlorpyriphos and dichlorvos decrease the strength of the aorta and therefore might influence the response of the aorta to mechanical loading induced by blood pressure.     

Superficial cell differentiation during embryonic and postnatal development of mouse urothelium

After drastic urothelial destruction around birth and around postnatal day 6, mouse urothelial renewal starts each time de novo. The differentiation of superficial cells during urothelial restoration was followed for the first time from embryonic day 15 to postnatal day 6 by the detection of differentiation markers: cytokeratins, uroplakins and apical membrane specialization. The differentiation markers of short-lived superficial cells were studied before and after urothelial destruction. Three distinctive types of superficial cells, typical for certain developmental period, were characterised: cells at low differentiation stage with microvilli and cilia, expressing CK7 and CK18, detected on embryonic day 15; cells at advanced differentiation stage with star-like arrangement of prominent membrane ridges, expressing CK7 and CK20, present between the two urothelial destruction events; highly differentiated cells with typically jagged apical surface, expressing CK7 and CK20, found twice during development. This cell type appears for the first time on embryonic day 18 as the terminal stage of embryonic differentiation. It was found again on postnatal day 6 as an initial stage of differentiation, leading toward terminally differentiated cells of the adult urothelium. Our work proves that apical membrane specialization is the most valuable differentiation marker of superficial cells.     

Golga5 is dispensable for mouse embryonic development and postnatal survival

Golgins are a family of coiled‐coil proteins located at the cytoplasmic surface of the Golgi apparatus and have been implicated in maintaining Golgi structural integrity through acting as tethering factors for retrograde vesicle transport. Whereas knockdown of several individual golgins in cultured cells caused Golgi fragmentation and disruption of vesicle trafficking, analysis of mutant mouse models lacking individual golgins have discovered tissue‐specific developmental functions. Recently, homozygous loss of function of GOLGA2, of which previous in vitro studies suggested an essential role in maintenance of Golgi structure and in mitosis, has been associated with a neuromuscular disorder in human patients, which highlights the need for understanding the developmental roles of the golgins in vivo. We report here generation of Golga5‐deficient mice using CRISPR/Cas9‐mediated genome editing. Although knockdown studies in cultured cells have implicated Golga5 in maintenance of Golgi organization, we show that Golga5 is not required for mouse embryonic development, postnatal survival, or fertility. Moreover, whereas Golga5 is structurally closely related to Golgb1, we show that inactivation of Golga5 does not enhance the severity of developmental defects in Golgb1‐deficient mice. The Golga5‐deficient mice enable further investigation of the roles and functional specificity of golgins in development and diseases.     

Effect of osmolarity on the zero-stress state and mechanical properties of aorta

Some pathological conditions may affect osmolarity, which can impact cell, tissue, and organ volume. The hypothesis of this study is that changes in osmolarity affect the zero-stress state and mechanical properties of the aorta. To test this hypothesis, a segment of mouse abdominal aorta was cannulated in vivo and mechanically distended by perfusion of physiological salt (NaCl) solutions with graded osmolarities from 145 to 562 mosM. The mechanical (circumferential stress, strain, and elastic modulus) and morphological (wall thickness and wall area) parameters in the loaded state were determined. To determine the osmolarity-induced changes of zero-stress state, the opening angle was observed by immersion of the sectors of mouse, rat, and pig thoracic aorta in NaCl solution with different osmolarities. Wall volume and tissue water content of the rings were also recorded at different osmolarities. Our results show that acute aortic swelling due to low osmolarity leads to an increase in wall thickness and area, a change in the stress-strain relationship, and an increase in the elastic modulus (stiffness) in mouse aorta. The opening angle, wall volume, and water content decreased significantly with increase in osmolarity. These findings suggest that acute aortic swelling and shrinking result in immediate mechanical changes in the aorta. Osmotic pressure-induced changes in the zero-stress state may serve to regulate mechanical homeostasis.     

Shh signaling from the nucleus pulposus is required for the postnatal growth and differentiation of the mouse intervertebral disc

Intervertebral discs (IVD) are essential components of the vertebral column. They maintain separation, and provide shock absorbing buffers, between adjacent vertebrae, while also allowing movements between them. Each IVD consists of a central semi-liquid nucleus pulposus (NP) surrounded by a multi-layered fibrocartilagenous annulus fibrosus (AF). Although the IVDs grow and differentiate after birth along with the vertebral column, little is known about the mechanism of this. Understanding the signals that control normal IVD growth and differentiation would also provide potential therapies for degenerative disc disease, which is the major cause of lower back pain and affects a large proportion of the population. In this work, we show that during postnatal growth of the mouse, Sonic hedgehog (Shh) signaling from the NP cells controls many aspects of growth and differentiation of both the NP cells themselves and of the surrounding AF, and that it acts, at least partly, by regulating other signaling pathways in the NP and AF. Recent studies have shown that the NP cells arise from the embryonic notochord, which acts as a major signaling center in the embryo. This work shows that this notochord-derived tissue continues to carry out a major signaling function in the postnatal body and that the IVDs are signaling centers, in addition to their already known functions in the mechanics of vertebral column function.     

Diaphragmatic activity is a determinant of postnatal lung growth

To test the hypothesis that activity of respiratory muscles determines regional growth of lung parenchyma, we studied the effects of unilateral diaphragmatic paralysis on contralateral/ipsilateral lung growth in cats and piglets. Five 10- to 12-wk-old cats and five 8-wk-old piglets underwent unilateral diaphragmatic paralysis by thoracic and cervical phrenectomy, respectively. Five to seven weeks after surgery, when the cats were killed for studies of lung growth, gain in body weight was the same as in five sham-operated controls. At this time, mean pleural pressure ipsilateral to the paralyzed hemidiaphragm was the same as contralateral mean pleural pressure during tidal breathing, and values did not differ from controls. However overall functional residual capacity was lower in the phrenectomized cats (35 +/- 4 ml) than in the controls (55 +/- 11 ml, P less than 0.01). Growth of contralateral lungs relative to ipsilateral lungs was greater in the phrenectomized cats than in the controls, as shown by ratios of contralateral/ipsilateral wet lung weight (1.44 vs. 1.34, P less than 0.01), maximum inflation volume (1.53 vs. 1.33, P less than 0.05), and total protein content (1.45 vs. 1.26, P less than 0.05). Ratios of total protein to DNA and RNA to DNA were unchanged. One week after surgery in the piglets, the ratio of contralateral/ipsilateral wet lung weight was increased (1.61 vs. 1.29, P less than 0.01) and total weight of both lungs was reduced. We conclude that regional growth of lung parenchyma by cell proliferation depends in part on regional distribution of respiratory muscle activity.     

Dephosphorylated NSSR1 is induced by androgen in mouse epididymis and phosphorylated NSSR1 is increased during sperm maturation

NSSR1 (Neural salient serine/arginine rich protein 1, alternatively SRp38) is a newly identified RNA splicing factor and predominantly expressed in neural tissues. Here, by Western blot analysis and immunofluorescent staining, we showed that the expression of dephosphorylated NSSR1 increased significantly during development of the caput epididymis. In adult mice, phosphorylated NSSR1 was mainly expressed in the apical side of epithelial cells, and dephosphorylated NSSR1 in caput epididymis was upregulated in a testosterone dependent manner. In addition, subcellular immunoreactive distribution of NSSR1 varied in different regions of the epididymis. With respect to the sperm, phosphorylated NSSR1 was detected in the mid-piece of the tail as well as the acrosome. Furthermore, NSSR1 was released from the sperm head during the capacitation and acrosome reaction. These findings for the first time provide the evidence for the potential roles of NSSR1 in sperm maturation and fertilization.     

An imprinted gene network that controls mammalian somatic growth is down-regulated during postnatal growth deceleration in multiple organs

In mammals, somatic growth is rapid in early postnatal life but decelerates with age and eventually halts, thus determining the adult body size of the species. This growth deceleration, which reflects declining proliferation, occurs simultaneously in multiple organs yet appears not to be coordinated by a systemic mechanism. We, therefore, hypothesized that growth deceleration results from a growth-limiting genetic program that is common to multiple tissues. Here, we identified a set of 11 imprinted genes that show down-regulation of mRNA expression with age in multiple organs. For these genes, Igf2, H19, Plagl1, Mest, Peg3, Dlk1, Gtl2, Grb10, Ndn, Cdkn1c, and SLC38a4, the declines show a temporal pattern similar to the decline in growth rate. All 11 genes have been implicated in the control of cell proliferation or somatic growth. Thus, our findings suggest that the declining expression of these genes contributes to coordinate growth deceleration in multiple tissues. We next hypothesized that the coordinate decline in expression of these imprinted genes is caused by altered methylation and consequent silencing of the expressed allele. Contrary to this hypothesis, the methylation status of the promoter regions of Mest, Peg3, and Plagl1 did not change with age. Our findings suggest that a set of growth-regulating imprinted genes is expressed at high levels in multiple tissues in early postnatal life, contributing to rapid somatic growth, but that these genes are subsequently downregulated in multiple tissues simultaneously, contributing to coordinate growth deceleration and cessation, thus imposing a fundamental limit on adult body size.     

Upregulation of erythropoietin receptor during postnatal and postpneumonectomy lung growth

Circulating erythropoietin (EPO) stimulates erythrocytosis, whereas organ-specific local EPO receptor (EPOR) expression has been linked to angiogenesis, tissue growth, and development. On the basis of the observation of concurrent enhancement of lung growth and erythrocyte production during exposure to chronic hypoxia, we hypothesized that a paracrine EPO system is involved in mediating lung growth. We analyzed EPOR protein expression in normal dog lung tissue during postnatal maturation and during compensatory lung growth after right pneumonectomy (PNX). Membrane-bound EPOR was significantly more abundant in the immature lung compared with mature lung and in the remaining lung 3 wk after PNX compared with matched sham controls. COOH-terminal cytosolic EPOR peptides, which were even more abundant than membrane-bound EPOR, were also upregulated in immature lung but differentially processed after PNX. Apoptosis was enhanced during both types of lung growth in direct relationship to cellular proliferation and EPOR expression. We conclude that both developmental and compensatory lung growth involve paracrine EPO signaling with parallel upregulation but differential processing of EPOR.     

The full-length isoform of the mouse pleckstrin homology domain-interacting protein (PHIP) is required for postnatal growth

PHIP was isolated as an insulin receptor substrate 1 (IRS-1) interacting protein. To date, the physiological roles of PHIP remain unknown. Here we show that mice lacking PHIP1, the full-length isoform of PHIP, are born at normal size but suffer a 40% growth deficit by weaning. PHIP1 mutant mice develop hypoglycemia and have an average lifespan of 4-5 weeks. PHIP1-deficient mouse embryonic fibroblasts (MEFs) grow markedly slower than wild-type MEFs, but exhibit normal AKT phosphorylation and an increased cell proliferation in response to IGF-1 treatment. Together these results suggest that PHIP1 regulates postnatal growth in an IGF-1/AKT pathway-independent manner.