Review and experimental evaluation of the embryonic development and evolutionary history of flipper development and hyperphalangy in dolphins (Cetacea: Mammalia).

Cetaceans are the only mammals to have evolved hyperphalangy, an increase in the number of phalanges beyond the mammalian plesiomorphic condition of three phalanges per digit. In this study, cetaceans were used as a novel model to review previous studies of mammalian hyperphalangy and contribute new experimental evidence as to the molecular origins of this phenotype in embryos of the pantropical spotted dolphin (Stenella attenuata). Results show embryos of dolphins, mice, and pigs share similar spatiotemporal patterns of signaling proteins known to shape limbs of mammals (e.g., FGF8, BMP2/4, WNT, GREM). However, fetal dolphins differ in that their interdigital tissues are retained, instead of undergoing apoptosis, and that multiple waves of interdigital signals likely contribute to the patterning of supernumerary joints and phalanges in adjacent digits. Integration of fossil and experimental evidence suggests that the presence of interdigital webbing within the fossils of semi-aquatic cetaceans, recovered from the Eocene Epoch (49Ma), was probably the result of BMP-antagonists counteracting interdigital apoptosis during embryonic limb development. Modifications to signals originating in these interdigital tissues likely contributed to the origin of an incipient form of hyperphalangy in obligatorily aquatic cetaceans about 35Ma. Finally, an extreme form of hyperphalangy, with six or more phalanges per digit, evolved independently in rorqual whales (Balaenopteridae) and delphinids, and was probably associated with a wave of signaling within the interdigital tissues.

MFehi adipose tissue macrophages compensate for tissue iron perturbations in mice.

Resident adipose tissue macrophages (ATMs) play multiple roles to maintain tissue homeostasis, such as removing excess free fatty acids and regulation of the extracellular matrix. The phagocytic nature and oxidative resiliency of macrophages not only allows them to function as innate immune cells but also to respond to specific tissue needs, such as iron homeostasis. MFehi ATMs are a subtype of resident ATMs that we recently identified to have twice the intracellular iron content as other ATMs and elevated expression of iron-handling genes. Although studies have demonstrated that iron homeostasis is important for adipocyte health, little is known about how MFehi ATMs may respond to and influence adipose tissue iron availability. Two methodologies were used to address this question: dietary iron supplementation and intraperitoneal iron injection. Upon exposure to high dietary iron, MFehi ATMs accumulated excess iron, whereas the iron content of MFelo ATMs and adipocytes remained unchanged. In this model of chronic iron excess, MFehi ATMs exhibited increased expression of genes involved in iron storage. In the injection model, MFehi ATMs incorporated high levels of iron, and adipocytes were spared iron overload. This acute model of iron overload was associated with increased numbers of MFehi ATMs; 17% could be attributed to monocyte recruitment and 83% to MFelo ATM incorporation into the MFehi pool. The MFehi ATM population maintained its low inflammatory profile and iron-cycling expression profile. These studies expand the field's understanding of ATMs and confirm that they can respond as a tissue iron sink in models of iron overload.

Development of the marsupial shoulder girdle complex: a case study in Monodelphis domestica.

During their embryogenesis, marsupials transiently develop a unique structure, the shoulder arch, which provides the structural support and muscle-attachments necessary for the newborn's crawl to the teat. One of the most pronounced and functionally important aspects of the shoulder arch is an enlarged coracoid. The goal of this study is to determine the molecular basis of shoulder arch formation in marsupials. To achieve this goal, this study investigates the relative expression of several genes with known roles in shoulder girdle morphogenesis in a marsupial-the opossum, Monodelphis domestica-and a placental, the mouse, Mus musculus. Results indicate that Hoxc6, a gene involved in coracoid patterning, is expressed for a longer period of time and at higher levels in opossum relative to mouse. Functional manipulation suggests that these differences in Hoxc6 expression are independent of documented differences in retinoic acid signaling in opossum and mouse forelimbs. Results also indicate that Emx2, a gene involved in scapular blade condensation, is upregulated in opossum relative to mouse. However, several other genes involved in shoulder girdle patterning (e.g., Gli3, Pax1, Pbx1, Tbx15) are comparably expressed in these species. These findings suggest that the upregulation of Hoxc6 and Emx2 occurs through independent genetic modifications in opossum relative to mouse. In summary, this study documents a correlation between gene expression and the divergent shoulder girdle morphogenesis of marsupial (i.e., opossum) and placental (i.e., mouse) mammals, and thereby provides a foundation for future research into the genetic basis of shoulder girdle morphogenesis in marsupials. Furthermore, this study supports the hypothesis that the mammalian shoulder girdle is a highly modular structure whose elements are relatively free to evolve independently.

Disparate Igf1 expression and growth in the fore- and hind limbs of a marsupial mammal (Monodelphis domestica).

Proper regulation of growth is essential to all stages of life, from development of the egg into an embryo to the maintenance of normal cell cycle progression in adults. However, despite growth's importance to basic biology and health, little is known about how mammalian growth is regulated. In this study, we investigated the molecular basis of the highly disparate growth of opossum fore- and hind limbs in utero. We first used a novel, opossum-specific microarray to identify several growth-related genes that are differentially expressed in opossum fore- and hind limbs of comparable developmental stages. These genes included Igf1. Given Igf1's role in the growth of other systems, we further investigated the role of Igf1 in opossum limb growth. Supporting the microarray results, RT-PCR indicated that Igf1 levels are approximately two times higher in opossum fore- than hind limbs. Consistent with this, while Igf1 transcripts were readily detectable in opossum forelimbs using whole-mount in situ hybridization, they were not detectable in opossum hind limbs. Furthermore, opossum limbs treated with exogenous Igf1 protein experienced significantly greater cellular proliferation and growth than control limbs in vitro. Taken together, results suggest that the differential expression of Igf1 in developing opossum limbs contributes to their divergent rate of growth, and the unique limb phenotype of opossum newborns. This study establishes the opossum limb as a new mammalian model system for study of organ growth.

Calcitriol and Levothyroxine Dosing for Patients With Pseudohypoparathyroidism.

Pseudohypoparathyroidism (PHP) is a rare hormone resistance syndrome caused by mutations in GNAS. This cross-sectional study investigated whether PHP patients with parathyroid hormone (PTH), thyrotropin (thyroid stimulating hormone; TSH), and free thyroxine (T4) levels at goal required higher doses of levothyroxine and calcitriol than recommended by current guidelines to overcome mineral ion abnormalities due to hormone resistance. Baseline demographic and clinical data of participants enrolled in PHP research studies between 2012-2021 were collected via retrospective chart review. Longitudinally, data were recorded at a maximum frequency of once a year starting at 1 year of age. The PTH at goal (PAG) group was defined as PTH < 150 pg/mL and calcium ≥ 8.4 mg/dL, and the TSH and free T4 at goal (TAG) group was defined as TSH < 5 mIU/L and free T4 ≥ 0.8 ng/dL. The PAG group (n = 74) was prescribed higher calcitriol doses than the PTH not at goal (PNAG) group (n = 50) (0.9 ±â€…1.1 vs 0.5 ±â€…0.9 mcg/day, P = 0.04) and 21% of individual patients were prescribed ≥ 1.5 mcg of calcitriol daily. This remained true after normalization for body weight (0.013 ±â€…0.015 vs 0.0067 ±â€…0.0095 mcg/kg/day, P = 0.008). There was no statistically significant difference in levothyroxine dosing between the TAG group (n = 122) and TSH and free T4 not at goal (TNAG) group (n = 45) when normalized for weight (2.0 ±â€…0.7 vs 1.8 ±â€…0.7 mcg/kg/day, P = 0.2). More than one-third of patients with PHP had PTH levels not at goal and some patients required calcitriol doses ≥ 1.5 mcg/day to meet current treatment goals.

Mathematical estimates of recovery after loss of activity: II. Long-range connectivity facilitates rapid functional recovery.

OBJECTIVE: To model the effects of system connectedness on recovery of dysfunctional tissues. DESIGN: One-dimensional elementary cellular automata models with small-world features, where the center-input for a few cells comes not from itself but, with a given probability, from another cell. This probability represents the connectivity of the network. The long-range connections are chosen randomly to survey the potential influences of distant information flowing into a local region. SETTING: MATLAB and Mathematica computing environments. PATIENTS: None. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We determined the recovery rate of the entropy after perturbing a uniformly dormant system. We observed that the recovery of normal activity after perturbation of a dormant system had the characteristics of an epidemic. Moreover, we found that the rate of recovery to normal steady-state activity increased rapidly even for small amounts of long-range connectivity. Findings obtained through numerical simulation were verified through analytical solutions. CONCLUSIONS: This study links our hypothesis that multiple organ function syndromes represent recoupling failure with a mathematical model showing the contribution of such coupling to reactivation of dormant systems. The implication is that strategies aimed not at target tissues or target organs but rather at restoring the quality and quantity of interconnections across those tissues and organs may be a novel therapeutic strategy.

Elevating adipose eosinophils in obese mice to physiologically normal levels does not rescue metabolic impairments.

OBJECTIVE: Obesity is a metabolic disorder that has reached epidemic proportions worldwide and leads to increased risk for diabetes, cardiovascular disease, asthma, certain cancers, and various other diseases. Obesity and its comorbidities are associated with impaired adipose tissue (AT) function. In the last decade, eosinophils have been identified as regulators of proper AT function. Our study aimed to determine whether normalizing the number of AT eosinophils in obese mice, to those of lean healthy mice, would reduce obesity and/or improve metabolic fitness. METHODS: C57BL/6J mice fed a high fat diet (HFD) were simultaneously given recombinant interleukin-5 (rIL5) for 8 weeks to increase AT eosinophils. Metabolic fitness was tested by evaluating weight gain, AT inflammation, glucose, lipid, and mixed-meal tolerance, AT insulin signaling, energy substrate utilization, energy expenditure, and white AT beiging capacity. RESULTS: Eosinophils were increased ∼3-fold in AT of obese HFD-fed mice treated with rIL5, and thus were restored to levels observed in lean healthy mice. However, there were no significant differences in rIL5-treated mice among the above listed comprehensive set of metabolic assays, despite the increased AT eosinophils. CONCLUSIONS: We have shown that restoring obese AT eosinophils to lean healthy levels is not sufficient to allow for improvement in any of a range of metabolic features otherwise impaired in obesity. Thus, the mechanisms that identified eosinophils as positive regulators of AT function, and therefore systemic health, are more complex than initially understood and will require further study to fully elucidate.

Role of lipids in the metabolism and activation of immune cells.

Immune cell plasticity has extensive implications in the pathogenesis and resolution of metabolic disorders, cancers, autoimmune diseases and chronic inflammatory disorders. Over the past decade, nutritional status has been discovered to influence the immune response. In metabolic disorders such as obesity, immune cells interact with various classes of lipids, which are capable of controlling the plasticity of macrophages and T lymphocytes. The purpose of this review is to discuss lipids and their impact on innate and adaptive immune responses, focusing on two areas: (1) the impact of altering lipid metabolism on immune cell activation, differentiation and function and (2) the mechanism by which lipids such as cholesterol and fatty acids regulate immune cell plasticity.

CC-chemokine receptor 7 (CCR7) deficiency alters adipose tissue leukocyte populations in mice.

The mechanism by which macrophages and other immune cells accumulate in adipose tissue (AT) has been an area of intense investigation over the past decade. Several different chemokines and their cognate receptors have been studied for their role as chemoattractants in promoting recruitment of immune cells to AT However, it is also possible that chemoattractants known to promote clearance of immune cells from tissues to regional lymph nodes might be a critical component to overall AT immune homeostasis. In this study, we evaluated whether CCR7 influences AT macrophage (ATM) or T-cell (ATT) accumulation. CCR7-/- and littermate wild-type (WT) mice were placed on low-fat diet (LFD) or high-fat diet (HFD) for 16 weeks. CCR7 deficiency did not impact HFD-induced weight gain, hepatic steatosis, or glucose intolerance. Although lean CCR7-/- mice had an increased proportion of alternatively activated ATMs, there were no differences in ATM accumulation or polarization between HFD-fed CCR7-/- mice and their WT counterparts. However, CCR7 deficiency did lead to the preferential accumulation of CD8+ ATT cells, which was further exacerbated by HFD feeding. Finally, expression of inflammatory cytokines/chemokines, such as Tnf, Il6, Il1ß, Ccl2, and Ccl3, was equally elevated in AT by HFD feeding in CCR7-/- and WT mice, while Ifng and Il18 were elevated by HFD feeding in CCR7-/- but not in WT mice. Together, these data suggest that CCR7 plays a role in CD8+ATT cell egress, but does not influence ATM accumulation or the metabolic impact of diet-induced obesity.

Iron homeostasis: a new job for macrophages in adipose tissue?

Elevated serum ferritin and increased cellular iron concentrations are risk factors for diabetes; however, the etiology of this association is unclear. Metabolic tissues such as pancreas, liver, and adipose tissue (AT), as well as the immune cells resident in these tissues, may be involved. Recent studies demonstrate that the polarization status of macrophages has important relevance to their iron-handling capabilities. Furthermore, a subset of macrophages in AT have elevated iron concentrations and a gene expression profile indicative of iron handling, a capacity diminished in obesity. Because iron overload in adipocytes increases systemic insulin resistance, iron handling by AT macrophages may have relevance not only to adipocyte iron stores but also to local and systemic insulin sensitivity.

The developmental reduction of the marsupial coracoid: a case study in Monodelphis domestica.

During their embryogenesis, marsupials develop a unique structure, the shoulder arch, which provides the structural and muscle-attachment support necessary for the newborn's crawl to the teat. One of the most pronounced and important aspects of the shoulder arch is an enlarged coracoid. After marsupial newborns reach the teat, the shoulder arch is remodeled and the coracoid is reduced to a small process on the scapula. Although an understanding of marsupial coracoid reduction has the potential to provide insights into both, marsupial evolution and the origin of mammals, little is known about the morphological and cellular processes controlling this process. To remedy this situation, this study examined the morphological and cellular mechanisms behind coracoid reduction in the gray short-tailed opossum, Monodelphis domestica. A quantitative, morphometric study of shoulder girdle development revealed that the coracoid is reduced in size relative to other aspects of the shoulder girdle by growing at a slower rate. Using a series of molecular assays for cell death, no evidence was found for programmed cell death playing a role in the reduction of coracoid size in marsupials (in contrast to hypotheses of previous researchers). Although it is likely the case that coracoid growth is reduced through a relatively lower rate of cellular proliferation, differences in proliferative rates in the coracoid and scapula were not great enough to be quantified using standard molecular assays.