Fetal Growth Plate Cartilage : Histological and Immunohistochemical Techniques.

Skeletal development is a tightly regulated process that primarily occurs through two distinct mechanisms. In intramembranous ossification, mesenchymal progenitors condense and transdifferentiate directly into osteoblasts, giving rise to the flat bones of the skull. The majority of the skeleton develops through endochondral ossification, in which mesenchymal progenitors give rise to a cartilaginous template that is gradually replaced by bone. The study of these processes necessitates a suitable animal model, a requirement to which the mouse is admirably suited. Their rapid reproductive ability, developmental and physiologic similarity to humans, and easily manipulated genetics all contribute to their widespread use. Outlined here are the most common histological and immunohistochemical techniques utilized in our laboratory for the isolation and analysis of specimens from the developing murine skeleton.

ERα Signaling in GHRH/Kiss1 Dual-Phenotype Neurons Plays Sex-Specific Roles in Growth and Puberty.

Gonadal steroids modulate growth hormone (GH) secretion and the pubertal growth spurt via undefined central pathways. GH-releasing hormone (GHRH) neurons express estrogen receptor α (ERα) and androgen receptor (AR), suggesting changing levels of gonadal steroids during puberty directly modulate the somatotropic axis. We generated mice with deletion of ERα in GHRH cells (GHRHΔERα), which displayed reduced body length in both sexes. Timing of puberty onset was similar in both groups, but puberty completion was delayed in GHRHΔERα females. Lack of AR in GHRH cells (GHRHΔAR mice) induced no changes in body length, but puberty completion was also delayed in females. Using a mouse model with two reporter genes, we observed that, while GHRHtdTom neurons minimally colocalize with Kiss1hrGFP in prepubertal mice, ∼30% of GHRH neurons coexpressed both reporter genes in adult females, but not in males. Developmental analysis of Ghrh and Kiss1 expression suggested that a subpopulation of ERα neurons in the arcuate nucleus of female mice undergoes a shift in phenotype, from GHRH to Kiss1, during pubertal transition. Our findings demonstrate that direct actions of gonadal steroids in GHRH neurons modulate growth and puberty and indicate that GHRH/Kiss1 dual-phenotype neurons play a sex-specific role in the crosstalk between the somatotropic and gonadotropic axes during pubertal transition.SIGNIFICANCE STATEMENT Late maturing adolescents usually show delayed growth and bone age. At puberty, gonadal steroids have stimulatory effects on the activation of growth and reproductive axes, but the existence of gonadal steroid-sensitive neuronal crosstalk remains undefined. Moreover, the neural basis for the sex differences observed in the clinical arena is unknown. Lack of ERα in GHRH neurons disrupts growth in both sexes and causes pubertal delay in females. Deletion of androgen receptor in GHRH neurons only delayed female puberty. In adult females, not males, a subset of GHRH neurons shift phenotype to start producing Kiss1. Thus, direct estrogen action in GHRH/Kiss1 dual-phenotype neurons modulates growth and puberty and may orchestrate the sex differences in endocrine function observed during pubertal transition.

Caregiver Experiences With an Innovative Farmers' Market Incentive Program for Children in Flint, Michigan.

An innovative farmers' market incentive program designed specifically for children was implemented to address persistent challenges with accessing fresh, nutrient-rich foods in a food desert community. The current study sought to qualitatively examine caregiver perceptions of the incentive program. Following distribution of farmers' market incentives to all children (ages 0 to 15 years) at 43 Flint-area early childcare facilities and elementary schools, researchers conducted semistructured interviews with 37 caregivers (mean age = 39.59 ± 11.73 years). The majority were female (87%) and African American (53%). Through these interviews, researchers explored family experiences with the farmers' market incentive program, as well as changes in environmental factors that may have resulted from program participation. Interviews were audio recorded and transcribed verbatim for textual analysis. Thematic analysis was used to identify patterns across transcripts and formulate emerging themes. Four recurrent themes emerged during interviews: (1) fruit and vegetable access, (2) child influence, (3) autonomous grocery shopping, and (4) program expansion. Interview participants indicated that the farmers' market incentive program was an effective tool to both encourage families to visit the farmers' market and purchase fresh foods there. Program design, particularly distribution to children, was credited with introducing families to the local farmers' market. The current study suggests that a farmers' market incentive program targeting children who reside in a food desert community may have meaningful impacts on access to fresh, nutrient-rich foods.

Suppressing Mitochondrial Respiration Is Critical for Hypoxia Tolerance in the Fetal Growth Plate.

Oxygen (O2) is both an indispensable metabolic substrate and a regulatory signal that controls the activity of Hypoxia-Inducible Factor 1α (Hif1a), a mediator of the cellular adaptation to low O2 tension (hypoxia). Hypoxic cells require Hif1a to survive. Additionally, Hif1a is an inhibitor of mitochondrial respiration. Hence, we hypothesized that enhancing mitochondrial respiration is detrimental to the survival of hypoxic cells in vivo. We tested this hypothesis in the fetal growth plate, which is hypoxic. Our findings show that mitochondrial respiration is dispensable for survival of growth plate chondrocytes. Furthermore, its impairment prevents the extreme hypoxia and the massive chondrocyte death observed in growth plates lacking Hif1a. Consequently, augmenting mitochondrial respiration affects the survival of hypoxic chondrocytes by, at least in part, increasing intracellular hypoxia. We thus propose that partial suppression of mitochondrial respiration is crucial during development to protect the tissues that are physiologically hypoxic from lethal intracellular anoxia.

Hypoxia-inducible factor 2α is a negative regulator of osteoblastogenesis and bone mass accrual.

Osteoblasts, which are the bone-forming cells, operate in a hypoxic environment. The transcription factors hypoxia-inducible factor-1α (HIF1) and HIF2 are key mediators of the cellular response to hypoxia. Both are expressed in osteoblasts. HIF1 is known to be a positive regulator of bone formation. Conversely, the role of HIF2 in the control osteoblast biology is still poorly understood. In this study, we used mouse genetics to demonstrate that HIF2 is an inhibitor of osteoblastogenesis and bone mass accrual. Moreover, we provided evidence that HIF2 impairs osteoblast differentiation at least in part, by upregulating the transcription factor Sox9. Our findings constitute a paradigm shift, as activation of the hypoxia-signaling pathway has traditionally been associated with increased bone formation through HIF1. Inhibiting HIF2 could thus represent a therapeutic approach for the treatment of the low bone mass observed in chronic diseases, osteoporosis, or aging.

The transcription factor GLI1 modulates the inflammatory response during pancreatic tissue remodeling.

Pancreatic cancer, one of the deadliest human malignancies, is almost uniformly associated with a mutant, constitutively active form of the oncogene Kras. Studies in genetically engineered mouse models have defined a requirement for oncogenic KRAS in both the formation of pancreatic intraepithelial neoplasias, the most common precursor lesions to pancreatic cancer, and in the maintenance and progression of these lesions. Previous work using an inducible model allowing tissue-specific and reversible expression of oncogenic Kras in the pancreas indicates that inactivation of this GTPase at the pancreatic intraepithelial neoplasia stage promotes pancreatic tissue repair. Here, we extend these findings to identify GLI1, a transcriptional effector of the Hedgehog pathway, as a central player in pancreatic tissue repair upon Kras inactivation. Deletion of a single allele of Gli1 results in improper stromal remodeling and perdurance of the inflammatory infiltrate characteristic of pancreatic tumorigenesis. Strikingly, this partial loss of Gli1 affects activated fibroblasts in the pancreas and the recruitment of immune cells that are vital for tissue recovery. Analysis of the mechanism using expression and chromatin immunoprecipitation assays identified a subset of cytokines, including IL-6, mIL-8, Mcp-1, and M-csf (Csf1), as direct GLI1 target genes potentially mediating this phenomenon. Finally, we demonstrate that canonical Hedgehog signaling, a known regulator of Gli1 activity, is required for pancreas recovery. Collectively, these data delineate a new pathway controlling tissue repair and highlight the importance of GLI1 in regulation of the pancreatic microenvironment during this cellular process.