Hox genes in development and beyond.

Hox genes encode evolutionarily conserved transcription factors that are essential for the proper development of bilaterian organisms. Hox genes are unique because they are spatially and temporally regulated during development in a manner that is dictated by their tightly linked genomic organization. Although their genetic function during embryonic development has been interrogated, less is known about how these transcription factors regulate downstream genes to direct morphogenetic events. Moreover, the continued expression and function of Hox genes at postnatal and adult stages highlights crucial roles for these genes throughout the life of an organism. Here, we provide an overview of Hox genes, highlighting their evolutionary history, their unique genomic organization and how this impacts the regulation of their expression, what is known about their protein structure, and their deployment in development and beyond.

Hox11-expressing interstitial cells contribute to adult skeletal muscle at homeostasis.

Interstitial stromal cells play critical roles in muscle development, regeneration and repair and we have previously reported that Hoxa11 and Hoxd11 are expressed in the interstitial cells of muscles attached to the zeugopod, and are crucial for the proper embryonic patterning of these muscles. Hoxa11eGFP expression continues in a subset of muscle interstitial cells through adult stages. The induction of Hoxa11-CreERT2-mediated lineage reporting (Hoxa11iTom) at adult stages in mouse results in lineage induction only in the interstitial cells. However, Hoxa11iTom+ cells progressively contribute to muscle fibers at subsequent stages. The contribution to myofibers exceeds parallel Pax7-CreERT2-mediated lineage labeling. Nuclear-specific lineage labeling demonstrates that Hoxa11-expressing interstitial cells contribute nuclear contents to myofibers. Crucially, at no point after Hoxa11iTom induction are satellite cells lineage labeled. When examined in vitro, isolated Hoxa11iTom+ interstitial cells are not capable of forming myotubes, but Hoxa11iTom+ cells can contribute to differentiating myotubes, supporting Hox-expressing interstitial cells as a new population of muscle progenitors, but not stem cells. This work adds to a small but growing body of evidence that supports a satellite cell-independent source of muscle tissue in vivo.

Early-life pulmonary viral infection leads to long-term functional and lower airway structural changes in the lungs.

Early-life respiratory virus infections have been correlated with enhanced development of childhood asthma. In particular, significant numbers of respiratory syncytial virus (RSV)-hospitalized infants go on to develop lung disease. It has been suggested that early-life viral infections may lead to altered lung development or repair that negatively impacts lung function later in life. Our data demonstrate that early-life RSV infection modifies lung structure, leading to decreased lung function. At 5 wk postneonatal RSV infection, significant defects are observed in baseline pulmonary function test (PFT) parameters consistent with decreased lung function as well as enlarged alveolar spaces. Lung function changes in the early-life RSV-infected group continue at 3 mo of age. The altered PFT and structural changes induced by early-life RSV were mitigated in TSLPR-/- mice that have previously been shown to have reduced immune cell accumulation associated with a persistent Th2 environment. Importantly, long-term effects were demonstrated using a secondary RSV infection 3 mo following the initial early-life RSV infection and led to significant additional defects in lung function, with severe mucus deposition within the airways, and consolidation of the alveolar spaces. These studies suggest that early-life respiratory viral infection leads to alterations in lung structure/repair that predispose to diminished lung function later in life.NEW & NOTEWORTHY These studies outline a novel finding that early-life respiratory virus infection can alter lung structure and function long-term. Importantly, the data also indicate that there are critical links between inflammatory responses and subsequent events that produce a more severe pathogenic response later in life. The findings provide additional data to support that early-life infections during lung development can alter the trajectory of airway function.

Characterization of a novel Hoxa5eGFP mouse line.

BACKGROUND: Hox genes encode transcription factors that are important for establishing the body plan. Hoxa5 is a member of the mammalian Hox5 paralogous group that regulates the patterning and morphology of the cervical-thoracic region of the axial skeleton. Hoxa5 also plays crucial functions in lung morphogenesis. RESULTS: We generated a Hoxa5eGFP reporter mouse line using CRISPR technology, allowing real-time visualization of Hoxa5 expression. Hoxa5eGFP recapitulates reported embryonic Hoxa5 mRNA expression patterns. Specifically, Hoxa5eGFP can be visualized in the developing mouse neural tube, somites, lung, diaphragm, foregut, and midgut, among other organs. In the stomach, posteriorly biased Hoxa5eGFP expression correlates with a drastic morphological reduction of the corpus in Hox5 paralogous mutants. Expression of Hoxa5eGFP in the lung continues in all lung fibroblast populations through postnatal and adult stages. CONCLUSIONS: We identified cell types that express Hoxa5 in postnatal and adult mouse lungs, including various fibroblasts and vascular endothelial cells. This reporter line will be a powerful tool for studies of the function of Hoxa5 during mouse development, homeostasis, and disease processes.

Hox genes maintain critical roles in the adult skeleton.

Hox genes are indispensable for the proper patterning of the skeletal morphology of the axial and appendicular skeleton during embryonic development. Recently, it has been demonstrated that Hox expression continues from embryonic stages through postnatal and adult stages exclusively in a skeletal stem cell population. However, whether Hox genes continue to function after development has not been rigorously investigated. We generated a Hoxd11 conditional allele and induced genetic deletion at adult stages to show that Hox11 genes play critical roles in skeletal homeostasis of the forelimb zeugopod (radius and ulna). Conditional loss of Hox11 function at adult stages leads to replacement of normal lamellar bone with an abnormal woven bone-like matrix of highly disorganized collagen fibers. Examining the lineage from the Hox-expressing mutant cells demonstrates no loss of stem cell population. Differentiation in the osteoblast lineage initiates with Runx2 expression, which is observed similarly in mutants and controls. With loss of Hox11 function, however, osteoblasts fail to mature, with no progression to osteopontin or osteocalcin expression. Osteocyte-like cells become embedded within the abnormal bony matrix, but they completely lack dendrites, as well as the characteristic lacuno-canalicular network, and do not express SOST. Together, our studies show that Hox11 genes continuously function in the adult skeleton in a region-specific manner by regulating differentiation of Hox-expressing skeletal stem cells into the osteolineage.

Bone morphology is regulated modularly by global and regional genetic programs.

Bone protrusions provide stable anchoring sites for ligaments and tendons and define the unique morphology of each long bone. Despite their importance, the mechanism by which superstructures are patterned is unknown. Here, we identify components of the genetic program that control the patterning of Sox9+/Scx+ superstructure progenitors in mouse and show that this program includes both global and regional regulatory modules. Using light-sheet fluorescence microscopy combined with genetic lineage labeling, we mapped the broad contribution of the Sox9+/Scx+ progenitors to the formation of bone superstructures. Then, by combining literature-based evidence, comparative transcriptomic analysis and genetic mouse models, we identified Gli3 as a global regulator of superstructure patterning, whereas Pbx1, Pbx2, Hoxa11 and Hoxd11 act as proximal and distal regulators, respectively. Moreover, by demonstrating a dose-dependent pattern regulation in Gli3 and Pbx1 compound mutations, we show that the global and regional regulatory modules work in a coordinated manner. Collectively, our results provide strong evidence for genetic regulation of superstructure patterning, which further supports the notion that long bone development is a modular process.This article has an associated 'The people behind the papers' interview.

Evolution of Hoxa11 regulation in vertebrates is linked to the pentadactyl state.

The fin-to-limb transition represents one of the major vertebrate morphological innovations associated with the transition from aquatic to terrestrial life and is an attractive model for gaining insights into the mechanisms of morphological diversity between species. One of the characteristic features of limbs is the presence of digits at their extremities. Although most tetrapods have limbs with five digits (pentadactyl limbs), palaeontological data indicate that digits emerged in lobed fins of early tetrapods, which were polydactylous. How the transition to pentadactyl limbs occurred remains unclear. Here we show that the mutually exclusive expression of the mouse genes Hoxa11 and Hoxa13, which were previously proposed to be involved in the origin of the tetrapod limb, is required for the pentadactyl state. We further demonstrate that the exclusion of Hoxa11 from the Hoxa13 domain relies on an enhancer that drives antisense transcription at the Hoxa11 locus after activation by HOXA13 and HOXD13. Finally, we show that the enhancer that drives antisense transcription of the mouse Hoxa11 gene is absent in zebrafish, which, together with the largely overlapping expression of hoxa11 and hoxa13 genes reported in fish, suggests that this enhancer emerged in the course of the fin-to-limb transition. On the basis of the polydactyly that we observed after expression of Hoxa11 in distal limbs, we propose that the evolution of Hoxa11 regulation contributed to the transition from polydactyl limbs in stem-group tetrapods to pentadactyl limbs in extant tetrapods.

Hox5 genes direct elastin network formation during alveologenesis by regulating myofibroblast adhesion.

Hox5 genes (Hoxa5, Hoxb5, Hoxc5) are exclusively expressed in the lung mesenchyme during embryogenesis, and the most severe phenotypes result from constitutive loss of function of all three genes. Because Hox5 triple null mutants exhibit perinatal lethality, the contribution of this paralogous group to postembryonic lung development is unknown. Intriguingly, expression of all three Hox5 genes peaks during the first 2 weeks after birth, reaching levels far exceeding those measured at embryonic stages, and surviving Hoxa5 single and Hox5 AabbCc compound mutants exhibit defects in the localization of alveolar myofibroblasts. To define the contribution of the entire Hox5 paralogous group to this process, we generated an Hoxa5 conditional allele to use with our existing null alleles for Hoxb5 and Hoxc5 Postnatally, mesenchymal deletion of Hoxa5 in an Hoxb5/Hoxc5 double-mutant background results in severe alveolar simplification. The elastin network required for alveolar formation is dramatically disrupted in Hox5 triple mutants, while the basal lamina, interstitial matrix, and fibronectin are normal. Alveolar myofibroblasts remain Pdgfrα+/SMA+ double positive and present in normal numbers, indicating that the irregular elastin network is not due to fibroblast differentiation defects. Rather, we observe that SMA+ myofibroblasts of Hox5 triple mutants are morphologically abnormal both in vivo and in vitro with highly reduced adherence to fibronectin. This loss of adhesion is a result of loss of the integrin heterodimer Itga5b1 in mutant fibroblasts. Collectively, these data show an important role for Hox5 genes in lung fibroblast adhesion necessary for proper elastin network formation during alveologenesis.

Hox5 Paralogous Genes Modulate Th2 Cell Function during Chronic Allergic Inflammation via Regulation of Gata3.

Allergic asthma is a significant health burden in western countries, and continues to increase in prevalence. Th2 cells contribute to the development of disease through release of the cytokines IL-4, IL-5, and IL-13, resulting in increased airway eosinophils and mucus hypersecretion. The molecular mechanisms behind the disease pathology remain largely unknown. In this study we investigated a potential regulatory role for the Hox5 gene family, Hoxa5, Hoxb5, and Hoxc5, genes known to be important in lung development within mesenchymal cell populations. We found that Hox5-mutant mice show exacerbated pathology compared with wild-type controls in a chronic allergen model, with an increased Th2 response and exacerbated lung tissue pathology. Bone marrow chimera experiments indicated that the observed enhanced pathology was mediated by immune cell function independent of mesenchymal cell Hox5 family function. Examination of T cells grown in Th2 polarizing conditions showed increased proliferation, enhanced Gata3 expression, and elevated production of IL-4, IL-5, and IL-13 in Hox5-deficient T cells compared with wild-type controls. Overexpression of FLAG-tagged HOX5 proteins in Jurkat cells demonstrated HOX5 binding to the Gata3 locus and decreased Gata3 and IL-4 expression, supporting a role for HOX5 proteins in direct transcriptional control of Th2 development. These results reveal a novel role for Hox5 genes as developmental regulators of Th2 immune cell function that demonstrates a redeployment of mesenchyme-associated developmental genes.

Mesenchymal Hox6 function is required for mouse pancreatic endocrine cell differentiation.

Despite significant advances in our understanding of pancreatic endocrine cell development, the function of the pancreatic mesodermal niche in this process is poorly understood. Here we report a novel role for mouse Hox6 genes in pancreatic organogenesis. Hox6 genes are expressed exclusively in the mesoderm of the developing pancreas. Genetic loss of all three Hox6 paralogs (Hoxa6, Hoxb6 and Hoxc6) leads to a dramatic loss of endoderm-derived endocrine cells, including insulin-secreting ß-cells, and to mild delays and disruptions in pancreatic branching and exocrine differentiation. Ngn3-expressing pan-endocrine progenitor cells are specified normally in Hox6 mutant pancreata, but fail to mature into hormone-producing cells. Reduced expression of Wnt5a is observed in mutant pancreatic mesenchyme, leading to subsequent loss of expression of the crucial Wnt inhibitors Sfrp3 and Dkk1 in endocrine progenitor cells. These results reveal a key role for Hox6 genes in establishing Wnt mesenchymal-epithelial crosstalk in pancreatic development.

Hox genes in the adult skeleton: Novel functions beyond embryonic development.

Hox genes encode evolutionarily conserved transcription factors that control skeletal patterning in the developing embryo. They are expressed in regionally restricted domains and function to regulate the morphology of specific vertebral and long bone elements. Recent work has provided evidence that Hox genes continue to be regionally expressed in adult tissues. Fibroblasts cultured from adult tissues show broadly maintained Hox gene expression patterns. In the adult skeleton, Hox genes are expressed in progenitor-enriched populations of mesenchymal stem/stromal cells (MSCs), and genetic loss-of-function analyses have provided evidence that Hox genes function during the fracture healing process. This review will highlight our current understanding of Hox expression in the adult animal and its function in skeletal regeneration. Developmental Dynamics 246:310-317, 2017. © 2016 Wiley Periodicals, Inc.

Hox11 genes are required for regional patterning and integration of muscle, tendon and bone.

Development of the musculoskeletal system requires precise integration of muscles, tendons and bones. The molecular mechanisms involved in the differentiation of each of these tissues have been the focus of significant research; however, much less is known about how these tissues are integrated into a functional unit appropriate for each body position and role. Previous reports have demonstrated crucial roles for Hox genes in patterning the axial and limb skeleton. Loss of Hox11 paralogous gene function results in dramatic malformation of limb zeugopod skeletal elements, the radius/ulna and tibia/fibula, as well as transformation of the sacral region to a lumbar phenotype. Utilizing a Hoxa11eGFP knock-in allele, we show that Hox11 genes are expressed in the connective tissue fibroblasts of the outer perichondrium, tendons and muscle connective tissue of the zeugopod region throughout all stages of development. Hox11 genes are not expressed in differentiated cartilage or bone, or in vascular or muscle cells in these regions. Loss of Hox11 genes disrupts regional muscle and tendon patterning of the limb in addition to affecting skeletal patterning. The tendon and muscle defects in Hox11 mutants are independent of skeletal patterning events as disruption of tendon and muscle patterning is observed in Hox11 compound mutants that do not have a skeletal phenotype. Thus, Hox genes are not simply regulators of skeletal morphology as previously thought, but are key factors that regulate regional patterning and integration of the musculoskeletal system.

In vivo structural and cellular remodeling of engineered bone-ligament-bone constructs used for anterior cruciate ligament reconstruction in sheep.

Anterior cruciate ligament (ACL) ruptures rank among the most prevalent and costly sports-related injuries. Current tendon grafts used for ACL reconstruction are limited by suboptimal biomechanical properties. We have addressed these issues by engineering multiphasic bone-ligament-bone (BLB) constructs that develop structural and mechanical properties similar to native ACL. The purpose of this study was to examine the acute remodeling process that occurs as the BLB grafts advance toward the adult ligament phenotype in vivo. Thus, we implanted BLB constructs fabricated from male cells into female host sheep and allowed 3, 7, 14, or 28 days (n = 4 at each time point) for recovery. To address whether or not graft-derived cells were even necessary, a subset of BLB constructs (n = 3) were acellularized, implanted, and allowed 28 days for recovery. At each recovery time point, the following histological analyses were performed: picrosirius red staining to assess collagen alignment and immunohistochemistry to assess both graft development and host immune response. Polymerase chain reaction (PCR) analysis, performed on every explanted BLB, was used to detect the presence of graft-derived male cells remaining in the constructs and/or migration into surrounding host tissue. The analysis of the PCR and histology samples revealed a rapid migration of host-derived macrophages and neutrophils into the graft at 3 days, followed by increased collagen density and alignment, vascularization, innervation, and near complete repopulation of the graft with host cells within 28 days. This study provides a greater understanding of the processes of ligament regeneration in our BLB constructs as they remodel toward the adult ligament phenotype.

Hox5 interacts with Plzf to restrict Shh expression in the developing forelimb.

To date, only the five most posterior groups of Hox genes, Hox9-Hox13, have demonstrated loss-of-function roles in limb patterning. Individual paralog groups control proximodistal patterning of the limb skeletal elements. Hox9 genes also initiate the onset of Hand2 expression in the posterior forelimb compartment, and collectively, the posterior HoxA/D genes maintain posterior Sonic Hedgehog (Shh) expression. Here we show that an anterior Hox paralog group, Hox5, is required for forelimb anterior patterning. Deletion of all three Hox5 genes (Hoxa5, Hoxb5, and Hoxc5) leads to anterior forelimb defects resulting from derepression of Shh expression. The phenotype requires the loss of all three Hox5 genes, demonstrating the high level of redundancy in this Hox paralogous group. Further analyses reveal that Hox5 interacts with promyelocytic leukemia zinc finger biochemically and genetically to restrict Shh expression. These findings, along with previous reports showing that point mutations in the Shh limb enhancer lead to similar anterior limb defects, highlight the importance of Shh repression for proper patterning of the vertebrate limb.

Sox9 plays multiple roles in the lung epithelium during branching morphogenesis.

Lung branching morphogenesis is a highly orchestrated process that gives rise to the complex network of gas-exchanging units in the adult lung. Intricate regulation of signaling pathways, transcription factors, and epithelial-mesenchymal cross-talk are critical to ensuring branching morphogenesis occurs properly. Here, we describe a role for the transcription factor Sox9 during lung branching morphogenesis. Sox9 is expressed at the distal tips of the branching epithelium in a highly dynamic manner as branching occurs and is down-regulated starting at embryonic day 16.5, concurrent with the onset of terminal differentiation of type 1 and type 2 alveolar cells. Using epithelial-specific genetic loss- and gain-of-function approaches, our results demonstrate that Sox9 controls multiple aspects of lung branching. Fine regulation of Sox9 levels is required to balance proliferation and differentiation of epithelial tip progenitor cells, and loss of Sox9 leads to direct and indirect cellular defects including extracellular matrix defects, cytoskeletal disorganization, and aberrant epithelial movement. Our evidence shows that unlike other endoderm-derived epithelial tissues, such as the intestine, Wnt/ß-catenin signaling does not regulate Sox9 expression in the lung. We conclude that Sox9 collectively promotes proper branching morphogenesis by controlling the balance between proliferation and differentiation and regulating the extracellular matrix.

Hox genes and limb musculoskeletal development.

In the musculoskeletal system, muscle, tendon, and bone tissues develop in a spatially and temporally coordinated manner, and integrate into a cohesive functional unit by forming specific connections unique to each region of the musculoskeletal system. The mechanisms of these patterning and integration events are an area of great interest in musculoskeletal biology. Hox genes are a family of important developmental regulators and play critical roles in skeletal patterning throughout the axial and appendicular skeleton. Unexpectedly, Hox genes are not expressed in the differentiated cartilage or other skeletal cells, but rather are highly expressed in the tightly associated stromal connective tissues as well as regionally expressed in tendons and muscle connective tissue. Recent work has revealed a previously unappreciated role for Hox in patterning all the musculoskeletal tissues of the limb. These observations suggest that integration of the musculoskeletal system is regulated, at least in part, by Hox function in the stromal connective tissue. This review will outline our current understanding of Hox function in patterning and integrating the musculoskeletal tissues.

Axial Hox9 activity establishes the posterior field in the developing forelimb.

Current models hold that the early limb field becomes polarized into anterior and posterior domains by the opposing activities of Hand2 and Gli3. This polarization is essential for the initiation of Shh expression in the posterior margin of the limb bud, but how this polarity is established is not clear. Here we show that initial anteroposterior polarization of the early forelimb field requires the function of all four Hox9 paralogs (Hoxa9, Hoxb9, Hoxc9, and Hoxd9). This is unexpected, given that only HoxA and HoxD AbdB group genes have been shown to play a role in forelimb patterning, regulating the activation and maintenance of Shh expression and subsequent proximal-distal patterning of the forelimb. Our analysis of Hox9 quadruple mutants demonstrates that Hox9 function is required for the expression of Hand2 in the posterior limb field. Subsequently, Gli3 expression is not repressed posteriorly, Shh expression is not initiated, and collinear expression of HoxA/D10-13 is not established, resulting in severely malformed forelimbs lacking all posterior, Shh-regulated elements. This Hox9 mutant phenotype is restricted to the forelimbs; mutant hindlimbs are normal, revealing fundamental differences in the patterning mechanisms governing the establishment of forelimb and hindlimb fields.

Hox genes and patterning the vertebrate body.

The diversity of vertebrate body plans is dizzying, yet stunning for the many things they have in common. Vertebrates have inhabited virtually every part of the earth from its coldest to warmest climates. They locomote by swimming, flying, walking, slithering, or climbing, or combinations of these behaviors. And they exist in many different sizes, from the smallest of frogs, fish and lizards to giraffes, elephants, and blue whales. Despite these differences, vertebrates follow a remarkably similar blueprint for the establishment of their body plan. Within the relatively small amount of time required to complete gastrulation, the process through which the three germ layers, ectoderm, mesoderm, and endoderm are created, the embryo also generates its body axis and is simultaneously patterned. For the length of this axis, the genes that distinguish the neck from the rib cage or the trunk from the sacrum are the Hox genes. In vertebrates, there was evolutionary pressure to maintain this set of genes in the organism. Over the past decades, much has been learned regarding the regulatory mechanisms that ensure the appropriate expression of these genes along the main body axes. Genetic functions continue to be explored though much has been learned. Much less has been discerned on the identity of co-factors used by Hox proteins for the specificity of transcriptional regulation or what downstream targets and pathways are critical for patterning events, though there are notable exceptions. Current work in the field is demonstrating that Hox genes continue to function in many organs long after directing early patterning events. It is hopeful continued research will shed light on remaining questions regarding mechanisms used by this important and conserved set of transcriptional regulators.

Partial functional redundancy between Hoxa5 and Hoxb5 paralog genes during lung morphogenesis.

Hox genes encode transcription factors governing complex developmental processes in several organs. A subset of Hox genes are expressed in the developing lung. Except for Hoxa5, the lack of overt lung phenotype in single mutants suggests that Hox genes may not play a predominant role in lung ontogeny or that functional redundancy may mask anomalies. In the Hox5 paralog group, both Hoxa5 and Hoxb5 genes are expressed in the lung mesenchyme whereas Hoxa5 is also expressed in the tracheal mesenchyme. Herein, we generated Hoxa5;Hoxb5 compound mutant mice to evaluate the relative contribution of each gene to lung development. Hoxa5;Hoxb5 mutants carrying the four mutated alleles displayed an aggravated lung phenotype, resulting in the death of the mutant pups at birth. Characterization of the phenotype highlighted the role of Hoxb5 in lung formation, the latter being involved in branching morphogenesis, goblet cell specification, and postnatal air space structure, revealing partial functional redundancy with Hoxa5. However, the Hoxb5 lung phenotypes were less severe than those seen in Hoxa5 mutants, likely because of Hoxa5 compensation. New specific roles for Hoxa5 were also unveiled, demonstrating the extensive contribution of Hoxa5 to the developing respiratory system. The exclusive expression of Hoxa5 in the trachea and the phrenic motor column likely underlies the Hoxa5-specific trachea and diaphragm phenotypes. Altogether, our observations establish that the Hoxa5 and Hoxb5 paralog genes shared some functions during lung morphogenesis, Hoxa5 playing a predominant role.