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.

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.

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.

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.

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.

EWS::FLI1 and HOXD13 Control Tumor Cell Plasticity in Ewing Sarcoma.

PURPOSE: Propagation of Ewing sarcoma requires precise regulation of EWS::FLI1 transcriptional activity. Determining the mechanisms of fusion regulation will advance our understanding of tumor progression. Here we investigated whether HOXD13, a developmental transcription factor that promotes Ewing sarcoma metastatic phenotypes, influences EWS::FLI1 transcriptional activity. EXPERIMENTAL DESIGN: Existing tumor and cell line datasets were used to define EWS::FLI1 binding sites and transcriptional targets. Chromatin immunoprecipitation and CRISPR interference were employed to identify enhancers. CUT&RUN and RNA sequencing defined binding sites and transcriptional targets of HOXD13. Transcriptional states were investigated using bulk and single-cell transcriptomic data from cell lines, patient-derived xenografts, and patient tumors. Mesenchymal phenotypes were assessed by gene set enrichment, flow cytometry, and migration assays. RESULTS: We found that EWS::FLI1 creates a de novo GGAA microsatellite enhancer in a developmentally conserved regulatory region of the HOXD locus. Knockdown of HOXD13 led to widespread changes in expression of developmental gene programs and EWS::FLI1 targets. HOXD13 binding was enriched at established EWS::FLI1 binding sites where it influenced expression of EWS::FLI1-activated genes. More strikingly, HOXD13 bound and activated EWS::FLI1-repressed genes, leading to adoption of mesenchymal and migratory cell states that are normally suppressed by the fusion. Single-cell analysis confirmed that direct transcriptional antagonism between HOXD13-mediated gene activation and EWS::FLI1-dependent gene repression defines the state of Ewing sarcoma cells along a mesenchymal axis. CONCLUSIONS: Ewing sarcoma tumors are comprised of tumor cells that exist along a mesenchymal transcriptional continuum. The identity of cells along this continuum is, in large part, determined by the competing activities of EWS::FLI1 and HOXD13. See related commentary by Weiss and Bailey, p. 4360.

The Lung Elastin Matrix Undergoes Rapid Degradation Upon Adult Loss of Hox5 Function.

Hox genes encode transcription factors that are critical for embryonic skeletal patterning and organogenesis. The Hoxa5, Hoxb5, and Hoxc5 paralogs are expressed in the lung mesenchyme and function redundantly during embryonic lung development. Conditional loss-of-function of these genes during postnatal stages leads to severe defects in alveologenesis, specifically in the generation of the elastin network, and animals display bronchopulmonary dysplasia (BPD) or BPD-like phenotype. Here we show the surprising results that mesenchyme-specific loss of Hox5 function at adult stages leads to rapid disruption of the mature elastin matrix, alveolar enlargement, and an emphysema-like phenotype. As the elastin matrix of the lung is considered highly stable, adult disruption of the matrix was not predicted. Just 2 weeks after deletion, adult Hox5 mutant animals show significant increases in alveolar space and changes in pulmonary function, including reduced elastance and increased compliance. Examination of the extracellular matrix (ECM) of adult Tbx4rtTA; TetOCre; Hox5a f a f bbcc lungs demonstrates a disruption of the elastin network although the underlying fibronectin, interstitial collagen and basement membrane appear unaffected. An influx of macrophages and increased matrix metalloproteinase 12 (MMP12) are observed in the distal lung 3 days after Hox5 deletion. In culture, fibroblasts from Hox5 mutant lungs exhibit reduced adhesion. These findings establish a novel role for Hox5 transcription factors as critical regulators of lung fibroblasts at adult homeostasis.

Squamous cell carcinoma subverts adjacent histologically normal epithelium to promote lateral invasion.

Recurrent and new tumors, attributed in part to lateral invasion, are frequent in squamous cell carcinomas and lead to poor survival. We identified a mechanism by which cancer subverts adjacent histologically normal epithelium to enable small clusters of cancer cells to burrow undetected under adjacent histologically normal epithelium. We show that suppression of DMBT1 within cancer promotes aggressive invasion and metastasis in vivo and is associated with metastasis in patients. Cancer cells via TGFß1 and TNFα also suppress DMBT1 in adjacent histologically normal epithelium, thereby subverting it to promote invasion of a small population of tumor cells. The sufficiency of DMBT1 in this process is demonstrated by significantly higher satellite tumor nests in Dmbt1-/- compared with wild-type mice. Moreover, in patients, invasion of small tumor nests under adjacent histologically normal epithelium is associated with increased risk for recurrence and shorter disease-free survival. This study demonstrates a crucial role of adjacent histologically normal epithelium in invasion and its important role in the tumor microenvironment and opens new possibilities for therapeutic strategies that reduce tumor recurrence.

Novel Lineage-Tracing System to Identify Site-Specific Ectopic Bone Precursor Cells.

Heterotopic ossification (HO) is a form of pathological cell-fate change of mesenchymal stem/precursor cells (MSCs) that occurs following traumatic injury, limiting range of motion in extremities and causing pain. MSCs have been shown to differentiate to form bone; however, their lineage and aberrant processes after trauma are not well understood. Utilizing a well-established mouse HO model and inducible lineage-tracing mouse (Hoxa11-CreERT2;ROSA26-LSL-TdTomato), we found that Hoxa11-lineage cells represent HO progenitors specifically in the zeugopod. Bioinformatic single-cell transcriptomic and epigenomic analyses showed Hoxa11-lineage cells are regionally restricted mesenchymal cells that, after injury, gain the potential to undergo differentiation toward chondrocytes, osteoblasts, and adipocytes. This study identifies Hoxa11-lineage cells as zeugopod-specific ectopic bone progenitors and elucidates the fate specification and multipotency that mesenchymal cells acquire after injury. Furthermore, this highlights homeobox patterning genes as useful tools to trace region-specific progenitors and enable location-specific gene deletion.

Ovarian Cells Have Increased Proliferation in Response to Heparin-Binding Epidermal Growth Factor as Collagen Density Increases.

It is well known that during ovarian cancer progression, the omentum transforms from a thin lacy organ to a thick tougher tissue. However, the mechanisms regulating this transformation and the implications of the altered microenvironment on ovarian cancer progression remain unclear. To address these questions, the global and local concentrations of collagen I were determined for normal and metastatic human omentum. Collagen I was increased 5.3-fold in omenta from ovarian cancer patients and localized to areas of activated fibroblasts rather than regions with a high density of cancer cells. Transforming growth factor beta 1 (TGFß1) was detected in ascites from ovarian cancer patients (4 ng/mL), suggesting a potential role for TGFß1 in the observed increase in collagen. Treatment with TGFß1 induced fibroblast activation, proliferation, and collagen deposition in mouse omental explants and an in vitro model with human omental fibroblasts. Finally, the impact of increased collagen I on ovarian cancer cells was determined by examining proliferation on collagen I gels formulated to mimic normal and cancerous omenta. While collagen density alone had no impact on proliferation, a synergistic effect was observed with collagen density and heparin-binding epidermal growth factor treatment. These results suggest that TGFß1 induces collagen deposition from the resident fibroblasts in the omentum and that this altered microenvironment impacts cancer cell response to growth factors found in ascites. Impact statement Using quantitative analysis of patient samples, in vitro models of the metastatic ovarian cancer microenvironment were designed with pathologically relevant collagen densities and growth factor concentrations. Studies in these models support a mechanism where transforming growth factor ß1 in the ascites fluid induces omental fibroblast proliferation, activation, and deposition of collagen I, which then impacts tumor cell proliferation in response to additional ascites growth factors such as heparin-binding epidermal growth factor. This approach can be used to dissect mechanisms involved in microenvironmental modeling in multiple disease applications.

Differential Contribution of Pancreatic Fibroblast Subsets to the Pancreatic Cancer Stroma.

BACKGROUND & AIMS: Although the healthy pancreas consists mostly of epithelial cells, pancreatic cancer and the precursor lesions known as pancreatic intraepithelial neoplasia, are characterized by an extensive accumulation of fibroinflammatory stroma that includes a substantial and heterogeneous fibroblast population. The cellular origin of fibroblasts within the stroma has not been determined. Here, we show that the Gli1 and Hoxb6 markers label distinct fibroblast populations in the healthy mouse pancreas. We then set out to determine whether these distinct fibroblast populations expanded during carcinogenesis. METHODS: We developed genetically engineered models using a dual-recombinase approach that allowed us to induce pancreatic cancer formation through codon-optimized Flp recombinase-driven epithelial recombination of Kirsten rat sarcoma viral oncogene homolog while labeling Gli1+ or Hoxb6+ fibroblasts in an inducible manner. By using these models, we lineage-traced these 2 fibroblast populations during the process of carcinogenesis. RESULTS: Although in the healthy pancreas Gli1+ fibroblasts and Hoxb6+ fibroblasts are present in similar numbers, they contribute differently to the stroma in carcinogenesis. Namely, Gli1+ fibroblasts expand dramatically, whereas Hoxb6+ cells do not. CONCLUSIONS: Fibroblasts present in the healthy pancreas expand during carcinogenesis, but with a different prevalence for different subtypes. Here, we compared Gli1+ and Hoxb6+ fibroblasts and found only Gli1+ expanded to contribute to the stroma during pancreatic carcinogenesis.

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.

Two CRISPR/Cas9-mediated methods for targeting complex insertions, deletions, or replacements in mouse.

Genetically modified model organisms are valuable tools for probing gene function, dissecting complex signaling networks, studying human disease, and more. CRISPR/Cas9 technology has significantly democratized and reduced the time and cost of generating genetically modified models to the point that small gene edits are now routinely and efficiently generated in as little as two months. However, generation of larger and more sophisticated gene-modifications continues to be inefficient. Alternative ways to provide the replacement DNA sequence, method of Cas9 delivery, and tethering the template sequence to Cas9 or the guide RNA (gRNA) have all been tested in an effort to maximize homology-directed repair for precise modification of the genome. We present two CRISPR/Cas9 methods that have been used to successfully generate large and complex gene-edits in mouse. In the first method, the Cas9 enzyme is used in conjunction with two sgRNAs and a long single-stranded DNA (lssDNA) template prepared by an alternative protocol. The second method utilizes a tethering approach to couple a biotinylated, double-stranded DNA (dsDNA) template to a Cas9-streptavidin fusion protein. •Alternative method for generating long, single-stranded DNA templates for CRISPR/Cas9 editing.•Demonstration that using two sgRNAs with Cas9-streptavidin/biotinylated-dsDNA is feasible for large DNA modifications.

Hox11 expressing regional skeletal stem cells are progenitors for osteoblasts, chondrocytes and adipocytes throughout life.

Multipotent mesenchymal stromal cells (MSCs) are required for skeletal formation, maintenance, and repair throughout life; however, current models posit that postnatally arising long-lived adult MSCs replace transient embryonic progenitor populations. We previously reported exclusive expression and function of the embryonic patterning transcription factor, Hoxa11, in adult skeletal progenitor-enriched MSCs. Here, using a newly generated Hoxa11-CreERT2 lineage-tracing system, we show Hoxa11-lineage marked cells give rise to all skeletal lineages throughout the life of the animal and persist as MSCs. Hoxa11 lineage-positive cells give rise to previously described progenitor-enriched MSC populations marked by LepR-Cre and Osx-CreER, placing them upstream of these populations. Our studies establish that Hox-expressing cells are skeletal stem cells that arise from the earliest stages of skeletal development and self-renew throughout the life of the animal.

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.

Anatomic Origin of Osteochondrogenic Progenitors Impacts Sensitivity to EWS-FLI1-Induced Transformation.

Ewing sarcomas predominantly arise in pelvic and stylopod bones (i.e., femur and humerus), likely as a consequence of EWS-FLI1 oncogene-induced transformation of mesenchymal stem/progenitor cells (MSCs). MSCs located in the embryonic superficial zone cells (eSZ) of limbs express anatomically distinct posterior Hox genes. Significantly, high expression of posterior HOXD genes, especially HOXD13, is a hallmark of Ewing sarcoma. These data drove our hypothesis that Hox genes in posterior skeleton MSCs contribute to Ewing sarcoma tumorigenesis. We isolated eSZ cells from stylopod and zeugopod (i.e., tibia/fibula, radius/ulna) bones, from wild-type and Hoxd13 mutant embryos, and tested the impact of EWS-FLI1 transduction on cell proliferation, gene expression, and tumorigenicity. Our data demonstrate that both stylopod and zeugopod eSZ cells tolerate EWS-FLI1 but that stylopod eSZ cells are relatively more susceptible, demonstrating changes in proliferation and gene expression consistent with initiation of malignant transformation. Significantly, loss of Hoxd13 had no impact, showing that it is dispensable for the initiation of EWS-FLI1-induced transformation in mouse MSCs. These findings show that MSCs from anatomically distinct sites are differentially susceptible to EWS-FLI1-induced transformation, supporting the premise that the dominant presentation of Ewing sarcoma in pelvic and stylopod bones is attributable to anatomically-defined differences in MSCs.

Development, repair, and regeneration of the limb musculoskeletal system.

The limb musculoskeletal system provides a primary means for locomotion, manipulation of objects and protection for most vertebrate organisms. Intricate integration of the bone, tendon and muscle tissues are required for function. These three tissues arise largely independent of one another, but the connections formed during later development are maintained throughout life and are re-established following injury. Each of these tissues also have mesenchymal stem/progenitor cells that function in maintenance and repair. Here in, we will review the major events in the development of limb skeleton, tendon, and muscle tissues, their response to injury, and discuss current knowledge regarding resident progenitor/stem cells within each tissue that participate in development, repair, and regeneration in vivo.

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.