Search

The International Society of Differentiation was born from the First International Conference on Cell Differentiation conceived by D.V. and held in Nice, France in 1971. The conference also resulted in the creation of the journal of the Society named Differentiation. The Society advocates for the field of differentiation through the journal Differentiation, organizing and supporting international scientific conferences, honoring scientific achievements, and supporting trainees.

Supplementary Table 1 from Misregulated Wnt/β-Catenin Signaling Leads to Ovarian Granulosa Cell Tumor Development

Misregulation of the Wnt/β-catenin signaling pathway is a hallmark of several forms of cancer. Components of the Wnt/β-catenin pathway are expressed in ovarian granulosa cells; nevertheless, its potential involvement in granulosa cell tumorigenesis has not been examined. To this end, human (n = 6) and equine (n = 18) granulosa cell tumors (GCT) were analyzed for β-catenin expression by immunohistochemistry. Unlike granulosa cells of normal ovaries, most (15 of 24) GCT samples showed nuclear localization of β-catenin, suggesting that activation of the Wnt/β-catenin pathway plays a role in the etiology of GCT. To confirm this hypothesis, Catnbflox(ex3)/+; Amhr2cre/+ mice that express a dominant stable β-catenin mutant in their granulosa cells were generated. These mice developed follicle-like structures containing disorganized, pleiomorphic granulosa by 6 weeks of age. Even in older mice, these follicle-like lesions grew no larger than the size of antral follicles and contained very few proliferating cells. Similar to corpora lutea, the lesions were highly vascularized, although they did not express the luteinization marker Cyp11a1. Catnbflox(ex3)/+; Amhr2cre/+ females were also found to be severely subfertile, and fewer corpora lutea were found to form in response to exogenous gonadotropin compared with control mice. In older mice, the ovarian lesions often evolved into GCT, indicating that they represent a pretumoral intermediate stage. The GCT in Catnbflox(ex3)/+; Amhr2cre/+ mice featured many histopathologic similarities to the human disease, and prevalence of tumor development attained 57% at 7.5 months of age. Together, these studies show a causal link between misregulated Wnt/β-catenin signaling and GCT development and provide a novel model system for the study of GCT biology.

PDF file - 249K, Expression of PR protein and its target genes in Wnt7acre+ Mig-6f/f mice after P4 treatment.

PDF file - 250K, Steroid hormone regulation of epithelial Mig-6 in the murine uterus.

Supplementary Figure Legend from Misregulated Wnt/β-Catenin Signaling Leads to Ovarian Granulosa Cell Tumor Development

Supplementary Figure 1 from Misregulated Wnt/β-Catenin Signaling Leads to Ovarian Granulosa Cell Tumor Development

Endometrial cancer is preceded by endometrial hyperplasia, unopposed estrogen exposure, and genetic alterations, but the precise causes of endometrial cancer remain uncertain. Mig-6, mainly known as a negative regulator of the EGF receptor, is an important mediator of progesterone signaling in the uterus, where it mediates tumor suppression by modulating endometrial stromal–epithelial communications. In this study, we investigated the function of Mig-6 in the uterine epithelium using a tissue-specific gene knockout strategy, in which floxed Mig-6 (Mig-6f/f) mice were crossed to Wnt7a-Cre mice (Wnt7acre+Mig-6f/f). Wnt7acre+Mig-6f/f mice developed endometrial hyperplasia and estrogen-dependent endometrial cancer, exhibiting increased proliferation in epithelial cells as well as apoptosis in subepithelial stromal cells. We documented increased expression of NOTCH1 and BIRC3 in epithelial cells of Wnt7acre+Mig-6f/f mice and decreased expression of the progesterone receptor (PR) in stromal cells. Progesterone therapy controls endometrial growth and prevents endometrial cancer, but the effectiveness of progesterone as a treatment for women with endometrial cancer is less clear. We noted that the hyperplasic phenotype of Wnt7acre+Mig-6f/f mice was prevented by progesterone treatment, whereas this treatment had no effect in PRcre/+Mig-6f/f mice where Mig-6 was deleted in both the epithelial and stromal compartments of the uterus. In contrast, activation of progesterone signaling in the stroma regulated proliferation and apoptosis in the epithelium via suppression of ERα signaling. In summary, our results establish that epithelial Mig-6 functions as a critical tumor suppressor that mediates the ability of progesterone to prevent the development of endometrial cancer. Cancer Res; 73(16); 5090–9. ©2013 AACR.

Lateral flight membranes, or patagia, have evolved repeatedly in diverse mammalian lineages. While little is known about patagium development, its recurrent evolution may suggest a shared molecular basis. By combining transcriptomics, developmental experiments, and mouse transgenics, we demonstrate that lateral Wnt5a expression in the marsupial sugar glider ( Petaurus breviceps ) promotes the differentiation of its patagium primordium. We further show that this function of Wnt5a reprises ancestral roles in skin morphogenesis predating mammalian flight and has been convergently used during patagium evolution in eutherian bats. Moreover, we find that many genes involved in limb development have been redeployed during patagium outgrowth in both the sugar glider and bat. Together, our findings reveal that deeply conserved genetic toolkits contribute to the evolutionary transition to flight in mammals.

Lateral flight membranes, or patagia, have evolved repeatedly in diverse mammalian lineages. While little is known about patagium development, its recurrent evolution may suggest a shared molecular basis. By combining transcriptomics, developmental experiments, and mouse transgenics, we demonstrate that lateral WNT5A expression in the marsupial sugar glider (Petaurus breviceps) promotes the differentiation of its patagium primordium. We further show that this function of WNT5A reprises ancestral roles in skin morphogenesis predating mammalian flight and has been convergently employed during patagium evolution in eutherian bats. Moreover, we find that many genes involved in limb development have been re-deployed during patagium outgrowth in both the sugar glider and bat. Taken together, our findings reveal that deeply conserved molecular toolkits underpin the evolutionary transition to flight in mammals.

Conditional overexpression of histone reader Tripartite motif containing protein 24 (TRIM24) in mouse mammary epithelia (Trim24COE) drives spontaneous development of mammary carcinosarcoma tumors, lacking ER, PR and HER2. Human carcinosarcomas or metaplastic breast cancers (MpBC) are a rare, chemorefractory subclass of triple-negative breast cancers (TNBC). Comparison of Trim24COE metaplastic carcinosarcoma morphology, TRIM24 protein levels and a derived Trim24COE gene signature reveals strong correlation with human MpBC tumors and MpBC patient-derived xenograft (PDX) models. Global and single-cell tumor profiling reveal Met as a direct oncogenic target of TRIM24, leading to aberrant PI3K/mTOR activation. Here, we find that pharmacological inhibition of these pathways in primary Trim24COE tumor cells and TRIM24-PROTAC treatment of MpBC TNBC PDX tumorspheres decreased cellular viability, suggesting potential in therapeutically targeting TRIM24 and its regulated pathways in TRIM24-expressing TNBC.

The cervical and anterior thoracic regions of mammals generally exhibit similar vertebral numbers and identities along the anterior-posterior axis. The position of the forelimbs along the axial skeleton is also generally conserved. In contrast, the number of lumbar and sacral vertebrae and pelvic position exhibit more variation, correlating with posture and locomotion. The molecular mechanisms that lead to these conserved and variable axial skeletal patterns between species are not fully understood. Here we use a human HOXB1-9 transgene to complement a HoxB1-9 deficiency in the mouse. In TgHOXB1-9 mice, human HOXB1, B2, B3, and B4 (HOXB1-4) genes were expressed in mouse embryos in patterns similar to mouse Hoxb1-4 genes. Human transgene expression rescued the cervical and anterior thoracic vertebral patterning defects of HoxB1-9

In eutherian and marsupial mammals, the site of embryo implantation and gestation is the uterus. Uterine morphologies vary between mammalian species. For example, laboratory mice have a bipartite uterus with two uterine horns and a single cervix, whereas humans have a simplex uterus with a single chamber and single cervix. The precursor tissue of the uterus, oviducts, and upper vagina is the Müllerian duct epithelium and its adjacent mesenchyme. Morphological variation between species is established during embryogenesis by species-specific differences in Müllerian duct fusion at the midline, growth, and differentiation. In humans, alterations in Müllerian duct development can lead to variations in uterine morphology that correlate with increased risks of miscarriage and infertility. Here we review the developmental genetic factors that regulate Müllerian duct development, uterine morphogenesis, and human uterine variation.

Female mice homozygous for an engineered Gnrhr E90K mutation have reduced gonadotropin-releasing hormone signaling, leading to infertility. Their ovaries have numerous antral follicles but no corpora lutea, indicating a block to ovulation. These mutants have high levels of circulating estradiol and low progesterone, indicating a state of persistent estrus. This mouse model provided a unique opportunity to examine the lack of cyclic levels of ovarian hormones on uterine gland biology. Although uterine gland development appeared similar to controls during prepubertal development, it was compromised during adolescence in the mutants. By age 20 weeks, uterine gland development was comparable to controls, but pathologies, including cribriform glandular structures, were observed. Induction of ovulations by periodic human chorionic gonadotropin treatment did not rescue postpubertal uterine gland development. Interestingly, progesterone receptor knockout mice, which lack progesterone signaling, also have defects in postpubertal uterine gland development. However, progesterone treatment did not rescue postpubertal uterine gland development. These studies indicate that chronically elevated levels of estradiol with low progesterone and therefore an absence of cyclic ovarian hormone secretion disrupts postpubertal uterine gland development and homeostasis.

Luteinizing hormone (LH) stimulates testosterone production from Leydig cells. Both LH and testosterone play important roles in spermatogenesis and male fertility. To identify LH - and testosterone - responsive transporter genes that play key roles in spermatogenesis, we performed large-scale gene expression analyses on testes obtained from adult control and Lhb knockout mice. We found a significant reduction in cystine/glutamate transporter encoding Slc7a11 mRNA in testes of Lhb null mice. We observed that Slc7a11/SLC7A11 expression was initiated pre-pubertally and developmentally regulated in mouse testis. Immunolocalization studies confirmed that SLC7A11 was mostly expressed in Sertoli cells in testes of control and germ cell-deficient mice. Western blot analyses indicated that SLC7A11 was significantly reduced in testes of mutant mice lacking either LH or androgen receptor selectively in Sertoli cells. Genetic and pharmacological rescue of Lhb knockout mice restored the testicular expression of Slc7a11 comparable to that observed in controls. Additionally, Slc7a11 mRNA was significantly suppressed upon Sertoli cell/testicular damage induced in mice by cadmium treatment. Knockdown of Slc7a11 in vitro in TM4 Sertoli cells or treatment of mice with sulfasalazine, a SLC7A11 inhibitor caused a significant reduction in intracellular cysteine and glutathione levels but glutamate content remained unchanged as determined by metabolomic analysis. Knockdown of Slc7a11 resulted in compensatory upregulation of other glutamate transporters belonging to the Slc1a family presumably to maintain intracellular glutamate levels. Collectively, our studies identified that SLC7A11 is an LH/testosterone-regulated transporter that is required for cysteine/glutathione but not glutamate homeostasis in mouse Sertoli cells.

Anti-Müllerian hormone (AMH) is a member of the Transforming Growth Factor-β family of secreted signaling proteins. AMH is expressed in Sertoli cells of the fetal and adult testes and granulosa cells of the postnatal ovary. AMH is required for the regression of the Müllerian ducts in mammalian fetuses during male differentiation. AMH signals through its Type II receptor, AMHR2. AMHR2 is expressed in mesenchyme adjacent to the Müllerian ducts, and in Sertoli, Leydig, and granulosa cells. Although AMH and AMHR2 genes have been identified in numerous vertebrate species, spontaneous or engineered mutations or variants have been found or created in only a few mammals and teleost fishes. AMH or AMHR2 mutations in mammals lead to the development of Persistent Müllerian Duct Syndrome (PMDS), a recessive condition in which affected males are fully virilized but retain Müllerian duct-derived tissues, including a uterus and oviducts, and in human and dog, undescended testes. Amh mutant female mice had accelerated ovarian primordial follicle recruitment, suggesting a role for AMH in regulating germ cells, amh and amhr2 mutations have also been experimentally generated in various teleost fishes. Depending on the fish species, loss of AMH signaling results in infertility, germ cell tumors, or male-to-female sex reversal. Here we compare the spectrum of phenotypes caused by AMH and AMHR2 mutations in a variety of vertebrate species. There are both common and unique phenotypes between species, highlighting the range of biological processes regulated by AMH signaling.

The LIM-homeodomain (LIM-HD) transcription factor Islet-1 (Isl1) interacts with the LIM domain-binding protein 1 (Ldb1) coregulator to control expression of key pancreatic β-cell genes. However, Ldb1 also has Isl1-independent effects, supporting that another LIM-HD factor interacts with Ldb1 to impact β-cell development and/or function. LIM homeobox 1 (Lhx1) is an Isl1-related LIM-HD transcription factor that appears to be expressed in the developing mouse pancreas and in adult islets. However, roles for this factor in the pancreas are unknown. This study aimed to determine Lhx1 interactions and elucidate gene regulatory and physiological roles in the pancreas. Co-immunoprecipitation using β-cell extracts demonstrated an interaction between Lhx1 and Isl1, and thus we hypothesized that Lhx1 and Isl1 regulate similar target genes. To test this, we employed siRNA-mediated Lhx1 knockdown in β-cell lines and discovered reduced Glp1R mRNA. Chromatin immunoprecipitation revealed Lhx1 occupancy at a domain also known to be occupied by Isl1 and Ldb1. Through development of a pancreas-wide knockout mouse model ( Lhx1∆Panc), we demonstrate that aged Lhx1∆Pancmice have elevated fasting blood glucose levels, altered intraperitoneal and oral glucose tolerance, and significantly upregulated glucagon, somatostatin, pancreatic polypeptide, MafB, and Arx islet mRNAs. Additionally, Lhx1∆Pancmice exhibit significantly reduced Glp1R, an mRNA encoding the insulinotropic receptor for glucagon-like peptide 1 along with a concomitant dampened Glp1 response and mild glucose intolerance in mice challenged with oral glucose. These data are the first to reveal that the Lhx1 transcription factor contributes to normal glucose homeostasis and Glp1 responses.

Morphogenetic events during the development of the fetal ovary are crucial to the establishment of female fertility. However, the effects of structural rearrangements of the ovary and surrounding reproductive tissues on ovary morphogenesis remain largely uncharacterized. Using tissue clearing and lightsheet microscopy, we found that ovary folding correlated with regionalization into cortex and medulla. Relocation of the oviduct to the ventral aspect of the ovary led to ovary encapsulation, and mutual attachment of the ovary and oviduct to the cranial suspensory ligament likely triggered ovary folding. During this process, the rete ovarii (RO) elaborated into a convoluted tubular structure extending from the ovary into the ovarian capsule. Using genetic mouse models in which the oviduct and RO are perturbed, we found the oviduct is required for ovary encapsulation. This study reveals novel relationships among the ovary and surrounding tissues and paves the way for functional investigation of the relationship between architecture and differentiation of the mammalian ovary.In humans and other mammals, the female reproductive organs, or ovaries, develop early in life, while the young are still in their mother’s womb. Ovaries contain several different compartments, including the ovarian follicles. These are small groups of cells that produce reproductive hormones, and each follicle also has the potential to produce one egg for fertilisation. The ovaries are further surrounded by different tissues that develop alongside them. These include the oviducts, which carry fertilised eggs from the ovaries into the womb, and ligaments, which anchor the ovaries to the wall of the body cavity. During the development of ovaries, ovarian follicles are sorted into two distinct groups. The first, called medullary follicles, are lost before puberty. The second group, or cortical follicles, remain in a state of ‘suspended animation’ until puberty. After that, they act as a ‘reserve’ of eggs for the rest of the reproductive lifespan. Once each cortical follicle has produced an egg, it is not replenished. This means that proper follicle sorting is crucial for establishing female fertility, and therefore the ability to conceive. The mechanisms behind follicle sorting, however, are still poorly understood. McKey et al. set out to determine how the ovary’s structure changed during its development. In the experiments, high-resolution microscopy techniques were used to reconstruct ovaries of mice in 3D across different stages of development. This revealed that the ends of each ovary started folding towards each other just before birth, and that the folding also happened at the same time as follicle sorting. Simultaneous changes in the shape and orientation of the ligaments suggested that these tissues might direct the folding, for example by pushing or pulling on the rest of the ovary. These results suggest that the changes in ovary structure in early life are critically linked to the establishment of the ovary’s egg reserves. McKey et al. hope that this study will pave the way to a better understanding of infertility and, ultimately, better treatments.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a range of symptoms in infected individuals, from mild respiratory illness to acute respiratory distress syndrome. A systematic understanding of host factors influencing viral infection is critical to elucidate SARS-CoV-2–host interactions and the progression of Coronavirus disease 2019 (COVID-19). Here, we conducted genome-wide CRISPR knockout and activation screens in human lung epithelial cells with endogenous expression of the SARS-CoV-2 entry factors ACE2 and TMPRSS2. We uncovered proviral and antiviral factors across highly interconnected host pathways, including clathrin transport, inflammatory signaling, cell-cycle regulation, and transcriptional and epigenetic regulation. We further identified mucins, a family of high molecular weight glycoproteins, as a prominent viral restriction network that inhibits SARS-CoV-2 infection in vitro and in murine models. These mucins also inhibit infection of diverse respiratory viruses. This functional landscape of SARS-CoV-2 host factors provides a physiologically relevant starting point for new host-directed therapeutics and highlights airway mucins as a host defense mechanism.

SummaryThe potential for variation and the capacity to evolve in response to ecological opportunity are important aspects of an adaptive radiation. Identifying the origin of phenotypic variation, in which natural selection might act upon, is a major goal of evolutionary developmental biology. The New World leaf-nosed bats (phyllostomids) are a textbook example of an adaptive radiation. Their cranial morphology is diverse along relative facial length, which is related to their diets. We previously used geometric morphometrics to reveal peramorphosis, a type of heterochrony, in the cranial evolution among phyllostomid bats. We then demonstrated that the mechanism of peramorphic diversity in phyllostomid rostrum length resulted from altered cellular proliferation. Here, we investigate the progenitors of the face, the cranial neural crest, and a key signaling pathway related to their proliferation and differentiation into mature tissues: the bone morphogenetic protein (BMP). With geometric morphometrics, immunofluorescence, and confocal imaging—in three phyllostomid species and one outgroup bat species—we show the molecular patterns that underlie the adaptive and innovative traits seen in phyllostomid bats. Then, with mouse genetics, we mimic the BMP molecular pattern observed in nectar feeding bats and recapitulate the elongated morphological variation in mice. Surprisingly, we also observe an expansion in the nose-tip of mice, akin to the expanding leaf-nose tissue in phyllostomid bats. These data, combined with the mouse genetics literature on BMP signaling, suggest the BMP developmental pathway plays a central role in shaping the craniofacial variation necessary for adaptation in bats. Further, we speculate that the BMP signaling pathway could underlie other bizarre facial phenotypes in mammals that are derived from frontonasal mesenchyme, such as the proboscis. Overall, this study combines a comparative framework to developmental data, with a genetic approach, to directly investigate the role of development on complex morphology.

Limb muscles are remarkably complex and evolutionarily labile. Although their anatomy is of great interest for studies of the evolution of form and function, their homologies among major amniote clades have remained obscure. Studies of adult musculature are inconclusive owing to the highly derived morphology of modern amniote limbs but correspondences become increasingly evident earlier in ontogeny. We followed the embryonic development of forelimb musculature in representatives of six major amniote clades and found, contrary to current consensus, that these early splitting patterns are highly conserved across Amniota. Muscle mass cleavage patterns and topology are highly conserved in reptiles including birds, irrespective of their skeletal modifications: the avian flight apparatus results from slight early topological modifications that are exaggerated during ontogeny. Therian mammals, while conservative in their cleavage patterns, depart drastically from the ancestral amniote musculoskeletal organization in terms of topology. These topological changes occur through extension, translocation and displacement of muscle groups later in development. Overall, the simplicity underlying the apparent complexity of forelimb muscle development allows us to resolve conflicting hypotheses about homology and to trace the history of each individual forelimb muscle throughout the amniote radiations.

We investigated genome folding across the eukaryotic tree of life. We find two types of three-dimensional(3D) genome architectures at the chromosome scale. Each type appears and disappears repeatedlyduring eukaryotic evolution. The type of genome architecture that an organism exhibits correlates with theabsence of condensin II subunits. Moreover, condensin II depletion converts the architecture of thehuman genome to a state resembling that seen in organisms such as fungi or mosquitoes. In this state,centromeres cluster together at nucleoli, and heterochromatin domains merge. We propose a physicalmodel in which lengthwise compaction of chromosomes by condensin II during mitosis determineschromosome-scale genome architecture, with effects that are retained during the subsequent interphase.This mechanism likely has been conserved since the last common ancestor of all eukaryotes., C.H. is supported by the Boehringer Ingelheim Fonds; C.H., Á.S.C., and B.D.R. are supported by an ERC CoG (772471, “CohesinLooping”); A.M.O.E. and B.D.R. are supported by the Dutch Research Council (NWO-Echo); and J.A.R. and R.H.M. are supported by the Dutch Cancer Society (KWF). T.v.S. and B.v.S. are supported by NIH Common Fund “4D Nucleome” Program grant U54DK107965. H.T. and E.d.W. are supported by an ERC StG (637597, “HAP-PHEN”). J.A.R., T.v.S., H.T., R.H.M., B.v.S., and E.d.W. are part of the Oncode Institute, which is partly financed by the Dutch Cancer Society. Work at the Center for Theoretical Biological Physics is sponsored by the NSF (grants PHY-2019745 and CHE-1614101) and by the Welch Foundation (grant C-1792). V.G.C. is funded by FAPESP (São Paulo State Research Foundation and Higher Education Personnel) grants 2016/13998-8 and 2017/09662-7. J.N.O. is a CPRIT Scholar in Cancer Research. E.L.A. was supported by an NSF Physics Frontiers Center Award (PHY-2019745), the Welch Foundation (Q-1866), a USDA Agriculture and Food Research Initiative grant (2017-05741), the Behavioral Plasticity Research Institute (NSF DBI-2021795), and an NIH Encyclopedia of DNA Elements Mapping Center Award (UM1HG009375). Hi-C data for the 24 species were created by the DNA Zoo Consortium (www.dnazoo.org). DNA Zoo is supported by Illumina, Inc.; IBM; and the Pawsey Supercomputing Center. P.K. is supported by the University of Western Australia. L.L.M. was supported by NIH (1R01NS114491) and NSF awards (1557923, 1548121, and 1645219) and the Human Frontiers Science Program (RGP0060/2017). The draft A. californica project was supported by NHGRI. J.L.G.-S. received funding from the ERC (grant agreement no. 740041), the Spanish Ministerio de Economía y Competitividad (grant no. BFU2016-74961-P), and the institutional grant Unidad de Excelencia María de Maeztu (MDM-2016-0687). R.D.K. is supported by NIH grant RO1DK121366. V.H. is supported by NIH grant NIH1P41HD071837. K.M. is supported by a MEXT grant (20H05936). M.C.W. is supported by the NIH grants R01AG045183, R01AT009050, R01AG062257, and DP1DK113644 and by the Welch Foundation. E.F. was supported by NHGRI

BackgroundSkull diversity in the neotropical leaf-nosed bats (Phyllostomidae) evolved through a heterochronic process called peramorphosis, with underlying causes varying by subfamily. The nectar-eating (subfamily Glossophaginae) and blood-eating (subfamily Desmondontinae) groups originate from insect-eating ancestors and generate their uniquely shaped faces and skulls by extending the ancestral ontogenetic program, appending new developmental stages and demonstrating peramorphosis by hypermorphosis. However, the fruit-eating phyllostomids (subfamilies Carollinae and Stenodermatinae) adjust their craniofacial development by speeding up certain developmental processes, displaying peramorphosis by acceleration. We hypothesized that these two forms of peramorphosis detected by our morphometric studies could be explained by differential growth and investigated cell proliferation during craniofacial morphogenesis.ResultsWe obtained cranial tissues from four wild-caught bat species representing a range of facial diversity and labeled mitotic cells using immunohistochemistry. During craniofacial development, all bats display a conserved spatiotemporal distribution of proliferative cells with distinguishable zones of elevated mitosis. These areas were identified as modules by the spatial distribution analysis. Ancestral state reconstruction of proliferation rates and patterns in the facial module between species provided support, and a degree of explanation, for the developmental mechanisms underlying the two models of peramorphosis. In the long-faced species,Glossophaga soricina, whose facial shape evolved by hypermorphosis, cell proliferation rate is maintained at lower levels and for a longer period of time compared to the outgroup speciesMiniopterus natalensis. In both species of studied short-faced fruit bats,Carollia perspicillataandArtibeus jamaicensis, which evolved under the acceleration model, cell proliferation rate is increased compared to the outgroup.ConclusionsThis is the first study which links differential cellular proliferation and developmental modularity with heterochronic developmental changes, leading to the evolution of adaptive cranial diversity in an important group of mammals.

Precise manipulation of gene expression with temporal and spatial control is essential for functional analysis and determining cell lineage relationships in complex biological systems. The Cre-loxP system is commonly used for gene manipulation at desired times and places. However, specificity is dependent on the availability of tissue- or cell-specific regulatory elements used in combination with Cre. Here we present CreLite, an optogenetically-controlled Cre system using red light in developing zebrafish embryos. Cre activity is disabled by splitting Cre and fusing with the Arabidopsis thaliana red light-inducible binding partners, PhyB and PIF6. Upon red light illumination, the PhyB-CreC and PIF6-CreN fusion proteins come together in the presence of the cofactor phycocyanobilin (PCB) to restore Cre activity. Red light exposure of zebrafish embryos harboring a Cre-dependent multi-color fluorescent protein reporter injected with CreLite mRNAs and PCB resulted in Cre activity as measured by the generation of multi-spectral cell labeling in several different tissues. Our data show that CreLite can be used for gene manipulations in whole embryos or small groups of cells at different developmental stages, and suggests CreLite may also be useful for temporal and spatial control of gene expression in cell culture, ex vivo organ culture, and other animal models. This article is protected by copyright. All rights reserved.

In mammals, the developing reproductive tract primordium of male and female fetuses consists of the Wolffian duct and the Müllerian duct (MD), two epithelial tube pairs surrounded by mesenchyme. During male development, mesenchyme-epithelia interactions mediate MD regression to prevent its development into a uterus, oviduct and upper vagina. It is well established that transforming growth factor-beta family member anti-Müllerian hormone (AMH) secreted from the fetal testis and its type 1 and 2 receptors expressed in MD mesenchyme regulate MD regression. However, little is known about the molecular network regulating downstream actions of AMH signaling. To identify potential AMH-induced genes and regulatory networks controlling MD regression in a global non-biased manner, we examined transcriptome differences in MD mesenchyme between males (AMH signaling on) and females (AMH signaling off) by RNA-Seq analysis of purified fetal MD mesenchymal cells. This analysis found 82 genes up-regulated in males during MD regression and identifiedOsterix (Osx)/Sp7, a key transcriptional regulator of osteoblast differentiation and bone formation, as a novel downstream effector of AMH signaling during MD regression. Osx/OSX was expressed in a male-specific pattern in MD mesenchyme during MD regression. OSX expression was lost in mutant males without AMH signaling. In addition, transgenic mice ectopically expressing human AMH in females induced a male pattern ofOsxexpression. Together these results indicate that AMH signaling is necessary and sufficient forOsxexpression in the MD mesenchyme. In addition, MD regression was delayed inOsxnull males, identifyingOsxas a new factor that regulates MD regression.SignificanceIn mammals, each embryo forms both male and female reproductive tract organ progenitor tissues. Anti-Müllerian hormone (AMH) secreted by fetal testes acts on mesenchyme cells adjacent to the Müllerian duct (MD) epithelium, the progenitor tissue of the female reproductive tract, to induce MD regression. While AMH and early AMH signaling components are elucidated, downstream gene networks directing this process are largely unknown. A global non-biased approach using whole transcriptome sequencing of fetal MD mesenchymal cells identified 82 factors as potential target genes of AMH includingOsterix (Osx). Our findings providein vivoevidenceOsxis an AMH-induced gene that regulates MD regression. Identification ofOsxmay provide key insights into gene regulatory networks underlying MD regression and male sex differentiation.

Endometrial or uterine glands secrete substances essential for uterine receptivity to the embryo, implantation, conceptus survival, and growth. Adenogenesis is the process of gland formation within the stroma of the uterus. In the mouse, uterine gland formation initiates at postnatal day (P) 5. Uterine gland morphology is poorly understood because it is primarily based on two-dimensional (2D) histology. To more fully describe uterine gland morphogenesis, we generated three-dimensional (3D) models of postnatal uterine glands from P0 to P21, based on volumetric imaging using light sheet microscopy. At birth (P0), there were no glands. At P8, we found bud- and teardrop-shaped epithelial invaginations. By P11, the forming glands were elongated epithelial tubes. By P21, the elongated tubes had a sinuous morphology. These morphologies are homogeneously distributed along the anterior–posterior axis of the uterus. To facilitate uterine gland analyses, we propose a novel 3D staging system of uterine gland morphology during development in the prepubertal mouse. We define five uterine gland stages: Stage 1: bud; Stage 2: teardrop; Stage 3: elongated; Stage 4: sinuous; and Stage 5: primary branches. This staging system provides a standardized key to assess and quantify prepubertal uterine gland morphology that can be used for studies of uterine gland development and pathology. In addition, our studies suggest that gland formation initiation occurs during P8 and P11. However, between P11 and P21 gland formation initiation stops and all glands elongate and become sinuous. We also found that the mesometrial epithelium develops a unique morphology we term the uterine rail.

BackgroundThe uterus is the location where multiple events occur that are required for the start of new life in mammals. The adult uterus contains endometrial or uterine glands that are essential for female fertility. In the mouse, uterine glands are located in the lateral and anti-mesometrial regions of the uterine horn. Previous 3D-imaging of the adult uterus, its glands, and implanting embryos has been performed by multiple groups, using fluorescent microscopy. Adenogenesis, the formation of uterine glands, initiates after birth. Recently, we created a 3D-staging system of mouse uterine gland development at postnatal time points, using light sheet fluorescent microscopy. Here, using a similar approach, we examine the morphological changes in the epithelium of the perinatal mouse uterus.ResultsThe uterine epithelium exhibits mesometrial-antimesometrial (dorsoventral) patterning as early as three days after birth (P3), marked by the presence of the mesometrially-positioned developing uterine rail. Uterine gland buds are present beginning at P4. Novel morphological epithelial structures, including a ventral ridge and uterine segments were identified.ConclusionsThe perinatal mouse uterine luminal epithelium develops mesometrial-antimesometrial (dorsal-ventral) morphologies at 3-4 days post-partum. Between 5-6 days post-partum uterine epithelial folds form, defining alternating left-right segments.Bullet pointsMorphological patterning events in the perinatal uterine epithelium are not well described.Light sheet microscopy was used to generate volumetric reconstructions of the perinatal mouse uterine epithelium.At postnatal day 3 (P3), the uterine epithelium shows the first signs of dorsoventral pattern, with the presence of the forming mesometrially-positioned uterine rail.The first morphological indication of uterine adenogenesis begins at P4.Novel morphological structures were identified from volumetric reconstructions, including the presence of a ventral ridge (another sign of dorsoventral pattern) and uterine segmentation.

Precise manipulation of gene expression with temporal and spatial control is essential for functional studies and the determination of cell lineage relationships in complex biological systems. The Cre-loxP system is commonly used for gene manipulation at desired times and places. However, specificity is dependent on the availability of tissue- or cell-specific regulatory elements used in combination with Cre or CreER (tamoxifen-inducible). Here we present CreLite, an optogenetically-controlled Cre system using red light in developing zebrafish embryos. Cre activity is disabled by splitting Cre and fusing the inactive halves with the Arabidopsis thaliana red light-inducible binding partners, PhyB and PIF6. In addition, PhyB-PIF6 binding requires phycocyanobilin (PCB), providing an additional layer of control. Upon exposure to red light (660 nm) illumination, the PhyB-CreC and PIF6-CreN fusion proteins come together in the presence of PCB to restore Cre activity. Red-light exposure of transgenic zebrafish embryos harboring a Cre-dependent multi-color fluorescent protein reporter (ubi:zebrabow) injected with CreLite mRNAs and PCB, resulted in Cre activity as measured by the generation of multi-spectral cell labeling in various tissues, including heart, skeletal muscle and epithelium. We show that CreLite can be used for gene manipulations in whole embryos or small groups of cells at different stages of development. CreLite provides a novel optogenetic tool for precise temporal and spatial control of gene expression in zebrafish embryos that may also be useful in cell culture, ex vivo organ culture, and other animal models for developmental biology studies.

Dead-End 1 (DND1) encodes an RNA binding protein critical for viable primordial germ cells in vertebrates. When introduced into cancer cell lines, DND1 suppresses cell proliferation and enhances apoptosis. However, the molecular function of mammalian wild-type DND1 has mostly been studied in cell lines and not verified in the organism. To facilitate study of wild-type DND1 function in mammalian systems, we generated a novel transgenic mouse line, LSL-FM-DND1 flox/+ , which conditionally expresses genetically engineered, FLAG-tagged and myc-tagged DND1 in a cell type-specific manner. We report that FLAG-myc-DND1 is indeed expressed in specific tissues of the mouse when LSL-FM-DND1 flox/+ is combined with mouse strains expressing Cre-recombinase. LSL-FM-DND1 flox/+ mice are fertile with no overt health effects. We expressed FLAG-myc-DND1 in the pancreas and found that chronic, ectopic expression of FLAG-myc-DND1 led to increase in fasting glucose levels in older mice. Thus, this novel LSL-FM-DND1 flox/+ mouse strain will facilitate studies on the biological and molecular function of wild-type DND1.

Mammalian embryos initially develop progenitor tissues for both male and female reproductive tract organs, known as the Wolffian ducts and the Müllerian ducts, respectively. Ultimately, each individual develops a single set of male or female reproductive tract organs. Therefore, an essential step for sex differentiation is the regression of one duct and growth and differentiation of the other duct. In males, this requires Müllerian duct regression and Wolffian duct growth and differentiation. Müllerian duct regression is induced by the expression of Amh, encoding anti-Müllerian hormone, from the fetal testes. Subsequently, receptor-mediated signal transduction in mesenchymal cells surrounding the Müllerian duct epithelium leads to duct elimination. The genes that induce Amh transcription and the downstream signaling that results from Amh activity form a pathway. However, the molecular details of this pathway are currently unknown. A set of essential genes for AMH pathway function has been identified. More recently, transcriptome analysis of male and female Müllerian duct mesenchyme at an initial stage of regression has identified new genes that may mediate elimination of the Müllerian system. The evidence taken together can be used to generate an initial gene regulatory network describing the Amh pathway for Müllerian duct regression. An Amh gene regulatory network will be a useful tool to study Müllerian duct regression, sex differentiation, and its relationship to environmental influences.

Defective anorectal and urogenital malformations are some of the most severe congenital anomalies encountered in children. Only a few molecular cues have been identified in early formation of the female urogenital system. Here we describe a novel long non-coding RNA molecule known as Leat1 (long non-coding RNA, EphrinB2 associated transcript 1). This lncRNA is syntenic with EfnB2 (which encodes EphrinB2) and expressed during embryonic development of the genital tubercle. While lncRNAs have varied functions, many are known to regulate their neighbouring genes. Eph/Ephrin bidirectional signaling molecules mediate many patterning pathways in early embryonic development, including cloacal septation and urethral development. Here we investigate the role of Leat1 and its possible regulation of EphrinB2 during development of the female reproductive tract. We show that a loss of Leat1 leads to reduced EfnB2 expression in the developing female genital tubercle, reduced anogenital distance and decreased fertility.

The Portuguese man of war, Physalia physalis, is one of the most conspicuous, but poorly understood members of the pleuston, a community of organisms that occupy a habitat at the sea-air interface. Physalia physalis is a siphonophore that uses a gas-filled float as a sail to catch the wind. The development, morphology, and colony organization of P. physalis is very different from all other siphonophores. Here, we look at live and fixed larval and juvenile specimens, and use optical projection tomography to build on existing knowledge about the morphology and development of this species. We also propose a framework for homologizing the axes with other siphonophores, and also suggest that the tentacle bearing zooids should be called tentacular palpons. Previous descriptions of P. physalis larvae, especially descriptions of budding order, were often framed with the mature colony in mind. However, we use the simpler organization of larvae and the juvenile specimens to inform our understanding of the morphology, budding order, and colony organization in the mature specimen. Finally, we review what is known about the ecology and lifecycle of P. physalis.

A simple cell or tissue lysate can provide a sufficient quality and amount of template DNA for polymerase chain reaction (PCR). In this protocol, a small piece from the tip of the tail is removed and processed using hot sodium hydroxide and Tris (HotSHOT).

DNA samples are prepared from mouse tail tips by Proteinase K digestion and subsequently extracted. The resulting preparation is suitable for use in Southern blotting or polymerase chain reaction (PCR).

SOX9 is a high mobility group transcription factor that is required in many biological processes, including cartilage differentiation, endoderm progenitor maintenance, hair differentiation, and testis determination. SOX9 has also been linked to colorectal, prostate, and lung cancer. We found that SOX9 is expressed in the epithelium of the adult mouse and human uterus, predominantly marking the uterine glands. To determine if SOX9 plays a role in the development of endometrial cancer we overexpressed Sox9 in the uterine epithelium using a progesterone receptor-Cre mouse model. Sox9 overexpression in the uterine epithelium led to the formation of simple and complex cystic glandular structures in the endometrium of aged-females. Histological analysis revealed that these structures appeared morphologically similar to structures present in patients with endometrial hyperplastic lesions and endometrial polyps that are thought to be precursors of endometrial cancer. The molecular mechanisms that cause the glandular epithelium to become hyperplastic, leading to endometrial cancer are still poorly understood. These findings indicate that chronic overexpression of Sox9 in the uterine epithelium can induce the development of endometrial hyperplastic lesions. Thus, SOX9 expression may be a factor in the formation of endometrial cancer.

Female mice homozygous for an engineered Gnrhr E90K mutation have reduced gonadotropin-releasing hormone signaling, leading to infertility. Their ovaries have numerous antral follicles but no corpora lutea, indicating a block to ovulation. These mutants have high levels of circulating estradiol and low progesterone, indicating a state of persistent estrus. This mouse model provided a unique opportunity to examine the lack of cyclic levels of ovarian hormones on uterine gland biology. Although uterine gland development appeared similar to controls during prepubertal development, it was compromised during adolescence in the mutants. By 20 weeks of age, uterine gland development was comparable to controls, but pathologies, including squamous neoplasia, tubal neoplasia, and cribriform glandular structures, were observed. Induction of ovulations by periodic human chorionic gonadotropin treatment did not rescue post-pubertal uterine gland development. Interestingly, progesterone receptor knockout mice, which lack progesterone signaling, also have defects in post-pubertal uterine gland development. However, progesterone treatment did not rescue post-pubertal uterine gland development. These studies indicate that chronically elevated levels of estradiol with low progesterone and therefore an absence of cyclic ovarian hormone secretion disrupts post-pubertal uterine gland development and homeostasis.

Disruption of endometrial gland formation or function can cause female infertility. Formation of endometrial glands via tubulogenesis of luminal epithelial cells requires the establishment and maintenance of cell polarity and cell adhesion. The FERM domain-containing protein Merlin coordinates epithelial cell polarity and cell adhesion and is critical for epithelial tissue function in the skin and kidney. We now demonstrate a requirement for Merlin in endometrial gland development. Conditional deletion of Merlin in the endometrium results in female infertility caused by the absence of gland formation. Interestingly, we observed glandular epithelial markers within discrete groups of cells in the Merlin-deficient luminal epithelium. Wnt signaling, a pathway necessary for endometrial gland development is maintained in Merlin-deficient endometrium, suggesting the glandular fate program is active. Instead, we observe increased levels of apical actin and markers indicative of high membrane tension on the basal surface of the Merlin-deficient luminal epithelium. These findings suggest that the structural integrity of the luminal epithelium during gland formation is required for appropriate endometrial tubulogenesis and tissue function. Moreover, our work implicates Merlin-dependent regulation of mechanical tension in the proper formation of endometrial gland architecture and function.

Fluorescent protein (FP)-expressing transgenic mice are powerful genetic resources for marking both live and fixed cells and tissues. Expression in live embryos requires only dissection and visualization using an appropriate microscope. Fixation can compromise FP activity. A simple tissue-clearing agent called Scale preserves FP activity while rendering the embryo or organ optically transparent.

The establishment of the head to tail axis at early stages of development is a fundamental aspect of vertebrate embryogenesis. In mice, experimental embryology, genetics and expression studies have suggested that the visceral endoderm, an extra-embryonic tissue, plays an important role in anteroposterior axial development. Here we show that absence of Wnt3 in the posterior visceral endoderm leads to delayed formation of the primitive streak and that interplay between anterior and posterior visceral endoderm restricts the position of the primitive streak. Embryos lacking Wnt3 in the visceral endoderm, however, appear normal by E9.5. Our results suggest a model for axial development in which multiple signals are required for anteroposterior axial development in mammals.