PBX1 and PBX3 transcription factors regulate SHH expression in the Frontonasal Ectodermal Zone through complementary mechanisms.

Sonic hedgehog (SHH) signaling from the frontonasal ectodermal zone (FEZ) is a key regulator of craniofacial morphogenesis. Along with SHH, pre-B-cell leukemia homeobox (PBX) transcription factors regulate midfacial development. PBXs act in the epithelium during fusion of facial primordia, but their specific interactions with SHH have not been fully investigated. We hypothesized that PBX1/3 regulate SHH expression in the FEZ by activating or repressing transcription. The hypothesis was tested by manipulating PBX1/3 expression in chick embryos and profiling epigenomic landscapes at early developmental stages. PBX1/3 expression was perturbed in the chick face beginning at stage 10 (HH10) using RCAS viruses, and the resulting SHH expression was assessed at HH22. Overexpressing PBX1 expanded SHH expression, while overexpressing PBX3 decreased SHH expression. Conversely, reducing PBX1 expression decreased SHH expression, but reducing PBX3 induced ectopic SHH expression. We performed ATAC-seq and mapped binding of PBX1 and PBX3 with ChIP-seq on the FEZ at HH22 to assess direct interactions of PBX1/3 with the SHH locus. These multi-omics approaches uncovered a 400 bp PBX1-enriched element within intron 1 of SHH (chr2:8,173,222-8,173,621). Enhancer activity of this element was demonstrated by electroporation of reporter constructs in ovo and luciferase reporter assays in vitro . When bound by PBX1, this element upregulates transcription, while it downregulates transcription when bound by PBX3. The present study identifies a cis- regulatory element, named SFE1, that interacts with PBX1/3 to modulate SHH expression in the FEZ and establishes that PBX1 and PBX3 play complementary roles in SHH regulation during embryonic development.

A Specialized Niche in the Pancreatic Microenvironment Promotes Endocrine Differentiation.

The interplay between pancreatic epithelium and the surrounding microenvironment is pivotal for pancreas formation and differentiation as well as adult organ homeostasis. The mesenchyme is the main component of the embryonic pancreatic microenvironment, yet its cellular identity is broadly defined, and whether it comprises functionally distinct cell subsets is not known. Using genetic lineage tracing, transcriptome, and functional studies, we identified mesenchymal populations with different roles during pancreatic development. Moreover, we showed that Pbx transcription factors act within the mouse pancreatic mesenchyme to define a pro-endocrine specialized niche. Pbx directs differentiation of endocrine progenitors into insulin- and glucagon-positive cells through non-cell-autonomous regulation of ECM-integrin interactions and soluble molecules. Next, we measured functional conservation between mouse and human pancreatic mesenchyme by testing identified mesenchymal factors in an iPSC-based differentiation model. Our findings provide insights into how lineage-specific crosstalk between epithelium and neighboring mesenchymal cells underpin the generation of different pancreatic cell types.

Ubiquitous overexpression of CXCL12 confers radiation protection and enhances mobilization of hematopoietic stem and progenitor cells.

C-X-C motif chemokine ligand 12 (CXCL12; aka SDF1α) is a major regulator of a number of cellular systems, including hematopoiesis, where it influences hematopoietic cell trafficking, proliferation, and survival during homeostasis and upon stress and disease. A variety of constitutive, temporal, ubiquitous, and cell-specific loss-of-function models have documented the functional consequences on hematopoiesis upon deletion of Cxcl12. Here, in contrast to loss-of-function experiments, we implemented a gain-of-function approach by generating a doxycycline-inducible transgenic mouse model that enables spatial and temporal overexpression of Cxcl12. We demonstrated that ubiquitous CXCL12 overexpression led to an increase in multipotent progenitors in the bone marrow and spleen. The CXCL12+ mice displayed reduced reconstitution potential as either donors or recipients in transplantation experiments. Additionally, we discovered that Cxcl12 overexpression improved hematopoietic stem and progenitor cell mobilization into the blood, and conferred radioprotection by promoting quiescence. Thus, this new CXCL12+ mouse model provided new insights into major facets of hematopoiesis and serves as a versatile resource for studying CXCL12 function in a variety of contexts.

Incomplete penetrance for isolated congenital asplenia in humans with mutations in translated and untranslated RPSA exons.

Isolated congenital asplenia (ICA) is the only known human developmental defect exclusively affecting a lymphoid organ. In 2013, we showed that private deleterious mutations in the protein-coding region of RPSA, encoding ribosomal protein SA, caused ICA by haploinsufficiency with complete penetrance. We reported seven heterozygous protein-coding mutations in 8 of the 23 kindreds studied, including 6 of the 8 multiplex kindreds. We have since enrolled 33 new kindreds, 5 of which are multiplex. We describe here 11 new heterozygous ICA-causing RPSA protein-coding mutations, and the first two mutations in the 5'-UTR of this gene, which disrupt mRNA splicing. Overall, 40 of the 73 ICA patients (55%) and 23 of the 56 kindreds (41%) carry mutations located in translated or untranslated exons of RPSA. Eleven of the 43 kindreds affected by sporadic disease (26%) carry RPSA mutations, whereas 12 of the 13 multiplex kindreds (92%) carry RPSA mutations. We also report that 6 of 18 (33%) protein-coding mutations and the two (100%) 5'-UTR mutations display incomplete penetrance. Three mutations were identified in two independent kindreds, due to a hotspot or a founder effect. Finally, RPSA ICA-causing mutations were demonstrated to be de novo in 7 of the 23 probands. Mutations in RPSA exons can affect the translated or untranslated regions and can underlie ICA with complete or incomplete penetrance.

Face morphogenesis is promoted by Pbx-dependent EMT via regulation of Snail1 during frontonasal prominence fusion.

Human cleft lip with or without cleft palate (CL/P) is a common craniofacial abnormality caused by impaired fusion of the facial prominences. We have previously reported that, in the mouse embryo, epithelial apoptosis mediates fusion at the seam where the prominences coalesce. Here, we show that apoptosis alone is not sufficient to remove the epithelial layers. We observed morphological changes in the seam epithelia, intermingling of cells of epithelial descent into the mesenchyme and molecular signatures of epithelial-mesenchymal transition (EMT). Utilizing mouse lines with cephalic epithelium-specific Pbx loss exhibiting CL/P, we demonstrate that these cellular behaviors are Pbx dependent, as is the transcriptional regulation of the EMT driver Snail1. Furthermore, in the embryo, the majority of epithelial cells expressing high levels of Snail1 do not undergo apoptosis. Pbx1 loss- and gain-of-function in a tractable epithelial culture system revealed that Pbx1 is both necessary and sufficient for EMT induction. This study establishes that Pbx-dependent EMT programs mediate murine upper lip/primary palate morphogenesis and fusion via regulation of Snail1. Of note, the EMT signatures observed in the embryo are mirrored in the epithelial culture system.

De novo, deleterious sequence variants that alter the transcriptional activity of the homeoprotein PBX1 are associated with intellectual disability and pleiotropic developmental defects.

We present eight patients with de novo, deleterious sequence variants in the PBX1 gene. PBX1 encodes a three amino acid loop extension (TALE) homeodomain transcription factor that forms multimeric complexes with TALE and HOX proteins to regulate target gene transcription during development. As previously reported, Pbx1 homozygous mutant mice (Pbx1-/-) develop malformations and hypoplasia or aplasia of multiple organs, including the craniofacial skeleton, ear, branchial arches, heart, lungs, diaphragm, gut, kidneys, and gonads. Clinical findings similar to those in Pbx mutant mice were observed in all patients with varying expressivity and severity, including external ear anomalies, abnormal branchial arch derivatives, heart malformations, diaphragmatic hernia, renal hypoplasia and ambiguous genitalia. All patients but one had developmental delays. Previously reported patients with congenital anomalies affecting the kidney and urinary tract exhibited deletions and loss of function variants in PBX1. The sequence variants in our cases included missense substitutions adjacent to the PBX1 homeodomain (p.Arg184Pro, p.Met224Lys, and p.Arg227Pro) or within the homeodomain (p.Arg234Pro, and p.Arg235Gln), whereas p.Ser262Glnfs*2, and p.Arg288* yielded truncated PBX1 proteins. Functional studies on five PBX1 sequence variants revealed perturbation of intrinsic, PBX-dependent transactivation ability and altered nuclear translocation, suggesting abnormal interactions between mutant PBX1 proteins and wild-type TALE or HOX cofactors. It is likely that the mutations directly affect the transcription of PBX1 target genes to impact embryonic development. We conclude that deleterious sequence variants in PBX1 cause intellectual disability and pleiotropic malformations resembling those in Pbx1 mutant mice, arguing for strong conservation of gene function between these two species.

Toward Microsurgical Correction of Cleft Lip Ex Utero through Restoration of Craniofacial Developmental Programs.

BACKGROUND: Cleft lip with or without cleft palate is present in approximately one in 500 to 700 live births, representing the most common congenital craniofacial anomaly. Previously, the authors developed a unique murine model with compound Pbx deficiency that exhibits fully penetrant cleft lip with or without cleft palate. To investigate the possibility of tissue repair at an early gestational stage, the authors designed a minimally invasive surgical approach suitable for intrauterine repair using Wnt9b-soaked collagen microspheres to restore craniofacial developmental programs for cleft correction. METHODS: Collagen microspheres with diameters ranging from 20 to 50 µm were fabricated to serve as a delivery vehicle for Wnt9b. At gestational day 11.5, wild-type and Pbx-deficient murine embryos were isolated. Microspheres soaked in murine purified Wnt9b protein were microsurgically implanted at the midface lambdoidal junction. Embryos were cultured in a 37°C modified whole-embryo culture system. RESULTS: Targeted release of Wnt9b resulted in augmented Wnt expression at the lambdoidal junction. Microsurgical implantation of Wnt9b-soaked microspheres resulted in cleft correction in 27.1 percent of the Pbx-deficient embryos. The difference in the ratio of the areas of clefting between implanted and nonimplanted embryos was significant (p < 0.05). CONCLUSIONS: Ex utero correction of cleft lip with or without cleft palate in the authors' murine model by means of microsurgical intervention and targeted delivery of Wnt proteins is an innovative and promising strategy. Although further refinement and optimization of this technique will be required to improve efficacy, the authors believe that this approach will open new avenues toward unconventional prenatal interventions for patients with cleft lip with or without cleft palate, and provide future approaches for prenatal repair of other congenital head and neck disorders.

Spleen hypoplasia leads to abnormal stress hematopoiesis in mice with loss of Pbx homeoproteins in splenic mesenchyme.

The spleen plays critical roles in immunity and also provides a permissive microenvironment for hematopoiesis. Previous studies have reported that the TALE-class homeodomain transcription factor Pbx1 is essential in hematopoietic stem and progenitor cells (HSPCs) for stem cell maintenance and progenitor expansion. However, the role of Pbx1 in the hematopoietic niche has not been investigated. Here we explored the effects that genetic perturbation of the splenic mesenchymal niche has on hematopoiesis upon loss of members of the Pbx family of homeoproteins. Splenic mesenchyme-specific inactivation of Pbx1 (SKO) on a Pbx2- or Pbx3-deficient genetic background (DKO) resulted in abnormal development of the spleen, which is dysmorphic and severely hypoplastic. This phenotype, in turn, affected the number of HSPCs in the fetal and adult spleen at steady state, as well as markedly impairing the kinetics of hematopoietic regeneration in adult mice after sub-lethal and lethal myelosuppressive irradiation. Spleens of mice with compound Pyx deficiency 8 days following sublethal irradiation displayed significant downregulation of multiple cytokine-encoding genes, including KitL/SCF, Cxcl12/SDF-1, IL-3, IL-4, GM-CSF/Csf2 IL-10, and Igf-1, compared with controls. KitL/SCF and Cxcl12/SDF-1 were recently shown to play key roles in the splenic niche in response to various haematopoietic stresses such as myeloablation, blood loss, or pregnancy. Our results demonstrate that, in addition to their intrinsic roles in HSPCs, non-cell autonomous functions of Pbx factors within the splenic niche contribute to the regulation of hematopoiesis, at least in part via the control of KitL/SCF and Cxcl12/SDF-1. Furthermore, our study establishes that abnormal spleen development and hypoplasia have deleterious effects on the efficiency of hematopoietic recovery after bone marrow injury.

A novel miRNA-mediated STOP sign in lung cancer: miR-340 inhibits the proliferation of lung cancer cells through p27(KIP1).

Oncosuppressor miRNAs inhibit cancer cell proliferation by targeting key components of the cell cycle machinery. In our recent report we showed that miR-340 is a novel tumor suppressor in non-small cell lung cancer. miR-340 inhibits neoplastic cell proliferation and induces p27(KIP1) by targeting multiple translational and post-translational regulators of this cyclin-dependent kinase inhibitor.

ESCRT-II/Vps25 constrains digit number by endosome-mediated selective modulation of FGF-SHH signaling.

Sorting and degradation of receptors and associated signaling molecules maintain homeostasis of conserved signaling pathways during cell specification and tissue development. Yet, whether machineries that sort signaling proteins act preferentially on different receptors and ligands in different contexts remains mysterious. Here, we show that Vacuolar protein sorting 25, Vps25, a component of ESCRT-II (Endosomal Sorting Complex Required for Transport II), directs preferential endosome-mediated modulation of FGF signaling in limbs. By ENU-induced mutagenesis, we isolated a polydactylous mouse line carrying a hypomorphic mutation of Vps25 (Vps25(ENU)). Unlike Vps25-null embryos we generated, Vps25(ENU/ENU) mutants survive until late gestation. Their limbs display FGF signaling enhancement and consequent hyperactivation of the FGF-SHH feedback loop causing polydactyly, whereas WNT and BMP signaling remain unperturbed. Notably, Vps25(ENU/ENU) Mouse Embryonic Fibroblasts exhibit aberrant FGFR trafficking and degradation; however, SHH signaling is unperturbed. These studies establish that the ESCRT-II machinery selectively limits FGF signaling in vertebrate skeletal patterning.

Transcription factor PREP1 induces EMT and metastasis by controlling the TGF-ß-SMAD3 pathway in non-small cell lung adenocarcinoma.

Pre-B-cell leukemia homeobox (Pbx)-regulating protein-1 (Prep1) is a ubiquitous homeoprotein involved in early development, genomic stability, insulin sensitivity, and hematopoiesis. Previously we have shown that Prep1 is a haploinsufficient tumor suppressor that inhibits neoplastic transformation by competing with myeloid ecotropic integration site 1 for binding to the common heterodimeric partner Pbx1. Epithelial-mesenchymal transition (EMT) is controlled by complex networks of proinvasive transcription factors responsive to paracrine factors such as TGF-ß. Here we show that, in addition to inhibiting primary tumor growth, PREP1 is a novel EMT inducer and prometastatic transcription factor. In human non-small cell lung cancer (NSCLC) cells, PREP1 overexpression is sufficient to trigger EMT, whereas PREP1 down-regulation inhibits the induction of EMT in response to TGF-ß. PREP1 modulates the cellular sensitivity to TGF-ß by inducing the small mothers against decapentaplegic homolog 3 (SMAD3) nuclear translocation through mechanisms dependent, at least in part, on PREP1-mediated transactivation of a regulatory element in the SMAD3 first intron. Along with the stabilization and accumulation of PBX1, PREP1 induces the expression of multiple activator protein 1 components including the proinvasive Fos-related antigen 1 (FRA-1) oncoprotein. Both FRA-1 and PBX1 are required for the mesenchymal changes triggered by PREP1 in lung tumor cells. Finally, we show that the PREP1-induced mesenchymal transformation correlates with significantly increased lung colonization by cells overexpressing PREP1. Accordingly, we have detected PREP1 accumulation in a large number of human brain metastases of various solid tumors, including NSCLC. These findings point to a novel role of the PREP1 homeoprotein in the control of the TGF-ß pathway, EMT, and metastasis in NSCLC.

miR-204 targeting of Ankrd13A controls both mesenchymal neural crest and lens cell migration.

Loss of cell adhesion and enhancement of cell motility contribute to epithelial-to-mesenchymal transition during development. These processes are related to a) rearrangement of cell-cell and cell-substrate adhesion molecules; b) cross talk between extra-cellular matrix and internal cytoskeleton through focal adhesion molecules. Focal adhesions are stringently regulated transient structures implicated in cell adhesion, spreading and motility during tissue development. Importantly, despite the extensive elucidation of the molecular composition of focal adhesions, the complex regulation of their dynamics is largely unclear. Here, we demonstrate, using live-imaging in medaka, that the microRNA miR-204 promotes both mesenchymal neural crest and lens cell migration and elongation. Overexpression of miR-204 results in upregulated cell motility, while morpholino-mediated ablation of miR-204 activity causes abnormal lens morphogenesis and neural crest cell mislocalization. Using a variety of in vivo and in vitro approaches, we demonstrate that these actions are mediated by the direct targeting of the Ankrd13A gene, which in turn controls focal cell adhesion formation and distribution. Significantly, in vivo restoration of abnormally elevated levels of Ankrd13A resulting from miR-204 inactivation rescued the aberrant lens phenotype in medaka fish. These data uncover, for the first time in vivo, the role of a microRNA in developmental control of mesenchymal cell migration and highlight miR-204 as a "master regulator" of the molecular networks that regulate lens morphogenesis in vertebrates.