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 common cis-regulatory variant impacts normal-range and disease-associated human facial shape through regulation of PKDCC during chondrogenesis.

Genome-wide association studies (GWAS) identified thousands of genetic variants linked to phenotypic traits and disease risk. However, mechanistic understanding of how GWAS variants influence complex morphological traits and can, in certain cases, simultaneously confer normal-range phenotypic variation and disease predisposition, is still largely lacking. Here, we focus on rs6740960, a single nucleotide polymorphism (SNP) at the 2p21 locus, which in GWAS studies has been associated both with normal-range variation in jaw shape and with an increased risk of non-syndromic orofacial clefting. Using in vitro derived embryonic cell types relevant for human facial morphogenesis, we show that this SNP resides in an enhancer that regulates chondrocytic expression of PKDCC - a gene encoding a tyrosine kinase involved in chondrogenesis and skeletal development. In agreement, we demonstrate that the rs6740960 SNP is sufficient to confer chondrocyte-specific differences in PKDCC expression. By deploying dense landmark morphometric analysis of skull elements in mice, we show that changes in Pkdcc dosage are associated with quantitative changes in the maxilla, mandible, and palatine bone shape that are concordant with the facial phenotypes and disease predisposition seen in humans. We further demonstrate that the frequency of the rs6740960 variant strongly deviated among different human populations, and that the activity of its cognate enhancer diverged in hominids. Our study provides a mechanistic explanation of how a common SNP can mediate normal-range and disease-associated morphological variation, with implications for the evolution of human facial features.

Conserved enhancers control notochord expression of vertebrate Brachyury.

The cell type-specific expression of key transcription factors is central to development and disease. Brachyury/T/TBXT is a major transcription factor for gastrulation, tailbud patterning, and notochord formation; however, how its expression is controlled in the mammalian notochord has remained elusive. Here, we identify the complement of notochord-specific enhancers in the mammalian Brachyury/T/TBXT gene. Using transgenic assays in zebrafish, axolotl, and mouse, we discover three conserved Brachyury-controlling notochord enhancers, T3, C, and I, in human, mouse, and marsupial genomes. Acting as Brachyury-responsive, auto-regulatory shadow enhancers, in cis deletion of all three enhancers in mouse abolishes Brachyury/T/Tbxt expression selectively in the notochord, causing specific trunk and neural tube defects without gastrulation or tailbud defects. The three Brachyury-driving notochord enhancers are conserved beyond mammals in the brachyury/tbxtb loci of fishes, dating their origin to the last common ancestor of jawed vertebrates. Our data define the vertebrate enhancers for Brachyury/T/TBXTB notochord expression through an auto-regulatory mechanism that conveys robustness and adaptability as ancient basis for axis development.

ESCRT-dependent control of craniofacial morphogenesis with concomitant perturbation of NOTCH signaling.

Craniofacial development is orchestrated by transcription factor-driven regulatory networks, epigenetic modifications, and signaling pathways. Signaling molecules and their receptors rely on endo-lysosomal trafficking to prevent accumulation on the plasma membrane. ESCRT (Endosomal Sorting Complexes Required for Transport) machinery is recruited to endosomal membranes enabling degradation of such endosomal cargoes. Studies in vitro and in invertebrate models established the requirements of the ESCRT machinery in membrane remodeling, endosomal trafficking, and lysosomal degradation of activated membrane receptors. However, investigations during vertebrate development have been scarce. By ENU-induced mutagenesis, we isolated a mouse line, Vps25ENU/ENU, carrying a hypomorphic allele of the ESCRT-II component Vps25, with craniofacial anomalies resembling features of human congenital syndromes. Here, we assessed the spatiotemporal dynamics of Vps25 and additional ESCRT-encoding genes during murine development. We show that these genes are ubiquitously expressed although enriched in discrete domains of the craniofacial complex, heart, and limbs. ESCRT-encoding genes, including Vps25, are expressed in both cranial neural crest-derived mesenchyme and epithelium. Unlike constitutive ESCRT mutants, Vps25ENU/ENU embryos display late lethality. They exhibit hypoplastic lower jaw, stunted snout, dysmorphic ear pinnae, and secondary palate clefting. Thus, we provide the first evidence for critical roles of ESCRT-II in craniofacial morphogenesis and report perturbation of NOTCH signaling in craniofacial domains of Vps25ENU/ENU embryos. Given the known roles of NOTCH signaling in the developing cranium, and notably the lower jaw, we propose that the NOTCH pathway partly mediates the craniofacial defects of Vps25ENU/ENU mouse embryos.

A spatio-temporally constrained gene regulatory network directed by PBX1/2 acquires limb patterning specificity via HAND2.

A lingering question in developmental biology has centered on how transcription factors with widespread distribution in vertebrate embryos can perform tissue-specific functions. Here, using the murine hindlimb as a model, we investigate the elusive mechanisms whereby PBX TALE homeoproteins, viewed primarily as HOX cofactors, attain context-specific developmental roles despite ubiquitous presence in the embryo. We first demonstrate that mesenchymal-specific loss of PBX1/2 or the transcriptional regulator HAND2 generates similar limb phenotypes. By combining tissue-specific and temporally controlled mutagenesis with multi-omics approaches, we reconstruct a gene regulatory network (GRN) at organismal-level resolution that is collaboratively directed by PBX1/2 and HAND2 interactions in subsets of posterior hindlimb mesenchymal cells. Genome-wide profiling of PBX1 binding across multiple embryonic tissues further reveals that HAND2 interacts with subsets of PBX-bound regions to regulate limb-specific GRNs. Our research elucidates fundamental principles by which promiscuous transcription factors cooperate with cofactors that display domain-restricted localization to instruct tissue-specific developmental programs.

Conserved enhancer logic controls the notochord expression of vertebrate Brachyury.

The cell type-specific expression of key transcription factors is central to development. Brachyury/T/TBXT is a major transcription factor for gastrulation, tailbud patterning, and notochord formation; however, how its expression is controlled in the mammalian notochord has remained elusive. Here, we identify the complement of notochord-specific enhancers in the mammalian Brachyury/T/TBXT gene. Using transgenic assays in zebrafish, axolotl, and mouse, we discover three Brachyury-controlling notochord enhancers T3, C, and I in human, mouse, and marsupial genomes. Acting as Brachyury-responsive, auto-regulatory shadow enhancers, deletion of all three enhancers in mouse abolishes Brachyury/T expression selectively in the notochord, causing specific trunk and neural tube defects without gastrulation or tailbud defects. Sequence and functional conservation of Brachyury-driving notochord enhancers with the brachyury/tbxtb loci from diverse lineages of fishes dates their origin to the last common ancestor of jawed vertebrates. Our data define the enhancers for Brachyury/T/TBXTB notochord expression as ancient mechanism in axis development.

Shaping faces: genetic and epigenetic control of craniofacial morphogenesis.

Major differences in facial morphology distinguish vertebrate species. Variation of facial traits underlies the uniqueness of human individuals, and abnormal craniofacial morphogenesis during development leads to birth defects that significantly affect quality of life. Studies during the past 40 years have advanced our understanding of the molecular mechanisms that establish facial form during development, highlighting the crucial roles in this process of a multipotent cell type known as the cranial neural crest cell. In this Review, we discuss recent advances in multi-omics and single-cell technologies that enable genes, transcriptional regulatory networks and epigenetic landscapes to be closely linked to the establishment of facial patterning and its variation, with an emphasis on normal and abnormal craniofacial morphogenesis. Advancing our knowledge of these processes will support important developments in tissue engineering, as well as the repair and reconstruction of the abnormal craniofacial complex.

Generation of Schwann cell derived melanocytes from hPSCs identifies pro-metastatic factors in melanoma.

The neural crest (NC) is highly multipotent and generates diverse lineages in the developing embryo. However, spatiotemporally distinct NC populations display differences in fate potential, such as increased gliogenic and parasympathetic potential from later migrating, nerve-associated Schwann cell precursors (SCPs). Interestingly, while melanogenic potential is shared by both early migrating NC and SCPs, differences in melanocyte identity resulting from differentiation through these temporally distinct progenitors have not been determined. Here, we leverage a human pluripotent stem cell (hPSC) model of NC temporal patterning to comprehensively characterize human NC heterogeneity, fate bias, and lineage development. We captured the transition of NC differentiation between temporally and transcriptionally distinct melanogenic progenitors and identified modules of candidate transcription factor and signaling activity associated with this transition. For the first time, we established a protocol for the directed differentiation of melanocytes from hPSCs through a SCP intermediate, termed trajectory 2 (T2) melanocytes. Leveraging an existing protocol for differentiating early NC-derived melanocytes, termed trajectory 1 (T1), we performed the first comprehensive comparison of transcriptional and functional differences between these distinct melanocyte populations, revealing differences in pigmentation and unique expression of transcription factors, ligands, receptors and surface markers. We found a significant link between the T2 melanocyte transcriptional signature and decreased survival in melanoma patients in the cancer genome atlas (TCGA). We performed an in vivo CRISPRi screen of T1 and T2 melanocyte signature genes in a human melanoma cell line and discovered several T2-specific markers that promote lung metastasis in mice. We further demonstrated that one of these factors, SNRPB, regulates the splicing of transcripts involved in metastasis relevant functions such as migration, cell adhesion and proliferation. Overall, this study identifies distinct developmental trajectories as a source of diversity in melanocytes and implicates the unique molecular signature of SCP-derived melanocytes in metastatic melanoma.

Loss of Extreme Long-Range Enhancers in Human Neural Crest Drives a Craniofacial Disorder.

Non-coding mutations at the far end of a large gene desert surrounding the SOX9 gene result in a human craniofacial disorder called Pierre Robin sequence (PRS). Leveraging a human stem cell differentiation model, we identify two clusters of enhancers within the PRS-associated region that regulate SOX9 expression during a restricted window of facial progenitor development at distances up to 1.45 Mb. Enhancers within the 1.45 Mb cluster exhibit highly synergistic activity that is dependent on the Coordinator motif. Using mouse models, we demonstrate that PRS phenotypic specificity arises from the convergence of two mechanisms: confinement of Sox9 dosage perturbation to developing facial structures through context-specific enhancer activity and heightened sensitivity of the lower jaw to Sox9 expression reduction. Overall, we characterize the longest-range human enhancers involved in congenital malformations, directly demonstrate that PRS is an enhanceropathy, and illustrate how small changes in gene expression can lead to morphological variation.

FaceBase 3: analytical tools and FAIR resources for craniofacial and dental research.

The FaceBase Consortium was established by the National Institute of Dental and Craniofacial Research in 2009 as a 'big data' resource for the craniofacial research community. Over the past decade, researchers have deposited hundreds of annotated and curated datasets on both normal and disordered craniofacial development in FaceBase, all freely available to the research community on the FaceBase Hub website. The Hub has developed numerous visualization and analysis tools designed to promote integration of multidisciplinary data while remaining dedicated to the FAIR principles of data management (findability, accessibility, interoperability and reusability) and providing a faceted search infrastructure for locating desired data efficiently. Summaries of the datasets generated by the FaceBase projects from 2014 to 2019 are provided here. FaceBase 3 now welcomes contributions of data on craniofacial and dental development in humans, model organisms and cell lines. Collectively, the FaceBase Consortium, along with other NIH-supported data resources, provide a continuously growing, dynamic and current resource for the scientific community while improving data reproducibility and fulfilling data sharing requirements.

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.

Functional characterization of a novel PBX1 de novo missense variant identified in a patient with syndromic congenital heart disease.

Pre-B cell leukemia factor 1 (PBX1) is an essential developmental transcription factor, mutations in which have recently been associated with CAKUTHED syndrome, characterized by multiple congenital defects including congenital heart disease (CHD). During analysis of a whole-exome-sequenced cohort of heterogeneous CHD patients, we identified a de novo missense variant, PBX1:c.551G>C p.R184P, in a patient with tetralogy of Fallot with absent pulmonary valve and extra-cardiac phenotypes. Functional analysis of this variant by creating a CRISPR-Cas9 gene-edited mouse model revealed multiple congenital anomalies. Congenital heart defects (persistent truncus arteriosus and ventricular septal defect), hypoplastic lungs, hypoplastic/ectopic kidneys, aplastic adrenal glands and spleen, as well as atretic trachea and palate defects were observed in the homozygous mutant embryos at multiple stages of development. We also observed developmental anomalies in a proportion of heterozygous embryos, suggestive of a dominant mode of inheritance. Analysis of gene expression and protein levels revealed that although Pbx1 transcripts are higher in homozygotes, amounts of PBX1 protein are significantly decreased. Here, we have presented the first functional model of a missense PBX1 variant and provided strong evidence that p.R184P is disease-causal. Our findings also expand the phenotypic spectrum associated with pathogenic PBX1 variants in both humans and mice.

'Building a perfect body': control of vertebrate organogenesis by PBX-dependent regulatory networks.

Pbx genes encode transcription factors that belong to the TALE (three-amino-acid loop extension) superclass of homeodomain proteins. We have witnessed a surge in information about the roles of this gene family as leading actors in the transcriptional control of development. PBX proteins represent a clear example of how transcription factors can regulate developmental processes by combinatorial properties, acting within multimeric complexes to implement activation or repression of transcription depending on their interaction partners. Here, we revisit long-emphasized functions of PBX transcription factors as cofactors for HOX proteins, major architects of the body plan. We further discuss new knowledge on roles of PBX proteins in different developmental contexts as upstream regulators of Hox genes-as factors that interact with non-HOX proteins and can work independently of HOX-as well as potential pioneer factors. Committed to building a perfect body, PBX proteins govern regulatory networks that direct essential morphogenetic processes and organogenesis in vertebrate development. Perturbations of PBX-dependent networks can cause human congenital disease and cancer.

Genetics of scapula and pelvis development: An evolutionary perspective.

In tetrapods, the scapular and pelvic girdles perform the important function of anchoring the limbs to the trunk of the body and facilitating the movement of each appendage. This shared function, however, is one of relatively few similarities between the scapula and pelvis, which have significantly different morphologies, evolutionary histories, embryonic origins, and underlying genetic pathways. The scapula evolved in jawless fish prior to the pelvis, and its embryonic development is unique among bones in that it is derived from multiple progenitor cell populations, including the dermomyotome, somatopleure, and neural crest. Conversely, the pelvis evolved several million years later in jawed fish, and it develops from an embryonic somatopleuric cell population. The genetic networks controlling the formation of the pelvis and scapula also share similarities and differences, with a number of genes shaping only one or the other, while other gene products such as PBX transcription factors act as hierarchical developmental regulators of both girdle structures. Here, we provide a detailed review of the cellular processes and genetic networks underlying pelvis and scapula formation in tetrapods, while also highlighting unanswered questions about girdle evolution and development.

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.

Pbx loss in cranial neural crest, unlike in epithelium, results in cleft palate only and a broader midface.

Orofacial clefting represents the most common craniofacial birth defect. Cleft lip with or without cleft palate (CL/P) is genetically distinct from cleft palate only (CPO). Numerous transcription factors (TFs) regulate normal development of the midface, comprising the premaxilla, maxilla and palatine bones, through control of basic cellular behaviors. Within the Pbx family of genes encoding Three Amino-acid Loop Extension (TALE) homeodomain-containing TFs, we previously established that in the mouse, Pbx1 plays a preeminent role in midfacial morphogenesis, and Pbx2 and Pbx3 execute collaborative functions in domains of coexpression. We also reported that Pbx1 loss from cephalic epithelial domains, on a Pbx2- or Pbx3-deficient background, results in CL/P via disruption of a regulatory network that controls apoptosis at the seam of frontonasal and maxillary process fusion. Conversely, Pbx1 loss in cranial neural crest cell (CNCC)-derived mesenchyme on a Pbx2-deficient background results in CPO, a phenotype not yet characterized. In this study, we provide in-depth analysis of PBX1 and PBX2 protein localization from early stages of midfacial morphogenesis throughout development of the secondary palate. We further establish CNCC-specific roles of PBX TFs and describe the developmental abnormalities resulting from their loss in the murine embryonic secondary palate. Additionally, we compare and contrast the phenotypes arising from PBX1 loss in CNCC with those caused by its loss in the epithelium and show that CNCC-specific Pbx1 deletion affects only later secondary palate morphogenesis. Moreover, CNCC mutants exhibit perturbed rostro-caudal organization and broadening of the midfacial complex. Proliferation defects are pronounced in CNCC mutants at gestational day (E)12.5, suggesting altered proliferation of mutant palatal progenitor cells, consistent with roles of PBX factors in maintaining progenitor cell state. Although the craniofacial skeletal abnormalities in CNCC mutants do not result from overt patterning defects, osteogenesis is delayed, underscoring a critical role of PBX factors in CNCC morphogenesis and differentiation. Overall, the characterization of tissue-specific Pbx loss-of-function mouse models with orofacial clefting establishes these strains as unique tools to further dissect the complexities of this congenital craniofacial malformation. This study closely links PBX TALE homeodomain proteins to the variation in maxillary shape and size that occurs in pathological settings and during evolution of midfacial morphology.

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.