An Alternative Splicing Program for Mouse Craniofacial Development.

Alternative splicing acts as a fundamental mechanism to increase the number of functional transcripts that can be derived from the genome - and its appropriate regulation is required to direct normal development, differentiation, and physiology, in many species. Recent studies have highlighted that mutation of splicing factors, resulting in the disruption of alternative splicing, can have profound consequences for mammalian craniofacial development. However, there has been no systematic analysis of the dynamics of differential splicing during the critical period of face formation with respect to age, tissue layer, or prominence. Here we used deep RNA sequencing to profile transcripts expressed in the developing mouse face for both ectodermal and mesenchymal tissues from the three facial prominences at critical ages for facial development, embryonic days 10.5, 11.5, and 12.5. We also derived separate expression data from the nasal pit relating to the differentiation of the olfactory epithelium for a total of 60 independent datasets. Analysis of these datasets reveals the differential expression of multiple genes, but we find a similar number of genes are regulated only via differential splicing, indicating that alternative splicing is a major source of transcript diversity during facial development. Notably, splicing changes between tissue layers and over time are more prevalent than between prominences, with exon skipping the most common event. We next examined how the variation in splicing correlated with the expression of RNA binding proteins across the various datasets. Further, we assessed how binding sites for splicing regulatory molecules mapped with respect to intron exon boundaries. Overall these studies help define an alternative splicing regulatory program that has important consequences for facial development.

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.

The molecular anatomy of mammalian upper lip and primary palate fusion at single cell resolution.

The mammalian lip and primary palate form when coordinated growth and morphogenesis bring the nasal and maxillary processes into contact, and the epithelia co-mingle, remodel and clear from the fusion site to allow mesenchyme continuity. Although several genes required for fusion have been identified, an integrated molecular and cellular description of the overall process is lacking. Here, we employ single cell RNA sequencing of the developing mouse face to identify ectodermal, mesenchymal and endothelial populations associated with patterning and fusion of the facial prominences. This analysis indicates that key cell populations at the fusion site exist within the periderm, basal epithelial cells and adjacent mesenchyme. We describe the expression profiles that make each population unique, and the signals that potentially integrate their behaviour. Overall, these data provide a comprehensive high-resolution description of the various cell populations participating in fusion of the lip and primary palate, as well as formation of the nasolacrimal groove, and they furnish a powerful resource for those investigating the molecular genetics of facial development and facial clefting that can be mined for crucial mechanistic information concerning this prevalent human birth defect.

Systems biology of facial development: contributions of ectoderm and mesenchyme.

The rapid increase in gene-centric biological knowledge coupled with analytic approaches for genomewide data integration provides an opportunity to develop systems-level understanding of facial development. Experimental analyses have demonstrated the importance of signaling between the surface ectoderm and the underlying mesenchyme are coordinating facial patterning. However, current transcriptome data from the developing vertebrate face is dominated by the mesenchymal component, and the contributions of the ectoderm are not easily identified. We have generated transcriptome datasets from critical periods of mouse face formation that enable gene expression to be analyzed with respect to time, prominence, and tissue layer. Notably, by separating the ectoderm and mesenchyme we considerably improved the sensitivity compared to data obtained from whole prominences, with more genes detected over a wider dynamic range. From these data we generated a detailed description of ectoderm-specific developmental programs, including pan-ectodermal programs, prominence- specific programs and their temporal dynamics. The genes and pathways represented in these programs provide mechanistic insights into several aspects of ectodermal development. We also used these data to identify co-expression modules specific to facial development. We then used 14 co-expression modules enriched for genes involved in orofacial clefts to make specific mechanistic predictions about genes involved in tongue specification, in nasal process patterning and in jaw development. Our multidimensional gene expression dataset is a unique resource for systems analysis of the developing face; our co-expression modules are a resource for predicting functions of poorly annotated genes, or for predicting roles for genes that have yet to be studied in the context of facial development; and our analytic approaches provide a paradigm for analysis of other complex developmental programs.

MicroRNA Profiling during Craniofacial Development: Potential Roles for Mir23b and Mir133b.

Defects in mid-facial development, including cleft lip/palate, account for a large number of human birth defects annually. In many cases, aberrant gene expression results in either a reduction in the number of neural crest cells (NCCs) that reach the frontonasal region and form much of the facial skeleton or subsequent failure of NCC patterning and differentiation into bone and cartilage. While loss of gene expression is often associated with developmental defects, aberrant upregulation of expression can also be detrimental. microRNAs (miRNAs) are a class of non-coding RNAs that normally repress gene expression by binding to recognition sequences located in the 3' UTR of target mRNAs. miRNAs play important roles in many developmental systems, including midfacial development. Here, we take advantage of high throughput RNA sequencing (RNA-seq) from different tissues of the developing mouse midface to interrogate the miRs that are expressed in the midface and select a subset for further expression analysis. Among those examined, we focused on four that showed the highest expression level in in situ hybridization analysis. Mir23b and Mir24.1 are specifically expressed in the developing mouse frontonasal region, in addition to areas in the perichondrium, tongue musculature and cranial ganglia. Mir23b is also expressed in the palatal shelves and in anterior epithelium of the palate. In contrast, Mir133b and Mir128.2 are mainly expressed in head and trunk musculature. Expression analysis of mir23b and mir133b in zebrafish suggests that mir23b is expressed in the pharyngeal arch, otic vesicle, and trunk muscle while mir133b is similarly expressed in head and trunk muscle. Functional analysis by overexpression of mir23b in zebrafish leads to broadening of the ethmoid plate and aberrant cartilage structures in the viscerocranium, while overexpression of mir133b causes a reduction in ethmoid plate size and a significant midfacial cleft. These data illustrate that miRs are expressed in the developing midface and that Mir23b and Mir133b may have roles in this developmental process.

The FaceBase Consortium: a comprehensive resource for craniofacial researchers.

The FaceBase Consortium, funded by the National Institute of Dental and Craniofacial Research, National Institutes of Health, is designed to accelerate understanding of craniofacial developmental biology by generating comprehensive data resources to empower the research community, exploring high-throughput technology, fostering new scientific collaborations among researchers and human/computer interactions, facilitating hypothesis-driven research and translating science into improved health care to benefit patients. The resources generated by the FaceBase projects include a number of dynamic imaging modalities, genome-wide association studies, software tools for analyzing human facial abnormalities, detailed phenotyping, anatomical and molecular atlases, global and specific gene expression patterns, and transcriptional profiling over the course of embryonic and postnatal development in animal models and humans. The integrated data visualization tools, faceted search infrastructure, and curation provided by the FaceBase Hub offer flexible and intuitive ways to interact with these multidisciplinary data. In parallel, the datasets also offer unique opportunities for new collaborations and training for researchers coming into the field of craniofacial studies. Here, we highlight the focus of each spoke project and the integration of datasets contributed by the spokes to facilitate craniofacial research.

A survey of software for genome-wide discovery of differential splicing in RNA-Seq data.

Alternative splicing is a major contributor to cellular diversity. Therefore the identification and quantification of differentially spliced transcripts in genome-wide transcript analysis is an important consideration. Here, I review the software available for analysis of RNA-Seq data for differential splicing and discuss intrinsic challenges for differential splicing analyses. Three approaches to differential splicing analysis are described, along with their associated software implementations, their strengths, limitations, and caveats. Suggestions for future work include more extensive experimental validation to assess accuracy of the software predictions and consensus formats for outputs that would facilitate visualizations, data exchange, and downstream analyses.

The TRC8 ubiquitin ligase is sterol regulated and interacts with lipid and protein biosynthetic pathways.

TRC8/RNF139 encodes an endoplasmic reticulum-resident E3 ubiquitin ligase that inhibits growth in a RING- and ubiquitylation-dependent manner. TRC8 also contains a predicted sterol-sensing domain. Here, we report that TRC8 protein levels are sterol responsive and that it binds and stimulates ubiquitylation of the endoplasmic reticulum anchor protein INSIG. Induction of TRC8 destabilized the precursor forms of the transcription factors SREBP-1 and SREBP-2. Loss of SREBP precursors was proteasome dependent, required a functional RING domain, occurred without generating processed nuclear forms, and suppressed SREBP target genes. TRC8 knockdown had opposite effects in sterol-deprived cells. In Drosophila, growth inhibition by DTrc8 was genetically suppressed by loss of specific Mprlp, Padlp N-terminal domain-containing proteins found in the COP9 signalosome and eIF3. DTrc8 genetically and physically interacted with two eIF3 subunits: eIF3f and eIF3h. Coimmunoprecipitation experiments confirmed these interactions in mammalian cells, and TRC8 overexpression suppressed polysome profiles. Moreover, high-molecular weight ubiquitylated proteins were observed in eIF3 immunoprecipitations from TRC8-overexpressing cells. Thus, TRC8 function may provide a regulatory link between the lipid and protein biosynthetic pathways.

Clonal analysis of Hedgehog signaling in Drosophila somatic tissues.

To fully understand how animals develop, it is often necessary to remove the function of a particular gene in a specific cell type or subset of cells. In Drosophila melanogaster, mosaic animals have been widely utilized to study cell fate, growth and patterning, and restriction of cell fate. This chapter describes using FLP recombinase to generate mosaic Drosophila, discussing the chromosomes and cross scheme, how to induce the clones, how to properly identify the appropriate progeny, and how to prepare and analyze the tissues, clones, and phenotypes. It then presents three examples, applying this technique to study Hedgehog signaling. The first example describes moderate-sized costal clones in imaginal discs, using green fluorescent protein (GFP) as a marker and dppLacZ and Engrailed expression as phenotypic reporters. The second describes filling the adult eye with roadkill mutant clones, using white as a marker and scoring morphology. The third describes clonal misexpression of a truncated form of Smoothened, using GFP and yellow as markers.

Roadkill attenuates Hedgehog responses through degradation of Cubitus interruptus.

The final step in Hedgehog (Hh) signal transduction is post-translational regulation of the transcription factor, Cubitus interruptus (Ci). Ci resides in the cytoplasm in a latent form, where Hh regulates its processing into a transcriptional repressor or its nuclear access as a transcriptional activator. Levels of latent Ci are controlled by degradation, with different pathways activated in response to different levels of Hh. Here, we describe the roadkill (rdx) gene, which is expressed in response to Hh. The Rdx protein belongs to a conserved family of proteins that serve as substrate adaptors for Cullin3-mediated ubiquitylation. Overexpression of rdx reduced Ci levels and decreased both transcriptional activation and repression mediated by Ci. Loss of rdx allowed excessive accumulation of Ci. rdx manipulation in the eye revealed a novel role for Hh in the organization and survival of pigment and cone cells. These studies identify rdx as a limiting factor in a feedback loop that attenuates Hh responses through reducing levels of Ci. The existence of human orthologs for Rdx raises the possibility that this novel feedback loop also modulates Hh responses in humans.

Communicating with Hedgehogs.

Signalling by secreted Hedgehog (Hh) proteins is important for the development of many tissues and organs. Damage to components of the Hh signal-transduction pathway can lead to birth defects and cancer. The Hh proteins are distributed in tissues in a gradient, and cells respond to different thresholds of Hh with distinct responses. The cellular machinery that is responsible for the unique molecular mechanisms of Hh signal transduction has been largely conserved during metazoan evolution.

Growth suppression induced by the TRC8 hereditary kidney cancer gene is dependent upon JAB1/CSN5.

TRC8 encodes an E3-ubiquitin ligase disrupted in a family with hereditary renal cell carcinoma (RCC). We previously reported that Drosophila Trc8 (DTrc8) overexpression inhibits growth and that human and fly proteins interact with with the COP9 signalosome (CSN) subunit JAB1/CSN5. However, further mechanistic evidence linking DTrc8 growth suppression to CSN5 was lacking. Here, we show that haploinsufficiency of CSN5, or a T100I point mutation (CSN5(3)), relieved growth suppression by DTrc8, whereas CSN5(1) (E160V) and CSN5(2) (G147D) mutations had no effect. The strength of yeast two-hybrid interactions between DTrc8 and CSN5 were in complete agreement with the observed phenotypes. DTrc8 overexpression resulted in elevated levels of CSN5 and CSN7, but had no effect on NEDD8-modified Cul-1. In contrast to CSN5, heterozygosity for CSN4null had no effect on the DTrc8 phenotype. We also looked for genetic interactions between DTrc8 and other MPN domain proteins in the CSN and 26S proteasome lid. CSN6 haploinsufficiency restored growth, whereas reduction of proteasome subunits RPN8 or RPN11 had no effect. DTrc8 expression increased the level of digitonin-extractable CSN complex, consistent with elevated levels of CSN5 and 7. Our genetic results confirm that DTrc8-induced growth suppression is CSN5 (and CSN6) dependent. While there was no obvious influence on CSN deneddylation activity, the increase in CSN subunits and holocomplex suggests that TRC8 modulates signalosome levels or compartmentalization.

Identification of a functional interaction between the transmembrane protein Smoothened and the kinesin-related protein Costal2.

The hedgehog (Hh) family of morphogens plays important instructional roles in the development of numerous metazoan structures. Consistent with the role Hh homologs play in cell fate determination, aberrant Hh signaling results in numerous human pathologies. Hh signal transduction is initiated when Hh binds to its receptor Patched (Ptc), activating the transmembrane protein Smoothened (Smo). Smo transmits its activation signal to a microtubule-associated Hedgehog signaling complex (HSC). At a minimum, the HSC consists of the Kinesin-related protein Costal2 (Cos2), the protein kinase Fused (Fu), and the transcription factor Cubitus interruptus (Ci). In response to HSC activation, the ratio between repressor and activator forms of Ci is altered, determining the expression levels of various Hh target genes. The steps between Smo activation and signaling to the HSC have not been described. Here, we describe a functional interaction between Smo and Cos2, which is necessary for Hh signaling. We propose that this interaction is direct and allows for activation of Ci in response to Hh. This work fills in the last major gap in our understanding of the Hh signal transduction pathway by suggesting that no intermediate signal is required to connect Smo to the HSC.

Smoothened translates Hedgehog levels into distinct responses.

In the Drosophila wing, Hedgehog is made by cells of the posterior compartment and acts as a morphogen to pattern cells of the anterior compartment. High Hedgehog levels instruct L3/4 intervein fate, whereas lower levels instruct L3 vein fate. Transcriptional responses to Hedgehog are mediated by the balance between repressor and activator forms of Cubitus interruptus, CiR and CiA. Hedgehog regulates this balance through its receptor, Patched, which acts through Smoothened and thence a regulatory complex that includes Fused, Costal, Suppressor of Fused and Cubitus interruptus. It is not known how the Hedgehog signal is relayed from Smoothened to the regulatory complex nor how responses to different levels of Hedgehog are implemented. We have used chimeric and deleted forms of Smoothened to explore the signaling functions of Smoothened. A Frizzled/Smoothened chimera containing the Smo cytoplasmic tail (FFS) can induce the full spectrum of Hedgehog responses but is regulated by Wingless rather than Hedgehog. Smoothened whose cytoplasmic tail is replaced with that of Frizzled (SSF) mimics fused mutants, interfering with high Hedgehog responses but with no effect on low Hedgehog responses. The cytoplasmic tail of Smoothened with no transmembrane or extracellular domains (SmoC) interferes with high Hedgehog responses and allows endogenous Smoothened to constitutively initiate low responses. SmoC mimics costal mutants. Genetic interactions suggest that SSF interferes with high signaling by titrating out Smoothened, whereas SmoC drives constitutive low signaling by titrating out Costal. These data suggest that low and high signaling (1) are qualitatively different, (2) are mediated by distinct configurations of the regulatory complex and (3) are initiated by distinct activities of Smoothened. We present a model where low signaling is initiated when a Costal inhibitory site on the Smoothened cytoplasmic tail shifts the regulatory complex to its low state. High signaling is initiated when cooperating Smoothened cytoplasmic tails activate Costal and Fused, driving the regulatory complex to its high state. Thus, two activities of Smoothened translate different levels of Hedgehog into distinct intracellular responses.

The TRC8 hereditary kidney cancer gene suppresses growth and functions with VHL in a common pathway.

VHL is part of an SCF related E3-ubiquitin ligase complex with 'gatekeeper' function in renal carcinoma. However, no mutations have been identified in VHL interacting proteins in wild type VHL tumors. We previously reported that the TRC8 gene was interrupted by a t(3;8) translocation in a family with hereditary renal and non-medullary thyroid cancer. TRC8 encodes a multi-membrane spanning protein containing a RING-H2 finger with in vitro ubiquitin ligase activity. We isolated the Drosophila homologue, DTrc8, and studied its function by genetic manipulations and a yeast 2-hybrid screen. Human and Drosophila TRC8 proteins localize to the endoplasmic reticulum. Loss of either DTrc8 or DVhl resulted in an identical ventral midline defect. Direct interaction between DTrc8 and DVhl was confirmed by GST-pulldown and co-immunoprecipitation experiments. CSN-5/JAB1 is a component of the COP9 signalosome, recently shown to regulate SCF function. We found that DTrc8 physically interacts with CSN-5 and that human JAB1 localization is dependent on VHL mutant status. Lastly, overexpression of DTrc8 inhibited growth consistent with its presumed role as a tumor suppressor gene. Thus, VHL, TRC8, and JAB1 appear to be linked both physically and functionally and all three may participate in the development of kidney cancer.

Homeotic gene function in the muscles of Drosophila larvae.

The segmental musculature of Drosophila melanogaster larvae consists of 24-30 muscles per segment. Unique patterns of muscles are found in the three thoracic segments and the first and last abdominal segments; the remaining abdominal segments share the same pattern. Mutations in Ultrabithorax (Ubx) cause partial transformation of the muscle pattern of larval abdominal segments towards metathorax. The muscles of the thorax are not affected. In the first two abdominal segments the changes include the loss of at least 11 ;abdominal' muscles and the gain of 11 ;thoracic' muscles. Less extensive transformations are seen in more posterior abdominal segments. Anterobithorax, bithorax, postbithorax and bithoraxoid mutations also induce transformations of the larval musculature. Each allelic group affects a domain that is a subset of the entire Ubx domain but these domains are not restricted to compartments or segments and may extend through as many as five segments. In the muscles the segmental distribution of Ubx antigen correlates with the segments affected by Ubx mutations. The different domains of Ubx in mesoderm and ectoderm argue that the segmental diversity of the muscle pattern is not simply induced by the overlying epidermis and that Ubx function in the mesoderm is required for the correct development of abdominal segments.