Erika Kague

Skeletogenic Fate of Zebrafish Cranial and Trunk Neural Crest

The neural crest (NC) is a major contributor to the vertebrate craniofacial skeleton, detailed in model organisms through embryological and genetic approaches, most notably in chick and mouse. Despite many similarities between these rather distant species, there are also distinct differences in the contribution of the NC, particularly to the calvariae of the skull. Lack of information about other vertebrate groups precludes an understanding of the evolutionary significance of these differences. Study of zebrafish craniofacial development has contributed substantially to understanding of cartilage and bone formation in teleosts, but there is currently little information on NC contribution to the zebrafish skeleton. Here, we employ a two–transgene system based on Cre recombinase to genetically label NC in the zebrafish. We demonstrate NC contribution to cells in the cranial ganglia and peripheral nervous system known to be NC–derived, as well as to a subset of myocardial cells. The indelible labeling also enables us to determine NC contribution to late–forming bones, including the calvariae. We confirm suspected NC origin of cartilage and bones of the viscerocranium, including cartilages such as the hyosymplectic and its replacement bones (hymandibula and symplectic) and membranous bones such as the opercle. The cleithrum develops at the border of NC and mesoderm, and as an ancestral component of the pectoral girdle was predicted to be a hybrid bone composed of both NC and mesoderm tissues. However, we find no evidence of a NC contribution to the cleithrum. Similarly, in the vault of the skull, the parietal bones and the caudal portion of the frontal bones show no evidence of NC contribution. We also determine a NC origin for caudal fin lepidotrichia; the presumption is that these are derived from trunk NC, demonstrating that these cells have the ability to form bone during normal vertebrate development.

Functionally conserved cis-regulatory elements of COL18A1 identified through zebrafish transgenesis

Type XVIII collagen is a component of basement membranes, and expressed prominently in the eye, blood vessels, liver, and the central nervous system. Homozygous mutations in COL18A1 lead to Knobloch Syndrome, characterized by ocular defects and occipital encephalocele. However, relatively little has been described on the role of type XVIII collagen in development, and nothing is known about the regulation of its tissue-specific expression pattern. We have used zebrafish transgenesis to identify and characterize cis-regulatory sequences controlling expression of the human gene. Candidate enhancers were selected from non-coding sequence associated with COL18A1 based on sequence conservation among mammals. Although these displayed no overt conservation with orthologous zebrafish sequences, four regions nonetheless acted as tissue-specific transcriptional enhancers in the zebrafish embryo, and together recapitulated the major aspects of col18a1 expression. Additional post-hoc computational analysis on positive enhancer sequences revealed alignments between mammalian and teleost sequences, which we hypothesize predict the corresponding zebrafish enhancers; for one of these, we demonstrate functional overlap with the orthologous human enhancer sequence. Our results provide important insight into the biological function and regulation of COL18A1, and point to additional sequences that may contribute to complex diseases involving COL18A1. More generally, we show that combining functional data with targeted analyses for phylogenetic conservation can reveal conserved cis-regulatory elements in the large number of cases where computational alignment alone falls short.

Osterix/Sp7 limits cranial bone initiation sites and is required for formation of sutures

During growth, individual skull bones overlap at sutures, where osteoblast differentiation and bone deposition occur. Mutations causing skull malformations have revealed some required genes, but many aspects of suture regulation remain poorly understood. We describe a zebrafish mutation in osterix/sp7, which causes a generalized delay in osteoblast maturation. While most of the skeleton is patterned normally, mutants have specific defects in the anterior skull and upper jaw, and the top of the skull comprises a random mosaic of bones derived from individual initiation sites. Osteoblasts at the edges of the bones are highly proliferative and fail to differentiate, consistent with global changes in gene expression. We propose that signals from the bone itself are required for orderly recruitment of precursor cells and growth along the edges. The delay in bone maturation caused by loss of Sp7 leads to unregulated bone formation, revealing a new mechanism for patterning the skull and sutures.

COL18A1 is highly expressed during human adipocyte differentiation and the SNP c.1136C > T in its "frizzled" motif is associated with obesity in diabetes type 2 patients

Collagen XVIII can generate two fragments, NC11-728 containing a frizzled motif which possibly acts in Wnt signaling and Endostatin, which is cleaved from the NC1 and is a potent inhibitor of angiogenesis. Collagen XVIII and Wnt signaling have recently been associated with adipogenic differentiation and obesity in some animal models, but not in humans. In the present report, we have shown that COL18A1 expression increases during human adipogenic differentiation. We also tested if polymorphisms in the Frizzled (c.1136C>T; Thr379Met) and Endostatin (c.4349G>A; Asp1437Asn) regions contribute towards susceptibility to obesity in patients with type 2 diabetes (113 obese, BMI > or =30; 232 non-obese, BMI < 30) of European ancestry. No evidence of association was observed between the allele c.4349G>A and obesity, but we observed a significantly higher frequency of homozygotes c.1136TT in obese (19.5%) than in non-obese individuals (10.9%) [P = 0.02; OR = 2.0 (95%CI: 1.07-3.73)], suggesting that the allele c.1136T is associated to obesity in a recessive model. This genotype, after controlling for cholesterol, LDL cholesterol, and triglycerides, was independently associated with obesity (P = 0.048), and increases the chance of obesity in 2.8 times. Therefore, our data suggest the involvement of collagen XVIII in human adipogenesis and susceptibility to obesity.

BMPs Regulate msx Gene Expression in the Dorsal Neuroectoderm of Drosophila and Vertebrates by Distinct Mechanisms

In a broad variety of bilaterian species the trunk central nervous system (CNS) derives from three primary rows of neuroblasts. The fates of these neural progenitor cells are determined in part by three conserved transcription factors: vnd/nkx2.2, ind/gsh and msh/msx in Drosophila melanogaster/vertebrates, which are expressed in corresponding non-overlapping patterns along the dorsal-ventral axis. While this conserved suite of “neural identity” gene expression strongly suggests a common ancestral origin for the patterning systems, it is unclear whether the original regulatory mechanisms establishing these patterns have been similarly conserved during evolution. In Drosophila, genetic evidence suggests that Bone Morphogenetic Proteins (BMPs) act in a dosage-dependent fashion to repress expression of neural identity genes. BMPs also play a dose-dependent role in patterning the dorsal and lateral regions of the vertebrate CNS, however, the mechanism by which they achieve such patterning has not yet been clearly established. In this report, we examine the mechanisms by which BMPs act on cis-regulatory modules (CRMs) that control localized expression of the Drosophila msh and zebrafish (Danio rerio) msxB in the dorsal central nervous system (CNS). Our analysis suggests that BMPs act differently in these organisms to regulate similar patterns of gene expression in the neuroectoderm: repressing msh expression in Drosophila, while activating msxB expression in the zebrafish. These findings suggest that the mechanisms by which the BMP gradient patterns the dorsal neuroectoderm have reversed since the divergence of these two ancient lineages.

Evaluation of cis-regulatory function in zebrafish

As increasing numbers of vertebrate genomes are sequenced, comparative genomics offers tremendous promise to unveil mechanisms of transcriptional gene regulation on a large scale. However, the challenge of analysing immense amounts of sequence data and relating primary sequence to function is daunting. Several teleost species occupy crucial niches in the world of comparative genomics, as experimental model organisms of wide utility and living roadmaps of molecular evolution. Extant species have evolved after a teleost-specific genome duplication, and offer the opportunity to examine the evolution of thousands of duplicate gene pairs. Transgenesis in zebrafish is being increasingly employed to functionally examine non-coding sequences, from fish and mammals. Here, we discuss current approaches to the study of gene regulation in teleosts, and the promise of future research.

Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences

The completion of the human genome sequence, along with that of many other species, has highlighted the challenge of ascribing specific function to non coding sequences. One prominent function carried out by the non coding fraction of the genome is to regulate gene transcription; however, there are no effective methods to broadly predict cis-regulatory elements from primary DNA sequence. We have developed an efficient protocol to functionally evaluate potential cis-regulatory elements through zebrafish transgenesis. Our approach offers significant advantages over cell-culture based techniques for developmentally important genes, since it provides information on spatial and temporal gene regulation. Conversely, it is faster and less expensive than similar experiments in transgenic mice, and we routinely apply it to sequences isolated from the human genome. Here we demonstrate our approach to selecting elements for testing based on sequence conservation and our protocol for cloning sequences and microinjecting them into zebrafish embryos.

Craniofrontonasal Syndrome Caused by Introduction of a Novel uATG in the 5′UTR of EFNB1

Craniofrontonasal syndrome (CFNS) is an X-linked disorder caused by EFNB1 mutations in which females are more severely affected than males. Severe male phenotypes are associated with mosaicism, supporting cellular interference for sex bias in this disease. Although many variants have been found in the coding region of EFNB1, only 2 pathogenic variants have been identified in the same nucleotide in 5′UTR, disrupting the stop codon of an upstream open reading frame (uORF). uORFs are known to be part of a wide range of post-transcriptional regulation processes, and just recently, their association with human diseases has come to light. In the present study, we analyzed EFNB1 in a female patient with typical features of CFNS. We identified a variant, located at c.-411, creating a new upstream ATG (uATG) in the 5′UTR of EFNB1, which is predicted to alter an existing uORF. Dual-luciferase reporter assays showed significant reduction in protein translation, but no difference in the mRNA levels. Our study demonstrates, for the first time, the regulatory impact of uATG formation on EFNB1 levels and suggests that this should be the target region in molecular diagnosis of CFNS cases without pathogenic variants in the coding and splice sites regions of EFNB1.

CD105 is regulated by hsa-miR-1287 and its expression is inversely correlated with osteopotential in SHED

A more accurate understanding of the molecular mechanisms and signaling pathways underpinning human mesenchymal stem cell (MSC) plasticity and differentiation properties is pivotal for accomplishing solid and diligent translation of MSC-based experimental therapeutics and clinical trials to broad clinical practice. In addition, this knowledge enables selection of MSC subpopulations with increased differentiation potential and/or use of exogenous factors to boost this potential. Here, we report that CD105 (ENG) is a predictive biomarker of osteogenic potential in two types of MSCs: stem cells from human exfoliated deciduous teeth (SHED) and human adipose-derived stem cells (hASC). We also validate that CD105 can be used to select and enrich for subpopulations of SHED and hASC with higher in vitro osteogenic potential. In addition, we show that hsa-mir-1287 regulates CD105 expression, and propose that fine-tuning hsa-mir-1287 levels could be used to control osteopotential in SHED. These findings provide better discernment of the molecular bases behind MSC osteogenic plasticity and open up new perspectives to leverage osteogenic potential in MSCs by modulation of a specific miRNA.