Project description:Oncogenic alterations to DNA are not transforming in all cellular contexts. This may be due to pre-existing transcriptional programs in the cell of origin. Here, we define anatomic position as a major determinant of why cells respond to specific oncogenes. Cutaneous melanoma arises throughout the body, whereas the acral subtype arises on the palms of the hands, soles of the feet, or under the nails3. We sequenced the DNA of cutaneous and acral melanomas from a large cohort of human patients and found a specific enrichment for BRAF mutations in cutaneous melanoma but CRKL amplifications in acral melanoma. We modeled these changes in transgenic zebrafish models and found that CRKL-driven tumors predominantly formed in the fins of the fish. The fins are the evolutionary precursors to tetrapod limbs, indicating that melanocytes in these acral locations may be uniquely susceptible to CRKL. RNA profiling of these fin/limb melanocytes, compared to body melanocytes, revealed a positional identity gene program typified by posterior HOX13 genes. This positional gene program synergized with CRKL to drive tumors at acral sites. Abrogation of this CRKL-driven program eliminated the anatomic specificity of acral melanoma. These data suggest that the anatomic position of the cell of origin endows it with a unique transcriptional state that makes it susceptible to only certain oncogenic insults.
Project description:Broadly we were interested in understanding how fin and body melanocytes were different. The goal of the experiment was to investigate the interaction between three different variables: (1) cell lineage: melanocyte vs bulk microenvironment, (2) anatomic location: fin vs body, (3) genotype: wildtype vs CRKL,GAB2,TERT overexpression with NF1 loss. We found that melanocytes have unique transcriptional programs based on their anatomic location and that these programs can regulate the response to oncogenic drivers like CRKL.
Project description:Teleost fish have the remarkable ability to regenerate their body parts including heart, spinal cord, and the caudal fin, while many higher vertebrates including us humans have only a limited ability. To facilitate molecular and genetic approaches for regeneration, we previously established an assay using the fin fold of early stage larvae, which regenerate their caudal fin folds as in adult regeneration. Here, we performed transcriptional profiling of regenerating larval fin folds and identified genes with differential expression during regeneration. Gene expression profiling of zebrafish larval fin-fold regeneration was performed by comparing amputated fin fold and uncut control. Keywords: Stress response, injury response.
Project description:Zebrafish have the remarkable ability to regenerate body parts including the heart, spinal cord and fins by a process referred to as epimorphic regeneration. Recent studies have illustrated that similar to adult zebrafish, early life stage-larvae also possess the ability to regenerate the caudal fin. A comparative genomic analysis was used to determine the degree of conservation in gene expression among the regenerating adult caudal fin, adult heart and larval fin. Results indicate that these tissues respond to amputation/injury with strikingly similar genomic responses. Comparative analysis revealed raldh2, a rate-limiting enzyme for the synthesis of Retinoic acid (RA), as one of the highly induced genes across the three regeneration platforms. Experiment Overall Design: The caudal fin of zebrafish larvae at 2days post fertilization were amputated. Caudal fin tissue at 2dpf and regenerating fins were isolated at 1, 2and 3 days post amputation. Three replicates were collected at each time point. 150 fins were pooled to comprise one replicate.
Project description:We compared transcriptional profiles of regenerating zebrafish caudal fins following fin amputation with profiles from uninjured zebrafish caudal fins
Project description:Teleost fish have the remarkable ability to regenerate their body parts including heart, spinal cord, and the caudal fin, while many higher vertebrates including us humans have only a limited ability. To facilitate molecular and genetic approaches for regeneration, we previously established an assay using the fin fold of early stage larvae, which regenerate their caudal fin folds as in adult regeneration. Here, we performed transcriptional profiling of regenerating larval fin folds and identified genes with differential expression during regeneration. Gene expression profiling of zebrafish larval fin-fold regeneration was performed by comparing amputated fin fold and uncut control. Keywords: Stress response, injury response. Two time points, 18-24 hours post amputation (hpa) and 48 hpa, of regenerating fin fold were analyzed. We performed one replicate per each time point. For microarray expression profiling, total RNA was extracted from regenerating and uncut caudal fin folds of AB strain larvae. Tail tissues of 16-24 hpa, 48 hpa, and uncut siblings of the respective stages including 3-5 posterior somite segments were collected on ice. Total RNA was extracted by using TRIzol reagent (Invitrogen, Carlsbad, California, United States) according to the manufacturerâs instruction. The quantity and quality of total RNA were assessed by absorbance at 260 nm and 280 nm and by gel electrophoresis. Approx. 9 μg of total RNA was recovered from ~250 tail tissues at 16-24 hpa or uncut control tissues; and approx. 5 μg, from ~130 tail tissues at 48 hpa or uncut control tissues. Probes for microarray analysis were labeled with cy3 (amputated fin fold at 16-24 hpa and uncut control at 48 hpa) or cy5 (uncut control at 16-24 hpa and amputated fin fold at 48 hpa), and used for hybridization.
Project description:Adult zebrafish are able to regenerate many organs such as their caudal fin in only few days post amputation. To explore the landscape and dynamic of the genes involed in regeneration, we performed a global transcriptomic analysis using RNA-seq during zebrafish caudal fin regeneration.
Project description:Previous studies of zebrafish caudal fin regeneration have shown that multiple genetic programs are moduled through regulatory factors. MicroRNAs are short highly conserved non-coding genes that suppress expression of target genes and thereby control multiple genetic programs. Given their important regulatory roles and evolutionary conservation, we hypothesize that microRNAs define a conserved genetic regulatory circuit important for appendage regeneration. We characterized microRNA expression during zebrafish caudal fin regeneration using small RNA sequencing. The stages of caudal fin regeneration were assayed for mRNA expression using mRNA sequencing. Small RNA and mRNA gene expression profiling during 0 and 4 days post amputation.
Project description:Previous studies of zebrafish caudal fin regeneration have shown that multiple genetic programs are moduled through regulatory factors. MicroRNAs are short highly conserved non-coding genes that suppress expression of target genes and thereby control multiple genetic programs. Given their important regulatory roles and evolutionary conservation, we hypothesize that microRNAs define a conserved genetic regulatory circuit important for appendage regeneration. We characterized microRNA expression during zebrafish caudal fin regeneration using small RNA sequencing. The stages of caudal fin regeneration were assayed for mRNA expression using mRNA sequencing.