Publications by authors named "Sebastian Dworkin"

34 Publications

"Delineating the roles of Grhl2 in craniofacial development through tissue-specific conditional deletion and epistasis approaches in mouse".

Dev Dyn 2021 Feb 26. Epub 2021 Feb 26.

Department of Physiology, Anatomy and Microbiology, La Trobe University, Melbourne, Victoria, Australia.

Background: The highly-conserved Grainyhead-like (Grhl) family of transcription factors play critical roles in the development of the neural tube and craniofacial skeleton. In particular, deletion of family member Grainyhead-like 2 (Grhl2) leads to mid-gestational embryonic lethality, maxillary clefting, abdominoschisis, and both cranial and caudal neural tube closure defects. These highly pleiotropic and systemic defects suggest that Grhl2 plays numerous critical developmental roles to ensure correct morphogenesis and patterning.

Results: Here, using four separate Cre-lox conditional deletion models, as well as one genetic epistasis approach (Grhl2 ;Edn1 double heterozygous mice) we have investigated tissue-specific roles of Grhl2 in embryonic development, with a particular focus on the craniofacial skeleton. We find that loss of Grhl2 in the pharyngeal epithelium (using the Shh driver) leads to low-penetrance micrognathia, whereas deletion of Grhl2 within the ectoderm of the pharynx (Nestin ) leads to small, albeit significant, differences in the proximal-distal elongation of both the maxilla and mandible. Loss of Grhl2 in endoderm (Sox17-2a ) resulted in noticeable lung defects and a single instance of secondary palatal clefting, although formation of other endoderm-derived organs such as the stomach, bladder and intestines was not affected. Lastly, deletion of Grhl2 in cells of the neural crest (Wnt1 ) did not lead to any discernible defects in craniofacial development, and similarly, our epistasis approach did not detect any phenotypic consequences of loss of a single allele of both Grhl2 and Edn1.

Conclusion: Taken together, our study identifies a pharyngeal-epithelium intrinsic, non cell-autonomous role for Grhl2 in the patterning and formation of the craniofacial skeleton, as well as an endoderm-specific role for Grhl2 in the formation and establishment of the mammalian lung. This article is protected by copyright. All rights reserved.
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http://dx.doi.org/10.1002/dvdy.322DOI Listing
February 2021

Effect of Pleomorphic Adenoma Gene 1 Deficiency on Selected Behaviours in Adult Mice.

Neuroscience 2021 02 17;455:30-38. Epub 2020 Dec 17.

Department of Physiology, Anatomy and Microbiology, La Trobe University, Bundoora, Victoria, Australia; Department of Biology, Catholic University of Leuven, Leuven, Belgium. Electronic address:

The proto-oncogene pleomorphic adenoma gene 1 (Plag1) encodes a zinc finger transcription factor. PLAG1 is part of the high motility group AT hook-2 (HGMA2)-PLAG1-insulin-like growth factor 2 (IGF2) pathway that, when disrupted, leads to Silver-Russell syndrome, a severe form of intrauterine growth restriction. With little known about PLAG1's role in normal physiology, this study is the first to characterise the behavioural phenotype of PLAG1-deficient mice. Mice were tested for differences in circadian locomotor activity and body temperature, sleep-like behaviour, anxiety-like behaviour, cognition, social behaviour, and sensorimotor gating. Overall, the behavioural phenotype of the Plag1 knock-out (KO) mice was mild: no significant differences were seen in circadian activity levels, locomotion, object recognition, spatial memory or sociability compared to wild-type mice. However, the cued test of fear conditioning, prepulse inhibition of the startle response and Preyer's reflex test suggest that Plag1 KO mice may have a hearing impairment. This implies that PLAG1 plays an important role in proper functioning and/or development of the neural circuitry behind the auditory processes or interacts with genes involved in those processes.
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http://dx.doi.org/10.1016/j.neuroscience.2020.12.003DOI Listing
February 2021

Recent Discoveries on the Involvement of Krüppel-Like Factor 4 in the Most Common Cancer Types.

Int J Mol Sci 2020 Nov 22;21(22). Epub 2020 Nov 22.

Institute of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, 1 Miecznikowa St., 02-096 Warsaw, Poland.

Krüppel-like factor 4 (KLF4) is a transcription factor highly conserved in evolution. It is particularly well known for its role in inducing pluripotent stem cells. In addition, KLF4 plays many roles in cancer. The results of most studies suggest that KLF4 is a tumor suppressor. However, the functioning of KLF4 is regulated at many levels. These include regulation of transcription, alternative splicing, miRNA, post-translational modifications, subcellular localization, protein stability and interactions with other molecules. Simple experiments aimed at assaying transcript levels or protein levels fail to address this complexity and thus may deliver misleading results. Tumor subtypes are also important; for example, in prostate cancer KLF4 is highly expressed in indolent tumors where it impedes tumor progression, while it is absent from aggressive prostate tumors. KLF4 is important in regulating response to many known drugs, and it also plays a role in tumor microenvironment. More and more information is available about upstream regulators, downstream targets and signaling pathways associated with the involvement of KLF4 in cancer. Furthermore, KLF4 performs critical function in the overall regulation of tissue homeostasis, cellular integrity, and progression towards malignancy. Here we summarize and analyze the latest findings concerning this fascinating transcription factor.
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http://dx.doi.org/10.3390/ijms21228843DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7700188PMC
November 2020

Transcriptome analysis of the epididymis from Plag1 deficient mice suggests dysregulation of sperm maturation and extracellular matrix genes.

Dev Dyn 2020 12 6;249(12):1500-1513. Epub 2020 Oct 6.

Department of Physiology, Anatomy and Microbiology, School of Life Sciences, La Trobe University, Bundoora, Victoria, Australia.

Background: The transcription factor pleomorphic adenoma gene 1 (PLAG1) is required for male fertility. Mice deficient in PLAG1 exhibit decreased sperm motility and abnormal epididymal tubule elongation and coiling, indicating impaired sperm maturation during epididymal transit. However, the downstream transcriptomic profile of the Plag1 knockout (KO; Plag1 ) murine epididymis is currently unknown.

Results: In this study, the PLAG1-dependent epididymal transcriptome was characterised using RNA sequencing. Several genes important for the control of sperm maturation, motility, capacitation and the acrosome reaction were dysregulated in Plag1 mice. Surprisingly, several cell proliferation genes were upregulated, and Ki67 analysis indicated that cell proliferation is aberrantly upregulated in the cauda epididymis stroma of Plag1 mice. Gene ontology analysis showed an overall upregulation of genes encoding extracellular matrix components, and an overall downregulation of genes encoding metalloendopeptidases in the epididymides from Plag1 mice.

Conclusion: Together, these results suggest a defect in the epididymal extracellular matrix in Plag1 mice. These results imply that in addition to maintaining epididymal integrity directly, PLAG1 may also regulate several genes involved in the regulation of sperm maturation and capacitation. Moreover, PLAG1 may also be involved in regulating tissue homeostasis and ensuring proper structure and maintenance of the extracellular matrix in the epididymis.
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http://dx.doi.org/10.1002/dvdy.254DOI Listing
December 2020

A rat model of valproate teratogenicity from chronic oral treatment during pregnancy.

Epilepsia 2020 06 16;61(6):1291-1300. Epub 2020 May 16.

Department of Medicine (Royal Melbourne Hospital), Melbourne Brain Centre, University of Melbourne, Parkville, Victoria, Australia.

Objective: Sodium valproate (VPA), the most effective antiepileptic drug for patients with genetic generalized epilepsy (GGE), is a potent human teratogen that increases the risk of a range of congenital malformations, including spina bifida. The mechanisms underlying this teratogenicity are not known, but may involve genetic risk factors. This study aimed to develop an animal model of VPA-induced birth defects.

Methods: We used three different rat strains: inbred Genetic Absence Epilepsy Rats From Strasbourg (GAERS), a model of GGE with absence seizures; inbred Non-Epileptic Controls (NEC); and outbred nonepileptic Wistars. Female rats were fed standard chow or VPA (20 g/kg food) mixed in standard chow for 2 weeks prior to conception, and then mated with same-strain males. Treatment continued throughout pregnancy. Fetuses were extracted via C-section on gestational day 21 and examined for birth defects, including external assessment and spinal measurements.

Results: VPA-exposed pups showed significant reductions in weight, length, and whole-body development compared with controls of all three strains (P < .0001). Gestational VPA treatment altered intravertebral distances, and resulted in underdeveloped vertebral arches between thoracic region T11 and caudal region C2 in most pups (GAERS, 100%; NEC, 95%; Wistar, 80%), more frequently than in controls (9%, 13%, 19%).

Significance: Gestational VPA treatment results in similar developmental and morphological abnormalities in three rat strains, including one with GGE, indicating that the genetic underpinnings of epilepsy do not contribute markedly to VPA-induced birth defects. This model may be used in future studies to investigate mechanisms involved in the pathogenesis of antiepileptic drug-induced birth defects.
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http://dx.doi.org/10.1111/epi.16536DOI Listing
June 2020

Inactivation of in GRHL2-deficient mouse embryos rescues mid-gestation viability and secondary palate closure.

Dis Model Mech 2020 03 25;13(3). Epub 2020 Mar 25.

Department of Medicine, Central Clinical School, Monash University, 99 Commercial Road, Melbourne, VIC 3004, Australia.

Cleft lip and palate are common birth defects resulting from failure of the facial processes to fuse during development. The mammalian grainyhead-like () genes play key roles in a number of tissue fusion processes including neurulation, epidermal wound healing and eyelid fusion. One family member, , is expressed in the epithelial lining of the first pharyngeal arch in mice at embryonic day (E)10.5, prompting analysis of the role of this factor in palatogenesis. -null mice die at E11.5 with neural tube defects and a cleft face phenotype, precluding analysis of palatal fusion at a later stage of development. However, in the first pharyngeal arch of -null embryos, dysregulation of transcription factors that drive epithelial-mesenchymal transition (EMT) occurs. The aberrant expression of these genes is associated with a shift in RNA-splicing patterns that favours the generation of mesenchymal isoforms of numerous regulators. Driving the EMT perturbation is loss of expression of the EMT-suppressing transcription factors and , which are direct GRHL2 targets. The expression of the miR-200 family of microRNAs, also GRHL2 targets, is similarly reduced, resulting in a 56-fold upregulation of expression, a major driver of mesenchymal cellular identity. The critical role of GRHL2 in mediating cleft palate in mice is evident, with rescue of both palatal and facial fusion seen in embryos. These findings highlight the delicate balance between GRHL2/ZEB1 and epithelial/mesenchymal cellular identity that is essential for normal closure of the palate and face. Perturbation of this pathway may underlie cleft palate in some patients.
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http://dx.doi.org/10.1242/dmm.042218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7104862PMC
March 2020

Interrogating the Grainyhead-like 2 (Grhl2) genomic locus identifies an enhancer element that regulates palatogenesis in mouse.

Dev Biol 2020 03 26;459(2):194-203. Epub 2019 Nov 26.

Department of Physiology, Anatomy and Microbiology, La Trobe University, Melbourne, Victoria, 3086, Australia. Electronic address:

The highly-conserved Grainyhead-like (Grhl) transcription factors are critical regulators of embryogenesis that regulate cellular survival, proliferation, migration and epithelial integrity, especially during the formation of the craniofacial skeleton. Family member Grhl2 is expressed throughout epithelial tissues during development, and loss of Grhl2 function leads to significant defects in neurulation, abdominal wall closure, formation of the face and fusion of the maxilla/palate. Whereas numerous downstream target genes of Grhl2 have been identified, very little is known about how this crucial developmental transcription factor itself is regulated. Here, using in silico and in utero expression analyses and functional deletion in mice, we have identified a novel 2.4 ​kb enhancer element (mm1286) that drives reporter gene expression in a pattern that strongly recapitulates endogenous Grhl2 in the craniofacial primordia, modulates Grhl2 expression in these tissues, and augments Grhl2-mediated closure of the secondary palate. Deletion of this genomic element, in the context of inactivation of one allele of Grhl2 (through generation of double heterozygous Grhl2;mm1286 mice), results in a significant predisposition to palatal clefting at birth. Moreover, we found that a highly conserved 325 bp region of mm1286 is both necessary and sufficient for mediating the craniofacial-specific enhancer activity of this region, and that an extremely well-conserved 12-bp sequence within this element (CTGTCAAACAGGT) substantially determines full enhancer function. Together, these data provide valuable new insights into the upstream genomic regulatory landscape responsible for transcriptional control of Grhl2 during palatal closure.
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http://dx.doi.org/10.1016/j.ydbio.2019.11.015DOI Listing
March 2020

Meta-Analysis of Dependent Transcriptional Networks: A Roadmap for Identifying Novel Conserved Genetic Pathways.

Genes (Basel) 2019 10 31;10(11). Epub 2019 Oct 31.

Department of Physiology, Anatomy and Microbiology, La Trobe University, Bundoora, VIC, 3086, Australia.

The () and vertebrate - () transcription factors are among the most critical genes for epithelial development, maintenance and homeostasis, and are remarkably well conserved from fungi to humans. Mutations affecting function lead to a myriad of developmental and adult onset epithelial disease, such as aberrant skin barrier formation, facial/palatal clefting, impaired neural tube closure, age-related hearing loss, ectodermal dysplasia, and importantly, cancers of epithelial origin. Recently, mutations in the family member have been shown to lead to both syndromic and non-syndromic facial and palatal clefting in humans, particularly the genetic disorder Van Der Woude Syndrome (VWS), as well as spina bifida, whereas mutations in mammalian lead to exencephaly and facial clefting. As transcription factors, Grhl proteins bind to and activate (or repress) a substantial number of target genes that regulate and drive a cascade of transcriptional networks. A multitude of large-scale datasets have been generated to explore the /-dependent transcriptome, following ablation or mis-regulation of /-function. Here, we have performed a meta-analysis of all 41 currently published and RNA-SEQ, and microarray datasets, in order to identify and characterise the transcriptional networks controlled by / genes across disparate biological contexts. Moreover, we have also cross-referenced our results with published ChIP and ChIP-SEQ datasets, in order to determine which of the critical effector genes are likely to be direct targets, based on genomic occupancy by / genes. Lastly, to interrogate the predictive strength of our approach, we experimentally validated the expression of the top 10 candidate target genes in epithelial development, in a zebrafish model lacking , and found that orthologues of seven of these (, , and ) were significantly down-regulated at 48 hours post-fertilisation. Therefore, our study provides a strong predictive resource for the identification of putative / effector target genes.
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http://dx.doi.org/10.3390/genes10110876DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6896185PMC
October 2019

Loss of GRHL3 leads to TARC/CCL17-mediated keratinocyte proliferation in the epidermis.

Cell Death Dis 2018 10 19;9(11):1072. Epub 2018 Oct 19.

Division of Cancer Research, Peter MacCallum Cancer Centre, Grattan Street, Melbourne, VIC, 3000, Australia.

Identifying soluble factors that influence epidermal integrity is critical for the development of preventative and therapeutic strategies for disorders such as ichthyosis, psoriasis, dermatitis and epidermal cancers. The transcription factor Grainyhead-like 3 (GRHL3) is essential for maintaining barrier integrity and preventing development of cutaneous squamous cell carcinoma (SCC); however, how loss of this factor, which in the skin is expressed exclusively within suprabasal epidermal layers triggers proliferation of basal keratinocytes, had thus far remained elusive. Our present study identifies thymus and activation-regulated chemokine (TARC) as a novel soluble chemokine mediator of keratinocyte proliferation following loss of GRHL3. Knockdown of GRHL3 in human keratinocytes showed that of 42 cytokines examined, TARC was the only significantly upregulated chemokine. Mouse skin lacking Grhl3 presented an inflammatory response with hallmarks of TARC activation, including heightened induction of blood clotting, increased infiltration of mast cells and pro-inflammatory T cells, increased expression of the pro-proliferative/pro-inflammatory markers CD3 and pSTAT3, and significantly elevated basal keratinocyte proliferation. Treatment of skin cultures lacking Grhl3 with the broad spectrum anti-inflammatory 5-aminosalicylic acid (5ASA) partially restored epidermal differentiation, indicating that abnormal keratinocyte proliferation/differentiation balance is a key driver of barrier dysfunction following loss of Grhl3, and providing a promising therapeutic avenue in the treatment of GRHL3-mediated epidermal disorders.
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http://dx.doi.org/10.1038/s41419-018-0901-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6195598PMC
October 2018

Lung morphogenesis is orchestrated through Grainyhead-like 2 (Grhl2) transcriptional programs.

Dev Biol 2018 11 5;443(1):1-9. Epub 2018 Sep 5.

ACRF Stem Cells and Cancer Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia; Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia. Electronic address:

The highly conserved transcription factor Grainyhead-like 2 (Grhl2) exhibits a dynamic expression pattern in lung epithelium throughout embryonic development. Using a conditional gene targeting approach to delete Grhl2 in the developing lung epithelium, our results demonstrate that Grhl2 plays multiple roles in lung morphogenesis that are essential for respiratory function. Loss of Grhl2 leads to impaired ciliated cell differentiation and perturbed formation of terminal saccules. Critically, a substantial increase in Sox9-positive distal tip progenitor cells was observed following loss of Grhl2, suggesting that Grhl2 plays an important role in branching morphogenesis. Gene transcription profiling of Grhl2-deficient lung epithelial cells revealed a significant down regulation of Elf5, a member of the Ets family of transcription factors. Furthermore, ChIP and comparative genomic analyzes confirmed that Elf5 is a direct transcriptional target of Grhl2. Taken together, these results support the hypothesis that Grhl2 controls normal lung morphogenesis by tightly regulating the activity of distal tip progenitor cells.
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http://dx.doi.org/10.1016/j.ydbio.2018.09.002DOI Listing
November 2018

Mis-expression of grainyhead-like transcription factors in zebrafish leads to defects in enveloping layer (EVL) integrity, cellular morphogenesis and axial extension.

Sci Rep 2017 12 14;7(1):17607. Epub 2017 Dec 14.

Department of Physiology, Anatomy and Microbiology, La Trobe University, Bundoora, VIC, 3086, Australia.

The grainyhead-like (grhl) transcription factors play crucial roles in craniofacial development, epithelial morphogenesis, neural tube closure, and dorso-ventral patterning. By utilising the zebrafish to differentially regulate expression of family members grhl2b and grhl3, we show that both genes regulate epithelial migration, particularly convergence-extension (CE) type movements, during embryogenesis. Genetic deletion of grhl3 via CRISPR/Cas9 results in failure to complete epiboly and pre-gastrulation embryonic rupture, whereas morpholino (MO)-mediated knockdown of grhl3 signalling leads to aberrant neural tube morphogenesis at the midbrain-hindbrain boundary (MHB), a phenotype likely due to a compromised overlying enveloping layer (EVL). Further disruptions of grhl3-dependent pathways (through co-knockdown of grhl3 with target genes spec1 and arhgef19) confirm significant MHB morphogenesis and neural tube closure defects. Concomitant MO-mediated disruption of both grhl2b and grhl3 results in further extensive CE-like defects in body patterning, notochord and somite morphogenesis. Interestingly, over-expression of either grhl2b or grhl3 also leads to numerous phenotypes consistent with disrupted cellular migration during gastrulation, including embryo dorsalisation, axial duplication and impaired neural tube migration leading to cyclopia. Taken together, our study ascribes novel roles to the Grhl family in the context of embryonic development and morphogenesis.
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http://dx.doi.org/10.1038/s41598-017-17898-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5730563PMC
December 2017

Stage-dependent therapeutic efficacy in PI3K/mTOR-driven squamous cell carcinoma of the skin.

Cell Death Differ 2018 06 13;25(6):1146-1159. Epub 2017 Dec 13.

Monash University Central Clinical School, Prahran, VIC, 3004, Australia.

Cutaneous squamous cell carcinoma (SCC) is a recurrent cancer that is prevalent in predisposed subjects such as immunosuppressed patients and patients being treated for other malignancies. Model systems to trial therapies at different stages of SCC development are lacking, therefore precluding efficient therapeutic interventions. Here, we have disrupted the expression of the tumor suppressor GRHL3 to induce loss of PTEN and activation of the PI3K/mTOR signaling pathway in mice and human skin, promoting aggressive SCC development. We then examined the potential for targeting PI3K/mTOR and an oncogenic driver miR-21, alone and in combination, for the prevention and treatment of SCC during the initiation, promotion/progression and establishment stages. Treatment with PI3K/mTOR inhibitors completely prevented tumor initiation, and these inhibitors significantly delayed the course of papilloma progression to malignancy. However, established SCC did not undergo any growth regression, indicating that this therapy is ineffective in established cancers. Mechanistically, the resistant SCCs displayed increased miR-21 expression in mice and humans where antagonists of miR-21 rescued expression levels of GRHL3/PTEN, but the combination of miR-21 antagonism with PI3K/mTOR inhibition resulted in acquired SCC resistance in part via c-MYC and OCT-4 upregulation. In conclusion, our data provide molecular evidence for the efficacy of targeting oncogenic drivers of SCC during the initiation and promotion stages and indicate that combination therapy may induce an aggressive phenotype when applied in the establishment stage.
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http://dx.doi.org/10.1038/s41418-017-0032-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5988694PMC
June 2018

Alternative splicing and start sites: Lessons from the Grainyhead-like family.

Dev Biol 2017 09 17;429(1):12-19. Epub 2017 Jun 17.

Division of Cancer Research, Peter MacCallum Cancer Centre, Grattan Street, Parkville, VIC 3052, Australia; Sir Peter MacCallum Department of Oncology, The University of Melbourne, VIC 3052, Australia. Electronic address:

The two main mechanisms that expand the proteomic output of eukaryotic genes are alternative splicing and alternative translation initiation signals. Despite being essential to generate isoforms of gene products that create functional diversity during development, the impact of these mechanisms on fine-tuning regulatory gene networks is still underappreciated. In this review, we use the Grainyhead-like (Grhl) family as a case study to illustrate the importance of isoforms when investigating transcription factor family function during development and disease, and highlight the potential for differential modulation of downstream target genes. We provide insights into the importance of considering alternative gene products when designing, undertaking, and analysing primary research, and the effect that isoforms may have on development. This review also covers known mutations in Grhl family members, and postulates how genetic changes may dictate transcriptional specificity between the Grhl family members. It also contrasts and compares the available literature on the function and importance of the Grhl isoforms, and highlights current gaps in our understanding of their regulatory gene networks in development and disease.
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http://dx.doi.org/10.1016/j.ydbio.2017.06.018DOI Listing
September 2017

Expression of CD133 and CD44 in glioblastoma stem cells correlates with cell proliferation, phenotype stability and intra-tumor heterogeneity.

PLoS One 2017 27;12(2):e0172791. Epub 2017 Feb 27.

Department of Pathology, University of Melbourne, Melbourne, Victoria, Australia.

Glioblastoma (GBM) is a heterogeneous tumor of the brain with a poor prognosis due to recurrence and drug resistance following therapy. Genome-wide profiling has revealed the existence of distinct GBM molecular subtypes that respond differently to aggressive therapies. Despite this, molecular subtype does not predict recurrence or drug resistance and overall survival is similar across subtypes. One of the key features contributing to tumor recurrence and resistance to therapy is proposed to be an underlying subpopulation of resistant glioma stem cells (GSC). CD133 expression has been used as a marker of GSCs, however recent evidence suggests the relationship between CD133 expression, GSCs and molecular subtype is more complex than initially proposed. The expression of CD133, Olig2 and CD44 was investigated using patient derived glioma stem-like cells (PDGCs) in vitro and in vivo. Different PDGCs exhibited a characteristic equilibrium of distinct CD133+ and CD44+ subpopulations and the influence of environmental factors on the intra-tumor equilibrium of CD133+ and CD44+ cells in PDGCs was also investigated, with hypoxia inducing a CD44+ to CD133+ shift and chemo-radiotherapy inducing a CD133+ to CD44+ shift. These data suggest that surveillance and modulation of intra-tumor heterogeneity using molecular markers at initial surgery and surgery for recurrent GBM may be important for more effective management of GBM.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0172791PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5328356PMC
September 2017

Grainyhead-like 3 (Grhl3) deficiency in brain leads to altered locomotor activity and decreased anxiety-like behaviors in aged mice.

Dev Neurobiol 2017 06 3;77(6):775-788. Epub 2017 Feb 3.

Department of Medicine, Monash University Central Clinical School, Prahran, Victoria, 3181, Australia.

The highly conserved Grainyhead-like (Grhl) family of transcription factors, comprising three members in vertebrates (Grhl1-3), play critical regulatory roles during embryonic development, cellular proliferation, and apoptosis. Although loss of Grhl function leads to multiple neural abnormalities in numerous animal models, a comprehensive analysis of Grhl expression and function in the mammalian brain has not been reported. Here they show that only Grhl3 expression is detectable in the embryonic mouse brain; particularly within the habenula, an organ known to modulate repressive behaviors. Using both Grhl3-knockout mice (Grhl3 ), and brain-specific conditional deletion of Grhl3 in adult mice (Nestin-Cre/Grhl3 ), they performed histological expression analyses and behavioral tests to assess long-term effects of Grhl3 loss on motor co-ordination, spatial memory, anxiety, and stress. They found that complete deletion of Grhl3 did not lead to noticeable structural or cell-intrinsic defects in the embryonic brain; however, aged Grhl3 conditional knockout (cKO) mice showed enlarged lateral ventricles and displayed marked changes in motor function and behaviors suggestive of decreased fear and anxiety. They conclude that loss of Grhl3 in the brain leads to significant alterations in locomotor activity and decreased self-inhibition, and as such, these mice may serve as a novel model of human conditions of impulsive behavior or hyperactivity. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 775-788, 2017.
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http://dx.doi.org/10.1002/dneu.22469DOI Listing
June 2017

Mice lacking the conserved transcription factor Grainyhead-like 3 (Grhl3) display increased apposition of the frontal and parietal bones during embryonic development.

BMC Dev Biol 2016 10 18;16(1):37. Epub 2016 Oct 18.

Department of Medicine, Monash University Central Clinical School, Prahran, VIC, 3004, Australia.

Background: Increased apposition of the frontal and parietal bones of the skull during embryogenesis may be a risk factor for the subsequent development of premature skull fusion, or craniosynostosis. Human craniosynostosis is a prevalent, and often serious embryological and neonatal pathology. Other than known mutations in a small number of contributing genes, the aetiology of craniosynostosis is largely unknown. Therefore, the identification of novel genes which contribute to normal skull patterning, morphology and premature suture apposition is imperative, in order to fully understand the genetic regulation of cranial development.

Results: Using advanced imaging techniques and quantitative measurement, we show that genetic deletion of the highly-conserved transcription factor Grainyhead-like 3 (Grhl3) in mice (Grhl3 ) leads to decreased skull size, aberrant skull morphology and premature apposition of the coronal sutures during embryogenesis. Furthermore, Grhl3 mice also present with premature collagen deposition and osteoblast alignment at the sutures, and the physical interaction between the developing skull, and outermost covering of the brain (the dura mater), as well as the overlying dermis and subcutaneous tissue, appears compromised in embryos lacking Grhl3. Although Grhl3 mice die at birth, we investigated skull morphology and size in adult animals lacking one Grhl3 allele (heterozygous; Grhl3 ), which are viable and fertile. We found that these adult mice also present with a smaller cranial cavity, suggestive of post-natal haploinsufficiency in the context of cranial development.

Conclusions: Our findings show that our Grhl3 mice present with increased apposition of the frontal and parietal bones, suggesting that Grhl3 may be involved in the developmental pathogenesis of craniosynostosis.
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http://dx.doi.org/10.1186/s12861-016-0136-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5070091PMC
October 2016

The Role of Sonic Hedgehog in Craniofacial Patterning, Morphogenesis and Cranial Neural Crest Survival.

J Dev Biol 2016 Aug 3;4(3). Epub 2016 Aug 3.

Department of Medicine, Monash University Central Clinical School, Prahran, Victoria 3004, Australia.

Craniofacial defects (CFD) are a significant healthcare problem worldwide. Understanding both the morphogenetic movements which underpin normal facial development, as well as the molecular factors which regulate these processes, forms the cornerstone of future diagnostic, and ultimately, preventative therapies. The soluble morphogen (), a vertebrate orthologue of Drosophila , is a key signalling factor in the regulation of craniofacial skeleton development in vertebrates, operating within numerous tissue types in the craniofacial primordia to spatiotemporally regulate the formation of the face and jaws. This review will provide an overview of normal craniofacial skeleton development, and focus specifically on the known roles of in regulating the development and progression of the first pharyngeal arch, which in turn gives rise to both the upper jaw (maxilla) and lower jaw (mandible).
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http://dx.doi.org/10.3390/jdb4030024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5831778PMC
August 2016

Identification of a Novel Proto-oncogenic Network in Head and Neck Squamous Cell Carcinoma.

J Natl Cancer Inst 2015 Sep 10;107(9). Epub 2015 Jun 10.

Department of Medicine, Monash University Central Clinical School, Prahran, Victoria 3004, Australia (SRG, MC, SS, DP, AA, SD, SMJ, CD); Department of Anatomical Pathology, Alfred Hospital, Prahran, Victoria 3004, Australia (CAM); Department of Hematology, Alfred Hospital, Prahran VIC 3181, Australia (SMJ).

Background: The developmental transcription factor Grainyhead-like 3 (GRHL3) plays a critical tumor suppressor role in the mammalian epidermis through direct regulation of PTEN and the PI3K/AKT/mTOR signaling pathway. GRHL3 is highly expressed in all tissues derived from the surface ectoderm, including the oral cavity, raising a question about its potential role in suppression of head and neck squamous cell carcinoma (HNSCC).

Methods: We explored the tumor suppressor role of Grhl3 in HNSCC using a conditional knockout (Grhl3 (∆/-) /K14Cre (+) ) mouse line (n = 26) exposed to an oral chemical carcinogen. We defined the proto-oncogenic pathway activated in the HNSCC derived from these mice and assessed it in primary human HNSCC samples, normal oral epithelial cell lines carrying shRNA to GRHL3, and human HNSCC cell lines. Data were analyzed with two-sided chi square and Student's t tests.

Results: Deletion of Grhl3 in oral epithelium in mice did not perturb PTEN/PI3K/AKT/mTOR signaling, but instead evoked loss of GSK3B expression, resulting in stabilization and accumulation of c-MYC and aggressive HNSCC. This molecular signature was also evident in a subset of primary human HNSCC and HNSCC cell lines. Loss of Gsk3b in mice, independent of Grhl3, predisposed to chemical-induced HNSCC. Restoration of GSK3B expression blocked proliferation of normal oral epithelial cell lines carrying shRNA to GRHL3 (cell no., Day 8: Scramble ctl, 616±21.8 x 10(3) vs GRHL3-kd, 1194±44 X 10(3), P < .001; GRHL3-kd vs GRHL3-kd + GSK3B, 800±98.84 X 10(3), P = .003) and human HNSCC cells.

Conclusions: We defined a novel molecular signature in mammalian HNSCC, suggesting new treatment strategies targeting the GRHL3/GSK3B/c-MYC proto-oncogenic network.
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http://dx.doi.org/10.1093/jnci/djv152DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4836819PMC
September 2015

Grainyhead-like 3 regulation of endothelin-1 in the pharyngeal endoderm is critical for growth and development of the craniofacial skeleton.

Mech Dev 2014 Aug 7;133:77-90. Epub 2014 Jun 7.

Department of Medicine, Monash University Central Clinical School, Prahran, VIC 3181, Australia; Alfred Hospital, Prahran, VIC 3181, Australia.

Craniofacial development is a highly conserved process that requires complex interactions between neural crest cells (NCCs) and pharyngeal tissues derived from all three germ layers. Signals emanating from the pharyngeal endoderm drive differentiation of NCCs into craniofacial cartilage, and disruption of this process underpins several human craniofacial defects (CFD). Here, we demonstrate that morpholino (MO)-mediated knockdown in zebrafish of the highly conserved transcription factor grainyhead-like 3 (grhl3), which is selectively expressed in the pharyngeal endoderm, leads to severe hypoplasia of the lower jaw cartilages. Phylogenetic analysis of conserved grhl-binding sites in gene regulatory regions identified endothelin-1 (edn1) as a putative direct grhl3 target gene, and this was confirmed by chromatin precipitation (ChIP) assays in zebrafish embryos. Injection of sub-phenotypic concentrations of MOs targeting both grhl3 and edn1 induced jaw abnormalities, and injection of edn1 mRNA into grhl3-morphants rescued both pharyngeal expression of the downstream effectors of edn1, and jaw cartilage formation. This study sheds new light on the role of endodermal endothelin-1 in vertebrate jaw development, and highlights potential new genetic defects that could underpin human CFD.
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http://dx.doi.org/10.1016/j.mod.2014.05.005DOI Listing
August 2014

Novel mechanisms that pattern and shape the midbrain-hindbrain boundary.

Cell Mol Life Sci 2013 Sep 10;70(18):3365-74. Epub 2013 Jan 10.

Department of Medicine, Monash University Central Clinical School, Melbourne, VIC, 3004, Australia.

The midbrain-hindbrain boundary (MHB) is a highly conserved vertebrate signalling centre, acting to pattern and establish neural identities within the brain. While the core signalling pathways regulating MHB formation have been well defined, novel genetic and mechanistic processes that interact with these core components are being uncovered, helping to further elucidate the complicated networks governing MHB specification, patterning and shaping. Although formation of the MHB organiser is traditionally thought of as comprising three stages, namely positioning, induction and maintenance, we propose that a fourth stage, morphogenesis, should be considered as an additional stage in MHB formation. This review will examine evidence for novel factors regulating the first three stages of MHB development and will explore the evidence for regulation of MHB morphogenesis by non-classical MHB-patterning genes.
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http://dx.doi.org/10.1007/s00018-012-1240-xDOI Listing
September 2013

CREB signalling in neural stem/progenitor cells: recent developments and the implications for brain tumour biology.

Bioessays 2012 Apr 13;34(4):293-300. Epub 2012 Feb 13.

Department of Pathology, The University of Melbourne, Parkville, Australia.

This paper discusses the evidence for the role of CREB in neural stem/progenitor cell (NSPC) function and oncogenesis and how these functions may be important for the development and growth of brain tumours. The cyclic-AMP response element binding (CREB) protein has many roles in neurons, ranging from neuronal survival to higher order brain functions such as memory and drug addiction behaviours. Recent studies have revealed that CREB also has a role in NSPC survival, differentiation and proliferation. Recent work has shown that over-expression of CREB in transgenic animals can impart oncogenic properties on cells in various tissues and that aberrant CREB expression is associated with tumours in patients. It is the central position of CREB, downstream of key developmental and growth signalling pathways, which give CREB the ability to influence a spectrum of cell activities, such as cell survival, growth and differentiation in both normal and cancer cells.
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http://dx.doi.org/10.1002/bies.201100133DOI Listing
April 2012

Midbrain-hindbrain boundary patterning and morphogenesis are regulated by diverse grainy head-like 2-dependent pathways.

Development 2012 Feb;139(3):525-36

Department of Medicine, Monash University Central Clinical School, Prahran VIC 3181, Australia.

The isthmic organiser located at the midbrain-hindbrain boundary (MHB) is the crucial developmental signalling centre responsible for patterning mesencephalic and metencephalic regions of the vertebrate brain. Formation and maintenance of the MHB is characterised by a hierarchical program of gene expression initiated by fibroblast growth factor 8 (Fgf8), coupled with cellular morphogenesis, culminating in the formation of the tectal-isthmo-cerebellar structures. Here, we show in zebrafish that one orthologue of the transcription factor grainy head-like 2 (Grhl2), zebrafish grhl2b plays a central role in both MHB maintenance and folding by regulating two distinct, non-linear pathways. Loss of grhl2b expression induces neural apoptosis and extinction of MHB markers, which are rescued by re-expression of engrailed 2a (eng2a), an evolutionarily conserved target of the Grhl family. Co-injection of sub-phenotypic doses of grhl2b and eng2a morpholinos reproduces the apoptosis and MHB marker loss, but fails to substantially disrupt formation of the isthmic constriction. By contrast, a novel direct grhl2b target, spec1, identified by phylogenetic analysis and confirmed by ChIP, functionally cooperates with grhl2b to induce MHB morphogenesis, but plays no role in apoptosis or maintenance of MHB markers. Collectively, these data show that MHB maintenance and morphogenesis are dissociable events regulated by grhl2b through diverse transcriptional targets.
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http://dx.doi.org/10.1242/dev.066522DOI Listing
February 2012

Targeting of the tumor suppressor GRHL3 by a miR-21-dependent proto-oncogenic network results in PTEN loss and tumorigenesis.

Cancer Cell 2011 Nov;20(5):635-48

Department of Medicine, Monash University Central Clinical School, Prahran, Victoria 3181, Australia.

Despite its prevalence, the molecular basis of squamous cell carcinoma (SCC) remains poorly understood. Here, we identify the developmental transcription factor Grhl3 as a potent tumor suppressor of SCC in mice, and demonstrate that targeting of Grhl3 by a miR-21-dependent proto-oncogenic network underpins SCC in humans. Deletion of Grhl3 in adult epidermis evokes loss of expression of PTEN, a direct GRHL3 target, resulting in aggressive SCC induced by activation of PI3K/AKT/mTOR signaling. Restoration of Pten expression completely abrogates SCC formation. Reduced levels of GRHL3 and PTEN are evident in human skin, and head and neck SCC, associated with increased expression of miR-21, which targets both tumor suppressors. Our data define the GRHL3-PTEN axis as a critical tumor suppressor pathway in SCC.
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http://dx.doi.org/10.1016/j.ccr.2011.10.014DOI Listing
November 2011

The planar cell polarity pathway in vertebrate epidermal development, homeostasis and repair.

Organogenesis 2011 Jul-Sep;7(3):202-8. Epub 2011 Jul 1.

Alfred Hospital and Monash University Central Clinical School, Prahran, VIC, Australia.

The planar cell polarity (PCP) pathway plays a critical role in diverse developmental processes that require coordinated cellular movement, including neural tube closure and renal tubulogenesis. Recent studies have demonstrated that this pathway also has emerging relevance to the epidermis, as PCP signaling underpins many aspects of skin biology and pathology, including epidermal development, hair orientation, stem cell division and cancer. Coordinated cellular movement required for epidermal repair in mammals is also regulated by PCP signaling, and in this context, a new PCP gene encoding the developmental transcription factor Grainyhead-like 3 (Grhl3) is critical. This review focuses on the role that PCP signaling plays in the skin across a variety of epidermal functions and highlights perturbations that induce epidermal pathologies.
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http://dx.doi.org/10.4161/org.7.3.18431DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3243033PMC
July 2012

Epidermal wound repair is regulated by the planar cell polarity signaling pathway.

Dev Cell 2010 Jul;19(1):138-47

Rotary Bone Marrow Research Laboratories, Parkville, Victoria 3050, Australia.

The mammalian PCP pathway regulates diverse developmental processes requiring coordinated cellular movement, including neural tube closure and cochlear stereociliary orientation. Here, we show that epidermal wound repair is regulated by PCP signaling. Mice carrying mutant alleles of PCP genes Vangl2, Celsr1, PTK7, and Scrb1, and the transcription factor Grhl3, interact genetically, exhibiting failed wound healing, neural tube defects, and disordered cochlear polarity. Using phylogenetic analysis, ChIP, and gene expression in Grhl3(-)(/-) mice, we identified RhoGEF19, a homolog of a RhoA activator involved in PCP signaling in Xenopus, as a direct target of GRHL3. Knockdown of Grhl3 or RhoGEF19 in keratinocytes induced defects in actin polymerization, cellular polarity, and wound healing, and re-expression of RhoGEF19 rescued these defects in Grhl3-kd cells. These results define a role for Grhl3 in PCP signaling and broadly implicate this pathway in epidermal repair.
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http://dx.doi.org/10.1016/j.devcel.2010.06.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2965174PMC
July 2010

Targeting CREB signalling in neurogenesis.

Expert Opin Ther Targets 2010 Aug;14(8):869-79

Bone Marrow Research Laboratories, Parkville 3050, Australia.

Importance Of The Field: Expansion and ultimately homeostasis of neural cell number is exquisitely regulated by molecular genetic networks operating in neural stem and progenitor cells (NSPCs) and in the neural stem cell niche during embryogenesis and in the adult brain. Transcription factors are crucial in orchestrating the correct cell-specific and temporal expression of all factors involved in these signalling networks. Aberrant expression of these factors can lead to abnormal brain development if this occurs during embryogenesis. In the adult brain, loss of neurogenic potential can lead to cognitive deficits and in combination with neural death can even contribute to neurodegenerative disease progress.

Areas Covered In This Review: This review focuses on a number of recent discoveries identifying the role of the transcription factor cAMP response element binding (CREB) protein in regulating brain development and neurogenesis in the adult brain.

What The Reader Will Gain: The significance of these discoveries is presented in the context of human brain disorders and how this knowledge could contribute to pharmacotherapeutic interventions targeting CREB signalling aimed at treating such diseases.

Take Home Message: Unravelling these precise molecular genetic networks is crucial to understanding how neural stem and progenitor cells function.
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http://dx.doi.org/10.1517/14728222.2010.501332DOI Listing
August 2010

cAMP response element binding protein is required for mouse neural progenitor cell survival and expansion.

Stem Cells 2009 Jun;27(6):1347-57

Differentiation and Transcription Laboratory, Trescowthick Research Laboratories, Peter MacCallum Cancer Centre, Victoria, Australia.

Development of the mammalian brain relies on the coordinated expansion of neural cells in a relatively short time, spanning for a period of only a few days in mice. The molecular networks regulating neural cell birth and expansion, termed neurogenesis, are still unresolved, although many studies using genetically modified mice have revealed a growing number of genes that are involved in regulating these processes. The cAMP response element binding protein (CREB) lies at the hub of a diverse array of intracellular signaling pathways and is a major transcriptional regulator of numerous functions in adult neural cells, including learning and memory and neuronal survival. Recent studies have shown that activated CREB is highly expressed in immature dividing cells in adult mouse and zebrafish brains and that CREB regulates neural stem/progenitor cells (NSPCs) proliferation in embryonic zebrafish brain. Using genetically modified mice, we show that deletion of CREB, without the concomitant loss of the related compensating factor cAMP response element modifier, leads to defects in neural progenitor cell expansion and survival. Cultured primary CREB(-/-) NSPCs exhibited decreased expression of several target genes important for neuronal survival and growth, including brain-derived neurotrophic factor and neural growth factor and showed that the survival and growth defect can be rescued by the addition of wild-type NSPC-conditioned medium. This is the first study showing a specific role for CREB in mammalian embryonic neurogenesis. This role appears to be mediated via the expression of factors important for NSPC survival and growth and suggests that CREB is an important signaling regulator within the developing neurogenic niche.
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http://dx.doi.org/10.1002/stem.56DOI Listing
June 2009

c-Myb is required for neural progenitor cell proliferation and maintenance of the neural stem cell niche in adult brain.

Stem Cells 2008 Jan 27;26(1):173-81. Epub 2007 Sep 27.

Differentiation and Transcription Laboratory, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia.

Ongoing production of neurons in adult brain is restricted to specialized neurogenic niches. Deregulated expression of genes controlling homeostasis of neural progenitor cell division and/or their microenvironment underpins a spectrum of brain pathologies. Using conditional gene deletion, we show that the proto-oncogene c-myb regulates neural progenitor cell proliferation and maintains ependymal cell integrity in mice. These two cellular compartments constitute the neurogenic niche in the adult brain. Brains devoid of c-Myb showed enlarged ventricular spaces, ependymal cell abnormalities, and reduced neurogenesis. Neural progenitor cells lacking c-Myb showed a reduced intrinsic proliferative capacity and reduction of Sox-2 and Pax-6 expression. These data point to an important role for c-Myb in the neurogenic niche of the adult brain.
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http://dx.doi.org/10.1634/stemcells.2007-0293DOI Listing
January 2008

A microenvironment-induced myeloproliferative syndrome caused by retinoic acid receptor gamma deficiency.

Cell 2007 Jun;129(6):1097-110

Trescowthick Research Laboratories, Peter MacCallum Cancer Centre, East Melbourne, Victoria, 3002, Australia.

Myeloproliferative syndromes (MPS) are a heterogeneous subclass of nonlymphoid hematopoietic neoplasms which are considered to be intrinsic to hematopoietic cells. The causes of MPS are largely unknown. Here, we demonstrate that mice deficient for retinoic acid receptor gamma (RARgamma), develop MPS induced solely by the RARgamma-deficient microenvironment. RARgamma(-/-) mice had significantly increased granulocyte/macrophage progenitors and granulocytes in bone marrow (BM), peripheral blood, and spleen. The MPS phenotype continued for the lifespan of the mice and was more pronounced in older mice. Unexpectedly, transplant studies revealed this disease was not intrinsic to the hematopoietic cells. BM from wild-type mice transplanted into mice with an RARgamma(-/-) microenvironment rapidly developed the MPS, which was partially caused by significantly elevated TNFalpha in RARgamma(-/-) mice. These data show that loss of RARgamma results in a nonhematopoietic cell-intrinsic MPS, revealing the capability of the microenvironment to be the sole cause of hematopoietic disorders.
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http://dx.doi.org/10.1016/j.cell.2007.05.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1974882PMC
June 2007

CREB activity modulates neural cell proliferation, midbrain-hindbrain organization and patterning in zebrafish.

Dev Biol 2007 Jul 27;307(1):127-41. Epub 2007 Apr 27.

Differentiation and Transcription Laboratory, Trescowthick Laboratories, Peter MacCallum Cancer Centre, Victoria, 3002, Australia.

Neural stem/progenitor cells (NPCs) self-renew and differentiate, generating neuronal and non-neuronal (glial) cell lineages. Although a number of factors, including transcription factors, have been shown to be important in the regulation of NPC proliferation and differentiation, the precise molecular networks remain to be identified. The cAMP Response Element-Binding protein (CREB) is a transcription factor important for neuronal survival, differentiation and plasticity. Recent work suggests that CREB activation, via serine phosphorylation in the kinase inducible domain, is important for neurogenesis in the adult rodent brain. We sought to further investigate CREB function in neurogenesis, using the zebrafish (Danio rerio). Structural and functional analysis of the zebrafish CREB orthologue showed high conservation with mammalian CREB. Activated (phosphorylated) CREB (pCREB) was localised to all known proliferation zones in the adult zebrafish brain, including actively cycling cells. Furthermore, we found that modulating CREB activity during early zebrafish development caused significant defects in neural proliferation, midbrain-hindbrain organization and body patterning. These findings reveal broader and stage-specific physiological roles of CREB function during vertebrate neural development and proliferation.
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http://dx.doi.org/10.1016/j.ydbio.2007.04.026DOI Listing
July 2007