Publications by authors named "Kimberly D Tremblay"

27 Publications

  • Page 1 of 1

Protein phosphatase 1 regulatory subunit 35 is required for ciliogenesis, notochord morphogenesis, and cell-cycle progression during murine development.

Dev Biol 2020 09 3;465(1):1-10. Epub 2020 Jul 3.

Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA, USA. Electronic address:

Protein phosphatases regulate a wide array of proteins through post-translational modification and are required for a plethora of intracellular events in eukaryotes. While some core components of the protein phosphatase complexes are well characterized, many subunits of these large complexes remain unstudied. Here we characterize a loss-of-function allele of the protein phosphatase 1 regulatory subunit 35 (Ppp1r35) gene. Homozygous mouse embryos lacking Ppp1r35 are developmental delayed beginning at embryonic day (E) 7.5 and have obvious morphological defects at later stages. Mutants fail to initiate turning and do not progress beyond the size or staging of normal E8.5 embryos. Consistent with recent in vitro studies linking PPP1R35 with the microcephaly protein Rotatin and with a role in centrosome formation, we show that Ppp1r35 mutant embryos lack primary cilia. Histological and molecular analysis of Ppp1r35 mutants revealed that notochord development is irregular and discontinuous and consistent with a role in primary cilia, that the floor plate of the neural tube is not specified. Similar to other mutant embryos with defects in centriole function, Ppp1r35 mutants displayed increased cell death that is prevalent in the neural tube and an increased number of proliferative cells in prometaphase. We hypothesize that loss of Ppp1r35 function abrogates centriole homeostasis, resulting in a failure to produce functional primary cilia, cell death and cell cycle delay/stalling that leads to developmental failure. Taken together, these results highlight the essential function of Ppp1r35 during early mammalian development and implicate this gene as a candidate for human microcephaly.
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http://dx.doi.org/10.1016/j.ydbio.2020.06.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7484031PMC
September 2020

On the origin of vertebrate body plan: Insights from the endoderm using the hourglass model.

Gene Expr Patterns 2020 09 27;37:119125. Epub 2020 Jun 27.

State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, 210096, China. Electronic address:

The vertebrate body plan is thought to be derived during the early Cambrian from a worm-like chordate ancestor. While all three germ layers were clearly involved in this innovation, the role of the endoderm remains elusive. According to the hourglass model, the optimal window for investigating the evolution of vertebrate endoderm-derived structures during cephalochordate development is from the Spemann's organizer stage to the opening of the mouth (Stages 1-7, described herein). Regulatory gene expression, examined during these stages, illustrate that the cephalochordate endoderm is patterned into 12 organ primordia. Early vertebrates inherited at least a portion of 6 of these primordia, while the remainder were lost. Of those that were preserved, we demonstrate that the vertebrate symmetric mouth was built on a vestige of the anterior pre-oral pit, that the pre-existing pharyngeal pouch in this chordate ancestor laid the foundation for the new neural crest cell (NCC)-derived vertebrate-type pharyngeal arches, that the thyroid evolved from the posterior endostyle primordim, that the pancreas was derived from the Pdx1-expressing diverticulum primordium, and the small and large intestines originated with the Cdx1-expressing hindgut rudiments. This investigation uncovers the evolutionary foundations of vertebrate endoderm-derived structures, and demonstrates that the number of organ primordia were reduced during evolution.
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http://dx.doi.org/10.1016/j.gep.2020.119125DOI Listing
September 2020

Nuclear-encoded mitochondrial ribosomal proteins are required to initiate gastrulation.

Development 2020 05 26;147(10). Epub 2020 May 26.

Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA 01003, USA

Mitochondria are essential for energy production and although they have their own genome, many nuclear-encoded mitochondrial ribosomal proteins (MRPs) are required for proper function of the organelle. Although mutations in MRPs have been associated with human diseases, little is known about their role during development. Presented here are the null phenotypes for 21 nuclear-encoded mitochondrial proteins and in-depth characterization of mouse embryos mutant for the Mrp genes , , , and Loss of each MRP results in successful implantation and egg-cylinder formation, followed by severe developmental delay and failure to initiate gastrulation by embryonic day 7.5. The robust and similar single knockout phenotypes are somewhat surprising given there are over 70 MRPs and suggest little functional redundancy. Metabolic analysis reveals that Mrp knockout embryos produce significantly less ATP than controls, indicating compromised mitochondrial function. Histological and immunofluorescence analyses indicate abnormal organelle morphology and stalling at the G2/M checkpoint in Mrp null cells. The nearly identical pre-gastrulation phenotype observed for many different nuclear-encoded mitochondrial protein knockouts hints that distinct energy systems are crucial at specific time points during mammalian development.
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http://dx.doi.org/10.1242/dev.188714DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7272334PMC
May 2020

Estrogen Acts Through Estrogen Receptor 2b to Regulate Hepatobiliary Fate During Vertebrate Development.

Hepatology 2020 Nov;72(5):1786-1799

Genetics Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA.

Background And Aims: During liver development, bipotent progenitor cells differentiate into hepatocytes and biliary epithelial cells to ensure a functional liver required to maintain organismal homeostasis. The developmental cues controlling the differentiation of committed progenitors into these cell types, however, are incompletely understood. Here, we discover an essential role for estrogenic regulation in vertebrate liver development to affect hepatobiliary fate decisions.

Approach And Results: Exposure of zebrafish embryos to 17β-estradiol (E2) during liver development significantly decreased hepatocyte-specific gene expression, liver size, and hepatocyte number. In contrast, pharmacological blockade of estrogen synthesis or nuclear estrogen receptor (ESR) signaling enhanced liver size and hepatocyte marker expression. Transgenic reporter fish demonstrated nuclear ESR activity in the developing liver. Chemical inhibition and morpholino knockdown of nuclear estrogen receptor 2b (esr2b) increased hepatocyte gene expression and blocked the effects of E2 exposure. esr2b mutant zebrafish exhibited significantly increased expression of hepatocyte markers with no impact on liver progenitors, other endodermal lineages, or vasculature. Significantly, E2-stimulated Esr2b activity promoted biliary epithelial differentiation at the expense of hepatocyte fate, whereas loss of esr2b impaired biliary lineage commitment. Chemical and genetic epistasis studies identified bone morphogenetic protein (BMP) signaling as a mediator of the estrogen effects. The divergent impact of estrogen on hepatobiliary fate was confirmed in a human hepatoblast cell line, indicating the relevance of this pathway for human liver development.

Conclusions: Our studies identify E2, esr2b, and downstream BMP activity as important regulators of hepatobiliary fate decisions during vertebrate liver development. These results have significant clinical implications for liver development in infants exposed to abnormal estrogen levels or estrogenic compounds during pregnancy.
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http://dx.doi.org/10.1002/hep.31184DOI Listing
November 2020

MCRS1 is essential for epiblast development during early mouse embryogenesis.

Reproduction 2020 01;159(1):1-13

Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, Massachusetts, USA.

Microspherule protein 1 (MCRS1, also known as MSP58) is an evolutionarily conserved protein that has been implicated in various biological processes. Although a variety of functions have been attributed to MCRS1 in vitro, mammalian MCRS1 has not been studied in vivo. Here we report that MCRS1 is essential during early murine development. Mcrs1 mutant embryos exhibit normal morphology at the blastocyst stage but cannot be recovered at gastrulation, suggesting an implantation failure. Outgrowth (OG) assays reveal that mutant blastocysts do not form a typical inner cell mass (ICM) colony, the source of embryonic stem cells (ESCs). Surprisingly, cell death and histone H4 acetylation analysis reveal that apoptosis and global H4 acetylation are normal in mutant blastocysts. However, analysis of lineage specification reveals that while the trophoblast and primitive endoderm are properly specified, the epiblast lineage is compromised and exhibits a severe reduction in cell number. In summary, our study demonstrates the indispensable role of MCRS1 in epiblast development during early mammalian embryogenesis.
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http://dx.doi.org/10.1530/REP-19-0334DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7431978PMC
January 2020

The elongation factor Elof1 is required for mammalian gastrulation.

PLoS One 2019 5;14(7):e0219410. Epub 2019 Jul 5.

Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA, United States of America.

Despite having been sequenced over a decade ago, the functional significance of much of the mammalian genome remains unknown. The mouse has become the preeminent mammalian model for identifying endogenous gene function in vivo. Here we characterize the phenotype of a loss-of function allele for the evolutionarily conserved transcription factor, Elongation Factor Homolog 1 (Elof1). Recent work utilizing the yeast homolog, Elf1, has demonstrated that Elf1 associates with the RNA polymerase II complex to promote elongation by relieving the association of the template DNA strand with bound histones. Loss of Elof1 results in developmental delay and morphological defects during early mouse development resulting in peri-gastrulation lethality. Although Elof1 is highly conserved we observe tissue specific expression during gastrulation and in adult murine tissues, suggesting there may be other genes with similar function in diverse tissues or that mElof1 has adopted lineage specific functions. To better understand its function in mammalian transcription, we examined splice variants and find that Elof1 regulates mutually exclusive exon use in vivo. Distinct from what has been demonstrated in yeast, we demonstrate that Elof1 is essential for viability during mammalian gastrulation which may be due to a role mediating tissue specific exclusive exon use, a regulatory function unique to higher eukaryotes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0219410PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6611630PMC
March 2020

A null allele of Dnaaf2 displays embryonic lethality and mimics human ciliary dyskinesia.

Hum Mol Genet 2019 08;28(16):2775-2784

Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA 01003, USA.

The dynein axonemal assembly factor (Dnaaf) protein family is involved in preassembly and stability of dynein arms before they are transported into the cilia. In humans, mutations in DNAAF genes lead to several diseases related to cilia defects such as primary ciliary dyskinesia (PCD; OMIM: 612518). Patients with PCD experience malfunctions in cilia motility, which can result in inflammation and infection of the respiratory tract among other defects. Previous studies have identified that a mutation in DNAAF2 results in PCD and that 40% of these patients also experience laterality defects. In an outbred genetic background, Dnaaf2 homozygotes die after birth and have left/right defects among other phenotypes. Here we characterize a novel null allele of Dnaaf2 obtained from the International Mouse Phenotyping Consortium. Our data indicate that on a defined C57bl/6NJ genetic background, homozygous Dnaaf2 mouse embryos fail to progress beyond organogenesis stages with many abnormalities including left-right patterning defects. These findings support studies indicating that hypomorphic mutations of human DNAAF2 can result in ciliary dyskinesia and identify Dnaaf2 as an essential component of cilia function in vivo.
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http://dx.doi.org/10.1093/hmg/ddz106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6688062PMC
August 2019

MED20 is essential for early embryogenesis and regulates NANOG expression.

Reproduction 2019 03;157(3):215-222

Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, Massachusetts, USA.

Mediator is an evolutionarily conserved multi-subunit complex, bridging transcriptional activators and repressors to the general RNA polymerase II (Pol II) initiation machinery. Though the Mediator complex is crucial for the transcription of almost all Pol II promoters in eukaryotic organisms, the phenotypes of individual Mediator subunit mutants are each distinct. Here, we report for the first time, the essential role of subunit MED20 in early mammalian embryo development. Although Med20 mutant mouse embryos exhibit normal morphology at E3.5 blastocyst stage, they cannot be recovered at early post-gastrulation stages. Outgrowth assays show that mutant blastocysts cannot hatch from the zona pellucida, indicating impaired blastocyst function. Assessments of cell death and cell lineage specification reveal that apoptosis, inner cell mass, trophectoderm and primitive endoderm markers are normal in mutant blastocysts. However, the epiblast marker NANOG is ectopically expressed in the trophectoderm of Med20 mutants, indicative of defects in trophoblast specification. These results suggest that MED20 specifically, and the Mediator complex in general, are essential for the earliest steps of mammalian development and cell lineage specification.
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http://dx.doi.org/10.1530/REP-18-0508DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6545164PMC
March 2019

Single-cell murine genetic fate mapping reveals bipotential hepatoblasts and novel multi-organ endoderm progenitors.

Development 2018 10 11;145(19). Epub 2018 Oct 11.

Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA 01003, USA

The definitive endoderm (DE) is the embryonic germ layer that forms the gut tube and associated organs, including thymus, lungs, liver and pancreas. To understand how individual DE cells furnish gut organs, genetic fate mapping was performed using the Cre-reporter paired with a tamoxifen-inducible DE-specific Cre-expressing transgene. We established a low tamoxifen dose that infrequently induced heritable expression in a single cell of individual E8.5 mouse embryos and identified clonal cell descendants at E16.5. As expected, only a fraction of the E16.5 embryos contained positive clonal descendants and a subset of these contained descendants in multiple organs, revealing novel ontogeny. Furthermore, immunohistochemical analysis was used to identify -positive hepatocytes and biliary epithelial cells, which are the cholangiocyte precursors, in each clonally populated liver. Together, these data not only uncover novel and suspected lineage relationships between DE-derived organs, but also illustrate the bipotential nature of individual hepatoblasts by demonstrating that single hepatoblasts contribute to both the hepatocyte and the cholangiocyte lineage .
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http://dx.doi.org/10.1242/dev.168658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6198474PMC
October 2018

Identification and fate mapping of the pancreatic mesenchyme.

Dev Biol 2018 03 9;435(1):15-25. Epub 2018 Jan 9.

Department of Veterinary&Animal Sciences, University of Massachusetts, Amherst, MA, USA. Electronic address:

The murine pancreas buds from the ventral embryonic endoderm at approximately 8.75 dpc and a second pancreas bud emerges from the dorsal endoderm by 9.0 dpc. Although it is clear that secreted signals from adjacent mesoderm-derived sources are required for both the appropriate emergence and further refinement of the pancreatic endoderm, neither the exact signals nor the requisite tissue sources have been defined in mammalian systems. Herein we use DiI fate mapping of cultured murine embryos to identify the embryonic sources of both the early inductive and later condensed pancreatic mesenchyme. Despite being capable of supporting pancreas induction from dorsal endoderm in co-culture experiments, we find that in the context of the developing embryo, the dorsal aortae as well as the paraxial, intermediate, and lateral mesoderm derivatives only transiently associate with the dorsal pancreas bud, producing descendants that are decidedly anterior to the pancreas bud. Unlike these other mesoderm derivatives, the axial (notochord) descendants maintain association with the dorsal pre-pancreatic endoderm and early pancreas bud. This fate mapping data points to the notochord as the likely inductive source in vivo while also revealing dynamic morphogenetic movements displayed by individual mesodermal subtypes. Because none of the mesoderm examined above produced the pancreatic mesenchyme that condenses around the induced bud to support exocrine and endocrine differentiation, we also sought to identify the mesodermal origins of this mesenchyme. We identify a portion of the coelomic mesoderm that contributes to the condensed pancreatic mesenchyme. In conclusion, we identify a portion of the notochord as a likely source of the signals required to induce and maintain the early dorsal pancreas bud, demonstrate that the coelomic mesothelium contributes to the dorsal and ventral pancreatic mesenchyme, and provide insight into the dynamic morphological rearrangements of mesoderm-derived tissues during early organogenesis stages of mammalian development.
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http://dx.doi.org/10.1016/j.ydbio.2018.01.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5937278PMC
March 2018

Patterning of the hepato-pancreatobiliary boundary by BMP reveals heterogeneity within the murine liver bud.

Hepatology 2018 07 9;68(1):274-288. Epub 2018 May 9.

Department of Veterinary and Animal Sciences, University of Massachusetts Amherst, Amherst, MA.

During development, the endoderm initiates organ-restricted gene expression patterns in a spatiotemporally controlled manner. This process, termed induction, requires signals from adjacent mesodermal derivatives. Fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) emanating from the cardiac mesoderm and the septum transversum mesenchyme (STM), respectively, are believed to be simultaneously and uniformly required to directly induce hepatic gene expression from the murine endoderm. Using small molecule inhibitors of BMP signals during liver bud induction in the developing mouse embryo, we found that BMP signaling was not uniformly required to induce hepatic gene expression. Although BMP inhibition caused an overall reduction in the number of induced hepatoblasts, the STM-bounded posterior liver bud demonstrated the most severe loss of the essential hepatic transcription factor, hepatocyte nuclear factor 4-α (HNF4α), whereas the sinus venosus-lined anterior liver bud was less affected. We found that the posterior liver bud progenitors were anteriorly displaced and aberrantly activated pancreatobiliary markers, including sex-determining region Y-box 9 (SOX9). Additionally, we found that ectopically expressed SOX9 inhibited HNF4α and that BMP was indirectly required for hepatoblast induction. Finally, because previous studies have demonstrated that FGF signals are essential for anterior but not posterior liver bud induction, we examined synchronous BMP and FGF inhibition and found this led to a nearly complete loss of hepatoblasts.

Conclusion: BMP signaling is required to maintain the hepato-pancreatobiliary boundary, at least in part, by indirectly repressing SOX9 in the hepatic endoderm. BMP and FGF signals are each required for the induction of spatially complementary subsets of hepatoblasts. These results underscore the importance of studying early inductive processes in the whole embryo. (Hepatology 2018;68:274-288).
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http://dx.doi.org/10.1002/hep.29769DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6033643PMC
July 2018

Towards Functional Annotation of the Preimplantation Transcriptome: An RNAi Screen in Mammalian Embryos.

Sci Rep 2016 11 21;6:37396. Epub 2016 Nov 21.

Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA 01003, USA.

With readily available transcriptome-wide data, understanding the role of each expressed gene is an essential next step. Although RNAi technologies allow for genome-wide screens in cell culture, these approaches cannot replace strategies for discovery in the embryo. Here we present, for the first time, a knockdown screen in mouse preimplantation embryos. Early mammalian development encompasses dynamic cellular, molecular and epigenetic events that are largely conserved from mouse to man. We assayed 712 genes for requirements during preimplantation. We identified 59 genes required for successful development or outgrowth and implantation. We have characterized each phenotype and revealed cellular, molecular, and lineage specific defects following knockdown of transcript. Induced network analyses demonstrate this as a valid approach to identify networks of genes that play important roles during preimplantation. Our approach provides a robust and efficient strategy towards identification of novel phenotypes during mouse preimplantation and facilitates functional annotation of the mammalian transcriptome.
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http://dx.doi.org/10.1038/srep37396DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5116644PMC
November 2016

DIGIT Is a Conserved Long Noncoding RNA that Regulates GSC Expression to Control Definitive Endoderm Differentiation of Embryonic Stem Cells.

Cell Rep 2016 10;17(2):353-365

Gastrointestinal Unit, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA. Electronic address:

Long noncoding RNAs (lncRNAs) exhibit diverse functions, including regulation of development. Here, we combine genome-wide mapping of SMAD3 occupancy with expression analysis to identify lncRNAs induced by activin signaling during endoderm differentiation of human embryonic stem cells (hESCs). We find that DIGIT is divergent to Goosecoid (GSC) and expressed during endoderm differentiation. Deletion of the SMAD3-occupied enhancer proximal to DIGIT inhibits DIGIT and GSC expression and definitive endoderm differentiation. Disruption of the gene encoding DIGIT and depletion of the DIGIT transcript reveal that DIGIT is required for definitive endoderm differentiation. In addition, we identify the mouse ortholog of DIGIT and show that it is expressed during development and promotes definitive endoderm differentiation of mouse ESCs. DIGIT regulates GSC in trans, and activation of endogenous GSC expression is sufficient to rescue definitive endoderm differentiation in DIGIT-deficient hESCs. Our study defines DIGIT as a conserved noncoding developmental regulator of definitive endoderm.
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http://dx.doi.org/10.1016/j.celrep.2016.09.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5120872PMC
October 2016

Tissue-specific regulation of Igf2r/Airn imprinting during gastrulation.

Epigenetics Chromatin 2015 14;8:10. Epub 2015 Mar 14.

Department of Veterinary and Animal Sciences, University of Massachusetts at Amherst, ISB 427M, 661 N. Pleasant Street, Amherst, MA 01003 USA.

Background: Appropriate epigenetic regulation of gene expression during lineage allocation and tissue differentiation is required for normal development. One example is genomic imprinting, which is defined as parent-of-origin mono-allelic gene expression. Imprinting is established largely due to epigenetic differences arriving in the zygote from sperm and egg haploid genomes. In the mouse, there are approximately 150 known imprinted genes, many of which occur in imprinted gene clusters that are regulated together. One imprinted cluster includes the maternally expressed Igf2r, Slc22a2, and Slc22a3 genes and the paternally expressed long non-coding RNA (lncRNA) Airn. Although it is known that Igf2r and Airn are reciprocally imprinted, the timing of imprinted expression and accompanying epigenetic changes have not been well characterized in vivo.

Results: Here we show lineage- and temporal-specific regulation of DNA methylation and histone modifications at the Igf2r/Airn locus correlating with differential establishment of imprinted expression during gastrulation. Our results show that Igf2r is expressed from both alleles in the E6.5 epiblast. After gastrulation commences, the locus becomes imprinted in the embryonic lineage with the lncRNA Airn expressed from the paternal allele and Igf2r restricted to maternal allele expression. We document differentially enriched allele-specific histone modifications in extraembryonic and embryonic tissues. We also document for the first time allele-specific spreading of DNA methylation during gastrulation concurrent with establishment of imprinted expression of Igf2r. Importantly, we show that imprinted expression does not change in the extraembryonic lineage even though maternal DMR2 methylation spreading does occur, suggesting distinct mechanisms at play in embryonic and extraembryonic lineages.

Conclusions: These results indicate that similar to preimplantation, gastrulation represents a window of dynamic lineage-specific epigenetic regulation in vivo.
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http://dx.doi.org/10.1186/s13072-015-0003-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4410455PMC
April 2015

FGF signaling is required for anterior but not posterior specification of the murine liver bud.

Dev Dyn 2015 Mar 23;244(3):431-43. Epub 2014 Oct 23.

Department of Veterinary & Animal Sciences, University of Massachusetts, Amherst, Massachusetts.

Background: The definitive endoderm arises as a naive epithelial sheet that produces the entire gut tube and associated organs including the liver, pancreas and lungs. Murine explant studies demonstrate that fibroblast growth factor (FGF) signaling from adjacent tissues is required to induce hepatic gene expression from isolated foregut endoderm. The requirement of FGF signaling during liver development is examined by means of small molecule inhibition during whole embryo culture.

Results: Loss of FGF signaling before hepatic induction results in morphological defects and gene expression changes that are confined to the anterior liver bud. In contrast the posterior portion of the liver bud remains relatively unaffected. Because FGF is thought to act as a morphogen during endoderm organogenesis, the ventral pancreas was also examined after FGF inhibition. Although the size of the ventral pancreas is not affected, loss of FGF signaling results in a significantly higher density of ventral pancreas cells.

Conclusions: The requirement for FGF-mediated induction of hepatic gene expression differs across the anterior/posterior axis of the developing liver bud. These results underscore the importance of studying tissue differentiation in the context of the whole embryo.
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http://dx.doi.org/10.1002/dvdy.24215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4344927PMC
March 2015

Arf4 is required for Mammalian development but dispensable for ciliary assembly.

PLoS Genet 2014 Feb 20;10(2):e1004170. Epub 2014 Feb 20.

Program in Molecular Medicine, University of Massachusetts Medical School, Biotech II, Worcester, Massachusetts, United States of America.

The primary cilium is a sensory organelle, defects in which cause a wide range of human diseases including retinal degeneration, polycystic kidney disease and birth defects. The sensory functions of cilia require specific receptors to be targeted to the ciliary subdomain of the plasma membrane. Arf4 has been proposed to sort cargo destined for the cilium at the Golgi complex and deemed a key regulator of ciliary protein trafficking. In this work, we show that Arf4 binds to the ciliary targeting sequence (CTS) of fibrocystin. Knockdown of Arf4 indicates that it is not absolutely required for trafficking of the fibrocystin CTS to cilia as steady-state CTS levels are unaffected. However, we did observe a delay in delivery of newly synthesized CTS from the Golgi complex to the cilium when Arf4 was reduced. Arf4 mutant mice are embryonic lethal and die at mid-gestation shortly after node formation. Nodal cilia appeared normal and functioned properly to break left-right symmetry in Arf4 mutant embryos. At this stage of development Arf4 expression is highest in the visceral endoderm but we did not detect cilia on these cells. In the visceral endoderm, the lack of Arf4 caused defects in cell structure and apical protein localization. This work suggests that while Arf4 is not required for ciliary assembly, it is important for the efficient transport of fibrocystin to cilia, and also plays critical roles in non-ciliary processes.
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http://dx.doi.org/10.1371/journal.pgen.1004170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3930517PMC
February 2014

Laser-mediated cell ablation during post-implantation mouse development.

Dev Dyn 2013 Oct 2;242(10):1202-9. Epub 2013 Sep 2.

Department of Veterinary and Animal Science, University of Massachusetts, Amherst, Massachusetts.

Background: Laser-mediated cell ablation is a powerful tool that has been used to understand cell fate in a variety of externally developing organisms but has not been used during mammalian post-implantation development.

Results: We describe a method pairing laser ablation with murine embryo culture and establish parameters that can be used to precisely ablate cells in the selected field with minimal disruption to adjacent cells or the underlying cell matrix. Ablation of a large domain of endoderm, followed by ~1 day of culture results in a phenotypically normal embryo and gut tube, indicating that laser ablation is compatible with normal development. We next focused on one of the three precursor populations that have been shown to produce the liver bud. Ablations of a single progenitor domain result in a unilateral delay in the liver bud while the contralateral side is unaffected.

Conclusions: We demonstrate that laser ablation is a specific and useful technique for studying cell fate in the mouse embryo. This method represents a powerful advance in developmental studies in the mouse and can be used to provide information on the specification of organs, differentiation, cell migration, and vital tissue interactions during development.
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http://dx.doi.org/10.1002/dvdy.24017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4090775PMC
October 2013

Visceral endoderm expression of Yin-Yang1 (YY1) is required for VEGFA maintenance and yolk sac development.

PLoS One 2013 15;8(3):e58828. Epub 2013 Mar 15.

Department of Veterinary and Animal Science, University of Massachusetts, Amherst, Massachusetts, USA.

Mouse embryos lacking the polycomb group gene member Yin-Yang1 (YY1) die during the peri-implantation stage. To assess the post-gastrulation role of YY1, a conditional knock-out (cKO) strategy was used to delete YY1 from the visceral endoderm of the yolk sac and the definitive endoderm of the embryo. cKO embryos display profound yolk sac defects at 9.5 days post coitum (dpc), including disrupted angiogenesis in mesoderm derivatives and altered epithelial characteristics in the visceral endoderm. Significant changes in both cell death and proliferation were confined to the YY1-expressing yolk sac mesoderm indicating that loss of YY1 in the visceral endoderm causes defects in the adjacent yolk sac mesoderm. Production of Vascular Endothelial Growth Factor A (VEGFA) by the visceral endoderm is essential for normal growth and development of the yolk sac vasculature. Reduced levels of VEGFA are observed in the cKO yolk sac, suggesting a cause for the angiogenesis defects. Ex vivo culture with exogenous VEGF not only rescued angiogenesis and apoptosis in the cKO yolk sac mesoderm, but also restored the epithelial defects observed in the cKO visceral endoderm. Intriguingly, blocking the activity of the mesoderm-localized VEGF receptor, FLK1, recapitulates both the mesoderm and visceral endoderm defects observed in the cKO yolk sac. Taken together, these results demonstrate that YY1 is responsible for maintaining VEGF in the developing visceral endoderm and that a VEGF-responsive paracrine signal, originating in the yolk sac mesoderm, is required to promote normal visceral endoderm development.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0058828PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3598950PMC
October 2013

A fate map of the murine pancreas buds reveals a multipotent ventral foregut organ progenitor.

PLoS One 2012 17;7(7):e40707. Epub 2012 Jul 17.

Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, Massachusetts, United States of America.

The definitive endoderm is the embryonic germ layer that gives rise to the budding endodermal organs including the thyroid, lung, liver and pancreas as well as the remainder of the gut tube. DiI fate mapping and whole embryo culture were used to determine the endodermal origin of the 9.5 days post coitum (dpc) dorsal and ventral pancreas buds. Our results demonstrate that the progenitors of each bud occupy distinct endodermal territories. Dorsal bud progenitors are located in the medial endoderm overlying somites 2-4 between the 2 and 11 somite stage (SS). The endoderm forming the ventral pancreas bud is found in 2 distinct regions. One territory originates from the left and right lateral endoderm caudal to the anterior intestinal portal by the 6 SS and the second domain is derived from the ventral midline of the endoderm lip (VMEL). Unlike the laterally located ventral foregut progenitors, the VMEL population harbors a multipotent progenitor that contributes to the thyroid bud, the rostral cap of the liver bud, ventral midline of the liver bud and the midline of the ventral pancreas bud in a temporally restricted manner. This data suggests that the midline of the 9.5 dpc thyroid, liver and ventral pancreas buds originates from the same progenitor population, demonstrating a developmental link between all three ventral foregut buds. Taken together, these data define the location of the dorsal and ventral pancreas progenitors in the prespecified endodermal sheet and should lead to insights into the inductive events required for pancreas specification.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0040707PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3398925PMC
March 2013

Yin-Yang1 is required for epithelial-to-mesenchymal transition and regulation of Nodal signaling during mammalian gastrulation.

Dev Biol 2012 Aug 2;368(2):273-82. Epub 2012 Jun 2.

Department of Veterinary and Animal Science, University of Massachusetts, Amherst, 661 North Pleasant Street, Amherst, MA 01003, United States.

The ubiquitously expressed Polycomb Group protein Yin-Yang1 (YY1) is believed to regulate gene expression through direct binding to DNA elements found in promoters or enhancers of target loci. Additionally, YY1 contains diverse domains that enable a plethora of protein-protein interactions, including association with the Oct4/Sox2 pluripotency complex and Polycomb Group silencing complexes. To elucidate the in vivo role of YY1 during gastrulation, we generated embryos with an epiblast specific deletion of Yy1. Yy1 conditional knockout (cKO) embryos initiate gastrulation, but both primitive streak formation and ingression through the streak is severely impaired. These streak descendants fail to repress E-Cadherin and are unable to undergo an appropriate epithelial to mesenchymal transition (EMT). Intriguingly, overexpression of Nodal and concomitant reduction of Lefty2 are observed in Yy1 cKO embryos, suggesting that YY1 is normally required for proper Nodal regulation during gastrulation. Furthermore, definitive endoderm is specified but fails to properly integrate into the outer layer. Although anterior neuroectoderm is specified, mesoderm production is severely restricted. We show that YY1 directly binds to the Lefty2 locus in E7.5 embryos and that pharmacological inhibition of Nodal signaling partially restores mesoderm production in Yy1 cKO mutant embryos. Our results reveal critical requirements for YY1 during several important developmental processes, including EMT and regulation of Nodal signaling. These results are the first to elucidate the diverse role of YY1 during gastrulation in vivo.
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http://dx.doi.org/10.1016/j.ydbio.2012.05.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3581338PMC
August 2012

Mechanism of a genetically encoded dark-to-bright reporter for caspase activity.

J Biol Chem 2011 Jul 10;286(28):24977-86. Epub 2011 May 10.

Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003, USA.

Fluorescent proteins have revolutionized modern biology with their ability to report the presence of tagged proteins in living systems. Although several fluorescent proteins have been described in which the excitation and emission properties can be modulated by external triggers, no fluorescent proteins have been described that can be activated from a silent dark state to a bright fluorescent state directly by the activity of an enzyme. We have developed a version of GFP in which fluorescence is completely quenched by appendage of a hydrophobic quenching peptide that tetramerizes GFP and prevents maturation of the chromophore. The fluorescence can be fully restored by catalytic removal of the quenching peptide, making it a robust reporter of proteolysis. We have demonstrated the utility of this uniquely dark state of GFP as a genetically encoded apoptosis reporter that monitors the function of caspases, which catalyze the fate-determining step in programmed cell death. Caspase Activatable-GFP (CA-GFP) can be activated both in vitro and in vivo, resulting in up to a 45-fold increase in fluorescent signal in bacteria and a 3-fold increase in mammalian cells. We used CA-GFP successfully to monitor real-time apoptosis in mammalian cells. This dark state of GFP may ultimately serve as a useful platform for probes of other enzymatic processes.
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http://dx.doi.org/10.1074/jbc.M111.221648DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3137071PMC
July 2011

Yin-yang1 is required in the mammalian oocyte for follicle expansion.

Biol Reprod 2011 Apr 1;84(4):654-63. Epub 2010 Dec 1.

Department of Veterinary and Animal Science, University of Massachusetts, Amherst, MA, USA.

The multifaceted polycomb group gene Yin-Yang1 (Yy1) has been implicated in a variety of transcriptional regulatory roles both as an activator and silencer of gene expression. Here we examine the role of Yy1 during oocyte growth by conditional deletion of the locus in the growing oocyte. Our results indicate that YY1 is required for oocyte maturation and granulosa cell expansion. In mutant oocytes, we observe severely reduced expression of both Gdf9 and Bmp15, suggesting a mechanism underlying the failure of granulosa cell expansion. Consequently, we observe infertility, failure of estrus cycling, and altered reproductive hormone levels in mutant females. Additionally, we find that YY1-deficient oocytes exhibit altered levels of several oocyte-specific factors, including Pou5f1, Figla, Lhx8, Oosp1, and Sohlh2. These results document YY1's involvement in folliculogenesis and ovarian function in the mouse and indicate that YY1 is required specifically in the oocyte for oocyte-granulosa cell communication.
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http://dx.doi.org/10.1095/biolreprod.110.087213DOI Listing
April 2011

Formation of the murine endoderm: lessons from the mouse, frog, fish, and chick.

Prog Mol Biol Transl Sci 2010 ;96:1-34

Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, Massachusetts, USA.

The mammalian definitive endoderm arises as a simple epithelial sheet. This sheet of cells will eventually produce the innermost tube that comprises the entire digestive tract from the esophagus to the colon as well as the epithelial component of the digestive and respiratory organs including the thymus, thyroid, lung, liver, gallbladder, and pancreas. Thus a wide array of tissue types are derived from the early endodermal sheet, and understanding the morphological and molecular mechanisms used to produce this tissue is integral to understanding the development of all these organs. The goal of this chapter is to summarize what is known about the morphological and molecular mechanisms used to produce this embryonic germ layer. Although this chapter mainly focuses on the mechanisms used to generate the murine endoderm, supportive or suggestive data from other species, including chick, frog (Xenopus laevis), and the Zebrafish (Danio rerio) are also examined.
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http://dx.doi.org/10.1016/B978-0-12-381280-3.00001-4DOI Listing
July 2011

Inducing the liver: understanding the signals that promote murine liver budding.

J Cell Physiol 2011 Jul;226(7):1727-31

Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, Massachusetts 01003, USA.

The endoderm emerges as an epithelial sheet that covers the surface of the developing murine embryo. This tissue will produce the entire gut tube as well as associated digestive and respiratory organs including the thyroid, thymus, lung, liver, and pancreas. The emergence of each endodermal organ occurs in a temporally distinct manner that is dependant upon reciprocal inductive interactions between the endoderm and the underlying mesoderm. The emergence of the hepatic endoderm, which occurs using a morphological process termed liver budding, initiates during early somitogenesis in the mouse at approximately 8.25 days post-coitum (dpc). Explant and transplant studies performed in chicken and mouse have demonstrated that secreted signals from adjacent mesodermal tissues initiate the hepatic gene program from ventral-fated endoderm. Here, we review the data in support of the roles of members of the fibroblast growth factor (FGF), bone morphogenetic protein (BMP), and Wnt signaling pathways in liver budding and discover that little is known about the precise endogenous signals involved in the molecular and morphological induction of liver budding in the mouse.
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http://dx.doi.org/10.1002/jcp.22409DOI Listing
July 2011

Identification of novel oocyte and granulosa cell markers.

Gene Expr Patterns 2009 Sep 16;9(6):404-10. Epub 2009 Jun 16.

Department of Veterinary and Animal Science, University of Massachusetts, Amherst, MA 01003, USA.

Here we present novel gene expression patterns in the ovary as part of an ongoing assessment of published micro-array data from mouse oocytes and embryos. We present the expression patterns of 13 genes that had been determined by micro-array to be expressed in the mature egg, but not during subsequent preimplantation development. In-situ hybridization of sectioned ovaries revealed that these genes were expressed in one of two distinct patterns: (1) oocyte-specific or (2) expressed in both the oocyte and surrounding granulosa cells. Despite the fact that micro-array data demonstrated expression in the egg, several of these genes are expressed at low levels in the oocyte, but strongly expressed in granulosa cells. Eleven of these genes have no reported function or expression during oogenesis, indicating that this approach is a necessary step towards functional annotation of the genome. Also of note is that while some of these gene products have been well characterized in other tissues and cell types, others are relatively unstudied in the literature. Our results provide novel gene expression information that may provide insights into the molecular mechanisms of follicular recruitment, oocyte maturation and ovulation and will direct further experimentation into the role these genes play during oogenesis.
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http://dx.doi.org/10.1016/j.gep.2009.06.004DOI Listing
September 2009

An FGF response pathway that mediates hepatic gene induction in embryonic endoderm cells.

Dev Cell 2006 Sep;11(3):339-48

Cell and Developmental Biology Program, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, Pennsylvania 19111, USA.

While particular combinations of mesodermal signals are known to induce distinct tissue-specific programs in the endoderm, there is little information about the response pathways within endoderm cells that control their specification. We have used signaling inhibitors on embryo tissue explants and whole-embryo cultures as well as genetic approaches to reveal part of an intracellular network by which FGF signaling helps induce hepatic genes and stabilize nascent hepatic cells within the endodermal epithelium. Specifically, we found that hepatic gene induction is elicited by an FGF/MAPK pathway. Although the PI3K pathway is activated in foregut endoderm cells, its inhibition does not block hepatic gene induction in explants; however, it does block tissue growth. We also found that at the onset of hepatogenesis, the FGF/MAPK and PI3K pathways do not crossregulate in the endoderm. The finding of separate pathways for endoderm tissue specification and growth provides insights for guiding cellular regeneration and stem cell differentiation.
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http://dx.doi.org/10.1016/j.devcel.2006.06.015DOI Listing
September 2006

Distinct populations of endoderm cells converge to generate the embryonic liver bud and ventral foregut tissues.

Dev Biol 2005 Apr;280(1):87-99

Cell and Developmental Biology Program, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA.

The location and movement of mammalian gut tissue progenitors, prior to the expression of tissue-specific genes, has been unknown, but this knowledge is essential to identify transitions that lead to cell type specification. To address this, we used vital dyes to label exposed anterior endoderm cells of early somite stage mouse embryos, cultured the embryos into the tissue bud phase of development, and determined the tissue fate of the dye labeled cells. This approach was performed at three embryonic stages that are prior to, or coincident with, foregut tissue patterning (1-3 somites, 4-6 somites, and 7-10 somites). Short-term labeling experiments tracked the movement of tissue progenitor cells during foregut closure. Surprisingly, we found that two distinct types of endoderm-progenitor cells, lateral and medial, arising from three spatially separated embryonic domains, converge to generate the epithelial cells of the liver bud. Whereas the lateral endoderm-progenitors give rise to descendants that are constrained in tissue fate and position along the anterior-posterior axis of the gut, the medial gut endoderm-progenitors give rise to descendants that stream along the anterior-posterior axis at the ventral midline and contribute to multiple gut tissues. The fate map reveals extensive morphogenetic movement of progenitors prior to tissue specification, it permits a detailed analysis of endoderm tissue patterning, and it illustrates that diverse progenitor domains can give rise to individual tissue cell types.
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http://dx.doi.org/10.1016/j.ydbio.2005.01.003DOI Listing
April 2005