92 research outputs found
The Boston University Jazz Big Band with the Jazz Workshop and Jazz Combo, December 10, 1993
No full textThis is the concert program of the The Boston University Jazz Big Band with the Jazz Workshop and Jazz Combo performance on Friday, December 10, 1993 at 8:00 p.m., at the Concert Hall, 855 Commonwealth Avenue. Works performed were Bluff Point by John LaBarbera, Summer Sauce by John LaBarbera, Dreamy by Errol Garner (arr. Sy Johnson), Morocco Bound by John Dankworth (arr. Kenny Wheeler), Relaxin' with Lee by Charlie Parker (arr. Rusty Dedrick), Echoes of Harlem by Duke Ellington (arr. Rusty Dedrick), Easy Livin' by Leo Robin and Ralph Rainger (arr. Jeff Holmes), Wild Flower by Wayne Shorter, Boogie Stop Shuffle by Charles Mingus, Soft Winds by Bennie Goodman, Maiden Voyage by Herbie Hancock, Mr. P. C. by John Coltrane, Nardis by Miles Davis, Out of the Night by Joe Henderson, and Ana Maria by Wayne Shorter. Digitization for Boston University Concert Programs was supported by the Boston University Humanities Library Endowed Fund
Mapping and charting the fate of the embryonic forearm
No full textThe radius and ulna, which make up the bones in the forearm, are fixed features of all
tetrapod pectoral limbs. The cellular and genetic networks that contribute to the
development of the radius and ulna are not well understood, although these bones are
susceptible to loss in congenital human syndromes and to the action of teratogens
such as thalidomide. In this thesis, I investigate the development of the radius and
ulna with a particular emphasis on differentiating their unique identities. Using a new
fate mapping approach with the Chameleon transgenic chicken line, I fate map the
early limb bud to show that the cells of the prospective radius and ulna are spatially
segregated and remain separate throughout development, challenging established
models that presume all bones are initially connected in early development. Based on
this fate map, I establish that the development of the chicken ulna is mostly in response
to paracrine signaling, unlike the chicken fibula which is mostly derived from SHH
expressing cells and thus subject to autocrine SHH signaling. I use this as evidence
that although zeugopod bones are clearly homologous according to the fossil record,
the gene regulatory networks which contribute to their development and evolution are
not fixed. I then use classical embryology experimental approaches to show that the
radius is vulnerable to loss when subject to SHH signaling and suggest a possible
framework for the aetiology of radial dysplasia. Finally, I evaluate the potential of the
chicken model to replicate muscular changes in human congenital limb differences
and propose that the muscles of the stylopod and zeugopod in chickens may be used
in the translation of human conditions
The genetic basis of previously unexplained aniridia
No full textClassical aniridia [MIM 106210] is a rare, panocular malformation characterised by a spectrum of iris and foveal hypoplasia, later complicated by cataracts, lens subluxation, glaucoma and keratopathy. It is overwhelmingly associated with PAX6 [MIM 607108] haploinsufficiency. PAX6 is a highly-conserved transcription factor that orchestrates eye development. Whilst the molecular genetic basis of aniridia and PAX6 disruption is broadly known, there remain unexplained areas that have guided the work in this thesis.
PAX6 missense variants can partially or fully phenocopy haploinsufficiency, causing classical aniridia. Why they also cause rare “worse than null” phenotypes is unclear. In order to study the effect of variant class on phenotype, I performed systematic, detailed ocular phenotyping followed by cluster analysis of PAX6 eye malformations. This compared all available missense variant cases (n=161) with a size-matched control of likely gene-disruptive (LGD) cases. Whilst LGD variants were almost universally associated with classical aniridia, the phenotypic spectrum of missense cases was wider and appeared to cluster separately, ranging in severity from isolated foveal hypoplasia (hypomorphic) to bilateral severe microphthalmia (worse than null). Recurrent substitutions of key residues in the DNA-binding paired domain of PAX6 accounted for the most severe cases, notably p.Ser54Arg and p.Asn124Lys. The impact of these variants was investigated in vitro, studying the binding of purified PAX6 paired domain to well-characterised PAX6 DNA targets, the LE9 and SIMO enhancers. Electromobility shift assays showed that binding was altered by p.Ser54Arg and p.Asn124Lys compared to wild type, though (unlike classical aniridia variants) not totally abrogated. Their impact differed between the two DNA targets studied. Whilst not explaining all of the observed genotype-phenotype correlation, these data suggest that the variants do alter protein-DNA interactions - consistent with results of protein modelling - and could affect the stoichiometry of PAX6 target gene activation.
I noted a striking similarity between the rare, severe PAX6 missense cases above and those seen in six families with heterozygous variants in MAB21L1 [MIM 601280], all resulting in Arg51 and Phe52 substitutions. All had aniridia and/or microphthalmia, 3/6 had both, and 2/6 had anomalous optic discs. MAB21L1 is involved in eye development downstream of PAX6, but was not known to cause aniridia. To characterise this further, ocular phenotyping of a CRIPSR-Cas9 gene-edited mouse strain carrying Mab21l1 p.Arg51Leu was performed. Adult mice heterozygous for this allele had a highly-penetrant excavated optic nerve anomaly. The phenotype overlaps with but is milder than the human counterpart. The homozygous animals showed severe microphthalmia. There are still questions to be answered regarding the mechanisms in both human and mouse, which may differ, but the human genetic data strongly support this being a new aniridia-associated gene with a gain of function effect.
A small proportion of aniridia is still genetically uncharacterised, indicating that there could be novel ways of inactivating PAX6 or additional disease loci. To address this, I undertook analysis of 37 unrelated mutation-negative individuals with classical aniridia (n=51 including relatives) using paired-end, short-read whole genome sequencing (WGS) technology. Following an in-house analysis pipeline, causative variants were identified in 22/37 cases (60%). 15/22 were structural variants, including: chromosome 11 inversions, one separating PAX6 from its critical downstream enhancers; deletions of this downstream enhancer region; whole/partial gene deletions of PAX6, and lastly three FOXC1 gene deletions. The remainder were intragenic PAX6 single nucleotide variants, including two deep intronic splice variants. RT-PCR analysis of RNA from patient-derived lymphoblastoid cell lines was performed in a subset, confirming missplicing in vitro. Notwithstanding historical differences in PAX6 screening, it is remarkable that over half the cohort had a disease mechanism involving either PAX6 or its regulatory region, highlighting the unusually strong disease-gene link.
In summary, this thesis fills gaps in our understanding of aniridia genetics through whole genome diagnosis of unsolved cases, underlining the importance of the wider PAX6 locus and adding to the known variants which inactivate it, as well as through characterisation of severe aniridia-microphthalmia phenotypes caused by rare missense variants in PAX6 and in the novel aniridia-associated gene MAB21L1
Mind the gap: exploring the genetic regulation behind optic fissure closure in chicken
No full textOptic fissure closure (OFC) is an evolutionarily conserved process during ocular development, in which opposing edges of the epithelial tissue in the ventral eye will fuse to close the optic fissure and form the complete circumferential retina. Failure of OFC results in ocular coloboma, a sight disorder which manifests as a keyhole-like gap at any point along the ventral eye. Notably, ocular coloboma exhibits genetic heterogeneity, with over 40 causative disease genes so far identified, and over 100 genomic loci associated with the condition. However, genetic causes for more than 80% of coloboma cases remain unknown, despite the application of whole exome and whole genome sequencing, suggesting non-coding cis-regulatory loci may harbour the causative genetic defect in some of these cases. In this project, I used chicken OFC as a model to explore stage-specific gene expression and to discover novel genomic loci regulating OFC.
By conducting bulk RNA-sequencing (RNA-seq) on fusing optic fissure tissue during OFC, a total of 106 genes were identified as fusion specific enriched genes using this approach, including previously known ocular coloboma associated genes (NTN1 and PAX2). To identify differentially accessible chromatin region specific to OFC, bulk Assay for Transposase-Accessible Chromatin (ATAC-seq) was conducted. 3869 open chromatin regions, known as peaks, with fusion specific enriched accessibility were identified genome-wide. Upon the integration of RNA-seq and ATAC-seq datasets, I discovered 105 fusion specific enriched peaks that were located in proximity to fusion specific enriched genes, providing candidate loci for the cis-regulation of OFC.
A NTN1 fluorescent OFC reporter chicken line (NTN1-T2A-GFP) was used to fluorescently label cells that directly mediate fusion, defined as the pioneer cells region. By comparing the transcriptome of NTN1-GFP positive pioneer cells to the neighbouring GFP negative cells, 433 genes were shown to be significantly enriched in the pioneer region. 5 genes with significantly enriched expression in the GFP-positive cells (GALNT6, CLYBL, DVL1, ZIC1, EGF) were validated in vivo by in-situ hybridisation with enriched expressions at the pioneer region. Functional annotation and protein network analysis of these cells showed overrepresentation of the pathway terms including MET/HGF and Akt-PI3K signaling. By incorporating the both the NTN-GFP positive cells transcriptome and in-situ hybridisation data with the lab-generated single-nuclei RNA-seq (snRNAseq) data, 2 clusters of cells in the pioneer region with different transcriptomic profiles were identified.
By conducting ATAC-footprinting analysis, TEAD1/3 was shown as the most differentially bound transcription factor (TF) during OFC. This echoes with previously published data, in which loss of function mutations on TEAD co-activator YAP1 resulted in ocular coloboma in humans and zebrafish. By further incorporating the existing RNA- seq data on the pioneer region into the ATAC-seq footprinting analysis, 42 peaks were predicted to harbour at least one binding site for pioneer cell enriched TFs and were situated next to a pioneer cell enriched gene, which were therefore considered as strong enhancers candidates regulating OFC.
In summary, this study provides insights to understand the gene regulation in OFC by generating the first published ATAC-seq dataset for the fusing optic fissure and revealed plausible candidates for ocular coloboma-causing loci. Secondly, expression of novel OFC-related genes and pathways were identified by a combination of bulk RNA-seq and sn-RNAseq, enhancing our understanding towards the overall OFC mechanism
The generation of a cell-based system to determine interaction partners and downstream responses for the essential tissue-fusion factor Netrin-1
No full textEpithelial fusion, where two opposing epithelial sheets converge and combine to create a
single continuous structure, occurs in the optic fissure (OF) during embryonic eye
development. The OF is a ventral gap that runs along the entire proximal-distal axis of the
developing optic cup. Fusion at the OF occurs around the 6th week of human development
and its dysregulation results in ocular coloboma, the most frequent congenital eye defect and
a leading cause of inherited childhood blindness in humans. The NTN1 gene encodes Netrin-
1, an extracellular laminin-related protein with evolutionary conserved roles as a facilitator of
tissue fusion in multiple developmental contexts, including the OF. Previous research on
Netrin-1 function has focused on its role in axon guidance, but emerging evidence has
established the basis for a broader role in the regulation of epithelial cell behaviours, such as
epithelial-to-mesenchymal transition, wound healing, and apoptosis, all features of tissue
fusion. How the secretion of Netrin-1 in these contexts regulates cell behaviour is currently
unknown, and is particularly restricted from a lack of knowledge of Netrin-1 binding partners
at the cell surface, and how these combine to induce specific cellular responses. The aim of
this study was therefore to develop a system to identify Netrin-1 protein binding partners and
assess these for their in vivo relevance to tissue fusion during optic fissure closure. An
inducible cell-system was generated to express Netrin-1 covalently linked to epitope tags to
facilitate the isolation of Netrin-1 with its binding partners using co-immunoprecipitation. This
thesis will describe the generation and validation of this system, describe steps to optimise its
application and the preliminary results obtained. It will also include data analysis from
experiments used to identify any transcriptional responses to the forced expression of Netrin-
1, testing the hypothesis that Netrin-1 is a regulator of epithelial-to-mesenchymal transition or
other cell behaviours relevant to tissue fusion. This work provides a crucial first step in the
discovery of Netrin-1 binding partners, and will be a valuable resource to determine to
functional roles of NTN1 during tissue fusion
Study of ophthalmo acromelic syndromes in human and mouse
No full textThe combination of severe ocular and distal limb malformations is rare.
Ophthalmo-acromelic syndrome (OAS; MIM 206920) is characterised by anophthalmia
with lower limb oligodactyly. To date <40 cases of this autosomal recessive disorder
have been reported. Genome-wide analysis of ~10,000 SNPs typed on two apparently
unrelated families - comprising a total of three affected individuals, four unaffected
siblings and their consanguineous parents - identified a large region of overlapping
autozygosity on chromosome 14q. Adding data from a third consanguineous family
gave a combined LOD score of >5 with no evidence of locus heterogeneity.
Collaborative data from a further 6 individuals refined the critical interval to a 3.4 Mb
region on chromosome14:69,652,605-73,059,612 Mb. To sequence all 19 known
protein-coding genes in the region, the 238 exons were ranked by evolutionary sequence
conservation and divided equally between the Edinburgh and Nijmegen groups.
Complete sequence coverage has been obtained for 61% of the “Edinburgh” exons but
no potentially causative mutations have been identified. Further mutation analysis of the
OAS locus is on-going.
Mice homozygous for the X-ray induced Mp mutation were reportedly
anophthalmic with hind limb oligodactyly and thus represented a potential model for
human OAS. This line was rederived in Edinburgh and phenotypic analysis of Mp/Mp
homozygotes showed runting, malformed pinnae with microphthalmia but not
anophthalmia. The apparent hind-limb oligodactyly was due to osseous syndactyly. Mp heterozygotes had milder microphthalmia and pinnae deformities, but lacked the
syndactyly. In both heterozygotes and homozygotes the eye malformations were fully
penetrant, pan-ocular and characterised by failure of both the ciliary apparatus and
vitreous body to form and abnormal retinal lamination.
Genome-wide microsatellite marker analysis showed linkage of the Mp
phenotype to chromosome 18. Fbn2 mapped within the linkage interval and was a good
candidate for Mp based on the finding of hind limb osseous syndactyly in Fbn2-null
mice. However, Fbn2-null mice have no eye phenotype. 3’-RACE identified that Mp
was as a 660 kb inversion affecting the 3’-regions of Fbn2 and the adjacent gene Isoc1.
This created two aberrant reciprocal fusion transcripts: Fbn2 exons 1-63 are fused to
Isoc1 exon 5; and Isoc1 exons 1-4 are fused to Fbn2 exons 64-65. This predicts
nonsense-mediated decay of the Isoc1 Mp transcript and production of a truncated Fbn2 Mp
protein.
Ocular development was analysed in homozygote and wild type embryos to
define the basis of the “worse than null phenotype” seen in Mp mice. RNA in situ
hybridisations (ISH) failed to detect expression of Isoc1 in the embryonic eye. In
contrast, normal expression of Fbn2 in the ciliary body and retina was consistent with
the Mp phenotype. A combination of EM and immunocytochemistry showed that
truncated Fbn2 (Fbn2Mp) was retained within the ER. Fbn2Mp co-localised with markers
of ER stress: Grp78 expression and UPR-specific Xbp1 splicing. Signalling by Wnt2b is thought to be critical for ciliary development and Lef1, a Wnt-responsive transcription
factor, showed increased and ectopic ocular expression in the region affected by ER
stress. Sox2 is a direct transcriptional target of Lef1 and we observed apparent ectopic
expression of Sox2 in the ciliary body. Throughout the developing retina in mutant
embryos we also observed individual cells that were ectopically expressing the
transcription factor Chx10 and other cells expressing the apoptotic marker Activated-
Caspase-3. The apoptotic marker did not specifically co-localise with Fbn2Mp.
Taken together, these findings suggest that the ocular malformations in Mp are a
direct result of the ER stress induced by Fbn2Mp in a specific group of cells in the early
ciliary body. The ER stress presumably halts post-translational modification of a
developmentally critical signaling molecule, possibly Wnt2b, which happens to be
expressed in the same cells. We have termed the resulting pathological mechanism a
synodiporic effect (synodiporia = the ones walking the street together or fellow
travellers). Such effects may have significant implications for human genetic disease
analysis, and may provide an explanation for other “worse than null” mutations
There are blossoms on Broadway when I'm walking with you [first line of chorus]
No full textintroduction and choruspiano and voice, ukulele, guitar, banjoads on inside front and on back covers for Famous Music stockJohns Hopkins University, Levy Sheet Music Collection, Box
158, Item 016aWords and Music by Leo Robin and Ralph Rainger. Piano Score by Mario Agnolucci.Edward Arnold, Shirley Ross, John Trent, William Frawley, Rufe Davis, Joe Weber, Lew Fields [in] Blossoms on Broadway. A B.P. Schulberg Production.unattrib. photo of Broadwa
There are blossoms on Broadway when I'm walking with you [first line of chorus]
No full textintroduction and choruspiano and voice, ukulele, guitar, banjoads on inside front and on back covers for Famous Music stockJohns Hopkins University, Levy Sheet Music Collection, Box
158, Item 016aWords and Music by Leo Robin and Ralph Rainger. Piano Score by Mario Agnolucci.Edward Arnold, Shirley Ross, John Trent, William Frawley, Rufe Davis, Joe Weber, Lew Fields [in] Blossoms on Broadway. A B.P. Schulberg Production.unattrib. photo of Broadwa
Cell adhesion marker expression dynamics during fusion of the optic fissure
No full textTissue fusion is a criHcal process that is repeated in mulHple contexts during embryonic development and shares common aYributes to processes such as wound healing and metastasis. Ocular coloboma is a developmental eye disorder that presents as a physical gap in the ventral eye, and is a major cause ofchildhood blindness. Coloboma results from fusion failure between opposing ventral reHnal epithelia, but there are major knowledge gaps in our understanding of this process at the molecular and cell behavioural levels. Here we catalogue the expression of cell adhesion proteins: N-cadherin, E-cadherin, R-cadherin, ZO-1, and the EMT transcripHonal acHvator and cadherin regulator SNAI2, in the developing chicken embryonic eye. We find that fusion pioneer cells at the edges of the fusing opHc fissure have unique and dynamic expression profiles for N-cad, E-cad and ZO-1, and that these are temporally preceded by expression ofSNAI2. This highlights the unique properHes of these cells and indicates that regulaHon of cell adhesion factors may be a criHcal process in opHc fissure closure
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