Regeneration is a remarkable yet highly variable trait across tissue types and animals. This contrast raises a central question in biology: is regenerative capacity truly lost in poorly regenerating animals, or can it be reactivated under the appropriate physiological conditions? In this thesis, I investigated whether specific factors can induce appendage regeneration in animals with poor regenerative capacity, and how this regeneration activation is coordinated at both the tissue and organismal levels.
\r\n\r\nI first tested this question in animal species across the animal phylogeny: jellyfish, fruit flies, and mice. Although these animals do not normally regenerate appendages, through screening, we observed initiation of appendage regeneration when they were administered leucine, glutamine, and insulin. Although the induced regeneration is not complete, the key finding is that providing nutrient factors is sufficient to activate regeneration across evolutionarily distant species. The cross-species response suggests that regenerative programs are not fundamentally lost during evolution, but instead remain latent and can be reactivated upon favorable metabolic conditions.
\r\n\r\nMotivated by the potential use of Drosophila for investigating novel and conserved pathways in regeneration induction, I further characterized the regeneration induction. Although limb regrowth could take months, the decision to initiate regeneration processes is made within hours after injury, manifested in altered wound healing. At day-scale, while tissues in the injured limb segment normally degenerate, tissues in nutrient-supplemented flies survive. At week-scale, regenerative growth is preceded by a dome-shaped protrusion at the injury site. Performing single-nucleus RNA sequencing in the limb at this stage, I find widespread transcriptional modulations across cell types, and distinct cell populations within the muscle and epithelial cells that are enriched in mitotic and morphogenetic genes. This evidence across multiple time scales reveals how nutrient supplements can remodel the cells to reverse tissue loss and reactivate tissue replenishment.
\r\n\r\nBecause nutrients act on the whole organism, I next investigated whether regeneration activation is accompanied by systemic remodeling, including changes at the organ level. Indeed, regenerating flies enlarge their gut, which is a known mechanism to adapt to increased demands. In parallel, flies that activate regeneration reduce ovary size and egg chamber maturation. Consistent with the trade-off between activating regeneration and sustaining reproduction, virgin flies are more responsive to treatment. To further dissect candidate systemic regulators, I performed whole-organism transcriptomic profiling to identify the earliest processes that accompany the reactivation of regeneration. This analysis reveals steroid hormone signaling as one of the most highly upregulated signaling pathways. Because steroid hormone signaling is sensitive to nutritional status and interacts closely with insulin signaling to regulate systemic metabolism, these findings suggest that nutrient-induced regeneration involves not only local tissue remodeling, but also systemic organ remodeling and organism-wide resource reallocation.
\r\n\r\nIn summary, my thesis identifies nutrient factors as a conserved strategy for inducing regeneration across evolutionarily distant species. By careful examination of gene-, cell- and tissue-level changes accompanying regeneration induction, I establish adult Drosophila as a tractable model to further dissect regeneration activation. Lastly, the role of nutrients further reveals that regeneration activation is a whole-body response that integrates nutrient sensing, systemic organ remodeling, and organism-wide resource reallocation.
", "doi": "10.7907/3q1n-yh19", "publication_date": "2027", "thesis_type": "phd", "thesis_year": "2027" }, { "id": "thesis:16911", "collection": "thesis", "collection_id": "16911", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12092024-181948039", "primary_object_url": { "basename": "DDH_2024_Thesis.pdf", "content": "final", "filesize": 5078904, "license": "other", "mime_type": "application/pdf", "url": "/16911/1/DDH_2024_Thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Development and Application of Proteomic and Genomic Methods in RNA Biology", "author": [ { "family_name": "Honson", "given_name": "Drew Daniel", "orcid": "0000-0002-4654-8974", "clpid": "Honson-Drew-Daniel" } ], "thesis_advisor": [ { "family_name": "Guttman", "given_name": "Mitchell", "orcid": "0000-0003-4748-9352", "clpid": "Guttman-M" } ], "thesis_committee": [ { "family_name": "Elowitz", "given_name": "Michael B.", "orcid": "0000-0002-1221-0967", "clpid": "Elowitz-M-B" }, { "family_name": "Fejes Toth", "given_name": "Katalin", "orcid": "0000-0001-6558-2636", "clpid": "Fejes-Toth-K" }, { "family_name": "Zernicka-Goetz", "given_name": "Magdalena", "orcid": "0000-0002-7004-2471", "clpid": "Zernicka-Goetz-M" }, { "family_name": "Guttman", "given_name": "Mitchell", "orcid": "0000-0003-4748-9352", "clpid": "Guttman-M" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "This thesis contains three interrelated projects. Chapter 1 describes the development of a novel RNA-proteomics method: RNA-antisense purification followed by mass spectrometry (RAP-MS 2.0). It contains results of a RAP-MS 2.0 study profiling the protein partners of eight RNAs (7SL, 7SK, RMRP, U1, U2, U6, U7, and Xist) as well as a detailed, step-by-step protocol for the new method. Chapter 2 describes a quality control method for Split and Pool Identification of RBP targets (SPIDR). It identifies an underappreciated failure point in SPIDR experiments (the equal loading of antibody-IDs onto beads), and describes a method for monitoring and resolving this issue. Chapter 3 describes the application of SPIDR to ribosome-associated proteins in human cells. The study both validates existing structures and identifies novel interactions between nucleolar proteins and immature ribosomal RNA, and between protein trafficking factors and the large ribosomal subunit.", "doi": "10.7907/08dq-1q63", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:16358", "collection": "thesis", "collection_id": "16358", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04182024-125718373", "primary_object_url": { "basename": "Strehle_Mackenzie_Thesis.pdf", "content": "final", "filesize": 28785152, "license": "other", "mime_type": "application/pdf", "url": "/16358/1/Strehle_Mackenzie_Thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Mechanisms of Xist-Mediated Gene Silencing During the Initiation and Maintenance of X Chromosome Inactivation", "author": [ { "family_name": "Strehle", "given_name": "Mackenzie", "orcid": "0000-0003-1410-8701", "clpid": "Strehle-Mackenzie" } ], "thesis_advisor": [ { "family_name": "Guttman", "given_name": "Mitchell", "orcid": "0000-0003-4748-9352", "clpid": "Guttman-M" } ], "thesis_committee": [ { "family_name": "Sternberg", "given_name": "Paul W.", "orcid": "0000-0002-7699-0173", "clpid": "Sternberg-P-W" }, { "family_name": "Zernicka-Goetz", "given_name": "Magdalena", "orcid": "0000-0002-7004-2471", "clpid": "Zernicka-Goetz-M" }, { "family_name": "Chong", "given_name": "Shasha", "orcid": "0000-0002-5372-311X", "clpid": "Chong-Shasha" }, { "family_name": "Guttman", "given_name": "Mitchell", "orcid": "0000-0003-4748-9352", "clpid": "Guttman-M" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "X chromosome inactivation (XCI) is a critical development process during which one of the two X chromosomes in female mammals is silenced to balance gene expression with males. XCI is initiated by upregulation of the long noncoding RNA (lncRNA) Xist from the future inactive X chromosome (Xi), which recruits a variety of proteins in cis to mediate transcriptional repression that is maintained throughout the lifetime of the organism. Recent studies have demonstrated that silencing following Xist expression is dependent on direct recruitment of the transcriptional silencing protein SHARP (also known as SPEN); however, the mechanism underlying formation of the Xi silencing compartment has remained poorly defined. Similarly, it has long been thought that maintenance of XCI occurs independently of Xist and depends on differential DNA methylation enrichment on the Xi, but the evidence in support of these views is lacking. Here, we show how low copy numbers of Xist can recruit SHARP in super-stoichiometric excess to initiate gene silencing on the X and mediate formation of the silent Xi compartment. We also provide preliminary evidence suggesting that maintenance of XCI is Xist independent, but dependent on DNA methylation and histone deacetylation. Together, these results offer a more holistic view of the molecular mechanisms underlying both initiation and maintenance XCI, as well as provide a framework for further investigation into lncRNA biology and epigenetic regulation more broadly.", "doi": "10.7907/nxq5-hj97", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:15214", "collection": "thesis", "collection_id": "15214", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05272023-062623589", "primary_object_url": { "basename": "Thesis_ChengShen_v3.pdf", "content": "final", "filesize": 14315052, "license": "other", "mime_type": "application/pdf", "url": "/15214/1/Thesis_ChengShen_v3.pdf", "version": "v8.0.0" }, "type": "thesis", "title": "Computational Imaging for Phase Retrieval and Biomedical Applications", "author": [ { "family_name": "Shen", "given_name": "Cheng", "orcid": "0000-0001-7136-4715", "clpid": "Shen-Cheng" } ], "thesis_advisor": [ { "family_name": "Yang", "given_name": "Changhuei", "orcid": "0000-0001-8791-0354", "clpid": "Yang-Changhuei" } ], "thesis_committee": [ { "family_name": "Wang", "given_name": "Lihong", "orcid": "0000-0001-9783-4383", "clpid": "Wang-Lihong" }, { "family_name": "Yang", "given_name": "Changhuei", "orcid": "0000-0001-8791-0354", "clpid": "Yang-Changhuei" }, { "family_name": "Bouman", "given_name": "Katherine L.", "orcid": "0000-0003-0077-4367", "clpid": "Bouman-K-L" }, { "family_name": "Zernicka-Goetz", "given_name": "Magdalena", "orcid": "0000-0002-7004-2471", "clpid": "Zernicka-Goetz-M" } ], "local_group": [ { "literal": "div_eng" } ], "abstract": "In conventional imaging, optimizing hardware is prioritized to enhance image quality directly. Digital signal processing is viewed as supplementary. Computational imaging intentionally distorts images through modulation schemes in illumination or sensing. Then its reconstruction algorithms extract desired object information from raw data afterwards. Co-designing hardware and algorithms reduces demands on hardware and achieves the same or even better image quality. Algorithm design is at the heart of computational imaging, with model-based inverse problem or data-driven deep learning methods as approaches. This thesis presents research work from both perspectives, with a primary focus on the phase retrieval issue in computational microscopy and the application of deep learning techniques to address biomedical imaging challenges.
\r\n\r\nThe first half of the thesis begins with Fourier ptychography, which was employed to overcome chromatic aberration problems in multispectral imaging. Then, we proposed a novel computational coherent imaging modality based on Kramers-Kronig relations, aiming to replace Fourier ptychography as a non-iterative method. While this approach showed promise, it lacks certain essential characteristics of the original Fourier ptychography. To address this limitation, we introduced two additional algorithms to form a whole package scheme. Through comprehensive evaluation, we demonstrated that the combined scheme outperforms Fourier ptychography in achieving high-resolution, large field-of-view, aberration-free coherent imaging.
\r\n\r\nThe second half of the thesis shifts focus to deep-learning-based methods. In one project, we optimized the scanning strategy and image processing pipeline of an epifluorescence microscope to address focus issues. Additionally, we leveraged deep-learning-based object detection models to automate cell analysis tasks. In another project, we predicted the polarity status of mouse embryos from bright field images using adapted deep learning models. These findings highlight the capability of computational imaging to automate labor-intensive processes, and even outperform humans in challenging tasks.
", "doi": "10.7907/pahb-cx81", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:15091", "collection": "thesis", "collection_id": "15091", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01232023-192745187", "primary_object_url": { "basename": "Thesis_RileyGalton_Edited.pdf", "content": "final", "filesize": 11362200, "license": "other", "mime_type": "application/pdf", "url": "/15091/1/Thesis_RileyGalton_Edited.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Co-Option of the piRNA Pathway to Regulate Neural Crest Specification", "author": [ { "family_name": "Galton", "given_name": "Riley", "orcid": "0000-0001-6777-2177", "clpid": "Galton-Riley" } ], "thesis_advisor": [ { "family_name": "Bronner", "given_name": "Marianne E.", "orcid": "0000-0003-4274-1862", "clpid": "Bronner-M-E" }, { "family_name": "Fejes T\u00f3th", "given_name": "Katalin", "orcid": "0000-0001-6558-2636", "clpid": "Fejes-T\u00f3th-K" } ], "thesis_committee": [ { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" }, { "family_name": "Bronner", "given_name": "Marianne E.", "orcid": "0000-0003-4274-1862", "clpid": "Bronner-M-E" }, { "family_name": "Fejes-T\u00f3th", "given_name": "Katalin", "orcid": "0000-0001-6558-2636", "clpid": "Fejes-T\u00f3th-K" }, { "family_name": "Aravin", "given_name": "Alexei A.", "orcid": "0000-0002-6956-8257", "clpid": "Aravin-A-A" }, { "family_name": "Zernicka-Goetz", "given_name": "Magdalena", "orcid": "0000-0002-7004-2471", "clpid": "Zernicka-Goetz-M" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "The piRNA pathway has persisted throughout evolution as an essential regulatory pathway to protect genomic integrity in the metazoan germline. It achieves this through the repression of transposable elements, or \u201cselfish genes,\u201d which would otherwise jump throughout the genome unchecked, causing genomic instability and infertility. While transposable elements are generally deleterious in nature, their persistence in our genome remains an important driver of evolution, both as an agent of mutation and source of raw genetic material. Thus, a delicate balance must be struck to both maintain genomic integrity for the next generation but still enable enough mutation to allow for adaptation. The arms race between the ever-adapting piRNA pathway and its transposon targets provides this balance, and for a long time the piRNA pathway was considered to be germline specific, since that is where both transposon and piRNA pathway activity is highest. It has since become clear that the piRNA pathway is also active in somatic tissue of several invertebrate species, and may even target host genes in some. Whether the piRNA pathway plays a role outside of the germline in vertebrates, however, has remained elusive.
\r\n\r\nIn this thesis, we demonstrate that the piRNA pathway is active in a vertebrate somatic cell type, the chick neural crest, where it has been co-opted into the gene regulatory network to repress the transposon-derived gene, ERNI. ERNI, in turn, supresses Sox2 when piRNA pathway protein Piwil1 is downregulated upon neural crest specification. Thus, the piRNA pathway functions to maintain Sox2 expression in the neural plate border stem cell niche, protecting its proliferative abilities and setting the timing of neural crest specification. We also provide preliminary evidence that the neural crest piRNA pathway might be conserved in other vertebrate species, and that two highly conserved transcription factors regulate its expression in the chick neural crest. Our work provides mechanistic insight into a novel function of the piRNA pathway as a regulator of somatic development in vertebrates, and raises the possibility that this ancient pathway may play a more significant role in evolution and transposon co-option by host genomes than previously thought.
", "doi": "10.7907/9rvr-rr55", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:16105", "collection": "thesis", "collection_id": "16105", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06112023-211027828", "type": "thesis", "title": "Stem Cell-Derived Embryo Models in Mouse and Human to Illuminate the \u201cBlack Box\u201d of Pre- to Post-Implantation Development", "author": [ { "family_name": "Jorgensen", "given_name": "Victoria Lynn", "orcid": "0000-0002-4205-6198", "clpid": "Jorgensen-Victoria-Lynn" } ], "thesis_advisor": [ { "family_name": "Zernicka-Goetz", "given_name": "Magdalena", "orcid": "0000-0002-7004-2471", "clpid": "Zernicka-Goetz-M" } ], "thesis_committee": [ { "family_name": "Guttman", "given_name": "Mitchell", "orcid": "0000-0003-4748-9352", "clpid": "Guttman-M" }, { "family_name": "Hay", "given_name": "Bruce A.", "orcid": "0000-0002-5486-0482", "clpid": "Hay-B-A" }, { "family_name": "Parker", "given_name": "Joseph", "orcid": "0000-0001-9598-2454", "clpid": "Parker-J" }, { "family_name": "Thomson", "given_name": "Matthew", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" }, { "family_name": "Zernicka-Goetz", "given_name": "Magdalena", "orcid": "0000-0002-7004-2471", "clpid": "Zernicka-Goetz-M" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Mammalian development is a complex and highly regulated process by which a single cell, the totipotent zygote, gives rise to all lineages of the future organism. While incredible advancements have been made to study and understand the earliest events of our life, many questions are still unanswered. Moreover, the most precarious stage of development, implantation, remains a \u201cblack box\u201d to researchers due to inaccessibility of the embryo within the uterus of the mother. In the last decade, however, the emergence of stem cell derived embryos represents an exciting alternative avenue to study these dynamic stages.
\r\n\r\nDuring my PhD, I worked to establish two pre-implantation stem cell models, one in human and one in mouse, to better understand the earliest days of mammalian development. These models replicate the blastocyst stage of development; at this point in time the embryo is ready to implant into the uterus and contains all embryonic and extra-embryonic tissues needed to form the future organism: the epiblast, the hypoblast, and the trophectoderm. Beginning with my human model, I demonstrate the ability of a single cell type, expanded potential stem cells (EPSCs), to give rise to structures that replicate the natural blastocyst in size, morphology, and initiation of lineage segregation. Furthermore, these human blastocyst-like structures can undergo the very beginning of post-implantation remodeling by forming an epiblast rosette and initiating lumenogenesis. Nevertheless, single cell RNA-seq (scRNA-seq) analysis reveals that lineages are not fully committed in this model, perhaps explaining why development is limited in these structures up to about Day 7/8. In the context of my mouse model, I combine not one but three distinct cell types to generate blastocyst-like structures: 1) wildtype embryonic stem cells (ESCs) to form the epiblast, 2) trophoblast stem cells (TSCs) to form the trophectoderm, and 3) Gata4-inducible ESCs to form the primitive endoderm. Again, these structures mimic the natural mouse blastocyst in morphology and lineage segregation and demonstrate the ability to transition to post-implantation stages. Development of the three blastocyst lineages was further confirmed via global scRNA-seq analysis comparing our Gata4i-Blastoids to natural embryos; importantly, however, this analysis also showed that differentiation of the mural trophectoderm, the tissue responsible for uterine invasion, is lacking in our stem cell model and likely explains the inability for these blastoids to implant in vivo.
\r\n\r\nAltogether, this dissertation explains key aspects of pre- to post-implantation development and highlights the incredible power of stem cell-derived embryos to self-organize into structures that closely mimic the natural embryo.
", "doi": "10.7907/t1fe-3915", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:14223", "collection": "thesis", "collection_id": "14223", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06012021-203020365", "primary_object_url": { "basename": "Shashank_Gandhi_Thesis_Final.pdf", "content": "final", "filesize": 80122487, "license": "other", "mime_type": "application/pdf", "url": "/14223/1/Shashank_Gandhi_Thesis_Final.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Molecular Mechanisms Underlying Cardiac Neural Crest Development in Avian Embryos", "author": [ { "family_name": "Gandhi", "given_name": "Shashank", "orcid": "0000-0002-4081-4338", "clpid": "Gandhi-Shashank" } ], "thesis_advisor": [ { "family_name": "Bronner", "given_name": "Marianne E.", "orcid": "0000-0003-4274-1862", "clpid": "Bronner-M-E" } ], "thesis_committee": [ { "family_name": "Rothenberg", "given_name": "Ellen V.", "orcid": "0000-0002-3901-347X", "clpid": "Rothenberg-E-V" }, { "family_name": "Thomson", "given_name": "Matthew", "orcid": "0000-0003-1021-1234", "clpid": "Thomson-M-W" }, { "family_name": "Parker", "given_name": "Joseph", "orcid": "0000-0001-9598-2454", "clpid": "Parker-J" }, { "family_name": "Zernicka-Goetz", "given_name": "Magdalena", "orcid": "0000-0002-7004-2471", "clpid": "Zernicka-Goetz-M" }, { "family_name": "Bronner", "given_name": "Marianne E.", "orcid": "0000-0003-4274-1862", "clpid": "Bronner-M-E" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "The neural crest is a multipotent, vertebrate-specific stem cell population that gives rise to diverse cell types in the developing embryo, including craniofacial cartilage, enteric ganglia, and cardiac septa. Neural crest cells that originate from a given axial level in the embryo give rise to a characteristic array of progeny and follow distinct pathways from those arising at other levels. One of these subpopulations, called the cardiac neural crest, originates in the dorsal hindbrain and migrates into the developing heart, where it forms the aorticopulmonary septum, cardiac ganglion, and part of the interventricular septum. Mutations in or loss of these cells causes heart defects that are among the most common birth defects in the general population. For my thesis, I sought to identify the mechanisms that underlie the formation of neural crest cells, and confer cardiac neural crest cells with their unique developmental potential.
\r\n\r\nTo enable interrogation of epistatic relationships between key neural crest genes during neural crest induction and crest specification, I first optimized the CRISPR-Cas9 system for genome editing in gastrula and neurula-stage chicken embryos. I then further improved the CRISPR toolbox by devising an all-in-one single-plasmid strategy that harnesses the self-cleavage properties of ribozymes for the simultaneous delivery of Cas9, gRNAs, and fluorescent reporters in transfected cells. This has enabled live tracking of wildtype and mutant neural crest cells as they migrate to their terminal locations.
\r\n\r\nPrior to their induction at the neural plate border, precursors in the neural plate border are transcriptionally primed toward multiple cell fates, including neural tube, neural crest, epidermis, and placode. While this priming has been thought to involve epigenetic regulation, chromatin remodeler genes have been overlooked in the context of neural crest formation given their concomitant expression in surrounding cell types. By combining single-cell transcriptional profiling of the early chick embryonic hindbrain with temporally-controlled knockouts, I uncovered a novel bimodal mechanism whereby the chromatin remodeler gene Hmga1 first regulates Pax7-dependent neural crest induction at the neural plate border, and later modulates Wnt signaling in the dorsal neural tube to control neural crest delamination. These results established Hmga1 as a direct regulator of neural crest induction and emigration.
\r\n\r\nFinally, given that amongst distinct neural crest subpopulations designated as cranial, cardiac/vagal, and trunk, only cardiac crest has the ability to contribute to heart development, and that neither trunk nor cranial neural crest subpopulations can rescue the loss of cardiac crest, I investigated the genetic logic that imbues cardiac crest with its unique ability to form cardiovascular derivatives. To this end, I combined surgical ablations, bulk and single-cell transcriptional profiling, RNA labeling, CRISPR-Cas9-mediated gene editing, transcription factor binding motif mutation analysis, and transgenic tissue grafting approaches to uncover and characterize a cardiac-neural-crest-specific subcircuit comprised of the transcription factors Sox8, Tgif1, and Ets1. I demonstrated that ectopic expression of this subcircuit in trunk neural crest cells reprogrammed them towards a cardiac-crest-like fate, and transplanting these reprogrammed cells in place of ablated cardiac crest restored cardiac-crest-like migration patterns and rescued outflow tract septation defects.
\r\n\r\nTaken together, my thesis work has not only built a genome engineering toolbox for a key model system in developmental biology, but has also expanded our understanding of the genetic circuits that govern the formation of the cardiac neural crest and underlie its unique ability to contribute to the heart.
", "doi": "10.7907/y1e4-d090", "publication_date": "2021", "thesis_type": "phd", "thesis_year": "2021" } ]