Biology subjects

Mendoza-Garcia, P.

Publications and source records attributed to Mendoza-Garcia, P..

3 recordsLinked to original sources

bioRxiv2022

Patient-associated mutations in Drosophila Alk perturb neuronal differentiation and promote survival

Activating Anaplastic Lymphoma Kinase (ALK) receptor tyrosine kinase (RTK) mutations occur in pediatric neuroblastoma and are associated with poor prognosis. To study ALK-activating mutations in a genetically controllable system we employed CRIPSR/Cas9, incorporating orthologues of the human oncogenic mutations ALKF1174L and ALKY1278S in the Drosophila Alk locus. AlkF1251L and AlkY1355S mutant Drosophila exhibit enhanced Alk signaling phenotypes, but unexpectedly depend on the Jelly belly (Jeb) ligand for activation. Both AlkF1251L and AlkY1355S mutant larval brains display hyperplasia, represented by increased numbers of Alk-positive neurons. Despite this hyperplasic phenotype, no brain tumors were observed in mutant animals. We show that hyperplasia in Alk mutants was not caused by significantly increased rates of proliferation, but rather by decreased levels of apoptosis in the larval brain. Using single-cell RNA sequencing (scRNA-seq), we identify perturbations during temporal fate specification in AlkY1355S mutant mushroom body lineages. These findings shed light on the role of Alk in neurodevelopmental processes and highlight the potential of activating Alk mutations to perturb specification and promote survival in neuronal lineages.

cancer biology↗

bioRxiv2021

Harnessing DSB repair to promote efficient homology-dependent and -independent prime editing

Prime editing recently emerged as a next-generation approach for precise genome editing. Here we exploit DNA double-strand break (DSB) repair to develop two novel strategies that install precise genomic insertions using an SpCas9 nuclease-based prime editor (PEn). We first demonstrate that PEn coupled to a regular prime editing guide RNA (pegRNA) efficiently promotes short genomic insertions through a homology-dependent DSB repair mechanism. While PEn editing lead to increased levels of by-products, it rescued pegRNAs that performed poorly with a nickase-based prime editor. We also present a small molecule approach that yielded increased product purity of PEn editing. Next, we developed a homology-independent PEn editing strategy by engineering a single primed insertion gRNA (springRNA) which installs genomic insertions at DSBs through the non-homologous end joining pathway (NHEJ). Lastly, we show that PEn-mediated insertions at DSBs prevent Cas9-induced large chromosomal deletions and provide evidence that continuous Cas9-mediated cutting is one of the mechanisms by which Cas9-induced large deletions arise. Altogether, this work expands the current prime editing toolbox by leveraging distinct DNA repair mechanisms including NHEJ, which represents the primary pathway of DSB repair in mammalian cells.

molecular biology↗

bioRxiv2021

DamID transcriptional profiling identifies the Snail/Scratch transcription factor Kahuli as Alk target in the Drosophila visceral mesoderm.

Development of the midgut visceral muscle of Drosophila crucially depends on Anaplastic Lymphoma Kinase (Alk) receptor tyrosine kinase (RTK) signalling, which is needed to specify founder cells (FCs) in the circular visceral mesoderm (VM). While activation of the Alk receptor by its ligand Jelly Belly (Jeb) is well characterized, only a small number of target molecules have been identified. Here, we assayed RNA polymerase II (Pol II) occupancy in VM cells by using the targeted DamID (TaDa) approach. To identify Alk targets we employed comparative analysis of embryos overexpressing Jeb versus embryos with abrogated Alk activity, revealing differential expression of a number of genes, including the Snail/Scratch family transcription factor Kahuli (Kah). Upon further in vivo validation, we confirmed that Alk signalling regulates Kah mRNA expression in the VM. We show that Kah mutants display defects in the formation of midgut constrictions, similar to that of pointed (pnt) mutants. Analysis of publicly available ChIP data defined a Kah target-binding site similar to that of Snail. In addition, we compared genes that were differentially expressed in Kah mutants with publicly available Kah- and Pnt-ChIP datasets identifying a set of common target genes putatively regulated by Kah and Pnt in midgut constriction. Taken together, we (i) report a rich dataset of Alk responsive loci in the embryonic VM, (ii) provide the first functional characterization of the Kah transcription factor, identifying a role in embryonic midgut constriction, and (iii) suggest a model in which Kah and Pnt cooperate in embryonic midgut morphogenesis.

developmental biology↗