Biology subjects

Bieli, D.

Publications and source records attributed to Bieli, D..

6 recordsLinked to original sources

bioRxiv2025

Genetic characterization of the apterous Life Span Enhancer in Drosophila melanogaster

The selector gene apterous (ap) of Drosophila melanogaster is best known for its prominent role in wing formation. apnull flies are viable but have no wings. However, apnull flies are associated with another phenotype whose analysis was largely neglected in the past 40 years: adults die precociously 2-3 days after eclosion. We have recently published a comprehensive analysis on the physiology of this striking phenotype. We showed that Ap protein is essential early in metamorphosis during hindgut remodeling where it is required for rectal papillae formation in the rectal ampulla. Wild-type flies have four rectal papillae that are involved in selective reabsorption of essential ions and metabolites from the primary urine produced by the Malpighian tubules. In apnull flies, rectal papillae formation is aborted prematurely. The corresponding papillar cells remain stuck in the rectal lumen. This leads to complete intestinal blockage and a cascade of pathologies resulting in premature death. In this study, we genetically identify and functionally dissect a novel tissue specific cis- regulatory element that directs Ap expression in the hindgut. This Life Span Enhancer (apLSE) maps to a [~]400 bp DNA fragment that is activated via only two small regulatory modules. Genetic analysis of apLSE alleles proves that they are essential for papilla formation and thus for adult survival. Our studies solve the 110-year-old enigma about the ap syndrome. They establish the apLSE as a key player linking hindgut morphogenesis and survival. We propose that several aspects of the ap syndrome are a consequence of drastic changes in endocrine homeostasis.

developmental biology↗

bioRxiv2023

Transcriptional control of compartmental boundary positioning during Drosophila wing development

The establishment of tissue axes is fundamental during embryonic development. In the Drosophila wing, the anterior/posterior (AP) and the dorsal/ventral (DV) compartment boundaries provide the basic coordinates around which the tissue develops. These boundaries arise as a result of two lineage decisions, the acquisition of posterior fate by the selector gene engrailed (en) and dorsal fate by the selector gene apterous (ap). While the en expression domain is set up during embryogenesis, ap expression begins only during early wing development. Thus, the correct establishment of the ap expression pattern relative to en must be tightly controlled. Here, we functionally investigate the transcriptional inputs integrated by the early ap enhancer (apE) and their requirement for correct boundary formation. Detailed mutational analyses using CRISPR/Cas revealed a role for apE in positioning the DV boundary relative to the AP boundary, with apE mutants often displaying mirror-image anterior wing duplications. We then designed and applied methods to accomplish tissue-specific enhancer disruption via dCas9 expression. This approach allowed us to dissect the spatiotemporal requirement for apE function, clarifying the mechanism by which apE misregulation leads to AP defects. Base-pair-resolution analyses of apE uncovered a single HOX binding site essential for wing development that, when mutated, led to wingless flies. We demonstrated that the transcription factors Pointed (Pnt), Homothorax (Hth), and Grain (Grn) are required for apE function, and the HOX gene Antennapedia (Antp) contributes to early wing development. Together, our results provide a comprehensive molecular basis of early ap activation and the developmental consequences of its misregulation, shedding light on how compartmental boundaries are set up during development.

developmental biology↗

bioRxiv2021

In vivo regulation of fluorescent fusion proteins by engineered kinases

Reversible protein phosphorylation by kinases in extensively used to control a plethora of processes essential for proper development and homeostasis of multicellular organisms. One main obstacle in studying the role of a defined kinase-substrate interaction is that kinases form complex signaling networks and most often phosphorylate multiple substrates involved in various cellular processes. In recent years, several new approaches have been developed to control the activity of a given kinase. However, most of them fail to regulate a single protein target, likely hiding the effect of a unique kinase-substrate by pleiotropic effects. To overcome this limitation, we have created protein binder-based engineered kinases for direct, robust and tissue-specific phosphorylation of target fluorescent protein fusions in vivo. We show that synthetic Rok kinases, based on the Drosophila ortholog of Rho-associated protein kinase (ROCK), are functional enzymes and can activate myosin II through phosphorylation of Sqh::GFP or Sqh::mCherry in different morphogenetic processes in a developing fly embryo. We next use the system to study the impact of actomyosin activation specifically in the developing tracheal branches and showed that ectopic activation of actomyosin with engineered Rok kinase did not prevent cell intercalation nor the formation of autocellular junctions. We assume that this approach can be adapted to other kinases and targets in various eukaryotic genetic systems.

developmental biology↗

bioRxiv2020

Asymmetric requirement of Dpp/BMP morphogen dispersal in the Drosophila wing disc

How morphogen gradients control patterning and growth in developing tissues remains largely unknown due to lack of tools manipulating morphogen gradients. Here, we generate two membrane-tethered protein binders that manipulate different aspects of Decapentaplegic (Dpp), a morphogen required for overall patterning and growth of the Drosophila wing. "HA trap" is based on a single-chain variable fragment (scFv) against the HA tag that traps HA-Dpp to mainly block its dispersal, while "Dpp trap" is based on a Designed Ankyrin Repeat Protein (DARPin) against Dpp that traps Dpp to block both its dispersal and signaling. Using these tools, we found that, while posterior patterning and growth require Dpp dispersal, anterior patterning and growth largely proceed without Dpp dispersal. We show that dpp transcriptional refinement from an initially uniform to a localized expression and persistent signaling in transient dpp source cells render the anterior compartment robust against the absence of Dpp dispersal. Furthermore, despite a critical requirement of dpp for the overall wing growth, neither Dpp dispersal nor direct signaling is critical for lateral wing growth after wing pouch specification. These results challenge the long-standing dogma that Dpp dispersal is strictly required to control and coordinate overall wing patterning and growth.

developmental biology↗

bioRxiv2018

DARPins recognizing mTFP1 as novel reagents for in vitro and in vivo protein manipulations

Over the last few years, protein-based affinity reagents have proven very helpful in cell and developmental biology. While many of these versatile small proteins can be expressed both in the intracellular and extracellular milieu in cultured cells and in living organisms, they can also be functionalized by fusing them to different protein domains in order to regulate or modulate their target proteins in diverse manners. For example, protein binders have been employed to degrade, trap, localize or enzymatically modify specific target proteins. Whereas binders to many endogenous proteins or small protein tags have been generated, also several affinity reagents against fluorescent proteins have been created and used to manipulate target proteins tagged with the corresponding fluorescent protein. Both of these approaches have resulted in improved methods for cell biological and developmental studies. While binders against GFP and mCherry have been previously isolated and validated, we now report the generation and utilization of designed ankyrin repeat proteins (DARPins) against the monomeric teal fluorescent protein 1 (mTFP1). Here we use the generated DARPins to delocalize Rab proteins to the nuclear compartment, in which they cannot fulfill their regular functions anymore. In the future, such manipulations might enable the production of acute loss-of-function phenotypes in different cell types or living organisms based on direct protein manipulation rather than on genetic loss-of-function analyses.\n\nSummary statementStructural characterization of two novel DARPins (designed ankyrin repeat proteins) recognizing the monomeric teal fluorescent protein 1 (mTFP1) and their functionalization for protein manipulation strategies in cultured cells and potentially in living organisms.

cell biology↗

bioRxiv2017

Junction-based lamellipodia drive endothelial cell rearrangements in vivo via a VE-cadherin/F-actin based oscillatory ratchet mechanism

Angiogenesis and vascular remodeling are driven by a wide range of endothelial cell behaviors, such as cell divisions, cell movements, cell shape and polarity changes. To decipher the cellular and molecular mechanism of cell movements, we have analyzed the dynamics of different junctional components during blood vessel anastomosis in vivo. We show that endothelial cell movements are associated with oscillating lamellipodia-like structures, which are orientated in the direction of these movements. These structures emerge from endothelial cell junctions and we thus call them junction-based lamellipodia (JBL). High-resolution time-lapse imaging shows that JBL are formed by F-actin based protrusions at the front end of moving cells. These protrusions also contain diffusely distributed VE-cadherin, whereas the junctional protein ZO-1 (Zona occludens 1) remains at the junction. Subsequently, a new junction is formed at the front of the JBL and the proximal junction is pulled towards the newly established distal junction. JBL function is highly dependent on F-actin dynamics. Inhibition of F-actin polymerization prevents JBL formation, whereas Rac-1 inhibition interferes with JBL oscillations. Both interventions disrupt endothelial junction formation and cell elongation. To examine the role of VE-cadherin (encoded by cdh5 gene) in this process, we generated a targeted mutation in VE-cadherin gene (cdh5ubs25), which prevents VE-cad/F-actin interaction. Although homozygous ve-cadherin mutants form JBL, these JBL are less dynamic and do not promote endothelial cell elongation. Taken together, our observations suggest a novel oscillating ratchet-like mechanism, which is used by endothelial cells to move along or over each other and thus provides the physical means for cell rearrangements.

developmental biology↗