Ernst H K Stelzer

Goethe-Universität Frankfurt am Main · Buchmann Institute for Molecular Life Sciences (BMLS)

A key problem in development is to understand how genes turn on or off at the right place and right time during embryogenesis. Such decisions are made by non-coding sequences called 'enhancers'. Much of our models of how enhancers work rely on the assumption that genes are activated de novo as stable domains of gene expressions that undergo little...

Background Organoids are morphologically heterogeneous three-dimensional cell culture systems and serve as an ideal model for understanding the principles of collective cell behaviour in mammalian organs during development, homeostasis, regeneration, and pathogenesis. To investigate the underlying cell organisation principles of organoids, we image...

During the mammalian preimplantation phase, cells undergo two subsequent cell fate decisions. During the first decision, the trophectoderm and the inner cell mass are formed. Subsequently, the inner cell mass segregates into the epiblast and the primitive endoderm. Inner cell mass organoids represent an experimental model system, mimicking the seco...

The p53 homolog TAp63α is the transcriptional key regulator of genome integrity in oocytes. After DNA damage, TAp63α is activated by multistep phosphorylation involving multiple phosphorylation events by the kinase CK1, which triggers the transition from a dimeric and inactive conformation to an open and active tetramer that initiates apoptosis. By...

Organoids are morphologically heterogeneous three-dimensional cell culture systems. To understand the cell organisation principles of their morphogenesis, we imaged hundreds of pancreas and liver organoids in parallel using light sheet and bright field microscopy for up to seven days. We quantified organoid behaviour at single-cell (microscale), in...

During mammalian blastocyst development, inner cell mass (ICM) cells differentiate into epiblast (Epi) or primitive endoderm (PrE). These two fates are characterized by the expression of the transcription factors NANOG and GATA6, respectively. Here, we investigate the spatio-temporal distribution of NANOG and GATA6 expressing cells in the ICM of th...

Thermal motions enable a particle to probe the optimal interaction state when binding to a cell membrane. However, especially on the scale of microseconds and nanometers, position and orientation fluctuations are difficult to observe with common measurement technologies. Here we show that it is possible to detect single binding events of IgG-coated...

Root branching is influenced by the soil environment and exhibits a high level of plasticity. We report that the radial positioning of emerging lateral roots is influenced by their hydrological environment during early developmental stages. New lateral root primordia have both a high degree of flexibility in terms of initiation and development angl...

In three-dimensional light microscopy, the heterogeneity of the optical density in a specimen ultimately limits the achievable penetration depth and hence the three-dimensional resolution. The most direct approach to reduce aberrations, improve the contrast and achieve an optimal resolution is to minimise the impact of changes of the refractive ind...

The genetic control of anterior brain development is highly conserved throughout animals. For instance, a conserved anterior gene regulatory network specifies the ancestral neuroendocrine center of animals and the apical organ of marine organisms. However, its contribution to the brain in non-marine animals has remained elusive. Here, we study the...

Animals must slow or halt locomotion to integrate sensory inputs or to change direction. In Caenorhabditis elegans, the GABAergic and peptidergic neuron RIS mediates developmentally timed quiescence. Here, we show RIS functions additionally as a locomotion stop neuron. RIS optogenetic stimulation caused acute and persistent inhibition of locomotion...

During the mammalian preimplantation phase, cells undergo two subsequent cell fate decisions. During the first cell fate decision, cells become either part of an outer trophectoderm or part of the inner cell mass. Subsequently, the inner cell mass (ICM) segregates into an embryonic and an extraembryonic lineage, giving rise to the epiblast and the...

Cell fate decisions such as apoptosis require cells to translate signaling input into a binary yes/no response. A tight control of the process is required to avoid loss of cells by accidental activation of cell death pathways. One particularly critical situation exists in primary oocytes because their finite number determines the reproductive capac...

The genetic control of anterior brain development is highly conserved throughout animals. For instance, a conserved anterior gene regulatory network specifies the ancestral neuroendocrine center of animals and the apical organ of marine organisms. However, its contribution to the brain in non-marine animals has remained elusive. Here, we study the...

Biglycan, a small leucine-rich proteoglycan, acts as a danger signal and is classically thought to promote macrophage recruitment via Toll-like receptors (TLR) 2 and 4. We have recently shown that biglycan signaling through TLR 2/4 and the CD14 co-receptor regulates inflammation, suggesting that TLR co-receptors may determine whether biglycan-TLR s...

In three-dimensional light microscopy, the heterogeneity of the optical density in a specimen ultimately limits the achievable penetration depth and hence the three-dimensional resolution. The most direct approach to reduce aberrations, improve the contrast, and achieve an optimal resolution is minimizing the impact of changes of the refractive ind...

Spheroids resemble features of tissues and serve as model systems to study cell–cell and cell–ECM interactions in non-adhesive three-dimensional environments. Although it is generally accepted that mature spheroids resemble tissue properties very well, no studies relate different phases in the spheroid formation processes that contribute to tissue...

During mammalian preimplantation, cells of the inner cell mass (ICM) adopt either an embryonic or an extraembryonic fate. This process is tightly regulated in space and time and has been studied previously in mouse embryos and embryonic stem cell models. Current research suggests that cell fates are arranged in a salt-and-pepper pattern of random c...

A key event in cellular physiology is the decision between membrane biogenesis and fat storage. Phosphatidic acid (PA) is an important intermediate at the branch point of these pathways and is continuously monitored by the transcriptional repressor Opi1 to orchestrate lipid metabolism. In this study, we report on the mechanism of membrane recogniti...

ELife digest Researchers frequently use model organisms, such as mice, zebrafish and various insect species, to understand biological processes – with the underlying idea that discoveries made can be applied to other species too. A common technique is genetic manipulation, in which a foreign gene is inserted into the chromosome of an organism. Thes...

Mating procedure results for seven of the thirteen functional AGOC sublines (Non-Lifeact) from the F3 to the F7 generation. Bold entries mark progeny that were used in the subsequent cross. F6-S, F7-O and F7-C are control crosses. No significant differences between the arithmetic means and the theoretical Mendelian ratios were found. See Source Dat...

All 25 vector sequences as Genebank (.gb) and Geneious (.geneious) files compressed into a single zipped folder (.zip).

Raw scores for all mating procedure result tables ordered by transgenic sublines.

Generations. In this table, the F0 to F7 and their characteristics are summarized.

Mating procedure results for six of the thirteen functional AGOC sublines (Lifeact only) from the F3 to the F7 generation. Bold entries mark progeny that were used in the subsequent cross. F6-S, F7-O and F7-C are control crosses. No significant differences between the arithmetic means and the theoretical Mendelian ratios were found. See Source Data...

F2 insert number determination cross. F2 (mO-mC) founder females were mated with wild-type males and the progeny were scored. Segregation of 60% or fewer transgenic descendants was defined as the criterion for one insert. No deviators could be identified.

Transgenic lines and sublines. In total, 7 transgenic lines with 21 sublines were created, that is, 6 proof-of-principle AGOC sublines, 13 functional AGOC sublines and 2 helper sublines. Two of the functional AGOC sublines have been analyzed with live imaging previously, live imaging data for three more is provided in this study.

Fluorescence stereo microscope filter sets. All components were obtained from AHF Analysentechnik, Tübingen, Germany.

F3 homozygous viability crosses. Two F3 (mO-mC) pre-recombination hemizygous siblings were mated and the progeny were scored. Segregation of 70% or more transgenic descendants was defined as the criterion for homozygous viability. Deviators are marked bold.

Insertion junctions. In the Junction column, the piggyBac TTAA insertion/excision target sequence is marked bold.

Mating procedure results for the two proof-of-principle AGOC #5 and #6 sublines from the F3 to the F7 generation with swapped genders as well as with an alternative Cre-expressing homozygous helper subline, ICE{HSP68’NLS-Cre} #2. Bold entries mark progeny that were used in the subsequent cross. F6-S, F7-O and F7-C are control crosses. No significan...

Vector summary. The 24 vectors used/created in this study listed in order of their type. Numbers in square brackets within the Source/molecular cloning column refer to the respective entry. See also (Figure 1—figure supplement 4).

Cloning and inverse PCR primer pairs. Primer pairs are listed in order of appearance in the Materials and methods section and Supplementary file 3. The Applied Biosciences web calculator (www6.appliedbiosystems.com/support/techtools/calc) was used to calculate the melting temperature TM. In case of primers with overhangs, the TM was only calculated...

Metadata and parameter for the long-term live-imaging datasets DS0001-0003.

The survival rate of cancer patients is steadily increasing, owing to more efficient therapies. Understanding the molecular mechanisms of chemotherapy-induced premature ovarian insufficiency (POI) could identify targets for prevention of POI. Loss of the primordial follicle reserve is the most important cause of POI, with the p53 family member p63...

A key event in cellular physiology is the decision between membrane biogenesis and fat storage. Phosphatidic acid (PA) is an important lipid intermediate and signaling lipid at the branch point of these pathways and constantly monitored by the transcriptional repressor Opi1 to orchestrate lipid metabolism. Here, we report on the mechanism of membra...

Movie S4 X‐ray computed tomography multiscan of 60‐d‐old PDR1 OE Petunia hybrida roots grown on clay+.

Movie S2 X‐ray computed tomography multiscan of 42‐d‐old PDR1 OE Petunia hybrida roots grown on clay+.

Movie S1 X‐ray computed tomography multiscan of 42‐d‐old wild‐type Petunia hybrida roots grown on clay+.

Fig. S1 Mycorrhization quantifications via the grid method and gene expression analyses on PhPT3 and PhPT5 in Petunia hybrida roots. Fig. S2 PDR1 OE effects on SL biosynthesis and P uptake in Petunia hybrida. Fig. S3 Semiquantitative SL quantification, 3H‐GR24 transport and leaf senescence in wild‐type and pdr1 ko Petunia hybrida leaves. Fig. S4...

Movie S3 X‐ray computed tomography multiscan of 60‐d‐old wild‐type Petunia hybrida roots grown on clay+.

Movie S5 Time lapse (35 h) of a developing lateral root from Arabidopsis thaliana acquired via light sheet fluorescence microscopy.

Strigolactones (SLs) are carotenoid‐derived phytohormones shaping plant architecture and inducing the symbiosis with endomycorrhizal fungi. In Petunia hybrida, SL transport within the plant and towards the rhizosphere is driven by the ABCG‐class protein PDR1. PDR1 expression is regulated by phytohormones and by the soil phosphate abundance, and thu...

The red flour beetle Tribolium castaneum has become the second most important insect model organism and is frequently used in developmental biology, genetics and pest-associated research. Consequently, the methodological arsenal increases continuously, but many routinely applied techniques for Drosophila melanogaster and other insect species are st...

Comparison of sequencing results from genomic DNA extracted either from the whole body or from one wing for SB and Prl. (A) Comparison for the SB strain. Vertical black bars represent sequence deviations. Two deviations were found within intron 2. (B) Comparison for the Prl strain. Vertical black bars represent sequence deviations. Two deviations w...

SB white allele (genomic DNA extracted from one wing; 8,506 bp). (TXT)

Comparison of survival rate when either one or both wings were dissected by two experimenters. (A) Quantification of surviving adults from five replicates with ten adults after the dissection of one wing. The sham control animals were also paralyzed, and their elytra were also lifted from the abdomen, but the wing was not dissected. No significant...

Primer group list. ATub’, alpha tubulin 1 upstream regulatory sequence; ExPCR, primer for extraction PCRs; FD, forward; RV, reverse. (XLSX)

Prl white allele (genomic DNA extracted from one wing; 8,372 bp). (TXT)

Comparison of the SB and Prl white gene sequences to the GA-2 standard. (A) Comparison for the SB allele. Vertical black bars represent sequence deviations, horizontal black lines show insertions / deletions. Compared to GA-2, the SB white allele has multiple insertions, deletions and substitutions mainly within intron 6. The deviations within the...

Successful PCRs on genomic DNA extracted from one or both wings by using a commercial DNA extraction kit. (A) Quantification of successful PCRs on the alpha-tubulin 1 upstream regulatory sequence from five replicates with ten adults each using genomic DNA extracted from one or both wings as template. No significant difference was found. Error bars...

During mammalian blastocyst development, inner cell mass (ICM) cells differentiate into epiblast (Epi) or primitive endoderm (PrE). These two fates are characterised by the transcription factors NANOG and GATA6, respectively. Here, we present quantitative three-dimensional single cell-based neighbourhood analyses to investigate the spatial distribu...

The adherence junction associated protein 1 (AJAP1, aka shrew-1) is presumably a type-I transmembrane protein localizing and interacting with the E-cadherin-catenin complex. In various tumors, AJAP1 expression is reduced or lost, including hepatocellular and esophageal squamous cell carcinoma, and glial-derived tumors. The aberrant expression of AJ...

Light-sheet-based fluorescence microscopy features optical sectioning in the excitation process. This reduces phototoxicity and photobleaching by up to four orders of magnitude compared with that caused by confocal fluorescence microscopy, simplifies segmentation and quantification for three-dimensional cell biology, and supports the transition fro...

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. The observation of Tribolium embryos with light sheet-based fluorescence microscopy has multiple advantages over conventional widefield and confocal fluorescence microscopy. Due to the unique prope...

Significance Structured illumination microscopy (SIM) is a superresolution technique that illuminates the specimen with a sine-modulated pattern. The frequency of the illumination pattern, i.e., the inverse of the resolution, is limited by the angular aperture of the objective lens. SIM in combination with light-sheet-based fluorescence microscopy...

We describe a method for the three-dimensional live imaging of filamentous fungi with light sheet-based fluorescence microscopy (LSFM). LSFM provides completely new opportunities to investigate the biology of fungal cells and other microorganisms with high spatial and temporal resolution. As an example, we study the established aging model Podospor...

Three-dimensional multicellular aggregates such as spheroids provide reliable in vitro substitutes for tissues. Quantitative characterization of spheroids at the cellular level is fundamental. We present the first pipeline that provides three-dimensional, high-quality images of intact spheroids at cellular resolution and a comprehensive image analy...

Dysregulation of the basal autophagic flux has been linked to several pathological conditions, including neurodegenerative diseases and cancer. In addition, autophagy has profound effects on the response of tumor cells to therapy. Hence, the search for pharmacological modulators of autophagy is of great clinical relevance. We established a drug scr...

Three-dimensional cell biology and histology of tissue sections strongly benefit from advanced light microscopy and optimized staining procedures to gather the full three-dimensional information. In particular, the combination of optical clearing with light sheet-based fluorescence microscopy simplifies fast high-quality imaging of thick biological...