By Michael Heide and Wieland B. Huttner

Introduction

The neocortex is the seat of our higher cognitive abilities that distinguish us from other mammals and that make us human (Rakic, 2009). One basis for this crucial feature is the increase in the size of the neocortex during hominin evolution, culminating in modern humans (Striedter, 2005, Azevedo et al., 2009, Kaas, 2013, Sousa et al., 2017, Molnar et al., 2019). A key issue in this context is the identification of genomic determinants that underlie the increased growth of the neocortex during brain development. Once candidate genes have been identified, the challenge then is to demonstrate their role in neocortex expansion in an appropriate system. Here, we summarize the background information that suggested the human-specific gene ARHGAP11B to be a candidate to have contributed to neocortex expansion and folding during human evolution, and explain the rationale for demonstrating this role in an ARHGAP11B-transgenic non-human primate model system, that is, fetuses of the common marmoset. Such functional, developmental and evolutionary studies have so far been rarely performed in transgenic non-human primates. In our recent publication, Heide et al. 2020, we show that the human-specific gene ARHGAP11B, when expressed to physiological levels in the fetal marmoset neocortex, indeed causes neocortex expansion and increases neocortex folding, implying that this gene significantly contributed to human neocortex evolution (Heide et al., 2020). Moreover, we hope that our findings provide evidence in support of the usefulness and feasibility of genetically modified non-human primates for neurodevelopmental and evolutionary studies, as well as other fields of neurobiology.

A need for genetically modifiable non-human primate models in neurobiological studies

Which systems are most appropriate for studying the development of the neocortex with the aim to gain insight into the development and evolution of the human neocortex? Commonly used animal models that lend themselves to genetic modifications have been smooth-brained (lissencephalic) rodents (mouse, rat) and the ferret, a carnivore with a folded (gyrencephalic) brain (Molnar et al., 2006, Sun and Hevner, 2014, Kawasaki, 2018, Zhao and Bhattacharyya, 2018). However, many findings about neocortical development obtained in these model systems cannot be translated as such to humans, as is exemplified, in particular, in the case of many mouse disease models (e.g., mouse models of primary microcephaly (Pinson et al., 2019)).

A recently emerged, promising in vitro model system of human neocortical development that overcomes some of these limitations are human brain organoids (Kadoshima et al., 2013, Lancaster et al., 2013, Camp et al., 2015, Qian et al., 2016, Quadrato et al., 2017). These are 3D cellular assemblies that model the tissue of certain brain regions over a particular developmental time window and recapitulate many aspects of the cytoarchitecture and cell-type complexity of the modelled brain tissue (Heide et al., 2018). Moreover, comparison of brain organoids of human vs. non-human primate origin (macaque, chimpanzee, orang-utan) has led to the identification of human-specific features of neocortical development (Otani et al., 2016, Mora-Bermudez et al., 2016, Pollen et al., 2019, Kanton et al., 2019). Importantly, brain organoids can easily be genetically modified (Fischer et al., 2019), and human brain organoids have been shown to recapitulate gene expression patterns of fetal human neocortex (Camp et al., 2015). However, despite its promise, the brain organoid model system still has certain limitations, such that is does not fully recapitulate the cytoarchitecture, cell-type composition and late and postnatal stages of neocortex development (Heide et al., 2018). Moreover, cell-type specification in brain organoids seems to be impaired due to cellular stress (Bhaduri et al., 2020).

Hence, fetal human brain tissue ex vivo (Rakic, 2006, Florio et al., 2015, Johnson et al., 2015, Pollen et al., 2015, Kalebic et al., 2019, Namba et al., 2020) and non-human primate models (Smart et al., 2002, Rakic, 2006, Kelava et al., 2012, Garcia-Moreno et al., 2012, Betizeau et al., 2013) have been increasingly used to overcome the limitations of the rodent and ferret models and of brain organoids for studying the development of the neocortex with the aim to gain insight into the development and evolution of the human neocortex. However, the use of fetal human brain tissue ex vivo is limited to early stages of neocortical development, and genetic modification approaches can only be conducted – for obvious ethical reasons – with ex vivo fetal human brain tissue. It is for these reasons that genetically modifiable non-human primate models have recently emerged as system of choice to gain insight into human neocortex development and evolution (Sasaki et al., 2009, Niu et al., 2010, Niu et al., 2014, Shi et al., 2019, Heide et al., 2020). A major advantage of non-human primate models is that in terms of morphology, cell-type composition, gene expression and interaction partners, these models are much closer to human neocortex development than the rodent and ferret models. Moreover, genetically modifiable non-human primate models are likely to have huge potential in generating models of neurological and neurodevelopmental disorders.

ARHGAP11B — a human-specific gene that may drive human neocortex expansion and folding?

The ARHGAP11B gene evolved ~5 mya, that is, after the split of the lineage leading to modern humans and the lineage leading to chimpanzee and bonobo, by segmental duplication of the widespread gene ARHGAP11A (Sudmant et al., 2010). In other words, ARHGAP11B is only found in humans and hence is a human-specific gene. However, ARHGAP11B is not a simple copy of ARHGAP11A, as the ARHGAP11B protein corresponds to only the N-terminal one quarter of the ARHGAP11A protein and, importantly, possesses a unique, human-specific 47 amino acid sequence in its C-terminal domain that is key for its function (Florio et al., 2015, Namba et al., 2020). This unique C-terminal protein sequence arises from a single C-to-G nucleotide substitution in ARHGAP11B that generated a novel splice donor site, resulting in the loss of 55 nucleotides upon mRNA splicing. This loss in turn causes a shift in the reading frame, leading to ARHGAP11B’s unique C-terminal 47 amino acid sequence (Florio et al., 2016). Previous functional analyses by strong overexpression of this gene in embryonic mouse (Florio et al., 2015) and ferret (Kalebic et al., 2018) neocortex showed increased numbers of basal progenitor cells and of upper-layer neurons, the class of progenitors and type of cortical neurons, respectively, that have been implicated in neocortex expansion (Lui et al., 2011, Fame et al., 2011, Borrell and Reillo, 2012, Betizeau et al., 2013, Florio and Huttner, 2014, Dehay et al., 2015, Lodato and Arlotta, 2015). Moreover, in the case of mouse, ARHGAP11B overexpression could induce folding of the normally smooth neocortex (Florio et al., 2015). These findings suggested that ARHGAP11B is a candidate gene to have contributed to neocortex expansion and folding during human evolution. However, the key question was whether this gene, when expressed to physiological levels and in a primate model that is evolutionarily closer to human than mouse or ferret, would increase neocortex size and folding.

Which genetically modifiable non-human primate model to choose?

We therefore searched for a genetically modifiable primate model that is evolutionarily as close as (ethically) possible to human and that possesses all relevant stem cell populations at the correct relative abundance in the developing neocortex, to allow us to explore the potential role of ARHGAP11B in the expansion of the neocortex during development and human evolution. This confined our choice to two non-human primate models, the common marmoset (Callithrix jacchus) and the macaque (eitherMacaca mulatta or Macaca fascicularis). We chose to focus on the marmoset, as this New World monkey, while exhibiting many of the features of the large and folded human neocortex, has – in contrast to the macaque – a small and unfolded neocortex, making it thus an ideal model to study ARHGAP11B’s potential contribution to neocortical expansion and folding.

ARHGAP11B increases fetal primate neocortex size and folding

In Heide et al. 2020, we generated, in collaboration with the research groups of Erika Sasaki and Hideyuki Okano in Japan who have pioneered the transgenic marmoset technology (Sasaki et al., 2009), ARHGAP11B-transgenic marmoset fetuses (Figure A) that expressed ARHGAP11B in the developing neocortex under the control of its own, human promoter (Heide et al., 2020).

In the neocortex of these ARHGAP11B-transgenic marmoset fetuses, ARHGAP11B was expressed to physiological levels, that is, to similar levels as in fetal human neocortex, and its expression within the neocortex was confined to neural stem and progenitor cells (Heide et al., 2020). Furthermore, this ARHGAP11B expression increased the abundance of basal radial glia (Heide et al., 2020) – the basal progenitor cell type thought to play a key role in neocortex expansion and folding, notably in human neocortex development (Lui et al., 2011, Borrell and Reillo, 2012 Florio and Huttner, 2014). This was accompanied by an increase in upper-layer neurons (Heide et al., 2020) — the cortical neuron type thought to strongly contribute to our higher cognitive abilities (Fame et al., 2011). At the supracellular level, this ARHGAP11B expression resulted in increased neocortex size and induced folding of the normally unfolded fetal marmoset neocortex (Heide et al., 2020) (Figure B). In summary, our study strongly suggests that ARHGAP11B did contribute to neocortex expansion and folding in the course of human evolution.

Ethical considerations

Experiments involving non-human primates have been under constant public and scientific debate, and it is perfectly legitimate to scrutinize whether or not such experiments are really necessary and to ask whether other animal model systems are available that are appropriate to address the same scientific questions. Furthermore, experiments involving non-human primates should be performed according to the highest ethical standards.

In our case, we reasoned that in order to answer the question whether the human-specific gene ARHGAP11B contributed to neocortex expansion and folding during human evolution, it was necessary to express ARHGAP11B in a suitable non-human primate model, that is, the common marmoset. We limited these experiments and analyses to fetal stages, for two main reasons. First, should ARHGAP11Bexpression increase neocortex size and neuron numbers as we anticipated, the consequences for the postnatal and adult marmosets with regard to behaviour and nervous system performance might be unpredictable. In this context, one should realize that increases in neocortex size and neuron numbers are not necessarily beneficial but can be the underlying cause of certain human diseases (e.g., megalencephaly, polymicrogyria), and accordingly could lead to suffering of the animals (e.g., from seizures). To avoid generating ARHGAP11B-expressing postnatal and adult marmosets, all ARHGAP11B-expressing marmoset fetuses analyzed in our study were newly and individually generated (rather than being descendants of an ARHGAP11B-expressing marmoset line). Second, our previous data had indicated that ARHGAP11B expression increased basal progenitors and upper-layer neurons during the development of the neocortex. This suggested that in our case an analysis of fetal stages of ARHGAP11B-expressing marmosets would be appropriate and constructive, and would allow closely monitoring neocortex development and analyzing it at the relevant stages.

We hope that our study shows that genetically modified non-human primate fetuses can be superior models to understand human neocortex development and evolution. Furthermore, our work will hopefully encourage other researchers from the present and other fields of neurobiology (including disease modelling) to study genetically modified non-human primate models in the future, as (i) such experiments can be performed in an ethically justifiable way (see above), (ii) the results may answer questions that other, non-primate models cannot answer, and (iii) the results most likely can be directly translated to humans.

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Join the Tissue Architecture Lab at the Novo Nordisk Foundation Center for Stem Cell Biology, DanStem at Copenhagen University!

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By Fei Zhang and Vivian F. Irish

Flowering plants, from giant sequoias to miniscule duckweed, all depend on the action of small populations of cells, called meristems, to grow.  Meristems contain stem cells that continue to proliferate to give rise to roots, shoots, leaves, and branches. However, there are situations in which meristematic cells cease their proliferative activity and terminally differentiate; this occurs in flowers, and floral specific gene networks have been identified that terminate stem cell activity as flower development ensues. A far less well studied example of meristem termination is the formation of thorns. A number of plant species equip themselves with thorns to deter herbivores yet little is known of how these weapons are made.

Over ten years ago, our group set out to understand how meristematic activity is arrested during thorn development. Citrus was selected as a model for thorn development for two reasons: agronomic importance and feasibility. Citrus is one of the most economically valuable fruit crops in the world, and thornlessness is a breeding target as thorns affect fruit harvesting efficiency and can physically damage fruits. In addition, research in citrus is practical: the genome sequences of several citrus species are available, there are naturally occurring citrus variants that lack thorns, and citrus is amenable to genetic transformation. As tractable mutants are key for functional genetics studies, we established a fast and efficient citrus genome editing system by using the YAO promoter to drive Cas9 expression and employed heat stress treatments to further increase CRISPR-mediated gene editing efficiency. This system has proven to be very effective and we can now inactivate up to six genes simultaneously in primary citrus transformants in just a few months.

In our recent paper, Zhang et al 2020, we first characterized thorn growth and defined stages from primordium initiation to terminal differentiation. At early stages, the thorn primordia appear dome-shaped, like that of typical shoot meristems. By stage 7, the thorn tip narrows and at stage 8, thorns elongate and become pointed. Furthermore, the expression of two meristem-expressed genes, SHOOT MERISTEMLESS (STM) and WUSCHEL (WUS) are downregulated at stage 8, with STM expression becoming confined to vascular tissues and WUS expression becoming undetectable. Cell division completely ceases by stage 13, and the thorn lignifies to produce the sharp hard stiletto-like tip.

The question that naturally arises is what terminates thorn meristem gene expression at stage 8. Using a thornless Mexican lime natural variant in comparison to normal Mexican limes, we performed transcriptome analyses of young shoots. We focused on differentially expressed transcription factors that are expressed preferentially in young thorns. One candidate gene, THORN IDENTITY1, encodes a TCP transcription factor, and its homologues control bud dormancy in other plant species and so was an excellent candidate for further analyses. Interestingly, TI1 expression starts at stage 8, and a CRISPR-induced mutation of TI1 leads to the transformation of some thorns into branches. TI1 has a paralogue in citrus, TI2, whose expression in thorns starts from stage 7, and a ti2 mutation also shows the thorn transformed into branch phenotype. Mutation of TI1 and TI2 together transforms nearly every thorn into a branch, resulting in a very bushy phenotype (Figure 1).

WUS expression is upregulated in ti1, ti2 and ti1ti2 mutants, suggesting that the TI genes may function through repressing WUS transcription. Supporting this, both TI1 and TI2 repress WUS promoter activity in transient expression assays, and TI1 directly binds to the WUS promoter as demonstrated by both chromatin immunoprecipitation and gel shift assays.  Interestingly, the citrus WUS promoter has a TCP consensus binding site that is not present in the model plant Arabidopsis thaliana, and we showed that mutation of this cis-element can abolish TI1-WUS promoter regulation. Finally, disruption of the TI1–WUS pathway by either ectopic expression of WUS or via a WUS promoter mutation affects thorn development, indicating that this pathway is critical for thorn growth.

In the classic textbook “Patterns in Plant Development”, by Taylor Steeves and Ian Sussex, thorns are viewed as “one of the most striking departures from typical shoot morphology”. Our work serves as a first step for understanding how such evolutionary novelties arise. With the identification of citrus thorn identity TI genes and a previously undescribed TI–WUS pathway for stem cell regulation, we now have a handle to explore the upstream and downstream regulators involved in weaponizing plants. It is also worth mentioning that thorns have evolved multiple times, and can exhibit very striking departures from shoots, for instance as in honey locust thorns (Figure 2). It still remains to be seen if different plant species redeploy the TI-WUS pathway for thorn development, or if different species armor themselves in unique ways.

Fei Zhang, Pascale Rossignol, Tengbo Huang, Yewei Wang, Alan May, Christopher Dupont, Vladimir Orbovic, Vivian F. Irish. 2020. ‘Reprogramming of Stem Cell Activity to Convert Thorns into Branches’. Current Biology 

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The Group of Nico Posnien at the Department of Developmental Biology of the Georg-August-University of Göttingen invites applications for a fully funded

PostDoc Position

starting from November 1, 2020 for the duration of one year. The salary is in accordance with the German public service salary scale (E13 TV-L) at 100% of the regular working hours (currently 39.8 hours per week).

Background and aim of the project:

We study the evolution of Drosophila head shape and compound eye size (e.g. see here) to contribute to a better understanding of the molecular basis of the morphological diversity. The PostDoc project will focus on interspecific differences in head development between D. americana and D. novamexicana and it builds on prior phenotypic and genetic data. The major aim of the project is revealing interspecific differences in developmental gene expression and chromatin accessibility using an already available extensive RNAseq and ATACseq dataset. Expression and accessibility data will be integrated with genome wide association mapping (GWAS) data (see here) to link variation in gene regulation to phenotypic differences. Additionally, the impact of various inversions that are present between D. americana and D. novamexicana (see here) on gene regulation and phenotypic differences will be tested.

Your profile:

• PhD in Biology, Bioinformatics or related fields
• Experience with Next Generation Sequencing (NGS) data is expected
• Background in Bioinformatics, Statistics and strong analytical skills is expected
• Experience with molecular biology (cloning, PCR) and/or Drosophila genetics is advantageous

What we offer:

The University of Göttingen is part of the interdisciplinary Göttingen Campus that offers a vital environment for basic research and a well-organized PostDoc community. The University of Göttingen is an equal opportunities employer and places particular emphasis on fostering career opportunities for women. Qualified women are therefore strongly encouraged to apply in fields in which they are underrepresented. The university has committed itself to being a family-friendly institution and supports their employees in balancing work and family life. The mission of the University is to employ a greater number of severely disabled persons. Applications from severely disabled persons with equivalent qualifications will be given preference.

Please send your application (motivation letter, CV, academic certificates and transcript of records, as well as contact information of 1-3 references) as a single pdf file to nposnie@gwdg.de.

Closing date for applications: August 23, 2020. For further information visit our website (https://www.posnien-lab.net/) or contact me via Email (nposnie@gwdg.de).

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We are seeking a highly motivated, organized and enthusiastic postdoctoral fellow or research associate to study embryonic kidney development and its relationship to developmental kidney diseases. We are interested in the role of Wnt signaling in shaping nephric tubules, utilizing Xenopus (frog) embryos and mammalian tissue culture as models. Current goals include: 1) Determining how junctions are formed during tubule formation; 2) Modeling how patient mutations result in human developmental kidney diseases; 3) Discovering novel components affecting nephron development; 4) Visualizing in vivo tube formation using advanced live imaging techniques; 5) Generating transgenic animals to visualize nephrogenesis in vivo

Current projects utilize developmental, molecular and cell biological approaches including imaging in living embryos. Applicants with a Ph.D., M.D. or equivalent and a strong background in Developmental Biology, Embryology, Cell Biology, Molecular Biology and/or Stem Cell Biology are highly encouraged to apply. Salary and benefits are commensurate with relevant experience. Review of applications will continue until the position has been filled.

Please send your CV, cover letter indicating current and future research interests, and the name/email address of three references to:

Rachel K. Miller, Ph.D.

Department of Pediatrics

The University of Texas Medical School at Houston

Email: Rachel.K.Miller@uth.tmc.edu

https://med.uth.edu/pediatrics/miller-lab/

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Registration is now open for our virtual networking event!

With COVID-19 cancelling conferences, researchers have lost one of their best ways to network. So we thought we’d try to help, aided by software that creates a virtual space for researchers to interact via video.

At this event you’ll meet other developmental biologists from across the world, discuss relevant topics and test your knowledge with a pub quiz.

The event will be split into four parts:

• Speed networking – meet the other attendants
• Discussion tables – topics picked by attendees, currently ranging from publishing to careers, starting your lab to promoting diversity (we welcome further suggestions and are looking for table chairs to lead the discussions – just email thenode@biologists.com)
• Pub quiz – how good is your devbio knowledge? Winning table gets a real life prize. Bring your own beer or coffee
• Informal hang out – once the structured sessions are over, we’ll leave the session open for people to hang out

We look forward to meeting you virtually!

Register here:

https://www.eventbrite.co.uk/e/the-node-10th-birthday-virtual-networking-event-tickets-113775309012

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An ERC funded research engineer position in Developmental and Cell Biology is available at the IBDM in Marseille in the group of Frank Schnorrer. The appointment will be for one year with a possible extension for up to four years.

We are looking for a highly motivated scientist, with experience in working at an English-speaking international research institute. The research project will require a particular knowledge of imaging, image processing, genetics and experience in developmental and cell biology in general.

The Schnorrer group is located at the Institute of Developmental Biology of Marseille (IBDM), an interdisciplinary research center with a strong focus on quantitative in vivo biology, microscopy and biophysics. Our group is specifically interested in how the contractile elements of muscles called sarcomeres are built during development and how they are maintained during the life of the fly.

See here for more information.

Please send your application, including a motivation letter, your CV and contact information for one or two references to: frank.schnorrer@univ-amu.fr

For more information and the application form see here.

Annoucement in pdf

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The Tata lab in the Department of Cell Biology, Duke University School of Medicine has two openings for postdoctoral researchers to study cellular plasticity mechanisms in lung injury repair and tumorigenesis. We seek to understand the genetic and epigenetic basis of organ regeneration and tumorigenesis. We study the properties of stem/progenitor cells in diverse epithelial tissues (with a primary focus on lung) and their relationships with neighboring tissues in pathophysiological conditions. We utilize in vivo mouse genetics, live imaging, 3D organoids, genome-wide Cas9/Crispr based functional genetic screening, and next generation sequencing technologies to study the behavior of tissues at single cell level. We offer an inspiring intellectual, collaborative and multidisciplinary research environment to support your career goals and provide access to state-of-the-art facilities. Candidates with background knowledge and hands-on experience in transcriptional regulation, 3D-organoids and bioinformatics skills are particularly welcome.

Requirements

– A PhD or MD/PhD (or equivalent) in biological sciences (cell & developmental biology or a related field).
– Strong research background in transcriptional regulation, cell biology, molecular biology, mouse models of cancer, and/or biochemistry. Prior experience in stem cells, Cas9/Crispr gene editing, 3D-organoid cultures, chromatin biology and bioinformatics is advantageous.
– Evidence of successful completion of a research project (publications)
– Ability to work independently; interpret, present and discuss experimental data
– Excellent communications skills

To apply, please submit a cover letter (less than a page) with a short summary of research interests, CV, and the contact information of 3 professional references to Dr. Purushothama Rao Tata (purushothamarao.tata@duke.edu) or apply at https://academicjobsonline.org/ajo/jobs/16539

Applications will be reviewed until positions are filled. The positions are available immediately.

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