Control of cardiac contractions using Cre-lox and degron strategies in zebrafish.

Cardiac contractions and hemodynamic forces are essential for organ development and homeostasis. Control over cardiac contractions can be achieved pharmacologically or optogenetically. However, these approaches lack specificity or require direct access to the heart. Here, we compare two genetic approaches to control cardiac contractions by modulating the levels of the essential sarcomeric protein Tnnt2a in zebrafish. We first recombine a newly generated tnnt2a floxed allele using multiple lines expressing Cre under the control of cardiomyocyte-specific promoters, and show that it does not recapitulate the tnnt2a/silent heart mutant phenotype in embryos. We show that this lack of early cardiac contraction defects is due, at least in part, to the long half-life of tnnt2a mRNA, which masks the gene deletion effects until the early larval stages. We then generate an endogenous Tnnt2a-eGFP fusion line that we use together with the zGRAD system to efficiently degrade Tnnt2a in all cardiomyocytes. Using single-cell transcriptomics, we find that Tnnt2a depletion leads to cardiac phenotypes similar to those observed in tnnt2a mutants, with a loss of blood and pericardial flow-dependent cell types. Furthermore, we achieve conditional degradation of Tnnt2a-eGFP by splitting the zGRAD protein into two fragments that, when combined with the cpFRB2-FKBP system, can be reassembled upon rapamycin treatment. Thus, this Tnnt2a degradation line enables non-invasive control of cardiac contractions with high spatial and temporal specificity and will help further understand how they shape organ development and homeostasis.

Pathway to Independence: the future of developmental biology.

In 2022, Development launched its Pathway to Independence (PI) Programme, aimed at supporting postdocs as they transition to their first independent position. We selected eight talented researchers as the first cohort of PI Fellows. In this article, each of our Fellows provides their perspective on the future of their field. Together, they paint an exciting picture of the current state of and open questions in developmental biology.

Biogenesis and function of ESCRT-dependent extracellular vesicles.

From bacteria to humans, cells secrete a large variety of membrane-bound extracellular vesicles. Only relatively recently has it however started to become clear that the exovesicular transport of proteins and RNAs is important for normal physiology and numerous pathological conditions. Extracellular vesicles can be formed through the release of the intralumenal vesicles of multivesicular endosomes as so-called exosomes, or through direct, ectosomal, budding from the cell surface. Through their ability to promote the bending of membranes away from the cytoplasm, the components of the Endosomal Sorting Complex Required for Transport (ESCRT) have been implicated in both exo- and ectosomal biogenesis. Studies of the ESCRT machinery may therefore provide important insights into the formation and function of extracellular vesicles. In the present review, we first describe the cell biological mechanisms through which ESCRT components contribute to the biogenesis of different types of extracellular vesicles. We then discuss how recent functional studies have started to uncover important roles of ESCRT-dependent extracellular vesicles in a wide variety of processes, including the transport of developmental signaling molecules and embryonic morphogenesis, the regulation of social behavior and host-pathogen interactions, as well as the etiology and progression of neurodegenerative pathologies and cancer.

egr3 is a mechanosensitive transcription factor gene required for cardiac valve morphogenesis.

Biomechanical forces, and their molecular transducers, including key mechanosensitive transcription factor genes, such as KLF2, are required for cardiac valve morphogenesis. However, klf2 mutants fail to completely recapitulate the valveless phenotype observed under no-flow conditions. Here, we identify the transcription factor EGR3 as a conserved biomechanical force transducer critical for cardiac valve formation. We first show that egr3 null zebrafish display a complete and highly penetrant loss of valve leaflets, leading to severe blood regurgitation. Using tissue-specific loss- and gain-of-function tools, we find that during cardiac valve formation, Egr3 functions cell-autonomously in endothelial cells, and identify one of its effectors, the nuclear receptor Nr4a2b. We further find that mechanical forces up-regulate egr3/EGR3 expression in the developing zebrafish heart and in porcine valvular endothelial cells, as well as during human aortic valve remodeling. Altogether, these findings reveal that EGR3 is necessary to transduce the biomechanical cues required for zebrafish cardiac valve morphogenesis, and potentially for pathological aortic valve remodeling in humans.

Multiple pkd and piezo gene family members are required for atrioventricular valve formation.

Cardiac valves ensure unidirectional blood flow through the heart, and altering their function can result in heart failure. Flow sensing via wall shear stress and wall stretching through the action of mechanosensors can modulate cardiac valve formation. However, the identity and precise role of the key mechanosensors and their effectors remain mostly unknown. Here, we genetically dissect the role of Pkd1a and other mechanosensors in atrioventricular (AV) valve formation in zebrafish and identify a role for several pkd and piezo gene family members in this process. We show that Pkd1a, together with Pkd2, Pkd1l1, and Piezo2a, promotes AV valve elongation and cardiac morphogenesis. Mechanistically, Pkd1a, Pkd2, and Pkd1l1 all repress the expression of klf2a and klf2b, transcription factor genes implicated in AV valve development. Furthermore, we find that the calcium-dependent protein kinase Camk2g is required downstream of Pkd function to repress klf2a expression. Altogether, these data identify, and dissect the role of, several mechanosensors required for AV valve formation, thereby broadening our understanding of cardiac valvulogenesis.

'Boots for my Sancho': structural vulnerability among Latin American day labourers in Berkeley, California.

This paper addresses the structural vulnerability of Latin American undocumented day labourers in Northern California, as it is expressed in conversations on street corners where they wait for work. The intimate aspects of migrant experience become exemplified in jokes about the Sancho - a hypothetical character who has moved in on a day labourer's family and who enjoys the money he sends home. Joking turns to more serious topics of nostalgia and tensions with family far away, elements that come together with the fears and threats of labour on the corner and affect the way day labourers see themselves. Sexuality is rearticulated in the absence of women and masculinity becomes enmeshed in the contingencies of unregulated work and long-term separation from the people the men support. Together, these elements result in the articulation of threat to the immigrant body itself, which is exemplified by anxieties over homosexual propositions on the corner.

Parental mutations influence wild-type offspring via transcriptional adaptation.

Transgenerational epigenetic inheritance (TEI) is mostly discussed in the context of physiological or environmental factors. Here, we show intergenerational and transgenerational inheritance of transcriptional adaptation (TA), a process whereby mutant messenger RNA (mRNA) degradation affects gene expression, in nematodes and zebrafish. Wild-type offspring of animals heterozygous for mRNA-destabilizing alleles display increased expression of adapting genes. Notably, offspring of animals heterozygous for nontranscribing alleles do not display this response. Germline-specific mutations are sufficient to induce TA in wild-type offspring, indicating that, at least for some genes, mutations in somatic tissues are not necessary for this process. Microinjecting total RNA from germ cells of TA-displaying heterozygous zebrafish can trigger TA in wild-type embryos and in their progeny, suggesting a model whereby mutant mRNAs in the germline trigger a TA response that can be epigenetically inherited. In sum, this previously unidentified mode of TEI reveals a means by which parental mutations can modulate the offspring's transcriptome.

Myosin1D is an evolutionarily conserved regulator of animal left-right asymmetry.

The establishment of left-right (LR) asymmetry is fundamental to animal development, but the identification of a unifying mechanism establishing laterality across different phyla has remained elusive. A cilia-driven, directional fluid flow is important for symmetry breaking in numerous vertebrates, including zebrafish. Alternatively, LR asymmetry can be established independently of cilia, notably through the intrinsic chirality of the acto-myosin cytoskeleton. Here, we show that Myosin1D (Myo1D), a previously identified regulator of Drosophila LR asymmetry, is essential for the formation and function of the zebrafish LR organizer (LRO), Kupffer's vesicle (KV). Myo1D controls the orientation of LRO cilia and interacts functionally with the planar cell polarity (PCP) pathway component VanGogh-like2 (Vangl2), to shape a productive LRO flow. Our findings identify Myo1D as an evolutionarily conserved regulator of animal LR asymmetry, and show that functional interactions between Myo1D and PCP are central to the establishment of animal LR asymmetry.

[The ESCRT complex: from endosomal transport to the development of multicellular organisms]. / Le complexe ESCRT : du transport endosomal au développement d'organismes multicellulaires.

Since its discovery more than 50 years ago, the endo-lysosomal system has emerged as a central integrator of different cellular activities. This vesicular trafficking apparatus governs processes as diverse as the transduction of stimuli by growth factor receptors, the recycling and secretion of signaling molecules and the regulation of cellular homeostasis through autophagy. Accordingly, dysfunctions of the vesicular transport machinery have been linked to a growing number of pathologies. In this review we take the "Endosomal Sorting Complex Required for Transport" (ESCRT) as an example to illustrate the multiple functions of an evolutionarily conserved endosomal transport machinery. We describe the major concepts that have emerged from the study of this machinery at the level of the development and the physiology of multi-cellular organisms. In particular, we highlight the essential contributions of ESCRT proteins on the regulation of three biological processes: the endocytic regulation of cell signaling, autophagy and its role in neuronal morphogenesis and finally the biogenesis and function of extracellular vesicles.

Companion Blood Cells Control Ovarian Stem Cell Niche Microenvironment and Homeostasis.

The extracellular matrix plays an essential role for stem cell differentiation and niche homeostasis. Yet, the origin and mechanism of assembly of the stem cell niche microenvironment remain poorly characterized. Here, we uncover an association between the niche and blood cells, leading to the formation of the Drosophila ovarian germline stem cell niche basement membrane. We identify a distinct pool of plasmatocytes tightly associated with the developing ovaries from larval stages onward. Expressing tagged collagen IV tissue specifically, we show that the germline stem cell niche basement membrane is produced by these "companion plasmatocytes" in the larval gonad and persists throughout adulthood, including the reproductive period. Eliminating companion plasmatocytes or specifically blocking their collagen IV expression during larval stages results in abnormal adult niches with excess stem cells, a phenotype due to aberrant BMP signaling. Thus, local interactions between the niche and blood cells during gonad development are essential for adult germline stem cell niche microenvironment assembly and homeostasis.

(Des)orden nacional: la construcción de la migración venezolana como una amenaza de salud y seguridad pública en Colombia / National Dis(order): The Construction of Venezuelan Migration as a Public Health and Security Threat in Colombia / (Des)ordem nacional: a construção da migração venezuelana como uma ameaça de saúde e segurança pública na Colômbia

Resumen Introducción: En este artículo se propone una reflexión sobre la construcción mediática y política de la imagen del migrante venezolano como amenaza a la salud y seguridad pública en Colombia. Desarrollo: A partir de 2015, la migración masiva de venezolanos enfrenta a la sociedad colombiana a una situación inédita en su historia reciente. En medio de un contexto atravesado por la hiperpolitización de las relaciones entre ambos países, miles de migrantes se han encontrado con la estigmatización en una sociedad que desarrolla mecanismos para contener las posibles 'amenazas' que representa su movilidad. Para tales fines, este artículo aborda tanto noticias e imágenes de diferentes medios de comunicación, como discursos políticos y noticias falsas que circulan mediante cadenas de WhatsApp que, en su conjunto, terminan por afianzar la percepción de amenaza en distintos sectores de la sociedad colombiana. Conclusiones: Las políticas de solidaridad y ayuda que pregona el Estado van de la mano con mensajes contradictorios producidos en los medios que, en últimas, le presentan al público a una población indiferenciada que pone en peligro al cuerpo de la nación.

Abstract Introduction: This article addresses the political and mediatized construction of the "Venezuelan migrant" as a threat to public health and security in Colombia. Development: Since 2015, massive migration from Venezuela has forced Colombian society into an unprecedented situation in its recent history. Amidst the hyper-politicization of bilateral relations between the countries, thousands of Venezuelan migrants have crossed the border, only to encounter stigmatization in a society that is in the process of developing different mechanisms to contain the supposed "danger" they represent. We use digital news reports and images published by media outlets, political speeches, and "fake news" messages disseminated through WhatsApp to show how this perception of threat consolidates. Conclusions: We suggest that the State's push to address the problem with solidarity and aid goes hand in hand with contradictory messages produced by the media, which present the Colombian public with an undifferentiated population that threatens the body of the nation.

Resumo Introdução: Neste artigo propomos uma reflexão sobre a construção mediática e política da imagem do "migrante venezuelano" como ameaça à saúde e segurança pública n Colômbia. Desenvolvimento: A partir do ano 2015 a migração massiva de venezuelanos enfrenta à sociedade colombiana a uma situação inédita em sua história recente. No meio de um contexto atravessado pela hiperpolitização das relações entre ambos os países, milhares de migrantes se têm encontrado com a estigmatização em uma sociedade que desenvolve mecanismos para conter as "ameaças" que representa sua mobilidade. Para esses fins, o artigo aborda tanto notícias e imagens de diferentes meios de comunicação, quanto discursos políticos e notícias falsas que circulam através de cadeias de WhatsApp, que em conjunto terminam por afiançar a percepção de ameaça em distintos setores da sociedade colombiana. Conclusões: Sugerimos que as políticas de solidariedade e ajuda que proclama o Estado vão da mão com mensagens contraditórios produzidos nos meios que, no final, apresentam ao publico uma população indiferenciada que põe em perigo ao corpo da nação.

Toracotomia em bloco em cães pré-medicados com acetilpromazina e anestesiados com tiopental sódico: efeitos sobre os gases sangüíneos e equilíbrio ácido-base / En bloc thoracotomy in dogs premedicated with acetylpromazine and anesthetized with thiopental sodium: effects on blood gases and acid-base-balance

A medição dos gases sangüíneos foi realizada em cães submetidos à técnica de toracotomia em bloco. A medicação pré-anestésica constou de acetilpromazina administrada pela via intravenosa. Com injeção intravenosa de tiopental sódico foram providas a indução, intubação orotraqueal e manutenção da anestesia cirúrgica. O traqueotubo foi conectado a um respirador mecânico pressométrico, que manteve o animal oxigenado durante a operação. Foram colhidas amostras de sangue arterial por punção da artéria femoral para determinação do pH, pressão de dióxido de carbono (PCO2), pressão de oxigênio (PO2), excesso de base (BE) e bicarbonato (HCO3-). Nos animais do grupo II, no final da cirurgia (Tempo 1) e 24 horas após (Tempo 2), foi feita anestesia local dos nervos intercostais junto das costelas seccionadas para comparação das prováveis alterações da mecânica respiratória. A técnica da toracotomia em bloco promoveu excelente exposição das vísceras do tórax, com recuperação satisfatória, sem desconforto e complicação pós-operatória.

Blood gas analysis were measured in dogs submitted to en bloc thoracotomy. Preanesthetic medication consisted of acepromazine maleate injected intravenously. The animal was anesthetized with thiopental sodium intravenously, and the surgical field prepared. The trachea was intubated and mechanical ventilator was used. A radical thoracotomy was performed. Arterial samples of blood were collected from the femoral artery. Arterial blood pH value, carbon dioxide pressure (PCO2), PO2 level, base excess and standard bicarbonato level (HCO3-) were determined. Local anesthesia of intercostal nerves of the thorax, dose to the thoracotomy, were also performed on dogs in group II at end of surgery (T1) and 24 hours (T2) after the surgery. The dog recovered satisfactonly and in the following 7 days had no observable discomfort. It is anticipated that the results of this study will be useful to those using the en bloc thoracotomy, which provided excellent exposure ot the thoracic viscera with no postoperative complications.