Wnt9 directs zebrafish heart tube assembly via a combination of canonical and non-canonical pathway signaling.

During zebrafish heart formation, cardiac progenitor cells converge at the embryonic midline where they form the cardiac cone. Subsequently, this structure transforms into a heart tube. Little is known about the molecular mechanisms that control these morphogenetic processes. Here, we use light-sheet microscopy and combine genetic, molecular biological and pharmacological tools to show that the paralogous genes wnt9a/b are required for the assembly of the nascent heart tube. In wnt9a/b double mutants, cardiomyocyte progenitor cells are delayed in their convergence towards the embryonic midline, the formation of the heart cone is impaired and the transformation into an elongated heart tube fails. The same cardiac phenotype occurs when both canonical and non-canonical Wnt signaling pathways are simultaneously blocked by pharmacological inhibition. This demonstrates that Wnt9a/b and canonical and non-canonical Wnt signaling regulate the migration of cardiomyocyte progenitor cells and control the formation of the cardiac tube. This can be partly attributed to their regulation of the timing of cardiac progenitor cell differentiation. Our study demonstrates how these morphogens activate a combination of downstream pathways to direct cardiac morphogenesis.

Blood Flow Suppresses Vascular Anomalies in a Zebrafish Model of Cerebral Cavernous Malformations.

RATIONALE: Pathological biomechanical signaling induces vascular anomalies including cerebral cavernous malformations (CCM), which are caused by a clonal loss of CCM1/KRIT1 (Krev interaction trapped protein 1), CCM2/MGC4607, or CCM3/PDCD10. Why patients typically experience lesions only in lowly perfused venous capillaries of the cerebrovasculature is completely unknown. OBJECTIVE: In contrast, animal models with a complete loss of CCM proteins lack a functional heart and blood flow and exhibit vascular anomalies within major blood vessels as well. This finding raises the possibility that hemodynamics may play a role in the context of this vascular pathology. METHODS AND RESULTS: Here, we used a genetic approach to restore cardiac function and blood flow in a zebrafish model of CCM1. We find that blood flow prevents cardiovascular anomalies including a hyperplastic expansion within a large Ccm1-deficient vascular bed, the lateral dorsal aorta. CONCLUSIONS: This study identifies blood flow as an important physiological factor that is protective in the cause of this devastating vascular pathology.

Asymmetric inheritance of the apical domain and self-renewal of retinal ganglion cell progenitors depend on Anillin function.

Divisions that generate one neuronal lineage-committed and one self-renewing cell maintain the balance of proliferation and differentiation for the generation of neuronal diversity. The asymmetric inheritance of apical domains and components of the cell division machinery has been implicated in this process, and might involve interactions with cell fate determinants in regulatory feedback loops of an as yet unknown nature. Here, we report the dynamics of Anillin - an essential F-actin regulator and furrow component - and its contribution to progenitor cell divisions in the developing zebrafish retina. We find that asymmetrically dividing retinal ganglion cell progenitors position the Anillin-rich midbody at the apical domain of the differentiating daughter. anillin hypomorphic conditions disrupt asymmetric apical domain inheritance and affect daughter cell fate. Consequently, the retinal cell type composition is profoundly affected, such that the ganglion cell layer is dramatically expanded. This study provides the first in vivo evidence for the requirement of Anillin during asymmetric neurogenic divisions. It also provides insights into a reciprocal regulation between Anillin and the ganglion cell fate determinant Ath5, suggesting a mechanism whereby the balance of proliferation and differentiation is accomplished during progenitor cell divisions in vivo.

Biasing amacrine subtypes in the Atoh7 lineage through expression of Barhl2.

Within the developing vertebrate retina, particular subtypes of amacrine cells (ACs) tend to arise from progenitors expressing the basic helix-loop-helix (bHLH) transcription factor, Atoh7, which is necessary for the early generation of retinal ganglion cells (RGCs). All ACs require the postmitotic expression of the bHLH pancreas transcription factor Ptf1a; however, Ptf1a alone is not sufficient to give subtype identities. Here we use functional and in vivo time-lapse studies in the zebrafish retina to investigate on the developmental programs leading to ACs specification within the subsequent divisions of Atoh7-positive progenitors. We find evidences that the homeobox transcription factor Barhl2 is an AC subtype identity-biasing factor that turns on within Atoh7-positive descendants. In vivo lineage tracing reveals that particular modes of cell division tend to generate Barhl2-positive precursors from sisters of RGCs. Additionally, Atoh7 indirectly impacts these division modes to regulate the right number of barhl2-expressing cells. We finally find that Atoh7 itself influences the subtypes of Barhl2-dependent ACs. Together, the results from our study uncover lineage-related and molecular logic of subtype specification in the vertebrate retina, by showing that specific AC subtypes arise via a particular mode of cell division and a transcriptional network cascade involving the sequential expression of first atoh7 followed by ptf1a and then barhl2.

Mechanosensitive Notch-Dll4 and Klf2-Wnt9 signaling pathways intersect in guiding valvulogenesis in zebrafish.

In the zebrafish embryo, the onset of blood flow generates fluid shear stress on endocardial cells, which are specialized endothelial cells that line the interior of the heart. High levels of fluid shear stress activate both Notch and Klf2 signaling, which play crucial roles in atrioventricular valvulogenesis. However, it remains unclear why only individual endocardial cells ingress into the cardiac jelly and initiate valvulogenesis. Here, we show that lateral inhibition between endocardial cells, mediated by Notch, singles out Delta-like-4-positive endocardial cells. These cells ingress into the cardiac jelly, where they form an abluminal cell population. Delta-like-4-positive cells ingress in response to Wnt9a, which is produced in parallel through an Erk5-Klf2-Wnt9a signaling cascade also activated by blood flow. Hence, mechanical stimulation activates parallel mechanosensitive signaling pathways that produce binary effects by driving endocardial cells toward either luminal or abluminal fates. Ultimately, these cell fate decisions sculpt cardiac valve leaflets.

The mechanobiology of zebrafish cardiac valve leaflet formation.

Over a lifetime, rhythmic contractions of the heart provide a continuous flow of blood throughout the body. An essential morphogenetic process during cardiac development which ensures unidirectional blood flow is the formation of cardiac valves. These structures are largely composed of extracellular matrix and of endocardial cells, a specialized population of endothelial cells that line the interior of the heart and that are subjected to changing hemodynamic forces. Recent studies have significantly expanded our understanding of this morphogenetic process. They highlight the importance of the mechanobiology of cardiac valve formation and show how biophysical forces due to blood flow drive biochemical and electrical signaling required for the differentiation of cells to produce cardiac valves.

Heg1 and Ccm1/2 proteins control endocardial mechanosensitivity during zebrafish valvulogenesis.

Endothelial cells respond to different levels of fluid shear stress through adaptations of their mechanosensitivity. Currently, we lack a good understanding of how this contributes to sculpting of the cardiovascular system. Cerebral cavernous malformation (CCM) is an inherited vascular disease that occurs when a second somatic mutation causes a loss of CCM1/KRIT1, CCM2, or CCM3 proteins. Here, we demonstrate that zebrafish Krit1 regulates the formation of cardiac valves. Expression of heg1, which encodes a binding partner of Krit1, is positively regulated by blood-flow. In turn, Heg1 stabilizes levels of Krit1 protein, and both Heg1 and Krit1 dampen expression levels of klf2a, a major mechanosensitive gene. Conversely, loss of Krit1 results in increased expression of klf2a and notch1b throughout the endocardium and prevents cardiac valve leaflet formation. Hence, the correct balance of blood-flow-dependent induction and Krit1 protein-mediated repression of klf2a and notch1b ultimately shapes cardiac valve leaflet morphology.

Early Commissural Diencephalic Neurons Control Habenular Axon Extension and Targeting.

Most neuronal populations form on both the left and right sides of the brain. Their efferent axons appear to grow synchronously along similar pathways on each side, although the neurons or their environment often differ between the two hemispheres [1-4]. How this coordination is controlled has received little attention. Frequently, neurons establish interhemispheric connections, which can function to integrate information between brain hemispheres (e.g., [5]). Such commissures form very early, suggesting their potential developmental role in coordinating ipsilateral axon navigation during embryonic development [4]. To address the temporal-spatial control of bilateral axon growth, we applied long-term time-lapse imaging to visualize the formation of the conserved left-right asymmetric habenular neural circuit in the developing zebrafish embryo [6]. Although habenular neurons are born at different times across brain hemispheres [7], we found that elongation of habenular axons occurs synchronously. The initiation of axon extension is not controlled within the habenular network itself but through an early developing proximal diencephalic network. The commissural neurons of this network influence habenular axons both ipsilaterally and contralaterally. Their unilateral absence impairs commissure formation and coordinated habenular axon elongation and causes their subsequent arrest on both sides of the brain. Thus, habenular neural circuit formation depends on a second intersecting commissural network, which facilitates the exchange of information between hemispheres required for ipsilaterally projecting habenular axons. This mechanism of network formation may well apply to other circuits, and has only remained undiscovered due to technical limitations.

Divergent Wnt8a gene expression in teleosts.

The analysis of genes in evolutionarily distant but morphologically similar species is of major importance to unravel the changes in genomes over millions of years, which led to gene silencing and functional diversification. We report the analysis of Wnt8a gene expression in the medakafish and provide a detailed comparison to other vertebrates. In all teleosts analyzed there are two paralogous Wnt8a copies. These show largely overlapping expression in the early developing zebrafish embryo, an evolutionarily distant relative of medaka. In contrast to zebrafish, we find that both maternal and zygotic expression of particularly one Wnt8a paralog has diverged in medaka. While Wnt8a1 expression is mostly conserved at early embryonic stages, the expression of Wnt8a2 differs markedly. In addition, both genes are distinctly expressed during organogenesis unlike the zebrafish homologs, which may hint at the emergence of functional diversification of Wnt8a ligands during evolution.

Tcf7l2 is required for left-right asymmetric differentiation of habenular neurons.

BACKGROUND: Although left-right asymmetries are common features of nervous systems, their developmental bases are largely unknown. In the zebrafish epithalamus, dorsal habenular neurons adopt medial (dHbm) and lateral (dHbl) subnuclear character at very different frequencies on the left and right sides. The left-sided parapineal promotes the elaboration of dHbl character in the left habenula, albeit by an unknown mechanism. Likewise, the genetic pathways acting within habenular neurons to control their asymmetric differentiated character are unknown. RESULTS: In a forward genetic screen for mutations that result in loss of habenular asymmetry, we identified two mutant alleles of tcf7l2, a gene that encodes a transcriptional regulator of Wnt signaling. In tcf7l2 mutants, most neurons on both sides differentiate with dHbl identity. Consequently, the habenulae develop symmetrically, with both sides adopting a pronounced leftward character. Tcf7l2 acts cell automously in nascent equipotential neurons, and on the right side, it promotes dHbm and suppresses dHbl differentiation. On the left, the parapineal prevents this Tcf7l2-dependent process, thereby promoting dHbl differentiation. CONCLUSIONS: Tcf7l2 is essential for lateralized fate selection by habenular neurons that can differentiate along two alternative pathways, thereby leading to major neural circuit asymmetries.