Wiebke Herzog

Adenohypophysis formation in the zebrafish and its dependence on sonic hedgehog

Formation of the adenohypophysis in mammalian embryos occurs via an invagination of the oral ectoderm to form Rathkes pouch, which becomes exposed to opposing dorsoventral gradients of signaling proteins governing specification of the different hormone-producing pituitary cell types. One signal promoting pituitary cell proliferation and differentiation to ventral cell types is Sonic hedgehog (Shh) from the oral ectoderm. To study pituitary formation and patterning in zebrafish, we cloned four cDNAs encoding different pituitary hormones, prolactin (prl), proopiomelancortin (pomc), thyroid stimulating hormone (tsh), and growth hormone (gh), and analyzed their expression patterns relative to that of the pituitary marker lim3. prl and pomc start to be expressed at the lateral edges of the lim3 expression domain, before pituitary cells move into the head. This indicates that patterning of the pituitary anlage and terminal differentiation of pituitary cells starts while cells are still organized in a placodal fashion at the anterior edge of the developing brain. Following the expression pattern of prl and pomc during development, we show that no pituitary-specific invagination equivalent to Rathkes pouch formation takes place. Rather, pituitary cells move inwards together with stomodeal cells during oral cavity formation, with medial cells of the placode ending up posterior and lateral cells ending up anterior, resulting in an anterior-posterior, rather than a dorsoventral, patterning of the adenohypophysis. Carrying out loss- and gain-of-function experiments, we show that Shh from the ventral diencephalon plays a crucial role during induction, patterning, and growth of the zebrafish adenohypophysis. The phenotypes are very similar to those obtained upon pituitary-specific inactivation or overexpression of Shh in mouse embryo, suggesting that the role of Shh during pituitary development has been largely conserved between fish and mice, despite the different modes of pituitary formation in the two vertebrate classes.

Fgf3 signaling from the ventral diencephalon is required for early specification and subsequent survival of the zebrafish adenohypophysis.

The pituitary gland consists of two major parts: the neurohypophysis, which is of neural origin; and the adenohypophysis, which is of non-neural ectodermal origin. Development of the adenohypophysis is governed by signaling proteins from the infundibulum, a ventral structure of the diencephalon that gives rise to the neurohypophysis. In mouse, the fibroblast growth factors Fgf8, Fgf10 and Fgf18 are thought to affect multiple processes of pituitary development: morphogenesis and patterning of the adenohypophyseal anlage; and survival, proliferation and differential specification of adenohypophyseal progenitor cells. Here, we investigate the role of Fgf3 during pituitary development in the zebrafish, analyzing lia/fgf3 null mutants. We show that Fgf3 signaling from the ventral diencephalon is required in a non-cell autonomous fashion to induce the expression of lim3, pit1 and other pituitary-specific genes in the underlying adenohypophyseal progenitor cells. Despite the absence of such early specification steps, fgf3 mutants continue to form a distinct pituitary anlage of normal size and shape, until adenohypophyseal cells die by apoptosis. We further show that Sonic Hedgehog (Shh) cannot rescue pituitary development, although it is able to induce adenohypophyseal cells in ectopic placodal regions of fgf3 mutants, indicating that Fgf3 does not act via Shh, and that Shh can act independently of Fgf3. In sum, our data suggest that Fgf3 signaling primarily promotes the transcriptional activation of genes regulating early specification steps of adenohypophyseal progenitor cells. This early specification seems to be essential for the subsequent survival of pituitary cells, but not for pituitary morphogenesis or pituitary cell proliferation.

Lymphatic vessels arise from specialized angioblasts within a venous niche

How cells acquire their fate is a fundamental question in developmental and regenerative biology. Multipotent progenitors undergo cell-fate restriction in response to cues from the microenvironment, the nature of which is poorly understood. In the case of the lymphatic system, venous cells from the cardinal vein are thought to generate lymphatic vessels through trans-differentiation. Here we show that in zebrafish, lymphatic progenitors arise from a previously uncharacterized niche of specialized angioblasts within the cardinal vein, which also generates arterial and venous fates. We further identify Wnt5b as a novel lymphatic inductive signal and show that it also promotes the ‘angioblast-to-lymphatic’ transition in human embryonic stem cells, suggesting that this process is evolutionarily conserved. Our results uncover a novel mechanism of lymphatic specification, and provide the first characterization of the lymphatic inductive niche. More broadly, our findings highlight the cardinal vein as a heterogeneous structure, analogous to the haematopoietic niche in the aortic floor.

Genetic Evidence for a Noncanonical Function of Seryl-tRNA Synthetase in Vascular Development

In a recent genetic screen, we identified mutations in genes important for vascular development and maintenance in zebrafish (Jin et al. Dev Biol. 2007;307:29–42). Thirty-two mutations at the adrasteia (adr) locus cause a pronounced dilatation of the aortic arch vessels as well as aberrant patterning of the hindbrain capillaries and, to a lesser extent, intersomitic vessels. This dilatation of the aortic arch vessels does not appear to be caused by increased cell proliferation but is dependent on vascular endothelial growth factor (Vegf) signaling. By positional cloning, we isolated seryl-tRNA synthetase (sars) as the gene affected by the adr mutations. Small interfering RNA knockdown experiments in human umbilical vein endothelial cell cultures indicate that SARS also regulates endothelial sprouting. These analyses of zebrafish and human endothelial cells reveal a new noncanonical function of Sars in endothelial development.

Bmp Signaling Exerts Opposite Effects on Cardiac Differentiation

Rationale: The importance for Bmp signaling during embryonic stem cell differentiation into myocardial cells has been recognized. The question when and where Bmp signaling in vivo regulates myocardial differentiation has remained largely unanswered. Objective: To identify when and where Bmp signaling regulates cardiogenic differentiation. Methods and Results: Here we have observed that in zebrafish embryos, Bmp signaling is active in cardiac progenitor cells prior to their differentiation into cardiomyocytes. Bmp signaling is continuously required during somitogenesis within the anterior lateral plate mesoderm to induce myocardial differentiation. Surprisingly, Bmp signaling is actively repressed in differentiating myocardial cells. We identified the inhibitory Smad6a, which is expressed in the cardiac tissue, to be required to inhibit Bmp signaling and thereby promote expansion of the ventricular myocardium. Conclusion: Bmp signaling exerts opposing effects on myocardial differentiation in the embryo by promoting as well as inhibiting cardiac growth.

The zebrafish common cardinal veins develop by a novel mechanism: lumen ensheathment

The formation and lumenization of blood vessels has been studied in some detail, but there is little understanding of the morphogenetic mechanisms by which endothelial cells (ECs) forming large caliber vessels aggregate, align themselves and finally form a lumen that can support blood flow. Here, we focus on the development of the zebrafish common cardinal veins (CCVs), which collect all the blood from the embryo and transport it back to the heart. We show that the angioblasts that eventually form the definitive CCVs become specified as a separate population distinct from the angioblasts that form the lateral dorsal aortae. The subsequent development of the CCVs represents a novel mechanism of vessel formation, during which the ECs delaminate and align along the inner surface of an existing luminal space. Thereby, the CCVs are initially established as open-ended endothelial tubes, which extend as single EC sheets along the flow routes of the circulating blood and eventually enclose the entire lumen in a process that we term ‘lumen ensheathment’. Furthermore, we found that the initial delamination of the ECs as well as the directional migration within the EC sheet depend on Cadherin 5 function. By contrast, EC proliferation within the growing CCV is controlled by Vascular endothelial growth factor C, which is provided by circulating erythrocytes. Our findings not only identify a novel mechanism of vascular lumen formation, but also suggest a new form of developmental crosstalk between hematopoietic and endothelial cell lineages.

The proneural gene ascl1a is required for endocrine differentiation and cell survival in the zebrafish adenohypophysis

Mammalian basic helix-loop-helix proteins of the achaete-scute family are proneural factors that, in addition to the central nervous system, are required for the differentiation of peripheral neurons and sensory cells, derivatives of the neural crest and placodal ectoderm. Here, in identifying the molecular nature of the pia mutation, we investigate the role of the zebrafish achaete-scute homologue ascl1a during development of the adenohypophysis, an endocrine derivative of the placodal ectoderm. Similar to mutants deficient in Fgf3 signaling from the adjacent ventral diencepahalon, pia mutants display failure of endocrine differentiation of all adenohypophyseal cell types. Shortly after the failed first phase of cell differentiation, the adenohypophysis of pia mutants displays a transient phase of cell death, which affects most, but not all adenohypophyseal cells. Surviving cells form a smaller pituitary rudiment, lack expression of specific adenohypophyseal marker genes (pit1, neurod), while expressing others (lim3, pitx3), and display an ultrastructure reminiscent of precursor cells. During normal development, ascl1a is expressed in the adenohypophysis and the adjacent diencephalon, the source of Fgf3 signals. However, chimera analyses show that ascl1a is required cell-autonomously in adenohypophyseal cells themselves. In fgf3 mutants, adenohypophyseal expression of ascl1a is absent, while implantation of Fgf3-soaked beads into pia mutants enhances ascl1a, but fails to rescue pit1 expression. Together, this suggests that Ascl1a might act downstream of diencephalic Fgf3 signaling to mediate some of the effects of Fgf3 on the developing adenohypophysis.

Angiogenesis in zebrafish

The vasculature consists of an extensively branched network of blood and lymphatic vessels that ensures the efficient circulation and thereby the supply of all tissues with oxygen and nutrients. Research within the last decade has tremendously advanced our understanding of how this complex network is formed, how angiogenic growth is controlled and how differences between individual endothelial cells contribute to achieving this complex pattern. The small size and the optical clarity of the zebrafish embryo in combination with the advancements in imaging technologies cleared the way for the zebrafish as an important in vivo model for elucidating the mechanisms of angiogenesis. In this review we discuss the recent contributions of the analysis of zebrafish vascular development on how vessels establish their characteristic morphology and become patent. We focus on the morphogenetic cellular behaviors as well as the molecular mechanisms that drive these processes in the developing zebrafish embryo.

The hormonal peptide Elabela guides angioblasts to the midline during vasculogenesis

A key step in the de novo formation of the embryonic vasculature is the migration of endothelial precursors, the angioblasts, to the position of the future vessels. To form the first axial vessels, angioblasts migrate towards the midline and coalesce underneath the notochord. Vascular endothelial growth factor has been proposed to serve as a chemoattractant for the angioblasts and to regulate this medial migration. Here we challenge this model and instead demonstrate that angioblasts rely on their intrinsic expression of Apelin receptors (Aplr, APJ) for their migration to the midline. We further show that during this angioblast migration Apelin receptor signaling is mainly triggered by the recently discovered ligand Elabela (Ela). As neither of the ligands Ela or Apelin (Apln) nor their receptors have previously been implicated in regulating angioblast migration, we hereby provide a novel mechanism for regulating vasculogenesis, with direct relevance to physiological and pathological angiogenesis. DOI: http://dx.doi.org/10.7554/eLife.06726.001

Endoglin controls blood vessel diameter through endothelial cell shape changes in response to haemodynamic cues

The hierarchical organization of properly sized blood vessels ensures the correct distribution of blood to all organs of the body, and is controlled via haemodynamic cues. In current concepts, an endothelium-dependent shear stress set point causes blood vessel enlargement in response to higher flow rates, while lower flow would lead to blood vessel narrowing, thereby establishing homeostasis. We show that during zebrafish embryonic development increases in flow, after an initial expansion of blood vessel diameters, eventually lead to vessel contraction. This is mediated via endothelial cell shape changes. We identify the transforming growth factor beta co-receptor endoglin as an important player in this process. Endoglin mutant cells and blood vessels continue to enlarge in response to flow increases, thus exacerbating pre-existing embryonic arterial–venous shunts. Together, our data suggest that cell shape changes in response to biophysical cues act as an underlying principle allowing for the ordered patterning of tubular organs.