Neural plate progenitors give rise to both anterior and posterior pituitary cells.

The pituitary is the master neuroendocrine gland, which regulates body homeostasis. It consists of the anterior pituitary/adenohypophysis harboring hormones producing cells and the posterior pituitary/neurohypophysis, which relays the passage of hormones from the brain to the periphery. It is accepted that the adenohypophysis originates from the oral ectoderm (Rathke's pouch), whereas the neural ectoderm contributes to the neurohypophysis. Single-cell transcriptomics of the zebrafish pituitary showed that cyp26b1-positive astroglial pituicytes of the neurohypophysis and prop1-positive adenohypophyseal progenitors expressed common markers implying lineage relatedness. Genetic tracing identifies that, in contrast to the prevailing dogma, neural plate precursors of zebrafish (her4.3+) and mouse (Sox1+) contribute to both neurohypophyseal and a subset of adenohypophyseal cells. Pituicyte-derived retinoic-acid-degrading enzyme Cyp26b1 fine-tunes differentiation of prop1+ progenitors into hormone-producing cells. These results challenge the notion that adenohypophyseal cells are exclusively derived from non-neural ectoderm and demonstrate that crosstalk between neuro- and adeno-hypophyseal cells affects differentiation of pituitary cells.

The adaptor protein Grb2b is an essential modulator for lympho-venous sprout formation in the zebrafish trunk.

Vegfc/Vegfr3 signaling is critical for lymphangiogenesis, the sprouting of lymphatic vessels. In zebrafish, cells sprouting from the posterior cardinal vein can either form lymphatic precursor cells or contribute to intersegmental vein formation. Both, the Vegfc-dependent differential induction of Prox1a in sprouting cells as well as a Notch-mediated pre-pattern within intersegmental vessels have been associated with the regulation of secondary sprout behavior. However, how exactly a differential lymphatic versus venous sprout cell behavior is achieved is not fully understood. Here, we characterize a zebrafish mutant in the adaptor protein Grb2b, and demonstrate through genetic interaction studies that Grb2b acts within the Vegfr3 pathway. Mutant embryos exhibit phenotypes that are consistent with reduced Vegfr3 signaling outputs prior to the sprouting of endothelial cells from the vein. During secondary sprouting stages, loss of grb2b leads to defective cell behaviors resulting in a loss of parachordal lymphangioblasts, while only partially affecting the number of intersegmental veins. A second GRB2 zebrafish ortholog, grb2a, contributes to the development of lymphatic structures in the meninges and in the head, but not in the trunk. Our results illustrate an essential role of Grb2b in vivo for cell migration to the horizontal myoseptum and for the correct formation of the lymphatic vasculature, while being less critically required in intersegmental vein formation. Thus, there appear to be higher requirements for Grb2b and therefore Vegfr3 downstream signaling levels in lymphatic versus vein precursor-generating sprouts.

Neuronal sFlt1 and Vegfaa determine venous sprouting and spinal cord vascularization.

Formation of organ-specific vasculatures requires cross-talk between developing tissue and specialized endothelial cells. Here we show how developing zebrafish spinal cord neurons coordinate vessel growth through balancing of neuron-derived Vegfaa, with neuronal sFlt1 restricting Vegfaa-Kdrl mediated angiogenesis at the neurovascular interface. Neuron-specific loss of flt1 or increased neuronal vegfaa expression promotes angiogenesis and peri-neural tube vascular network formation. Combining loss of neuronal flt1 with gain of vegfaa promotes sprout invasion into the neural tube. On loss of neuronal flt1, ectopic sprouts emanate from veins involving special angiogenic cell behaviours including nuclear positioning and a molecular signature distinct from primary arterial or secondary venous sprouting. Manipulation of arteriovenous identity or Notch signalling established that ectopic sprouting in flt1 mutants requires venous endothelium. Conceptually, our data suggest that spinal cord vascularization proceeds from veins involving two-tiered regulation of neuronal sFlt1 and Vegfaa via a novel sprouting mode.

Divergence of zebrafish and mouse lymphatic cell fate specification pathways.

In mammals, the homeodomain transcription factor Prox1 acts as the central regulator of lymphatic cell fate. Its restricted expression in a subset of cardinal vein cells leads to a switch towards lymphatic specification and hence represents a prerequisite for the initiation of lymphangiogenesis. Murine Prox1-null embryos lack lymphatic structures, and sustained expression of Prox1 is indispensable for the maintenance of lymphatic cell fate even at adult stages, highlighting the unique importance of this gene for the lymphatic lineage. Whether this pre-eminent role of Prox1 within the lymphatic vasculature is conserved in other vertebrate classes has remained unresolved, mainly owing to the lack of availability of loss-of-function mutants. Here, we re-examine the role of Prox1a in zebrafish lymphangiogenesis. First, using a transgenic reporter line, we show that prox1a is initially expressed in different endothelial compartments, becoming restricted to lymphatic endothelial cells only at later stages. Second, using targeted mutagenesis, we show that Prox1a is dispensable for lymphatic specification and subsequent lymphangiogenesis in zebrafish. In line with this result, we found that the functionally related transcription factors Coup-TFII and Sox18 are also dispensable for lymphangiogenesis. Together, these findings suggest that lymphatic commitment in zebrafish and mice is controlled in fundamentally different ways.

Ccbe1 regulates Vegfc-mediated induction of Vegfr3 signaling during embryonic lymphangiogenesis.

The VEGFC/VEGFR3 signaling pathway is essential for lymphangiogenesis (the formation of lymphatic vessels from pre-existing vasculature) during embryonic development, tissue regeneration and tumor progression. The recently identified secreted protein CCBE1 is indispensible for lymphangiogenesis during development. The role of CCBE1 orthologs is highly conserved in zebrafish, mice and humans with mutations in CCBE1 causing generalized lymphatic dysplasia and lymphedema (Hennekam syndrome). To date, the mechanism by which CCBE1 acts remains unknown. Here, we find that ccbe1 genetically interacts with both vegfc and vegfr3 in zebrafish. In the embryo, phenotypes driven by increased Vegfc are suppressed in the absence of Ccbe1, and Vegfc-driven sprouting is enhanced by local Ccbe1 overexpression. Moreover, Vegfc- and Vegfr3-dependent Erk signaling is impaired in the absence of Ccbe1. Finally, CCBE1 is capable of upregulating the levels of fully processed, mature VEGFC in vitro and the overexpression of mature VEGFC rescues ccbe1 loss-of-function phenotypes in zebrafish. Taken together, these data identify Ccbe1 as a crucial component of the Vegfc/Vegfr3 pathway in the embryo.

A fisheye view on lymphangiogenesis.

Zebrafish have been widely used to study vasculogenesis and angiogenesis, and the vascular system is one of the most intensively studied organ systems in teleosts. It is a little surprising, therefore, that the development of the zebrafish lymphatic network has only been investigated in any detail for less than a decade now. In those last few years, however, significant progress has been made. Due to favorable imaging possibilities within the early zebrafish embryo, we have a very good understanding of what cellular behavior accompanies the formation of the lymphatic system and which cells within the vasculature are destined to contribute to lymphatic vessels. The migration routes of future lymphatic endothelial cells have been monitored in great detail, and a number of transgenic lines have been developed that help to distinguish between arterial, venous, and lymphatic fates in vivo. Furthermore, both forward and reverse genetic tools have been systematically employed to unravel which genes are involved in the process. Not surprisingly, a number of known players were identified (such as vegfc and flt4), but work on zebrafish has also distinguished genes and proteins that had not previously been connected to lymphangiogenesis. Here, we will review these topics and also compare the equivalent stages of lymphatic development in zebrafish and mice. We will, in addition, highlight some of those studies in zebrafish that have helped to identify and to further characterize human disease conditions.

Mutations in KIF11 cause autosomal-dominant microcephaly variably associated with congenital lymphedema and chorioretinopathy.

We have identified KIF11 mutations in individuals with syndromic autosomal-dominant microcephaly associated with lymphedema and/or chorioretinopathy. Initial whole-exome sequencing revealed heterozygous KIF11 mutations in three individuals with a combination of microcephaly and lymphedema from a microcephaly-lymphedema-chorioretinal-dysplasia cohort. Subsequent Sanger sequencing of KIF11 in a further 15 unrelated microcephalic probands with lymphedema and/or chorioretinopathy identified additional heterozygous mutations in 12 of them. KIF11 encodes EG5, a homotetramer kinesin motor. The variety of mutations we have found (two nonsense, two splice site, four missense, and six indels causing frameshifts) are all predicted to have an impact on protein function. EG5 has previously been shown to play a role in spindle assembly and function, and these findings highlight the critical role of proteins necessary for spindle formation in CNS development. Moreover, identification of KIF11 mutations in patients with chorioretinopathy and lymphedema suggests that EG5 is involved in the development and maintenance of retinal and lymphatic structures.

CCBE1 is essential for mammalian lymphatic vascular development and enhances the lymphangiogenic effect of vascular endothelial growth factor-C in vivo.

RATIONALE: Collagen- and calcium-binding EGF domains 1 (CCBE1) has been associated with Hennekam syndrome, in which patients have lymphedema, lymphangiectasias, and other cardiovascular anomalies. Insight into the molecular role of CCBE1 is completely lacking, and mouse models for the disease do not exist. OBJECTIVE: CCBE1 deficient mice were generated to understand the function of CCBE1 in cardiovascular development, and CCBE1 recombinant protein was used in both in vivo and in vitro settings to gain insight into the molecular function of CCBE1. METHODS AND RESULTS: Phenotypic analysis of murine Ccbe1 mutant embryos showed a complete lack of definitive lymphatic structures, even though Prox1(+) lymphatic endothelial cells get specified within the cardinal vein. Mutant mice die prenatally. Proximity ligation assays indicate that vascular endothelial growth factor receptor 3 activation appears unaltered in mutants. Human CCBE1 protein binds to components of the extracellular matrix in vitro, and CCBE1 protein strongly enhances vascular endothelial growth factor-C-mediated lymphangiogenesis in a corneal micropocket assay. CONCLUSIONS: Our data identify CCBE1 as a factor critically required for budding and migration of Prox-1(+) lymphatic endothelial cells from the cardinal vein. CCBE1 probably exerts these effects through binding to components of the extracellular matrix. CCBE1 has little lymphangiogenic effect on its own but dramatically enhances the lymphangiogenic effect of vascular endothelial growth factor-C in vivo. Thus, our data suggest CCBE1 to be essential but not sufficient for lymphangiogenesis.