Frédéric Clotman

Intrahepatic bile ducts develop according to a new mode of tubulogenesis regulated by the transcription factor SOX9.

BACKGROUND & AIMS A number of diseases are characterized by defective formation of the intrahepatic bile ducts. In the embryo, hepatoblasts differentiate to cholangiocytes, which give rise to the bile ducts. Here, we investigated duct development in mouse liver and characterized the role of the SRY-related HMG box transcription factor 9 (SOX9). METHODS We identified SOX9 as a new biliary marker and used it in immunostaining experiments to characterize bile duct morphogenesis. The expression of growth factors was determined by in situ hybridization and immunostaining, and their role was studied on cultured hepatoblasts. SOX9 function was investigated by phenotyping mice with a liver-specific inactivation of Sox9. RESULTS Biliary tubulogenesis started with formation of asymmetrical ductal structures, lined on the portal side by cholangiocytes and on the parenchymal side by hepatoblasts. When the ducts grew from the hilum to the periphery, the hepatoblasts lining the asymmetrical structures differentiated to cholangiocytes, thereby allowing formation of symmetrical ducts lined only by cholangiocytes. We also provide evidence that transforming growth factor-beta promotes differentiation of the hepatoblasts lining the asymmetrical structures. In the absence of SOX9, the maturation of asymmetrical structures into symmetrical ducts was delayed. This was associated with abnormal expression of CCAAT/Enhancer Binding Protein alpha and Homolog of Hairy/Enhancer of Split-1, as well as of the transforming growth factor-beta receptor type II, which are regulators of biliary development. CONCLUSIONS Our results suggest that biliary development proceeds according to a new mode of tubulogenesis characterized by transient asymmetry and whose timing is controlled by SOX9.

Mice lacking factor VII develop normally but suffer fatal perinatal bleeding

Blood coagulation in vivo is initiated by factor VII (FVII) binding to its cellular receptor tissue factor (TF). FVII is the only known ligand for TF, so it was expected that FVII-deficient embryos would have a similar phenotype to TF-deficient embryos, which have defective vitello-embryonic circulation and die around 9.5 days of gestation. Surprisingly, we find that FVII-deficient (FVII−/−) embryos developed normally. FVII−/− mice succumbed perinatally because of fatal haemorrhaging from normal blood vessels. At embryonic day 9.5, maternal–fetal transfer of FVII was undetectable and survival of embryos did not depend on TF–FVII-initiated fibrin formation. Thus, the TF−/− embryonic lethal and the FVII−/− survival-phenotypes suggest a role for TF during embryogenesis beyond fibrin formation.

Molecular interrogation of hypothalamic organization reveals distinct dopamine neuronal subtypes

The hypothalamus contains the highest diversity of neurons in the brain. Many of these neurons can co-release neurotransmitters and neuropeptides in a use-dependent manner. Investigators have hitherto relied on candidate protein-based tools to correlate behavioral, endocrine and gender traits with hypothalamic neuron identity. Here we map neuronal identities in the hypothalamus by single-cell RNA sequencing. We distinguished 62 neuronal subtypes producing glutamatergic, dopaminergic or GABAergic markers for synaptic neurotransmission and harboring the ability to engage in task-dependent neurotransmitter switching. We identified dopamine neurons that uniquely coexpress the Onecut3 and Nmur2 genes, and placed these in the periventricular nucleus with many synaptic afferents arising from neuromedin S+ neurons of the suprachiasmatic nucleus. These neuroendocrine dopamine cells may contribute to the dopaminergic inhibition of prolactin secretion diurnally, as their neuromedin S+ inputs originate from neurons expressing Per2 and Per3 and their tyrosine hydroxylase phosphorylation is regulated in a circadian fashion. Overall, our catalog of neuronal subclasses provides new understanding of hypothalamic organization and function.

Control of hepatic differentiation by activin/TGFbeta signaling.

During liver development, liver progenitors called hepatoblasts differentiate into hepatocytesor biliary cells. Recently, we showed that the segregation between hepatocytes and biliarycells is dependent on a gradient of Activin/TGFβ signaling, and that Activin/TGFβ signalingis controlled in fetal liver by transcription factors of the Onecut family. Here, we discusscandidate factors possibly involved in the formation of the Activin/TGFβ signaling gradient,how this gradient could integrate into a network of signaling pathways modulatinghepatoblast differentiation, and the implications for human liver disease and therapy.

Increased protein glycation in fructosamine-3-kinase-deficient mice

Amines, including those present on proteins, spontaneously react with glucose to form fructosamines in a reaction known as glycation. In the present paper, we have explored, through a targeted gene inactivation approach, the role of FN3K (fructosamine 3-kinase), an intracellular enzyme that phosphorylates free and protein-bound fructose-epsilon-lysines and which is potentially involved in protein repair. Fn3k-/- mice looked healthy and had normal blood glucose and serum fructosamine levels. However, their level of haemoglobin-bound fructosamines was approx. 2.5-fold higher than that of control (Fn3k+/+) or Fn3k+/- mice. Other intracellular proteins were also significantly more glycated in Fn3k-/- mice in erythrocytes (1.8-2.2-fold) and in brain, kidney, liver and skeletal muscle (1.2-1.8-fold), indicating that FN3K removes fructosamines from intracellular proteins in vivo. The urinary excretion of free fructose-epsilon-lysine was 10-20-fold higher in fed mice compared with mice starved for 36 h, and did not differ between fed Fn3k+/+ and Fn3k-/- mice, indicating that food is the main source of urinary fructose-epsilon-lysine in these mice and that FN3K does not participate in the metabolism of food-derived fructose-epsilon-lysine. However, in starved animals, the urinary excretion of fructose-epsilon-lysine was 2.5-fold higher in Fn3k-/- mice compared with Fn3k+/+ or Fn3k+/- mice. Furthermore, a marked increase (5-13-fold) was observed in the concentration of free fructose-epsilon-lysine in tissues of fed Fn3k-/- mice compared with control mice, indicating that FN3K participates in the metabolism of endogenously produced fructose-epsilon-lysine. Taken together, these data indicate that FN3K serves as a protein repair enzyme and also in the metabolism of endogenously produced free fructose-epsilon-lysine.

Hepatic artery malformations associated with a primary defect in intrahepatic bile duct development.

BACKGROUND/AIMS The portal tracts contain bile ducts associated with branches of the portal vein and of the hepatic artery. Hepatic artery malformations are found in diseases in which fetal biliary structures persist after birth (ductal plate malformations). Here we investigated how hepatic artery malformations relate to abnormal bile duct development. METHODS Hepatic artery and biliary development was analyzed in fetuses with Jeune syndrome or Meckel syndrome, which show ductal plate malformations. We also analyzed hepatic artery development in transgenic mice which exhibit biliary anomalies following inactivation of the genes for hepatocyte nuclear factor (HNF)-6 or HNF-1beta, two transcription factors expressed in biliary cells, but not in arteries. RESULTS We show that arterial anomalies occurred in fetuses with Jeune syndrome or Meckel syndrome. We provide the first description of hepatic artery branch development in the mouse and show that inactivation of the Hnf6 or Hnf1beta gene results in anomalies of the hepatic artery branches. In the transgenic mice and in the human syndromes, the biliary anomalies preceded the arterial anomalies. CONCLUSIONS A primary defect in biliary epithelial cells is associated with hepatic artery malformations in mice. Our data provide a model to interpret and study hepatic artery anomalies in humans.

Renshaw cell interneuron specialization is controlled by a temporally restricted transcription factor program

The spinal cord contains a diverse array of physiologically distinct interneuron cell types that subserve specialized roles in somatosensory perception and motor control. The mechanisms that generate these specialized interneuronal cell types from multipotential spinal progenitors are not known. In this study, we describe a temporally regulated transcriptional program that controls the differentiation of Renshaw cells (RCs), an anatomically and functionally discrete spinal interneuron subtype. We show that the selective activation of the Onecut transcription factors Oc1 and Oc2 during the first wave of V1 interneuron neurogenesis is a key step in the RC differentiation program. The development of RCs is additionally dependent on the forkhead transcription factor Foxd3, which is more broadly expressed in postmitotic V1 interneurons. Our demonstration that RCs are born, and activate Oc1 and Oc2 expression, in a narrow temporal window leads us to posit that neuronal diversity in the developing spinal cord is established by the composite actions of early spatial and temporal determinants.

Transcription factor HNF-6/OC-1 inhibits the stimulation of the HNF-3α/Foxa1 gene by TGF-β in mouse liver

A network of liver‐enriched transcription factors controls differentiation and morphogenesis of the liver. These factors interact via direct, feedback, and autoregulatory loops. Previous work has suggested that hepatocyte nuclear factor (HNF)‐6/OC‐1 and HNF‐3α/FoxA1 participate coordinately in this hepatic network. We investigated how HNF‐6 controls the expression of Foxa1. We observed that Foxa1 expression was upregulated in the liver of Hnf6−/− mouse embryos and in bipotential mouse embryonic liver (BMEL) cell lines derived from embryonic Hnf6−/− liver, suggesting that HNF‐6 inhibits the expression of Foxa1. Because no evidence for a direct repression of Foxa1 by HNF‐6 was found, we postulated the existence of an indirect mechanism. We found that the expression of a mediator and targets of the transforming growth factor beta (TGF‐β) signaling was increased both in Hnf6−/− liver and in Hnf6−/− BMEL cell lines. Using these cell lines, we demonstrated that TGF‐β signaling was increased in the absence of HNF‐6, and that this resulted from upregulation of TGF‐β receptor II expression. We also found that TGF‐β can stimulate the expression of Foxa1 in Hnf6+/+ cells and that inhibition of TGF‐β signaling in Hnf6−/− cells down‐regulates the expression of Foxa1. In conclusion, we propose that Foxa1 upregulation in the absence of HNF‐6 results from increased TGF‐β signaling via increased expression of the TGF‐β receptor II. We further conclude that HNF‐6 inhibits Foxa1 by inhibiting the activity of the TGF‐β signaling pathway. This identifies a new mechanism of interaction between liver‐enriched transcription factors whereby one factor indirectly controls another by modulating the activity of a signaling pathway. (HEPATOLOGY 2004;40:1266–1274.)

Dynamic expression of the Onecut transcription factors HNF-6, OC-2 and OC-3 during spinal motor neuron development

The Onecut (OC) transcription factors, namely Hepatocyte nuclear factor 6 (HNF-6), OC-2 and OC-3, are transcriptional activators expressed in liver, pancreas and nervous system during development. Although their expression and roles in endoderm-derived tissues and in the trigeminal ganglia have been investigated, their expression in the CNS has not been described yet. In this study, we report a qualitative and quantitative expression profile of the OC factors in the developing spinal motor neurons (MN). We provide evidence that OC expression is initiated in newly-born MN. At later stages, they are differentially and dynamically expressed in subsets of differentiating motor neuron within the four motor columns. We also show that the expression profile of HNF-6 in spinal MN is conserved in chick embryos. Together, our data unveil a complex and dynamic expression profile of the OC proteins in spinal MN, which suggests that these factors may participate in regulatory networks that control different steps of motor neuron development.

Onecut transcription factors act upstream of Isl1 to regulate spinal motoneuron diversification

During development, spinal motoneurons (MNs) diversify into a variety of subtypes that are specifically dedicated to the motor control of particular sets of skeletal muscles or visceral organs. MN diversification depends on the coordinated action of several transcriptional regulators including the LIM-HD factor Isl1, which is crucial for MN survival and fate determination. However, how these regulators cooperate to establish each MN subtype remains poorly understood. Here, using phenotypic analyses of single or compound mutant mouse embryos combined with gain-of-function experiments in chick embryonic spinal cord, we demonstrate that the transcriptional activators of the Onecut family critically regulate MN subtype diversification during spinal cord development. We provide evidence that Onecut factors directly stimulate Isl1 expression in specific MN subtypes and are therefore required to maintain Isl1 production at the time of MN diversification. In the absence of Onecut factors, we observed major alterations in MN fate decision characterized by the conversion of somatic to visceral MNs at the thoracic levels of the spinal cord and of medial to lateral MNs in the motor columns that innervate the limbs. Furthermore, we identify Sip1 (Zeb2) as a novel developmental regulator of visceral MN differentiation. Taken together, these data elucidate a comprehensive model wherein Onecut factors control multiple aspects of MN subtype diversification. They also shed light on the late roles of Isl1 in MN fate decision.