Mette C. Jørgensen

Retinoic Acid Signaling Organizes Endodermal Organ Specification along the Entire Antero-Posterior Axis

Background Endoderm organ primordia become specified between gastrulation and gut tube folding in Amniotes. Although the requirement for RA signaling for the development of a few individual endoderm organs has been established a systematic assessment of its activity along the entire antero-posterior axis has not been performed in this germ layer. Methodology/Principal Findings RA is synthesized from gastrulation to somitogenesis in the mesoderm that is close to the developing gut tube. In the branchial arch region specific levels of RA signaling control organ boundaries. The most anterior endoderm forming the thyroid gland is specified in the absence of RA signaling. Increasing RA in anterior branchial arches results in thyroid primordium repression and the induction of more posterior markers such as branchial arch Hox genes. Conversely reducing RA signaling shifts Hox genes posteriorly in endoderm. These results imply that RA acts as a caudalizing factor in a graded manner in pharyngeal endoderm. Posterior foregut and midgut organ primordia also require RA, but exposing endoderm to additional RA is not sufficient to expand these primordia anteriorly. We show that in chick, in contrast to non-Amniotes, RA signaling is not only necessary during gastrulation, but also throughout gut tube folding during somitogenesis. Our results show that the induction of CdxA, a midgut marker, and pancreas induction require direct RA signaling in endoderm. Moreover, communication between CdxA + cells is necessary to maintain CdxA expression, therefore synchronizing the cells of the midgut primordium. We further show that the RA pathway acts synergistically with FGF4 in endoderm patterning rather than mediating FGF4 activity. Conclusions/Significance Our work establishes that retinoic acid (RA) signaling coordinates the position of different endoderm organs along the antero-posterior axis in chick embryos and could serve as a basis for the differentiation of specific endodermal organs from ES cells.

An Improved Method for Three-dimensional Reconstruction of Protein Expression Patterns in Intact Mouse and Chicken Embryos and Organs

We have developed a wholemount immunofluorescence protocol for the simultaneous detection of up to three proteins in mouse and chicken embryos. Combined with Murrays clearing reagent (BABB) and microscope objectives with long working ranges and high numerical apertures mounted on a confocal microscope, cellular resolution can be obtained in depths offering the possibility of examining expression patterns in entire organs or embryos. Three-dimensional projections of the optical confocal sections can be computed with computer software allowing rotation around any axis. The protocol is robust and we find that most antibodies working on tissue sections also work with this protocol. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Mind bomb 1 is required for pancreatic β-cell formation

During early pancreatic development, Notch signaling represses differentiation of endocrine cells and promotes proliferation of Nkx6-1+Ptf1a+ multipotent progenitor cells (MPCs). Later, antagonistic interactions between Nkx6 transcription factors and Ptf1a function to segregate MPCs into distal Nkx6-1−Ptf1a+ acinar progenitors and proximal Nkx6-1+Ptf1a− duct and β-cell progenitors. Distal cells are initially multipotent, but evolve into unipotent, acinar cell progenitors. Conversely, proximal cells are bipotent and give rise to duct cells and late-born endocrine cells, including the insulin producing β-cells. However, signals that regulate proximodistal (P-D) patterning and thus formation of β-cell progenitors are unknown. Here we show that Mind bomb 1 (Mib1) is required for correct P-D patterning of the developing pancreas and β-cell formation. We found that endoderm-specific inactivation of Mib1 caused a loss of Nkx6-1+Ptf1a− and Hnf1β+ cells and a corresponding loss of Neurog3+ endocrine progenitors and β-cells. An accompanying increase in Nkx6-1−Ptf1a+ and amylase+ cells, occupying the proximal domain, suggests that proximal cells adopt a distal fate in the absence of Mib1 activity. Impeding Notch-mediated transcriptional activation by conditional expression of dominant negative Mastermind-like 1 (Maml1) resulted in a similarly distorted P-D patterning and suppressed β-cell formation, as did conditional inactivation of the Notch target gene Hes1. Our results reveal iterative use of Notch in pancreatic development to ensure correct P-D patterning and adequate β-cell formation.

Ptf1a-mediated control of Dll1 reveals an alternative to the lateral inhibition mechanism

Neurog3-induced Dll1 expression in pancreatic endocrine progenitors ostensibly activates Hes1 expression via Notch and thereby represses Neurog3 and endocrine differentiation in neighboring cells by lateral inhibition. Here we show in mouse that Dll1 and Hes1 expression deviate during regionalization of early endoderm, and later during early pancreas morphogenesis. At that time, Ptf1a activates Dll1 in multipotent pancreatic progenitor cells (MPCs), and Hes1 expression becomes Dll1 dependent over a brief time window. Moreover, Dll1, Hes1 and Dll1/Hes1 mutant phenotypes diverge during organ regionalization, become congruent at early bud stages, and then diverge again at late bud stages. Persistent pancreatic hypoplasia in Dll1 mutants after eliminating Neurog3 expression and endocrine development, together with reduced proliferation of MPCs in both Dll1 and Hes1 mutants, reveals that the hypoplasia is caused by a growth defect rather than by progenitor depletion. Unexpectedly, we find that Hes1 is required to sustain Ptf1a expression, and in turn Dll1 expression in early MPCs. Our results show that Ptf1a-induced Dll1 expression stimulates MPC proliferation and pancreatic growth by maintaining Hes1 expression and Ptf1a protein levels.

Cloning and DNA-binding properties of the rat pancreatic β-cell-specific factor Nkx6.1

The homeodomain (HD) protein Nkx6.1 is the most β‐cell‐specific transcription factor known in the pancreas and its function is critical for the formation of the insulin‐producing β‐cells. However, the target genes, DNA‐binding site, and transcriptional properties of Nkx6.1 are unknown. Using in vitro binding site selection we have identified the DNA sequence of the Nkx6.1 binding site to be TTAATTG/A. A reporter plasmid containing four copies of this sequence is activated by an Nkx6.1HD/VP16 fusion construct. Full‐length Nkx6.1 fails to activate this reporter plasmid in spite of robust interaction with the binding site in vitro. Stable expression of Nkx6.1 in the glucagon‐producing α‐cell‐like MSL‐G‐AN cells induces expression of the endogenous insulin gene in a subset of the cell population. The expression of other known β‐cell‐specific factors such as Pax4, Pax6, Pdx1, GLUT2 and GLP1‐R is unchanged by the introduction of Nkx6.1.

The Prdm13 histone methyltransferase encoding gene is a Ptf1a-Rbpj downstream target that suppresses glutamatergic and promotes GABAergic neuronal fate in the dorsal neural tube.

The basic helix-loop-helix (bHLH) transcriptional activator Ptf1a determines inhibitory GABAergic over excitatory glutamatergic neuronal cell fate in progenitors of the vertebrate dorsal spinal cord, cerebellum and retina. In an in situ hybridization expression survey of PR domain containing genes encoding putative chromatin-remodeling zinc finger transcription factors in Xenopus embryos, we identified Prdm13 as a histone methyltransferase belonging to the Ptf1a synexpression group. Gain and loss of Ptf1a function analyses in both frog and mice indicates that Prdm13 is positively regulated by Ptf1a and likely constitutes a direct transcriptional target. We also showed that this regulation requires the formation of the Ptf1a-Rbp-j complex. Prdm13 knockdown in Xenopus embryos and in Ptf1a overexpressing ectodermal explants lead to an upregulation of Tlx3/Hox11L2, which specifies a glutamatergic lineage and a reduction of the GABAergic neuronal marker Pax2. It also leads to an upregulation of Prdm13 transcription, suggesting an autonegative regulation. Conversely, in animal caps, Prdm13 blocks the ability of the bHLH factor Neurog2 to activate Tlx3. Additional gain of function experiments in the chick neural tube confirm that Prdm13 suppresses Tlx3(+)/glutamatergic and induces Pax2(+)/GABAergic neuronal fate. Thus, Prdm13 is a novel crucial component of the Ptf1a regulatory pathway that, by modulating the transcriptional activity of bHLH factors such as Neurog2, controls the balance between GABAergic and glutamatergic neuronal fate in the dorsal and caudal part of the vertebrate neural tube.

Generation and Characterization of Monoclonal Antibodies against the Transcription Factor Nkx6.1

We present the generation of a panel of monoclonal antibodies (F55A10, F55A12, F64A6B4, and F65A2) against the homeodomain transcription factor Nkx6.1, one of the essential transcription factors that regulates the multistep differentiation process of precursor cells into endocrine β-cells in the pancreas. Expression of Nkx6.1 can be detected in developing pancreatic epithelium and in adult insulin-producing β-cells, making this transcription factor a unique β-cell marker. For production of monoclonal antibodies, RBF mice were immunized with a GST-Nkx6.1 fusion protein containing a 66-amino acid C-terminal fragment of rat Nkx6.1. Four clones were established as stable hybridoma cell lines and the produced antibodies were of the mouse IgG1/κ subtype. When applied for immunohistochemistry on frozen sections of adult mouse pancreas, monoclonal antibodies stain specifically the β-cells in the endocrine islets of Langerhans with patterns comparable to that of a previously produced polyclonal rabbit serum. Monoclonal antibodies can be divided into two groups that appear to recognize different epitopes, as determined by competition ELISA. The presented antibodies are useful tools for the further characterization of the role and function of Nkx6.1 in pancreatic development, especially for use in double-labeling experiments with existing polyclonal rabbit antibodies. (J Histochem Cytochem 54:567-574, 2006)

Specificity of Four Monoclonal Anti-Nkx6-1 Antibodies

The homeodomain transcription factor Nkx6-1 is essential for proper motor neuron development and development of insulin-producing pancreatic β-cells. Nkx6-1 is closely related to Nkx6-2 and Nkx6-3, and all three are expressed in the developing central nervous system and in the developing foregut. Immunohistochemical detection of protein expression is an important tool for description of the temporal differences in expression patterns. When several gene family members like the Nkx6 factors have overlapping or juxtaposed expression domains, there is an elevated risk of unrecognized cross-reactivity, and it is therefore crucial to determine the specificities of antibodies against such targets. In this study we have determined the epitope consensus sequences of four monoclonal antibodies against Nkx6-1 using SPOT membranes, and we refined the results by combined peptide recognition and blocking assays. We show that two of the monoclonal anti-Nkx6-1 antibodies specifically recognize Nkx6-1 and do not cross-react to Nkx6-2 and Nkx6-3. The other two monoclonal anti-Nkx6-1 antibodies are specific to Nkx6-1 in mice but do not recognize Nkx6-1 in chicken and human.

Notch Controls Multiple Pancreatic Cell Fate Regulators Through Direct Hes1-mediated Repression

Notch signaling and its effector Hes1 regulate multiple cell fate choices in the developing pancreas, but few direct target genes are known. Here we use transcriptome analyses combined with chromatin immunoprecipitation with next-generation sequencing (ChIP-seq) to identify direct target genes of Hes1. ChIP-seq analysis of endogenous Hes1 in 266-6 cells, a model of multipotent pancreatic progenitor cells, revealed high-confidence peaks associated with 354 genes. Among these were genes important for tip/trunk segregation such as Ptf1a and Nkx6-1, genes involved in endocrine differentiation such as Insm1 and Dll4, and genes encoding non-pancreatic basic-Helic-Loop-Helix (bHLH) factors such as Neurog2 and Ascl1. Surprisingly, we find that Hes1 binds a large number of loci previously reported to bind Ptf1a, including a site downstream of the Nkx6-1 gene. Notably, we find a number of Hes1 bound genes that are upregulated by γ-secretase inhibition in pancreas explants independently of Neurog3 function, including the tip progenitor/acinar genes; Ptf1a, Gata4, Bhlha15, and Gfi1. Together, our data suggest that Notch signaling suppress the tip cell fate by Hes1-mediated repression of the tip-specific gene regulatory network module that includes transcriptional regulators such as Ptf1a, Gata4, Mist1, and Gfi1. Our data also uncover new molecular targets of Notch signaling that may be important for controlling cell fate choices in pancreas development.

Dll1 and Jag1 are Differentially Required to Specify Proximal and Distal Pancreatic Duct Compartments

Pancreatic β-cells arise from bipotent trunk progenitors (TrPCs) that are specified from multipotent pancreatic progenitors (MPCs) in a Notch-dependent manner. The time window during which Notch signaling is required to specify TrPCs and the identity of the Notch ligands required for this patterning process remain obscure. Here we show that blocking Notch signal transduction before E13 drives progenitors to an acinar fate while blockade after E13 results in amplified and accelerated endocrine differentiation. Mapping of Dll1 and Jag1 expression using IF and novel targeted reporters revealed that uniform Jag1 expression in E10.5 MPCs becomes restricted to distal Ptf1a+ tip progenitors (TiPCs) during PD patterning. Conversely, Dll1 is expressed in scattered MPCs, TrPCs, and TiPCs, and in Neurog3+ endocrine precursors at all stages. Endodermal deletion of Jag1 delays resolution of tip and trunk domains, and at E15.5 distal Sox9+ TrPCs are replaced by Ptf1a+ Mist1+ cells. Remarkably, this occurred with negligible impact on pancreas size and gross morphology of the ductal tree despite the replacement of TrPCs with TiPCs in the duct-lining epithelium. Some Sox9+ progenitors remain proximally in the prospective main duct and loss of β-cells is not complete. However, endodermal deletion of Dll1 and Jag1 ablated the remaining Sox9+ TrPCs and β-cells entirely at E15.5, while normal specification of Sox9+ TrPCs was seen in Dll1 single mutants. Together, our results reveal a shift in competence of Hnf1β+ progenitors around E13 and show that multiple cellular sources provide Notch ligand input to specify different parts of the pancreatic epithelium prior to E13.Summary Notch signaling controls proliferation of multipotent pancreatic progenitor cells (MPCs) and their segregation into bipotent progenitors (BPs) and unipotent pro-acinar cells (PACs). Here we uncover fast ultradian oscillations in the ligand Dll1, and the transcriptional effector Hes1, which proved crucial for MPC expansion. Conversely Jag1, a uniformly expressed ligand, curbed MPC growth, but as expression later segregated to PACs it proved critical for specifying all but the most proximal 5% of BPs, while BPs were entirely lost in Jag1, Dll1 double mutants. Moreover, experimentally induced changes in Hes1 oscillation parameters was associated with selective adoption of BP or PAC fates. Anatomically, ductal morphogenesis and organ architecture is minimally perturbed in Jag1 mutants until later stages, when ductal remodeling fails and signs of acinar-to-ductal metaplasia appear. Our study uncovers oscillating Notch activity in the developing pancreas, which along with modulation by Jag1 is required to coordinate MPC growth and fate.Abstract Notch signaling governs the proliferation of multipotent pancreatic progenitor cells (MPCs) and their segregation into proximal duct/endocrine bipotent progenitors (BPs), and distal unipotent pro-acinar cells (PACs). However, it is unclear which ligands are involved and when they act. Here we show antagonistic effects of Jag1 and Dll1 on MPC proliferation. Furthermore, blocking Notch signaling before E13 shunts MPCs into the distal PAC fate, while later inactivation shunts BPs to endocrine differentiation. All BPs are eliminated in Jag1, Dll1 double mutants, with Jag1 expression in PACs proving critical for specification all but the most proximal 5% of BPs. Hes1 expression is elevated in E12.5 Jag1 mutant pancreas and release from the multipotent state is delayed. However, by E14.5 Hes1 expression becomes attenuated, coincident with the biased adoption of a PAC fate. Remarkably, ductal morphogenesis and organ architecture are minimally perturbed in the absence of Jag1 until later stages, when ductal remodeling fails and signs of acinar-to-ductal metaplasia appear. Our study uncovers that an interplay between Jag1 and Dll1 control the multipotent state and that they together specify the entire pancreatic duct cell lineage.