The otic placode is a transient embryonic structure that gives rise to the inner ear. Although inductive signals for otic placode formation have been characterized, less is known about the molecules that respond to these signals within otic primordia. Here, we identify a mutation in zebrafish, hearsay, which disrupts the initiation of placode formation. We show that hearsay disrupts foxi1, a forkhead domain-containing gene, which is expressed in otic precursor cells before placodes become visible; foxi1 appears to be the earliest marker known for the otic anlage. We provide evidence that foxi1 regulates expression of pax8, indicating a very early role for this gene in placode formation. In addition, foxi1 is expressed in the developing branchial arches, and jaw formation is disrupted in hearsay mutant embryos.

The formation of the otic placode is a complex process requiring multiple inductive signals. In zebrafish, fgf3 and fgf8, dlx3b and dlx4b, and foxi1 have been identified as the earliest-acting genes in this process. fgf3 and fgf8 are required as inductive signals, whereas dlx3b, dlx4b, and foxi1 appear to act directly within otic primordia. We have investigated potential interactions among these genes. Depletion of either dlx3b and dlx4b or foxi1 leads to a delay of pax2a expression in the otic primordia and reduction of the otic vesicle. Depletion of both foxi1 and dlx3b results in a complete ablation of otic placode formation. A strong synergistic interaction is also observed among foxi1, fgf3, and fgf8, and a weaker interaction among dlx3b, fgf3, and fgf8. Misexpression of foxi1 can induce expression of pax8, an early marker for the otic primordia, in embryos treated with an inhibitor of fibroblast growth factor (FGF) signaling. Conversely, morpholino knockdown of foxi1 blocks ectopic pax8 expression and otic vesicle formation induced by misexpression of fgf3 and/or fgf8. The observed genetic interactions suggest a model in which foxi1 and dlx3b/dlx4b act in independent pathways together with distinct phases of FGF signaling to promote otic placode induction and development. Developmental Dynamics 230:419 -433, 2004.

Light regulates various aspects of plant growth, and the photoreceptor phytochrome B (phyB) mediates many responses to red light. In a screen for Arabidopsis mutants with phenotypes similar to those of phyB mutants, we isolated two new elf3 mutants. One has weaker morphological phenotypes than previously identified elf3 alleles, but still abolishes circadian rhythms under continuous light. Like phyB mutants, elf3 mutants have elongated hypocotyls and petioles, flower early, and have defects in the red light response. However, we found that elf3 mutations have an additive interaction with a phyB null mutation, with phyA or hy4 null mutations, or with a PHYB overexpression construct, and that an elf3 mutation does not prevent nuclear localization of phyB. These results suggest that either there is substantial redundancy in phyB and elf3 function, or the two genes regulate distinct signaling pathways.

In 404 Lepob/ob F2 progeny of a C57BL/6J (B6) x DBA/2J (DBA) intercross, we mapped a DBA-related quantitative trait locus (QTL) to distal Chr1 at 169.6 Mb, centered about D1Mit110, for diabetes-related phenotypes that included blood glucose, HbA1c, and pancreatic islet histology. The interval was refined to 1.8 Mb in a series of B6.DBA congenic/subcongenic lines also segregating for Lepob. The phenotypes of B6.DBA congenic mice include reduced β-cell replication rates accompanied by reduced β-cell mass, reduced insulin/glucose ratio in blood, reduced glucose tolerance, and persistent mild hypoinsulinemic hyperglycemia. Nucleotide sequence and expression analysis of 14 genes in this interval identified a predicted gene that we have designated “Lisch-like” (Ll) as the most likely candidate. The gene spans 62.7 kb on Chr1qH2.3, encoding a 10-exon, 646–amino acid polypeptide, homologous to Lsr on Chr7qB1 and to Ildr1 on Chr16qB3. The largest isoform of Ll is predicted to be a transmembrane molecule with an immunoglobulin-like extracellular domain and a serine/threonine-rich intracellular domain that contains a 14-3-3 binding domain. Morpholino knockdown of the zebrafish paralog of Ll resulted in a generalized delay in endodermal development in the gut region and dispersion of insulin-positive cells. Mice segregating for an ENU-induced null allele of Ll have phenotypes comparable to the B.D congenic lines. The human ortholog, C1orf32, is in the middle of a 30-Mb region of Chr1q23-25 that has been repeatedly associated with type 2 diabetes.

Nkx2.2 and NeuroD1 are two critical regulators of pancreatic ␤ cell development. Nkx2.2 is a homeodomain transcription factor that is essential for islet cell type specification and mature ␤ cell function. NeuroD1 is a basic helix-loop-helix transcription factor that is critical for islet ␤ cell maturation and maintenance. Although both proteins influence ␤ cell development directly downstream of the endocrine progenitor factor, neurogenin3 (Ngn3), a connection between the two proteins in the regulation of ␤ cell fate and function has yet to be established. In this study, we demonstrate that Nkx2.2 transcriptional activity is required to facilitate the activation of NeuroD1 by Ngn3. Furthermore, Nkx2.2 is necessary to maintain high levels of NeuroD1 expression in developing mouse and zebrafish islets and in mature ␤ cells. Interestingly, Nkx2.2 regulates NeuroD1 through two independent promoter elements, one that is bound and activated directly by Nkx2.2 and one that appears to be regulated by Nkx2.2 through an indirect mechanism. Together, these findings suggest that Nkx2.2 coordinately activates NeuroD1 with Ngn3 within the endocrine progenitor cell and also plays a role in the maintenance of NeuroD1 expression to regulate ␤ cell function in the mature islet. Collectively, these findings further define the conserved regulatory networks involved in islet ␤ cell formation and function.The pancreas is an intricate organ composed of exocrine tissue that secretes digestive enzymes into pancreatic ducts, and the endocrine islets of Langerhans that produce the metabolic hormones insulin, glucagon, somatostatin, pancreatic polypeptide, and ghrelin. The different pancreatic cell lineages arise during the critical developmental events referred to as the primary and secondary transitions (Ref. 1; reviewed in Ref. 2). The primary transition occurs between embryonic day (e) 8.5 and e11.5 2 and encompasses the initial patterning and specification of the pancreatic endoderm, which originates from the foregut. The secondary transition is the critical stage between e12.5 and e15.5 when endocrine and exocrine progenitors expand and a large second wave of differentiation is initiated. The secondary transition also marks an increase in the expression and/or relocalization of a number of transcription factors that are important in pancreatic development, including Pdx1, Ptf1a, Ngn3, Nkx2.2, NeuroD1, Pax4, Pax6, and Nkx6.1 (reviewed in Ref. 3). A large number of studies of these transcription factors have yielded a timeline of gene expression and determined which cell lineages are regulated by each transcription factor (2-7). Notably, temporal and spatial changes in many of the transcription factor expression profiles can re-program a progenitor or precursor cell to alter, prevent, or initiate endocrine differentiation (3, 8 -12). The cumulative findings of these studies have illustrated that the regulation of islet cell differentiation depends on complex relationships between the transcriptional regulatory cascades. These regulato...

We have identified three novel members of the zebrafish forkhead class I gene family, which we have named foxi2, foxi3a, and foxi3b. We have reported previously the identification of zebrafish foxi1, which is required for otic placode and jaw development. Expression analysis shows that foxi2 is expressed within the chordamesoderm during early somitogenesis and the retina and pharyngeal arches during later stages. The foxi3a and foxi3b genes likely represent a recently duplicated pair, and they are similarly expressed in epidermal mucous cells throughout embryogenesis and early larval stages. None of these newly identified FoxI genes are expressed in otic precursor cells and, therefore, are unlikely to share functional overlap with foxi1 in the development of the inner ear. In addition to these zebrafish FoxI paralogs, we have identified 16 new FoxI sequences in species ranging from Ciona intestinalis to Homo sapiens. We present an extensive phylogenetic analysis of the FoxI class that includes these new sequences together with those previously reported. This analysis supports the existence of three subfamilies within the FoxI class, each containing at least one zebrafish member. Developmental Dynamics 228:301-307, 2003.

SUMMARYAlthough the liver and ventral pancreas are thought to arise from a common multipotent progenitor pool, it is unclear whether these progenitors of the hepatopancreas system are specified by a common genetic mechanism. Efforts to determine the role of Hnf1b and Wnt signaling in this crucial process have been confounded by a combination of factors, including a narrow time frame for hepatopancreas specification, functional redundancy among Wnt ligands, and pleiotropic defects caused by either severe loss of Wnt signaling or Hnf1b function. Using a novel hypomorphic hnf1ba zebrafish mutant that exhibits pancreas hypoplasia, as observed in HNF1B monogenic diabetes, we show that hnf1ba plays essential roles in regulating β-cell number and pancreas specification, distinct from its function in regulating pancreas size and liver specification, respectively. By combining Hnf1ba partial loss of function with conditional loss of Wnt signaling, we uncover a crucial developmental window when these pathways synergize to specify the entire ventrally derived hepatopancreas progenitor population. Furthermore, our in vivo genetic studies demonstrate that hnf1ba generates a permissive domain for Wnt signaling activity in the foregut endoderm. Collectively, our findings provide a new model for HNF1B function, yield insight into pancreas and β-cell development, and suggest a new mechanism for hepatopancreatic specification.

Sensory placodes are ectodermal thickenings that give rise to elements of the vertebrate cranial sensory nervous system, including the inner ear and nose. Although mutations have been described in humans, mice and zebrafish that perturb ear and nose development, no mutation is known to prevent sensory placode formation. Thus, it has been postulated that a functional redundancy exists in the genetic mechanisms that govern sensory placode development. We describe a zebrafish deletion mutation, b380, which results in a lack of both otic and olfactory placodes.The b380 deletion removes several known genes and expressed sequence tags, including dlx3 and dlx7, two transcription factors that share a homoeobox domain similar in sequence to the Drosophila Distal-less gene. dlx3 and dlx7 are expressed in an overlapping pattern in the regions that produce the otic and olfactory placodes in zebrafish. We present evidence suggesting that it is specifically the removal of these two genes that leads to the otic and olfactory phenotype of b380 mutants. Using morpholinos, antisense oligonucleotides that effectively block translation of target genes, we find that functional reduction of both dlx genes contributes to placode loss. Expression patterns of the otic marker pax2.1, olfactory marker anxV and eya1, a marker of both placodes, in morpholino-injected embryos recapitulate the reduced expression of these genes seen in b380 mutants. We also examine expression of dlx3 and dlx7 in the morpholino-injected embryos and present evidence for existence of auto- and cross-regulatory control of expression among these genes.We demonstrate that dlx3 is necessary and sufficient for proper otic and olfactory placode development. However, our results indicate that dlx3 and dlx7 act in concert and their importance in placode formation is only revealed by inactivating both paralogs.