Deletion of the Ebf1, a mouse deafness gene, causes a dramatic increase in hair cells and support cells of the organ of Corti.

Following up on our previous observation that early B cell factor (EBF) sites are enriched in open chromatin of the developing sensory epithelium of the mouse cochlea, we investigated the effect of deletion of Ebf1 on inner ear development. We used a Cre driver to delete Ebf1 at the otocyst stage before development of the cochlea. We examined the cochlea at postnatal day (P) 1 and found that the sensory epithelium had doubled in size but the length of the cochlear duct was unaffected. We also found that deletion of Ebf1 led to ectopic sensory patches in the Kölliker's organ. Innervation of the developing organ of Corti was disrupted with no obvious spiral bundles. The ectopic patches were also innervated. All the extra hair cells (HCs) within the sensory epithelium and Kölliker's organ contained mechanoelectrical transduction channels, as indicated by rapid uptake of FM1-43. The excessive numbers of HCs were still present in the adult Ebf1 conditional knockout (cKO) animal. The animals had significantly elevated auditory brainstem response thresholds, suggesting that this gene is essential for hearing development.

Peptidylglycine α-amidating monooxygenase is required for atrial secretory granule formation.

The discovery of atrial secretory granules and the natriuretic peptides stored in them identified the atrium as an endocrine organ. Although neither atrial nor brain natriuretic peptide (ANP, BNP) is amidated, the major membrane protein in atrial granules is peptidylglycine α-amidating monooxygenase (PAM), an enzyme essential for amidated peptide biosynthesis. Mice lacking cardiomyocyte PAM (PamMyh6-cKO/cKO) are viable, but a gene dosage-dependent drop in atrial ANP and BNP content occurred. Ultrastructural analysis of adult PamMyh6-cKO/cKO atria revealed a 13-fold drop in the number of secretory granules. When primary cultures of Pam0-Cre-cKO/cKO atrial myocytes (no Cre recombinase, PAM floxed) were transduced with Cre-GFP lentivirus, PAM protein levels dropped, followed by a decline in ANP precursor (proANP) levels. Expression of exogenous PAM in PamMyh6-cKO/cKO atrial myocytes produced a dose-dependent rescue of proANP content; strikingly, this response did not require the monooxygenase activity of PAM. Unlike many prohormones, atrial proANP is stored intact. A threefold increase in the basal rate of proANP secretion by PamMyh6-cKO/cKO myocytes was a major contributor to its reduced levels. While proANP secretion was increased following treatment of control cultures with drugs that block the activation of Golgi-localized Arf proteins and COPI vesicle formation, proANP secretion by PamMyh6-cKO/cKO myocytes was unaffected. In cells lacking secretory granules, expression of exogenous PAM led to the accumulation of fluorescently tagged proANP in the cis-Golgi region. Our data indicate that COPI vesicle-mediated recycling of PAM from the cis-Golgi to the endoplasmic reticulum plays an essential role in the biogenesis of proANP containing atrial granules.

Identifying roles for peptidergic signaling in mice.

Despite accumulating evidence demonstrating the essential roles played by neuropeptides, it has proven challenging to use this information to develop therapeutic strategies. Peptidergic signaling can involve juxtacrine, paracrine, endocrine, and neuronal signaling, making it difficult to define physiologically important pathways. One of the final steps in the biosynthesis of many neuropeptides requires a single enzyme, peptidylglycine α-amidating monooxygenase (PAM), and lack of amidation renders most of these peptides biologically inert. PAM, an ancient integral membrane enzyme that traverses the biosynthetic and endocytic pathways, also affects cytoskeletal organization and gene expression. While mice, zebrafish, and flies lacking Pam (PamKO/KO ) are not viable, we reasoned that cell type-specific elimination of Pam expression would generate mice that could be screened for physiologically important and tissue-specific deficits. Conditional PamcKO/cKO mice, with loxP sites flanking the 2 exons deleted in the global PamKO/KO mouse, were indistinguishable from wild-type mice. Eliminating Pam expression in excitatory forebrain neurons reduced anxiety-like behavior, increased locomotor responsiveness to cocaine, and improved thermoregulation in the cold. A number of amidated peptides play essential roles in each of these behaviors. Although atrial natriuretic peptide (ANP) is not amidated, Pam expression in the atrium exceeds levels in any other tissue. Eliminating Pam expression in cardiomyocytes increased anxiety-like behavior and improved thermoregulation. Atrial and serum levels of ANP fell sharply in PAM myosin heavy chain 6 conditional knockout mice, and RNA sequencing analysis identified changes in gene expression in pathways related to cardiac function. Use of this screening platform should facilitate the development of therapeutic approaches targeted to peptidergic pathways.

A Novel Pattern of Yolk Processing in Developing Snake Eggs (Colubridae: Lampropeltini) and its Functional and Evolutionary Implications.

Early amniotic vertebrates evolved large-yolked eggs that permitted production of well-developed, terrestrial hatchlings. This reproductive pattern required new mechanisms for cellularizing the yolk and mobilizing it for embryonic use. In birds, cells that line the yolk sac cavity phagocytose and digest the yolk material, a pattern that is commonly assumed to be universal among oviparous amniotes. However, recent evidence challenges the assumption that all squamate reptiles conform to the avian developmental pattern. In this paper, scanning electron microscopy and histology were used to study mechanisms of yolk processing in two colubrid snakes, the kingsnake Lampropeltis getula and the milksnake L. triangulum. Endodermal cells from the yolk sac splanchnopleure proliferate massively as they invade the yolk sac cavity, forming elaborate chains of interlinked cells. These cells grow in size as they phagocytose yolk material. Subsequently, vitelline capillaries invade the masses of yolk-laden cells and become coated with the endodermal cells, forming an elaborate meshwork of cell-coated strands. The close association of cells, yolk, and blood vessels allows yolk material to be cellularized, digested, and transported for embryonic use. The overall pattern is like that of the corn snake Pantherophis guttatus, but contrasts markedly with that of birds. Given recent evidence that this developmental pattern may also occur in certain lizards, we postulate that it is ancestral for squamates. Studies of lizards, crocodilians, and turtles are needed to clarify the evolutionary history of this pattern and its implications for the evolution of the amniotic (terrestrial) vertebrate egg.

Cell-type specific knockout of peptidylglycine α-amidating monooxygenase reveals specific behavioral roles in excitatory forebrain neurons and cardiomyocytes.

Neuropeptides and peptide hormones play a crucial role in integrating the many factors that affect physiologic and cognitive processes. The potency of many of these peptides requires an amidated amino acid at the C-terminus; a single enzyme, peptidylglycine α-amidating monooxygenase (PAM), catalyzes this modification. Anxiety-like behavior is known to be altered in mice with a single functional Pam allele (Pam+/- ) and in mice unable to express Pam in excitatory forebrain neurons (PamEmx1-cKO/cKO ) or in cardiomyocytes (PamMyh6-cKO/cKO ). Examination of PAM-positive and glutamic acid decarboxylase 67 (GAD)-positive cells in the amygdala of PamEmx1-cKO/cKO mice demonstrated the absence of PAM in pyramidal neurons and its continued presence in GAD-positive interneurons, suggestive of altered excitatory/inhibitory balance. Additional behavioral tests were used to search for functional alterations in these cell-type specific knockout mice. PamEmx1-cKO/cKO mice exhibited a less focused search pattern for the Barnes Maze escape hole than control or PamMyh6-cKO/cKO mice. While wildtype mice favor interacting with novel objects as opposed to familiar objects, both PamEmx1-cKO/cKO and PamMyh6-cKO/cKO mice exhibited significantly less interest in the novel object. Since PAM levels in the central nervous system of PamMyh6-cKO/cKO mice are unaltered, the behavioral effect observed in these mice may reflect their inability to produce atrial granules and the resulting reduction in serum levels of atrial natriuretic peptide. In the sociability test, male mice of all three genotypes spent more time with same-sex stranger mice; while control females showed no preference for stranger mice, female PamEmx1-cKO/cKO mice showed preference for same-sex stranger mice in all trials.

Morphological features of the yolk processing pattern in the eastern fence lizard, Sceloporus undulatus (Phrynosomatidae).

Features of embryonic development in birds traditionally have been assumed to be shared by sauropsids in general. Herein, we document a pattern of yolk processing and cellularization in the Eastern fence lizard (Sceloporus undulatus) that is fundamentally different from that of birds. In the avian pattern, cells of the yolk sac lining phagocytose, and digest yolk material. These cells release products of digestion into underlying blood vessels for transport back to the embryo. In contrast, microscopic examination of the developing eggs of S. undulatus reveals that the yolk mass is converted into vascularized, "spaghetti-like" strands that fill the yolk sac cavity. Three successive developmental stages are involved. First, the liquid yolk is invaded by proliferating endodermal cells, which phagocytose and digest the yolk material. These cells form clumps that progressively fill the yolk sac cavity. Second, small blood vessels derived from the yolk sac vasculature invade the yolk sac cavity. Third, the endodermal cells become organized in monolayers around these vessels. This arrangement provides a means by which large numbers of endodermal cells can digest yolk, with each cell being positioned to release products of digestion into an adjacent blood vessel for transport to the embryo. The mechanism of yolk processing in this lizard species is similar to that of recently studied snakes. From its phylogenetic distribution, we infer that this pattern probably is ancestral for squamate sauropsids.

Morphological specializations of the yolk sac for yolk processing in embryonic corn snakes (Pantherophis guttatus: Colubridae).

Non-avian reptiles commonly are assumed to be like birds in their overall patterns of development. However, colubrid corn snakes (Pantherophis guttatus) have mechanisms of yolk cellularization and processing that are entirely different from the avian pattern. In birds, a vascular "yolk sac" surrounds and digests the liquid yolk. In contrast, in corn snakes, the yolk material is converted into vascularized cords of yolk-filled cells. In this study, we used stereomicroscopy, histology, and scanning electron microscopy to analyze this unusual developmental pattern in corn snakes. Our observations reveal that the yolk sac cavity is invaded by endodermal cells that proliferate, absorb yolk spheres, and form aggregates of interconnected cells within the liquid yolk mass. As development proceeds, small blood vessels arise from the yolk sac omphalopleure, penetrate into the yolk mass, and become tightly encased in the endodermal cells. The entire vitellus ultimately becomes converted into a mass of vascularized, "spaghetti-like" strands of yolk-laden cells. The resulting arrangement allows yolk to be digested intracellularly and yolk products to be transported to the developing embryo. Indirect evidence for this pattern in other species raises the possibility that it is ancestral for squamates and quite possibly Reptilia in general.