David A. Rubin

Zebrafish Express the Common Parathyroid Hormone/Parathyroid Hormone-related Peptide Receptor (PTH1R) and a Novel Receptor (PTH3R) That Is Preferentially Activated by Mammalian and Fugufish Parathyroid Hormone-related Peptide

To further explore the evolution of receptors for parathyroid hormone (PTH) and PTH-related peptide (PTHrP), we searched for zebrafish (z) homologs of the PTH/PTHrP receptor (PTH1R). In mammalian genes encoding this receptor, exons M6/7 and M7 are highly conserved and separated by 81–84 intronic nucleotides. Genomic polymerase chain reaction using degenerate primers based on these exons led to two distinct DNA fragments comprising portions of genes encoding the zPTH1R and the novel zPTH3R. Sequence comparison of both full-length teleost receptors revealed 69% similarity (61% identity), but less homology with zPTH2R. When compared with hPTH1R, zPTH1R showed 76% and zPTH3R 67% amino acid sequence similarity; similarity with hPTH2R was only 59% for both teleost receptors. When expressed in COS-7 cells, zPTH1R bound [Tyr34]hPTH-(1–34)-amide (hPTH), [Tyr36]hPTHrP-(1–36)-amide (hPTHrP), and [Ala29,Glu30,Ala34,Glu35, Tyr36]fugufish PTHrP-(1–36)-amide (fuguPTHrP) with a high apparent affinity (IC50: 1.2–3.5 nm), and was efficiently activated by all three peptides (EC50: 1.1–1.7 nm). In contrast, zPTH3R showed higher affinity for fuguPTHrP and hPTHrP (IC50: 2.1–11.1 nm) than for hPTH (IC50: 118.2–127.0 nm); cAMP accumulation was more efficiently stimulated by fugufish and human PTHrP (EC50: 0.47 ± 0.27 and 0.45 ± 0.16, respectively) than by hPTH (EC50: 9.95 ± 1.5 nm). Agonist-stimulated total inositol phosphate accumulation was observed with zPTH1R, but not zPTH3R.

A G Protein-coupled Receptor from Zebrafish Is Activated by Human Parathyroid Hormone and Not by Human or Teleost Parathyroid Hormone-related Peptide IMPLICATIONS FOR THE EVOLUTIONARY CONSERVATION OF CALCIUM-REGULATING PEPTIDE HORMONES

Genomic and cDNA clones encoding portions of a putative catfish parathyroid hormone (PTH) 2 receptor (PTH2R) led to the isolation of a cDNA encoding a full-length zebrafish PTH2R (zPTH2R). The zPTH2R shared 63 and 60% amino acid sequence identity with human and rat PTH2Rs, respectively, 47–52% identity with mammalian and frog PTH/PTHrP receptors (PTH1R), and less than 37% with other members of this family of G protein-coupled receptors. COS-7 cells expressing zPTH2R(43), a 5′ splice variant that lacked 17 amino acids in the amino-terminal extracellular domain, showed cAMP accumulation when challenged with [Tyr34]hPTH(1–34)-amide (hPTH) (EC50, 1.64 ± 0.95 nm) and [Ile5,Trp23,Tyr36]hPTHrP-(1–36)-amide ([Ile5, Trp23]hPTHrP) (EC50, 46.8 ± 12.1 nm) but not when stimulated with [Tyr36]hPTHrP-(1–36)-amide (hPTHrP), [Trp23,Tyr36]hPTHrP-(1–36)-amide ([Trp23]hPTHrP), or [Ala29,Glu30,Ala34,Glu35,Tyr36]fugufish PTHrP-(1–36)amide (fuguPTHrP). FuguPTHrP also failed to activate the human PTH2R but had similar efficiency and efficacy as hPTH and hPTHrP when tested with cells expressing the human PTH1R. Agonist-dependent activation of zPTH2R was less efficient than that of zPTH2R(43), and both receptor variants showed no cAMP accumulation when stimulated with either secretin, growth hormone-releasing hormone, or calcitonin. The zPTH2R thus has ligand specificity similar to that of the human homolog, which raises the possibility that a PTH-like molecule exists in zebrafish, species which lack parathyroid glands.

Evaluating the Ligand Specificity of Zebrafish Parathyroid Hormone (PTH) Receptors: Comparison of PTH, PTH-Related Protein, and Tuberoinfundibular Peptide of 39 Residues

Homologs of mammalian PTH1 and PTH2 receptors, and a novel PTH3 receptor have been identified in zebrafish (zPTH1, zPTH2, and zPTH3). zPTH1 receptor ligand specificity is similar to that of mammalian PTH1 receptors. The zPTH2 receptor is selective for PTH over PTH-related protein (PTHrP); however, PTH produces only modest cAMP accumulation. A PTH2 receptor-selective peptide, tuberoinfundibular peptide of 39 residues (TIP39), has recently been purified from bovine hypothalamus. The effect of TIP39 has not previously been examined on zebrafish receptors. The zPTH3 receptor was initially described as PTHrP selective based on comparison with the effects of human PTH. We have now examined the ligand specificity of the zebrafish PTH-recognizing receptors expressed in COS-7 cells using a wide range of ligands. TIP39 is a potent agonist for stimulation of cAMP accumulation at two putative splice variants of the zPTH2 receptor (EC50, 2.6 and 5.2 nm); in comparison, PTH is a partial agonist[ maximal effect (Emax) o...

Identification and Characterization of the Murine and Human Gene Encoding the Tuberoinfundibular Peptide of 39 Residues

By screening public databases, we identified human and mouse genomic DNA clones that encode the tuberoinfundibular peptide of 39 residues (TIP39). The TIP39 precursor is encoded by at least three exons; a noncoding exon U1, exon 1 encoding residues -61 (initiator methionine) to -19 of the leader sequence, and exon 2 encoding residues -18 to -1 and residues +1 to +39. Secreted human TIP39 is identical to the previously isolated bovine TIP39, whereas mouse TIP39 differs by four amino acids. Phylogenetic analyses suggested that TIP39, PTH, and PTHrP may have evolved from a common ancestor. Synthetic human and mouse TIP39 showed indistinguishable potencies [EC(50): 0.54 (human) vs. 0.74 nM (mouse)] at the human PTH2-receptor stably expressed in LLCPK(1) cells; furthermore, TIP-(9-39) was an inhibitor of cAMP accumulation stimulated by either [Tyr(34)]PTH(1-34)amide or human/bovine TIP39. In the mouse, an approximately 4.5-kb mRNA encoding TIP39 was identified by Northern blot analysis in testis and, less abundantly, in liver and kidney, whereas other tissues revealed additional smaller transcripts. In situ hybridizations revealed TIP39 expression in seminiferous tubuli and several brain regions, including nucleus ruber, nucleus centralis pontis, and nucleus subparafascicularis thalami. Because PTH2 receptor expression was previously shown to be highest in brain, pancreas, and testis, our findings are consistent with the notion that TIP39 is a neuropeptide which may also have a role in spermatogenesis.

Identification, functional characterization, and developmental expression of two nonallelic parathyroid hormone (PTH)/PTH-related peptide receptor isoforms in Xenopus laevis (Daudin).

Complementary DNAs encoding two nonallelic PTH/PTH-related peptide (PTHrP) receptor (PPR) isoforms, xPPR-A and xPPR-B, were isolated from a kidney complementary DNA library of the tetraploid African clawed frog Xenopus laevis. Both isoforms differ in their coding region by 19 amino acids, and lack the region corresponding to the mammalian exon E2. When expressed in mammalian COS-7 cells, both receptor isoforms bound radiolabeled PTH-(1–34) and PTHrP-(1–36) analogs with comparable affinity, and both unlabeled peptides equivalently stimulated the accumulation of cAMP. xPPR-A also mediated inositol phosphate turnover in COS cells and stimulated channel-mediated current changes in voltage clamp experiments after injection into oocytes. Using ribonuclease protection analysis, significant xPPR-A messenger RNA expression was first detected in neurula stage embryos, which subsequently increased approximately 30-fold during tadpole development. Expression reached a maximum at the metamorphotic climax, when isoform...

The effect of perch availability during pullet rearing and egg laying on musculoskeletal health of caged White Leghorn hens

A major skeletal problem of conventionally caged hens is increased susceptibility to osteoporosis mainly due to lack of exercise. Osteoporosis is characterized by a progressive decrease in mineralized structural bone. Whereas considerable attention has been given to enriching laying cages, little research has been conducted on providing caged pullets with furnishments, in particular perches. The objective of the current study was to determine if metal perches during all or part of the life cycle of White Leghorns affected hen musculoskeletal health, especially at end of lay. Treatments during the pullet phase (hatch to 16.9 wk) entailed cages with and without perches. Four treatments were used during the laying phase (17 to 71 wk of age). Treatment 1 chickens never had access to perches at any point during their life cycle, typical of egg industry practices in the United States for conventional cages. Treatment 2 chickens had access to perches only during the egg-laying phase, which was from 17 to 71 wk of age. Treatment 3 chickens had access to perches only during the pullet phase (0 to 16.9 wk of age). Treatment 4 chickens had perch access throughout their entire life cycle (0 to 71 wk of age). Musculoskeletal health was assessed by measuring muscle weights, bone mineralization, bone fracture incidence, and keel bone deviations. Muscle deposition of 71-wk-old hens increased when given access to perches as pullets. Bone mineralization of 71-wk-old hens also increased if given perch access as adults. However, the disadvantage of the adult perch was the higher incidence of keel deviations and keel fractures at end of lay. The increase in bone mineralization of the keel bone as a result of perch access during the pullet and laying phases was not great enough to prevent a higher incidence of keel bone fractures at end of lay. Perch redesign and placement of perches within the cage to minimize keel fractures and deviations are possible solutions.

Early access to perches in caged White Leghorn pullets

Osteoporosis, a progressive decrease in mineralized structural bone, causes 20 to 35% of all mortalities in caged White Leghorn hens. Previous research has focused on manipulating the egg laying environment to improve skeletal health, with little research on the pullet. The objective of the current study was to determine the effect of perch access on pullet health, bone mineralization, muscle deposition, and stress in caged White Leghorns. From 0 to 17 wk of age, half of the birds were placed in cages with 2 round metal perches, while the other half did not have perches (controls). Bone mineralization and bone size traits were determined in the tibia, femur, sternum, humerus, ulna, radius, and phalange (III carpometacarpal) using dual energy x-ray absorptiometry. Muscle weights were obtained for the breast and left leg (drum and thigh). A sample of pullets from each cage was evaluated for foot health, BW, right adrenal weight, and packed cell volume. Most measurements were taken at 3, 6, and 12 wk of age. Access to perches did not affect breast muscle weight, percentage breast muscle, percentage leg muscle, bone mineral density, bone length, bone width, adrenal weight, packed cell volume, and hyperkeratosis of the foot-pad and toes. There were no differences in BW, bone mineral content, and leg muscle weight at 3 and 6 wk of age. However, at 12 wk of age, BW (P = 0.025), bone mineral content of the tibia, sternum, and humerus (P = 0.015), and the left leg muscle weight (P = 0.006) increased in pullets with access to perches as compared with controls. These results suggest that perch access has beneficial effects on pullet health by stimulating leg muscle deposition and increasing the mineral content of certain bones without causing a concomitant decrease in bone mineral density.

Evolution of the vertebrate pth2 (tip39) gene family and the regulation of PTH type-2 Receptor (pth2r) and its endogenous ligand pth2 by Hedgehog signaling in zebrafish development.

In mammals, parathyroid hormone (PTH), secreted by parathyroid glands, increases calcium levels in the blood from reservoirs in bone. While mammals have two PTH receptor genes, PTH1R and PTH2R, zebrafish has three receptors, pth1r, pth2r, and pth3r. PTH can activate all three zebrafish Pthrs while PTH2 (alias tuberoinfundibular peptide 39, TIP39) preferentially activates zebrafish and mammalian PTH2Rs. We know little about the roles of the PTH2/PTH2R system in the development of any animal. To determine the roles of PTH2 and PTH2R during vertebrate development, we evaluated their expression patterns in developing zebrafish, observed their phylogenetic and conserved synteny relationships with humans, and described the genomic organization of pth2, pth2r, and pth2r splice variants. Expression studies showed that pth2 is expressed in cells adjacent to the ventral part of the posterior tuberculum in the diencephalon, whereas pth2r is robustly expressed throughout the central nervous system. Otic vesicles express both pth2 and pth2r, but heart expresses only pth2. Analysis of mutants showed that hedgehog (Hh) signaling regulates the expression of pth2 transcripts more than that of nearby gnrh2-expressing cells. Genomic analysis showed that a lizard, chicken, and zebra finch lack a PTH2 gene, which is associated with an inversion breakpoint. Likewise, chickens lack PTH2R, while humans lack PTH3R, a case of reciprocally missing ohnologs (paralogs derived from a genome duplication). The considerable evolutionary conservation in genomic structure, synteny relationships, and expression of zebrafish pth2 and pth2r provides a foundation for exploring the endocrine roles of this system in developing vertebrate embryos.

The effect of perches in cages during pullet rearing and egg laying on hen performance, foot health, and plumage

Enrichment of pullet cages with perches has not been studied. Our objective was to determine if access to metal perches during all or part of the life cycle of caged White Leghorns affected egg traits, foot health, and feather condition. Treatment 1 represented control chickens that never had access to perches during their life cycle. Treatment 2 hens had perches only during the egg laying phase of the life cycle (17 to 71 wk of age), whereas treatment 3 chickens had perches during the pullet phase (0 to 16.9 wk of age). Treatment 4 chickens always had access to perches (0 to 71 wk of age). Comparisons between chickens that always had perches with controls that never had perches showed similar performance relative to egg production, cracked eggs, egg weight, shell weight, % shell, and shell thickness. More dirty eggs occurred in laying cages with perches. Feed usage increased resulting in poorer feed efficiency in hens with perch exposure during the pullet phase with no effect during egg laying. Perches did not affect hyperkeratosis of toes and feet. The back claw at 71 wk of age broke less if hens had prior experience with perches during the pullet phase. In contrast, during egg laying, the back claw at 71 wk of age broke more due to the presence of perches in laying cages. Perches in laying cages resulted in shorter trimmed claws and improved back feather scores, but caused poorer breast and tail feather scores. In conclusion, enriching conventional cages with perches during the entire life cycle resulted in similar hen performance compared with controls. Fewer broken back claws but poorer feed efficiency occurred because of prior experience with perches as pullets. Perch presence during egg laying improved back feather scores with more trimmed nails but caused more dirty eggs, broken back claws, and poorer breast and tail feather scores. Although perches allow chickens to express their natural perching instinct, it was not without causing welfare problems.

Duplicated zebrafish co-orthologs of parathyroid hormone-related peptide (PTHrP, Pthlh) play different roles in craniofacial skeletogenesis

In mammals, parathyroid hormone-related peptide (PTHrP, alias PTH-like hormone (Pthlh)) acts as a paracrine hormone that regulates the patterning of cartilage, bone, teeth, pancreas, and thymus. Beyond mammals, however, little is known about the molecular genetic mechanisms by which Pthlh regulates early development. To evaluate conserved pathways of craniofacial skeletogenesis, we isolated two Pthlh co-orthologs from the zebrafish (Danio rerio) and investigated their structural, phylogenetic, and syntenic relationships, expression, and function. Results showed that pthlh duplicates originated in the teleost genome duplication. Zebrafish pthlha and pthlhb were maternally expressed and showed overlapping and distinct zygotic expression patterns during skeletal development that mirrored mammalian expression domains. To explore the regulation of duplicated pthlh genes, we studied their expression patterns in mutants and found that both sox9a and sox9b are upstream of pthlha in arch and fin bud cartilages, but only sox9b is upstream of pthlha in the pancreas. Morpholino antisense knockdown showed that pthlha regulates both sox9a and sox9b in the pharyngeal arches but not in the brain or otic vesicles and that pthlhb does not regulate either sox9 gene, which is likely related to its highly degraded nuclear localization signal. Knockdown of pthlha but not pthlhb caused runx2b overexpression in craniofacial cartilages and premature bone mineralization. We conclude that in normal cartilage development, sox9 upregulates pthlh, which downregulates runx2, and that the duplicated nature of all three of these genes in zebrafish creates a network of regulation by different co-orthologs in different tissues.