The broad goal of this proposal is to define signaling pathways that direct craniofacial development, with the long-term goal of diagnosing and treating abnormalities. Specific focus is on the jaws, derived from the neural crest, and on the mouth. The mouth develops from the """"""""extreme anterior domain"""""""" (EAD), where the ectoderm and endoderm directly juxtapose. The Bradykinin pathway, previously described only in adults, is locally necessary in the EAD, for mouth formation. However, this pathway also has more global effects on face formation. I hypothesize that the EAD is a craniofacial organizer that regulates mouth and cranial neural crest development, through graded Bradykinin signaling, and culminating in NO production. There are two Aims.
Aim 1 will characterize the effects of CPN loss of function on neural crest formation. CPN is a Bradykinin pathway mediator, expressed in the EAD, and loss of function results in abnormal mouth and neural crest tissues. I hypothesize that CPN signaling regulates cranial neural crest determination and serves as a migration stop signal at the facial midline. These hypotheses will be tested after local loss of CPN function using antisense oligonucleotides and a face transplant assay developed in the Sive lab. The effect on neural crest migration will use lineage labeling with fluorescent, injected tracers.
Aim will determine the role of Bradykinin signaling on mouth and neural crest formation. CPN processes the ligand Bradykinin to desArgBradykinin. Both are active ligands and lead to nitric oxide NO production. I hypothesize that Bradykinins act in a concentration-dependent manner, through the Bradykinin receptor, to determine the mouth and cranial neural crest. These hypotheses will be tested by implanting beads loaded with Bradykinin peptides into loss of function embryos, and analyzing correction of defects at different distances from the bead. The spectrum of tissues that can respond to Bradykinin signaling will be determined by analysis of Bradykinin receptor function. This study uses the frog Xenopus, an ideal model for analysis of craniofacial defects, as embryos develop outside the mother, and as the developing face is readily accessible. Hundreds of embryos can be obtained, and these allow rapid assays, since mouth and jaw precursors form 36 hours after fertilization, and are functional by 3 days. The large embryos allow micromanipulation, and gain and loss of function assays can be performed. Facial structure and regulatory genes appear conserved between Xenopus and mammals, suggesting that the information gained from this study will be directly relevant to human biology. Craniofacial anomalies are prevalent, appearing in 1 out of 700 live births, yet most have unknown cause. This project is exciting since Bradykinin signaling in facial development has not previously been described, neither has the role of the EAD as a facial organizer. These studies interface with the NIDCR mission, and have potential to provide etiologies for craniofacial defects and suggest future early, minimally invasive treatments.

Public Health Relevance

The proposed study will investigate the mechanisms by which the face forms, focusing on the Bradykinin pathway, that is known to control blood pressure in adults, but has not been previously been described as functional in developing embryos. I will also examine the function of a small region of the developing face, the extreme anterior domain (EAD), which has the capacity to organize a much larger region into mouth and jaws. This is an exciting and important project that will increase understanding of why craniofacial abnormalities occur so frequently, and in the future, what non-surgical therapies may be considered in their treatment.

National Institute of Health (NIH)
National Institute of Dental & Craniofacial Research (NIDCR)
Individual Predoctoral NRSA for M.D./Ph.D. Fellowships (ADAMHA) (F30)
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NIDCR Special Grants Review Committee (DSR)
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Frieden, Leslie A
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Harvard University
Anatomy/Cell Biology
Schools of Medicine
United States
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Chen, Justin; Jacox, Laura A; Saldanha, Francesca et al. (2017) Mouth development. Wiley Interdiscip Rev Dev Biol 6:
Jacox, Laura; Chen, Justin; Rothman, Alyssa et al. (2016) Formation of a ""Pre-mouth Array"" from the Extreme Anterior Domain Is Directed by Neural Crest and Wnt/PCP Signaling. Cell Rep 16:1445-1455
Jacox, Laura; Sindelka, Radek; Chen, Justin et al. (2014) The extreme anterior domain is an essential craniofacial organizer acting through Kinin-Kallikrein signaling. Cell Rep 8:596-609
Jacox, Laura A; Dickinson, Amanda J; Sive, Hazel (2014) Facial transplants in Xenopus laevis embryos. J Vis Exp :