# Novel gene regulatory networks underlying two specialized modes of craniofacial development in a new model system of pupfishes

> **NIH NIH K99** · UNIVERSITY OF CALIFORNIA BERKELEY · 2024 · $148,176

## Abstract

Project Summary
The study of the genetic basis of craniofacial adaptations in naturally divergent groups of species can provide
an opportunity to uncover novel gene networks and genome regulatory elements with clinical relevance. One of
the most remarkable examples of craniofacial diversification across vertebrates is represented in teleost fishes,
often associated with their diverse and highly specialized modes of feeding. In my early postdoctoral career, I
leveraged a recently established evolutionary model of Cyprinodon pupfishes and established it as a new model
system in eco-evo-devo1. This radiation is endemic to San Salvador Island (SSI), and includes a generalist
pupfish (Cyprinodon variegatus), and two trophic specialists, the scale-eater C. desquamator, with longer oral
jaws, increased oral tooth number, and larger mandibular muscles and the molluscivore, C. brontotheroides, with
a shorter jaw with and a novel maxillary extension. Only a few mutations are found to be fixed between
specialists2, with almost all of them found in regulatory or intronic regions, suggesting that the genetic changes
underlying SSI pupfish craniofacial divergence lie in regions of the genome that affect gene expression control.
I hypothesize that temporal and spatial changes in the expression of novel craniofacial candidate genes
within the specialist’s gene regulatory networks (i.e., gba3, pycr3, or galr2a) are caused by dynamics
differences in the chromatin state of their regulatory regions during development, which in turns,
underpins the divergent craniofacial morphological development observed in SSI pupfishes. To test this,
I will combine tissue-specific differential expression analysis (Aim 1) with genome-wide studies of chromatin
accessibility (Aim 2) in three developmental stages (embryonic pharyngula, hatched larvae, and metamorphic
fry) from divergent and non-divergent tissues among SSI pupfish species (oral jaws vs. tail caudal region). In
Aim 1, I will identify tissue-specific and specialist-specific spatiotemporal gene regulatory networks (GRNs) by
analyzing tissue-specific differential gene expression (DGE) (Aim 1.1) and alternative splicing identification (Aim
1.2) between SSI pupfish’s transcriptomes, life stages, and tissues. In Aim 2, I will explore chromatin state
dynamics by analyzing tissue-specific chromatin accessibility (ATAC-seq) across SSI species, life stages, and
tissues. I will integrate the spatiotemporal dynamics of DGE and alternative splice variants with chromatin
accessibility analyses to build a predictive model to identify and test novel craniofacial gene networks and
regulatory regions crucial for pupfish craniofacial divergence (Aim 3.1). The top 5 novel craniofacial candidate
genes within the inferred GRN and with paired differences in chromatin accessibility will be tested using
HCR (Aim 3.1), CRISPR-Cas9 gene editing (Aim 3.2), and Tol2 transgenesis (Aim 3.3). This study will reveal
the different levels of genome regul...

## Key facts

- **NIH application ID:** 10949484
- **Project number:** 1K99DE034058-01
- **Recipient organization:** UNIVERSITY OF CALIFORNIA BERKELEY
- **Principal Investigator:** Maria Fernanda Palominos Chacon
- **Activity code:** K99 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $148,176
- **Award type:** 1
- **Project period:** 2024-08-08 → 2026-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10949484

## Citation

> US National Institutes of Health, RePORTER application 10949484, Novel gene regulatory networks underlying two specialized modes of craniofacial development in a new model system of pupfishes (1K99DE034058-01). Retrieved via AI Analytics 2026-06-12 from https://api.ai-analytics.org/grant/nih/10949484. Licensed CC0.

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