Distinct Functional and Temporal Requirements for Zebrafish <i>Hdac1</i> during Neural Crest-Derived Craniofacial and Peripheral Neuron Development

<div><p>The regulation of gene expression is accomplished by both genetic and epigenetic means and is required for the precise control of the development of the neural crest. In <i>hdac1^b382</i> mutants, craniofacial cartilage development is defective in two distinct ways. First, fewer <i>hoxb3a, dlx2</i> and <i>dlx3-</i>expressing posterior branchial arch precursors are specified and many of those that are consequently undergo apoptosis. Second, in contrast, normal numbers of progenitors are present in the anterior mandibular and hyoid arches, but chondrocyte precursors fail to terminally differentiate. In the peripheral nervous system, there is a disruption of enteric, DRG and sympathetic neuron differentiation in <i>hdac1^b382</i> mutants compared to wildtype embryos. Specifically, enteric and DRG-precursors differentiate into neurons in the anterior gut and trunk respectively, while enteric and DRG neurons are rarely present in the posterior gut and tail. Sympathetic neuron precursors are specified in <i>hdac1^b382</i> mutants and they undergo generic neuronal differentiation but fail to undergo noradrenergic differentiation. Using the HDAC inhibitor TSA, we isolated enzyme activity and temporal requirements for HDAC function that reproduce <i>hdac1^b382</i> defects in craniofacial and sympathetic neuron development. Our study reveals distinct functional and temporal requirements for zebrafish <i>hdac1</i> during neural crest-derived craniofacial and peripheral neuron development.</p></div

Differential temporal requirements for HDAC function during craniofacial development.

<p>A–D and A’–D’ Wild-type embryos treated with TSA for 24 hpf at different stages of embryonic development. After treatment periods other than 48–72hpf, embryos were washed to remove TSA and then allowed to develop until 3.5 dpf. Embryos were fixed at 3.5 dpf and then stained with alcian blue. A–D lateral views, A’-D’ ventral views. A–A’ are DMSO-treated controls, B–B’ 16–40 hpf TSA-treated embryos, C–C’ 28–52 hpf TSA-treated embryos, D-D’ 48–72 hpf TSA-treated embryos. A’’-D’’ schematic with summary of craniofacial defects at different TSA treatment concentrations. M, mandibular; H, hyoid; cb1-5, cerato-branchials 1-5; BA, branchial arches;+++ wild-type,++ reduced in size compared to wild-type,+Severely reduced compared to wild-type, +/− severely reduced or absent altogether.</p

Effect of 800 nM of TSA on sympathetic neuron <i>th</i> expression.

<p>Percentages reflect the proportion of embryos with <i>th</i>-expression to the total number of treated embryos expressed as a percentage.</p

Craniofacial progenitor differentiation defects in the mandibular and hyoid arches of <i>hdac1^b382</i>mutants.

<p>A, C, E, G wild-type, B, D, F, H <i>hdac1^b382</i> mutants; A, B Lateral views of the head with <i>dlx2</i> expression labeling developing jaw elements in 48 hpf embryos. C, D, Lateral views of the head with <i>dlx3</i> expression labeling developing jaw elements in 48 hpf embryos. E, F ventral views of the head in 68 hpf embryos expressing <i>col2a1</i> in different jaw structures, <i>col2a1</i>. G, H ventral views of the head of 68 hpf embryos expressing <i>sox9a</i> in different jaw elements. M, mandibular; H, hyoid; BA, branchial arches.</p

Neural crest-derived posterior branchial arch progenitor specification is defective in <i>hdac1^b382</i> mutants.

<p>A, C, E, G, I, K wild-type embryos, B, D, F, H, J, L <i>hdac1^b382</i> mutants. A, B Dorsal view of embryos at 25 hpf with <i>dlx2</i> expression labeling the mandibular, hyoid and branchial arch precursor populations. C, D Lateral views of the head region at 25 hpf of <i>crestin</i> expression in the head region, black arrowheads indicate branchial arch populations. E, F, Lateral views of the head region at 25 hpf of <i>hoxb3a</i> expression in the hind-brain and branchial arch precursors. G, H, Lateral views of the head region at 96 hpf of <i>col2a1</i> expression in mandibular, hyoid and branchial arches. I, J, Lateral views of the head region at 56 hpf of TUNEL-positive staining in the head and jaw regions. K, L, Side views of the head region at 32 hpf with <i>tbx1</i> expression highlighting the endodermal pouches. M, mandibular; H, hyoid; BA, branchial arches.</p

Temporal requirements of HDAC function during craniofacial development.

<p>Embryos were assigned the following groups based on the severity of craniofacial malformations when compared to wild-type embryos;+++ wild-type, +++/− Mild malformation,++ Moderate malformation,+ Severe malformation, - Jaw elements absent or not stained.</p><p>Percentages reflect proportion of embryos with alcian blue stained cartilage elements to the total number of treated embryos expressed as a percentage.</p

Effect of 400, 600 and 800 nM of TSA on craniofacial development.

<p>Embryos were assigned the following groups based on the severity of craniofacial malformations when compared to wild-type embryos;+++ wild-type, +++/− Mild malformation,++ Moderate malformation,+Severe malformation, - Jaw elements absent or not stained.</p><p>Percentages reflect proportion of embryos with alcian blue stained cartilage element to the total number of treated embryos expressed as a percentage.</p

Effect of HDAC inhibition on sympathetic neuron differentiation is reversible.

<p>A, B wild-type embryos treated with DMSO (A) and TSA (B) continuously from 28–52 hpf and then fixed at 52 hpf and stained for <i>th</i> expression, black arrowheads indicate sympathetic neurons. C–E wild-type embryos treated under three different conditions, C with DMSO between 28–72 hpf, D with TSA between 28–72 hpf, and E with TSA between 28–52 hpf after which the TSA is washed out and then the embryos are treated in DMSO between 52–72 hpf. All embryos were fixed at 72 hpf and then stained for <i>th</i> expression. Black arrowheads indicate <i>th</i>-xpressing sympathetic neurons, or their absence.</p

Treatment with the HDAC inhibitor TSA can reproduce the <i>hdac1^b382</i> mutant phenotype.

<p>A–d Lateral views of wild-type embryos treated with DMSO, 400 nM, 600 nM and 800 nM TSA from 16–24 hpf after which embryos were fixed and stained for <i>dlx2</i> expression. A–D and A’–D’ Alcian blue stained 3.5 dpf wild-type embryos under different TSA treatment conditions, all embryos were treated between 16 hpf and 3.5 dpf after which embryos were fixed and then stained with alcian blue; A–A’ DMSO controls, B–B’ 400 nM TSA, C–C’ 600 nM TSA, D–D’ 800 nM TSA. A–D lateral view; A’–D’ ventral views. A’’–D’’ schematic with summary of craniofacial defects at different TSA treatment conditions. M, mandibular; H, hyoid; cb1-5, cerato-branchials 1-5; BA, branchial arches,+++ wild-type,++ reduced in size compared to wild-type,+severely reduced compared to wild-type, +/− severely reduced or absent altogether.</p

Sensory, enteric and sympathetic neuron development in <i>hdac1^b382</i> mutants.

<p>A–D Anti-Hu/16A11 staining of wild-type and <i>hdac1^b382</i> mutants at 3dpf and 4dpf. Black arrowheads indicate normally positioned DRG in wild-type and <i>hdac1^b382</i> mutants. White arrowheads indicate examples of DRG neurons in ectopic locations. Black asterisks (A-D and inset) indicate the regions in the tail of wild-type and <i>hdac1^b382</i> mutants where DRG are present. White asterisks (B and inset, D) indicate the regions in the tail of <i>hdac1^b382</i> mutants where DRG are absent. E-G a cross sections of wild-type (E) and examples of 2 sections (F, G) of <i>hdac1^b382</i> mutants illustrating that DRG neurons in mutant embryos are often ectopically localized. H, I wild-type and <i>hdac1^b382</i> mutants with <i>sox10</i> expression in the tail region at 52 hpf, black arrowheads in I indicate distinct foci of <i>sox10</i> expression in mutant in locations where DRG would normally reside. J, K cross-sections of the anterior gut in wild-type and <i>hdac1^b382</i> mutants stained with anti-Hu/16A11 labeling enteric neurons at 4 dpf (white arrowheads). L, M <i>th</i> expression in the cervical sympathetic ganglion (black arrowhead) in wild-type and <i>hdac1^b382</i> mutants at 72 hpf. N, O, <i>phox2b</i> labeling of sympathetic neuron precursors (black arrowheads) in wild-type and <i>hdac1^b382</i> mutants at 56 hpf. P, Q, <i>dbh</i> staining of sympathetic neurons (black arrowheads) in wild-type and <i>hdac1^b382</i> mutants at 48 hpf. R, S, Anti Hu/16A11 staining of wild-type and <i>hdac1^b382</i> mutants at 3dpf with black arrowheads indicating sympathetic neurons.</p